PubAnnotation

Id	Subject	Object	Predicate	Lexical cue
T13	0-99	Sentence	denotes	The current emergence caused by SARS-Cov-2 is dramatically changing the everyday life of all of us.
T14	100-232	Sentence	denotes	Due to the high globalization, new viruses can spread all over the world much faster than ever, infecting the communities worldwide.
T15	233-327	Sentence	denotes	The current technologies and measurements are able to sensibly slow down the infection spread.
T16	328-477	Sentence	denotes	However, their cost is tremendously high, impacting the healthcare systems and causing the shutdown of industries and the lockdown of the population.
T17	478-746	Sentence	denotes	The impact of SARS-Cov-2 on the worldwide economy is estimated to be a 2–3% decline, making this the biggest crisis since the world wars.1 A possible vaccine is hopefully expected to come in 1–2 years, originating a buffer period pretty much uncertain for many people.
T18	747-861	Sentence	denotes	Vaccination is the only way known to accelerate the flock immunity without causing further death by this pandemic.
T19	862-1114	Sentence	denotes	Contemporary history has seen the spread of other viral pandemics such as H2N2 flu (1956–1958), H3N3 flu (1968), HIV (peak reached between 2005 and 2012), SARS (2009), while MERS (2012 to now) and Ebola (1975 to now) viruses are in a prepandemic phase.
T20	1115-1242	Sentence	denotes	The continuous colonization of wild nature lands may touch unknown virus reservoirs causing the spread of contagious epidemics.
T21	1243-1577	Sentence	denotes	Due to these facts, there is a clear urgency in the development of viral treatments to avoid the risk of new pandemics.2 In particular, the possibility to have smart antiviral tools able to efficiently disinfect surfaces, block the viral spreading, enhance the survival of infected people, and boost immunization are highly desirable.
T22	1578-1667	Sentence	denotes	In particular, more investments in the next years are expected in the antiviral research.
T23	1668-2283	Sentence	denotes	Hard nanomaterials (HNMs) have been extensively studied for many types of applications including drug delivery, bioimaging, and biosensing.3−5 The use of nanomaterials in biomedical research is highly developed, reaching in some cases clinical approval.6 Despite that, nanomaterials have been mainly developed for cancer therapy, while scarce attention has been spent on their application in viral infections.7 The continuous virology research has more and more increased into viral replication machinery, allowing the preparation and rationalization of more sophisticated vaccine formulations and viral inhibitors.
T24	2284-2420	Sentence	denotes	The use of HNMs may be one of the keys to provide more effective biomedical agents with a wide spectrum of activity in viral pandemics.8
T25	2421-2521	Sentence	denotes	An increasing number of reports describe how HNMs can be successfully applied to block viral spread.
T26	2522-2681	Sentence	denotes	HNMs can be used at different stages of viral infection: blocking viral entry, hampering interaction with infected host cells, and modulating immune responses.
T27	2682-2904	Sentence	denotes	Due to their core composition (e.g., metal oxides, noble metals), HNMs can have an important antiviral activity inactivating some specific proteins of the capsid or dysregulating radical homeostasis in the virus particles.
T28	2905-3067	Sentence	denotes	Additionally, surface functionalization can sensibly increase HNM antiviral activity, enabling the mimicking of host cells or enhancing the targeting efficiency.9
T29	3068-3180	Sentence	denotes	In this review, we will critically analyze different strategies for the application of HNMs as antiviral agents.
T30	3181-3289	Sentence	denotes	The rational and the synthetic strategies will be highlighted and correlated to different relevant examples.
T31	3290-3400	Sentence	denotes	Particular attention will be paid on the mechanisms of antiviral action and on HNM applicability and efficacy.
T32	3401-3564	Sentence	denotes	For the sake of clarity, this review is divided into three parts, namely: blocking viral entry, antiviral activity in host cells, and stimulation of immune system.
T33	3565-3618	Sentence	denotes	We have focused on the different stages of infection.
T34	3619-3803	Sentence	denotes	In the section dedicated to blocking viral entry, the application of HNMs for surface disinfection and inactivation of the virus prior to interaction with host cells will be described.
T35	3804-4023	Sentence	denotes	Subsequently, the interaction of HNMs after internalization into host cells will be addressed, stressing their antiviral delivery features and their activity in viral replication blockage, leading to host cell survival.
T36	4024-4165	Sentence	denotes	Then, activation of immune response induced by HNMs, triggering the innate and the adaptive (e.g., nanovaccines) immunity, will be presented.
T37	4166-4333	Sentence	denotes	The different adopted strategies will be correlated to the nanomaterial core (e.g., composition, size, shape) and surface (e.g., chemistry, surface charge) properties.
T38	4334-4589	Sentence	denotes	Finally, limits (e.g., unknown long-term toxicity), advantages (e.g., high and wide spectrum virucidal activity), and perspectives will be discussed with particular attention to the applications in viral pandemics (e.g., HIV, SARS, and influenza viruses).
T39	4590-4696	Sentence	denotes	This review is addressed to material and biomaterials scientists who are interested in antiviral research.
T40	4697-4861	Sentence	denotes	We acknowledge that application of HNMs as antiviral agents is still in the early stages; however, we believe that the research on this topic is going to grow soon.
T41	4862-4995	Sentence	denotes	Thus, with this contribution we genuinely hope to inspire researchers in the preparation of smart and efficient HNM antiviral agents.
T42	4997-5017	Sentence	denotes	Blocking Viral Entry
T43	5018-5098	Sentence	denotes	The last decades have been characterized by increasing investigation on viruses.
T44	5099-5277	Sentence	denotes	In particular, their surface charge, protein composition, and host cell entry mechanism have been elucidated, allowing to formulate the first-generation wide spectrum antivirals.
T45	5278-5397	Sentence	denotes	Blocking the viral entry is one of the most common known antimicrobial procedure to stop infections at the early stage.
T46	5398-5524	Sentence	denotes	In this context, antiviral materials have been used for surface disinfection or for epidemic limitation in humans and animals.
T47	5525-5690	Sentence	denotes	Due to their high surface to volume ratio, composition, and tunable surface chemistry, HNMs are now more and more studied as powerful agents in blocking viral entry.
T48	5691-6024	Sentence	denotes	As for other biological interactions, the attachment and entry of viruses into host cells are mediated by multivalent interactions between the surface of the virus and cell surface receptors.10 Nanomaterials can display multivalency that makes them able to compete with the host cells on virus attachment, limiting their infectivity.
T49	6025-6110	Sentence	denotes	The mechanism of antiviral actions relies on the inactivation of the capsid proteins.
T50	6111-6310	Sentence	denotes	As a matter of fact, HNMs have been used for: (1) blocking target proteins for viral entry, (2) capsid protein oxidation, (3) mimicking cell surface, and (4) mechanical rupture of viruses (Figure 1).
T51	6311-6463	Sentence	denotes	These strategies target proteins and mechanisms of entry common in most of the viruses, thus allowing the preparation of wide spectrum antiviral agents.
T52	6464-6571	Sentence	denotes	In this section, the application of different HNMs as powerful inhibitors of viral entry will be discussed.
T53	6572-6741	Sentence	denotes	Figure 1 Illustration of the main mechanisms of blocking virus entry into host cells: capsid denaturation, mimicking cell surface, and mechanical breaking of the virus.
T54	6743-6762	Sentence	denotes	Noble Nanoparticles
T55	6763-6943	Sentence	denotes	Noble nanoparticles (NPs), made of gold and silver, are attractive as antiviral agents for their surface functionalization versatility and their capacity to cleave disulfide bonds.
T56	6944-7030	Sentence	denotes	Their use in disinfection has been extensively studied for different types of viruses.
T57	7031-7187	Sentence	denotes	The morphology and the size of the NPs play a crucial role in their ability to efficiently interact with the capsids and in their toxicity for the organism.
T58	7188-7314	Sentence	denotes	These nanomaterials are characterized by a very large specific surface area (inversely proportional to the particle diameter).
T59	7315-7473	Sentence	denotes	As the particle size becomes smaller and smaller, the percentage of surface atoms increases, creating many unsaturated bonds due to lack of neighboring atoms.
T60	7474-7553	Sentence	denotes	As a consequence, AgNPs and AuNPs have unstable atoms with high surface energy.
T61	7554-7666	Sentence	denotes	This kind of structure provides a lot of contact adsorption sites and reaction points for further modifications.
T62	7667-7772	Sentence	denotes	These chemical features allow to easily combine surface NP atoms with other atoms through chemical bonds.
T63	7773-7904	Sentence	denotes	Besides the composition of the metal core, several studies have pointed out the importance of the control of the surface chemistry.
T64	7905-8054	Sentence	denotes	The surface groups can: (1) stabilize NPs in the biological media, (2) insert targeting agents, and (3) enhance the circulation time inside the body.
T65	8055-8234	Sentence	denotes	Antiviral efficiency can be also enhanced by the multivalency effect, where highly branched ligands are used to locally augment the local concentration of the targeting molecules.
T66	8235-8386	Sentence	denotes	In this section the main strategies and results for silver (AgNPs) and gold (AuNPs) nanoparticles in blocking viral entry will be critically discussed.
T67	8388-8408	Sentence	denotes	Silver Nanoparticles
T68	8409-8540	Sentence	denotes	Many studies have shown that naked AgNPs have a good effect on the control and prevention of a variety of viral diseases (Table 1).
T69	8541-8605	Sentence	denotes	However, the antiviral mechanism of nanosilver is still unclear.
T70	8606-8671	Sentence	denotes	The antiviral action is associated with the following mechanisms:
T71	8672-8852	Sentence	denotes	Nanosilver can prevent the virus from entering the host cells and inhibit the virus from binding to the cell receptor, thereby stopping the virus from infecting the targeted cells.
T72	8853-9000	Sentence	denotes	AgNPs may be able to bind the viral surface protein and inhibit the interaction between the virus and the cell membrane receptors (Figure 2, left).
T73	9001-9225	Sentence	denotes	However, it has been also reported that AgNPs can inactivate the virus through denaturation of surface proteins containing cysteine and methionine residues present on the viral capsid, in a similar way reported for bacteria.
T74	9226-9673	Sentence	denotes	For example, AgNPs smaller than 10 nm were shown to interact with the sulfur-bearing residues of gp120 glycoprotein knobs distributed on the lipid membrane of HIV-1 virus, preventing the virus from binding to CD4 receptor site on the host cells, thus inhibiting the viral infection.11 By means of a viral adsorption assay, it was shown that the AgNP mechanism of anti-HIV action is based on the inhibition of the initial stages of the HIV-1 cycle.
T75	9674-9926	Sentence	denotes	To demonstrate that the antiviral effect of AgNPs is due to the particle structure rather than to silver ions present in solution, the antiviral activity of silver sulfadiazine (AgSD) and silver nitrate (known antibacterial silver salts) was evaluated.
T76	9927-10317	Sentence	denotes	Both salts showed a much lower therapeutic index than AgNPs in vitro, indicating that silver ions themselves are less efficient.12 These results point out that the antiviral efficacy is not only related to the dose of Ag+ ions present in solution but is also regulated by different other parameters (e.g., size, charge, and surface functionalization) associated with the nanosize dimension.
T77	10318-10667	Sentence	denotes	For instance, in the case of Herpesviridae and Paramyxoviridae viruses (both enveloped viruses with embedded viral-encoded glycoproteins), AgNPs can effectively reduce their infectivity, by blocking the interaction between the viral particles and the host cells with an antiviral activity strictly dependent on the size and ζ potential of the AgNPs.
T78	10668-10766	Sentence	denotes	As a general observation, it was reported that smaller nanoparticles have better antiviral effect.
T79	10767-11163	Sentence	denotes	This effect was associated with the increase of the surface area, where smaller-sized AgNPs could bind more efficiently to the viral particles exerting a higher antiviral activity.13 Another study reported the impairment of Peste des petits ruminants virus (PPRV) replication after incubating infectious viral particles with AgNPs, which did not exhibit any virucidal effect even up to 900 μg/mL.
T80	11164-11300	Sentence	denotes	This result suggested that the anti-PPRV activity of the AgNPs is due to the inhibitory effect on viral replication in the target cells.
T81	11301-11407	Sentence	denotes	AgNPs do not prevent the binding of PPRV to host cells, but inhibit the entry of viruses into these cells.
T82	11408-11576	Sentence	denotes	AgNPs can also interact with the surface and core of PPRV, but this interaction cannot kill the virus directly.12 The same results were then confirmed on other viruses.
T83	11577-11687	Sentence	denotes	AgNPs with a diameter of 25 nm inhibited Vaccinia virus replication by preventing viral entry into host cells.
T84	11688-11854	Sentence	denotes	However, AgNPs cannot prevent the virus from adsorbing onto the cells, and this virus is still infectious, indicating that AgNPs lack a direct virus-killing effect.13
T85	11855-12290	Sentence	denotes	Figure 2 Potential antiviral mechanism of AgNPs. (1) AgNPs interact with viral envelope and/or viral surface proteins; (2) AgNPs interact with cell membranes and block viral penetration; (3) AgNPs block cellular pathways of viral entry; (4) AgNPs interact with viral genome; (5) AgNPs interact with viral factors necessary for viral replication; and (6) AgNPs interact with cellular factors necessary for productive viral replication.
T86	12291-12332	Sentence	denotes	Reproduced with permission from ref (14).
T87	12333-12367	Sentence	denotes	Copyright 2016 Taylor and Francis.
T88	12368-12432	Sentence	denotes	Table 1 Antiviral AgNPs and Their Possible Mechanisms of Action
T89	12433-12504	Sentence	denotes	virus shape size (nm) active concentration mechanism of action ref
T90	12505-12567	Sentence	denotes	HIV-1 spherical 1–10 25 μg/mL interaction with gp120 (11)
T91	12568-12629	Sentence	denotes	HIV-1 IIIB – 30–50 440 μg/mL interaction with gp120 (19)
T92	12630-12755	Sentence	denotes	HSV-1, HSV-2, and HPIV-3 – 20–50 not available possible interaction directly with the viral envelope or its protein (20)
T93	12756-12863	Sentence	denotes	Adenovirus type 3 spherical 5–18 25 μg/mL direct destruction of virus particles and DNA structure (15)
T94	12864-13025	Sentence	denotes	H1N1 influenza A virus spherical 5–20 12.5 μg/mL inhibition of respiratory enzymes and electron transport components and interference with DNA function (21)
T95	13026-13133	Sentence	denotes	HBV spherical 10–50 5 μM interaction with double-stranded DNA and/or binding with viral particles (16)
T96	13134-13214	Sentence	denotes	PPRV spherical 5–30 11.1 μg/mL interaction with virus surface and core (12)
T97	13215-13305	Sentence	denotes	Vaccinia virus spherical 25 not available preventing viral entry into host cells (13)
T98	13306-13406	Sentence	denotes	Monkey pox virus (MPV) – 10–80 12.5 μg/mL blocking virus-host cell binding and penetration (22)
T99	13407-13499	Sentence	denotes	Tacaribe virus (TCRV) – 5–10 25 μg/mL inactivation of virus particles before entry (23)
T100	13500-13611	Sentence	denotes	Poliovirus spherical 4–9 3.1 ppm preventing viral particles from binding to the receptors of RD cells (24)
T101	13612-13725	Sentence	denotes	TGEV spherical <20 12.5 μg/mL direct interaction with TGEV surface protein, such as TGEV S glycoprotein (18)
T102	13726-13745	Sentence	denotes	linear 60000–80000
T103	13746-13943	Sentence	denotes	linear 20000–30000 Alternatively, nanosilver can be combined with viral nucleic acids to change the capsid structure, affect the replication of viral genetic material, and make the virus inactive.
T104	13944-14379	Sentence	denotes	For example, TEM analyses have shown that NPs can cause a change of the structure of the Ad3 virus from a hexahedral shape to an irregular shape, destroying its fibers and capsid proteins, leading to inhibition of the virus from binding to the host cells and destroying the DNA structure, preventing adenoviral infection.15 Nanosilver can also bind directly to the double-stranded DNA of hepatitis B virus to inhibit its replication.16
T105	14380-14497	Sentence	denotes	In other studies, it has been demonstrated that silver ions released from nanosilver can directly damage the viruses.
T106	14498-14567	Sentence	denotes	Based in this property, an interesting application has been proposed.
T107	14568-14696	Sentence	denotes	AgNPs were used as a coating on polyurethane condoms, effectively inhibiting the activity of HIV and herpes simplex virus (HSV).
T108	14697-14857	Sentence	denotes	The hypothesized mechanism is that silver ions are transferred directly from oxidized NPs to biological targets, such as viral membrane proteins gp120 and gp41.
T109	14858-14981	Sentence	denotes	In addition, a small amount of silver ion is also released from the coated contraceptives to improve the antiviral level.17
T110	14982-15208	Sentence	denotes	Although the studies on naked AgNPs to reduce viral infectivity have shown their potential as broad-spectrum antiviral agents, the understanding of the specific antiviral action mechanism still needs to be elucidated in depth.
T111	15209-15494	Sentence	denotes	Many studies have shown that the antiviral performance of naked AgNPs is related to their size, and smaller nanoparticles have better antiviral activities.16 In addition to particle size, the antiviral action of AgNP morphology has also attracted interest to fight against coronavirus.
T112	15495-15690	Sentence	denotes	AgNPs and two types of silver nanowires were able to significantly cause an inhibitory effect on coronavirus transmissible gastroenteritis (TGEV)-induced host cell infection and TGEV replication.
T113	15691-15852	Sentence	denotes	The mechanism is likely based on a direct interaction of AgNPs with TGEV surface proteins (e.g., TGEV glycoproteins) to inhibit the beginning of viral infection.
T114	15853-16029	Sentence	denotes	It is possible that AgNPs and Ag nanowires alter the structure of some surface proteins of TGEV and then inhibit their recognition and adhesion to the cellular receptor pAPN.18
T115	16030-16235	Sentence	denotes	Although the potential of AgNPs as antiviral agents has been commonly recognized, unfortunately, their wide biological applications are limited by the risks of self-aggregation and environmental pollution.
T116	16236-16466	Sentence	denotes	Silver ions can be released from the surface of AgNPs and potentially pollute the environment, and their agglomeration into bulkier particles or fibers may change their biological characteristics, diminishing the antiviral effect.
T117	16467-16721	Sentence	denotes	In several cases, it has been reported that naked AgNPs may affect human health.25 Therefore, research and development of AgNPs whose surface is modified or stabilized by protecting molecular layers is an urgent need to overcome these problems (Table 2).
T118	16722-16802	Sentence	denotes	Poly(N-vinyl-2-pyrrolidone) (PVP) is the most commonly used stabilizer of AgNPs.
T119	16803-17408	Sentence	denotes	The PVP-coated AgNPs are able to inhibit the activities of HIV-1, herpes simplex 2 virus (HSV-2), and respiratory syncytial virus (RSV).11,26,27 But compared to foamy carbon, small-sized PVP and BSA-coated AgNPs showed poor antiviral activity to the HIV-1 virus.11 For RSV, PVP-coated AgNPs have a specific binding capacity to the viral surface, evidencing a regular spatial arrangement and a clear interaction with G-protein.26 In addition, to improve the stability of AgNPs, their surface modification with antiviral drugs was proved to reduce the drug resistance caused by the drugs administered alone.
T120	17409-17506	Sentence	denotes	Tannic acid-modified AgNPs showed good antiviral effects on HSV-2 infection in vitro and in vivo.
T121	17507-17600	Sentence	denotes	The viral infection was inhibited only when these NPs directly interacted with HSV-2 virions.
T122	17601-17687	Sentence	denotes	Indeed, the pretreatment of host cells with such AgNPs did inhibit the entry of HSV-2.
T123	17688-17925	Sentence	denotes	Due to the high affinity of tannins to proteins and sugars, tannic acid can bind glycoproteins on the surface of viruses to make them inert, impairing glycoprotein function and preventing viruses from attaching and entering host cells.28
T124	17926-18001	Sentence	denotes	Table 2 Surface-Modified Antiviral AgNPs and Possible Mechanisms of Action
T125	18002-18060	Sentence	denotes	virus shape size (nm) coating mechanism of action ref
T126	18061-18140	Sentence	denotes	HIV-1 spherical 1–10 foamy carbon, PVP and BSA interaction with gp120 (11)
T127	18141-18263	Sentence	denotes	RSV spherical – PVP, BSA, and recombinant F protein (RF 412) interaction with the G-protein on the virus surface (26)
T128	18264-18382	Sentence	denotes	H1N1 influenza virus spherical 2–5 Oseltamivir inhibition of the activity of neuraminidase and hemagglutinin (34)
T129	18383-18399	Sentence	denotes	Amantadine (35)
T130	18400-18415	Sentence	denotes	Zanamivir (36)
T131	18416-18481	Sentence	denotes	inhibition of accumulation of reactive oxygen species (ROS)
T132	18482-18597	Sentence	denotes	HAV, NoV and CoxB4 spherical – polyphosphonium-oligochitosans preventing viral attachment and penetration (29)
T133	18598-18691	Sentence	denotes	MPV spherical 10–80 polysaccharide blocking virus-host cell binding and penetration (22)
T134	18692-18781	Sentence	denotes	TCRV spherical 10 polysaccharide inactivation of virus particles prior to entry (23)
T135	18782-18886	Sentence	denotes	HSV-1 spherical 4 mercaptoethanesulfonate competition for the binding of the virus to the cell (30)
T136	18887-19024	Sentence	denotes	Enterovirus 71 (EV71) spherical 2–5 PEI and antiviral siRNA inhibition of the accumulation of ROS and activation of AKT and p53 (37)
T137	19025-19221	Sentence	denotes	HSV-2 spherical 13, 33, 46 tannic acid direct interaction and blocking of virus attachment, penetration and spread (28) The surface modification can also exert a synergistic antiviral effect.
T138	19222-19360	Sentence	denotes	AgNPs decorated with polyphosphonium-oligochitosan (PQPOC) exhibited moderate to excellent antiviral activity against HAV, NoV, and CoxB4.
T139	19361-19470	Sentence	denotes	In addition, AgNPs could interact with the virion glycoproteins and prevent viral attachment and penetration.
T140	19471-19712	Sentence	denotes	PQPOC can also serve as an effective virus inhibitor by blocking the interaction of the targeted virus with the host through the electrostatic interaction between the cationic polymers and the negatively charged binding sites of the virus.29
T141	19713-19809	Sentence	denotes	Surface-modified AgNPs can also prevent viral infection by competitive adsorption on host cells.
T142	19810-19986	Sentence	denotes	The process of infection of cells by herpes simplex virus type 1 (HSV-1) involves the interaction between viral envelope glycoproteins and heparan sulfate (HS) on cell surface.
T143	19987-20189	Sentence	denotes	Therefore, researchers designed AgNPs capped with mercaptoethanesulfonate (Ag-MES) to compete with the cellular HS through the sulfonate end groups, thereby blocking the virus from entering the cells.30
T144	20190-20696	Sentence	denotes	A few years ago, it was shown that curcumin could prevent the replication and the budding of RSV,31 but the disadvantage of poor solubility and low bioavailability limited its clinical application.32 Curcumin was used as a reducing and capping agent to prepare stable curcumin AgNPs (cAgNPs) under physiological conditions. cAgNPs could reduce cytopathic effects induced by RSV and showed efficient antiviral activity against infection by directly inactivating the virus prior to entry into the host cells.
T145	20697-20782	Sentence	denotes	Its antiviral effect was higher than curcumin alone or unmodified AgNPs (Figure 3).33
T146	20783-20916	Sentence	denotes	Figure 3 Schematic representation of the synthesis of cAgNPs (A) and a proposed inhibition mode of cAgNPs against RSV infection (B).
T147	20917-21095	Sentence	denotes	The inhibition mode of (B) shows that cAgNPs can reduce the binding ability of virus with the binding centers on the surface of cells (b) as compared to those without cAgNPs (a).
T148	21096-21137	Sentence	denotes	Reproduced with permission from ref (33).
T149	21138-21177	Sentence	denotes	Copyright 2016 Royal Chemistry Society.
T150	21178-21335	Sentence	denotes	Alternatively, Zhu et al. prepared AgNPs surface-modified with oseltamivir, amantadine, and zanamivir (Ag@OTV,34 Ag@AM,35 and Ag@ZNV36), by chemical methods.
T151	21336-21456	Sentence	denotes	The results showed that these nanoparticles can directly interact with the virions, resulting in viral function damages.
T152	21457-21540	Sentence	denotes	Overall different studies have reported the capacity of AgNPs to block viral entry.
T153	21541-21739	Sentence	denotes	However, there is not a concerted antiviral mechanism, but their activity differs from case to case, based on viral particle adsorption, capsid structure alteration, or surface protein denaturation.
T154	21740-21970	Sentence	denotes	For AgNPs, the antiviral activity can be associated with different parameters including size, shape, surface charge, and functionalization but also to the topical release of silver ions able to disturb the viral cycle replication.
T155	21971-22148	Sentence	denotes	As described before, bare AgNPs can be used as disinfectant agents, however their use in biological media is limited by their low colloidal stability and potential cytotoxicity.
T156	22149-22333	Sentence	denotes	Surface functionalization can alleviate cytotoxicity, but it can also mask the nanoparticle surface, reducing their affinity for viral particles, thus reducing AgNP antiviral activity.
T157	22334-22459	Sentence	denotes	For these reasons, AgNPs at the moment could find application mainly for surface disinfection and for topical administration.
T158	22460-22550	Sentence	denotes	Further studies are needed to prepare safer AgNP formulations for systemic administration.
T159	22551-22804	Sentence	denotes	In particular, the clarification of the antiviral mechanisms and the use of surface functional groups able to stabilize AgNPs in biological fluids without affecting their prominent antiviral activity are probably the most important challenges to tackle.
T160	22806-22824	Sentence	denotes	Gold Nanoparticles
T161	22825-23055	Sentence	denotes	Compared to AgNPs, AuNPs exhibit reduced toxicity on healthy cells, making them more attractive for in vivo and clinical applications.38 Indeed, AuNPs have been successfully tested as inhibitors of viral entry into the host cells.
T162	23056-23253	Sentence	denotes	AuNPs interact with hemagglutinin (HA), where Au is able to oxidize the disulfide bond of this glycoprotein causing its inactivation, thus impeding the membrane fusion of the virus with host cells.
T163	23254-23715	Sentence	denotes	Targeting HA has emerged as an alternative strategy to the actual therapies (e.g., matrix protein 2 and neuramidase), especially to pandemic viruses that show an accelerated mutation speed of their surface proteins, hence a resistance to conventional treatments increasing their infectivity and mortality.38 This strategy has been applied to influenza (e.g., H1N1, HCV) and herpes viruses.39−44 The activity of AuNPs is proportional to the surface area exposed.
T164	23716-23833	Sentence	denotes	As a consequence, the size and the morphology of these metal NPs play a substantial role in their antiviral activity.
T165	23834-24152	Sentence	denotes	Recently, Kim et al. have reported that porous AuNPs are able to inhibit influenza A infection more efficiently than nonporous AuNPs.39 This effect has been associated with the higher surface area of the porous material that favors their interaction with capsids and thus increases their antiviral activity (Figure 4).
T166	24153-24262	Sentence	denotes	Figure 4 Schematic illustration of inactivation of influenza A virus (IAV) treated with porous AuNP (PoGNP).
T167	24263-24339	Sentence	denotes	PoGNP interacts with IAV surface proteins and cleaves their disulfide bonds.
T168	24340-24395	Sentence	denotes	Inactivated viruses exhibit lower infectivity to cells.
T169	24396-24476	Sentence	denotes	Reproduced with permission under a Creative Commons CC-BY license from ref (39).
T170	24477-24529	Sentence	denotes	Copyright 2020 BioMed Central Ltd., Springer Nature.
T171	24530-24671	Sentence	denotes	Besides the per se antiviral activity, AuNP surface modifications have been developed in order to enhance their overall therapeutic benefits.
T172	24672-24790	Sentence	denotes	The engineering of tailored AuNPs with selected ligands has allowed the preparation of efficient antiviral nanoagents.
T173	24791-24921	Sentence	denotes	The target ligands can be introduced directly during the particle synthesis via ligand exchange reactions or ligand modifications.
T174	24922-25409	Sentence	denotes	For instance, direct reduction of gold ions in the presence of gallic acid produced homogeneous AuNPs able to sensibly reduce herpes simplex virus infection in vitro.40 Compared to free ligand NPs, functionalized AuNPs benefit from the multivalency effect and higher circulation times, decreasing the needed therapeutic concentrations.39 Functionalized AuNPs can present organic groups that mimic host cell surfaces or other specific molecular patterns that selectively target the virus.
T175	25410-25532	Sentence	denotes	Normally, negative charges are used to mimic cell surfaces and favor the interaction between the particles and the capsid.
T176	25533-26024	Sentence	denotes	In particular, sulfonates and organic sulfates have been used for their capacity to attract the virus via capsid protein interaction and block the HA activity.41 AuNPs functionalized with sulfonates showed an increasing inhibition of influenza A compared to the nanoparticles capped with succinic acid.42 This study also demonstrated that there is not a correlation between the negative charge and the antiviral activity, but instead the inhibition depends mainly on the organic groups used.
T177	26025-26213	Sentence	denotes	Thiol-capped AuNPs also displayed powerful inactivation of bovine viral diarrhea virus in vitro.43 Multivalency has been exploited in more complex systems using dendrons as capping agents.
T178	26214-26396	Sentence	denotes	This strategy allows to generate higher concentrations of the target ligand in close proximity to the AuNPs and to increase the binding efficiency of the nanoparticles to the capsid.
T179	26397-26513	Sentence	denotes	The driving force of the antiviral efficiency relies on the concentration of the targeting agent onto the particles.
T180	26514-26691	Sentence	denotes	Sulfonated dendrons were grafted to AuNPs via a sulfide bond and tested for HIV inhibition.44 The results showed that the decorated AuNPs exerted a higher affinity to the virus.
T181	26692-26932	Sentence	denotes	Additionally, comparing AuNPs functionalized with different generation dendrons, those with a third generation displayed the highest inhibition performance with an IC50 below 0.1 μmol/mL, thus making them attractive for in vivo translation.
T182	26933-27399	Sentence	denotes	It is worth noting that the inhibition efficiency is strictly dependent on the available sulfonate groups present on the surface of the NPs, making crucial a thorough characterization of the material.44 The size of the AuNPs clearly plays an important role in the concentration of targeting ligands exposed per particle.45 Indeed, too big NPs have a limited surface area, while too small would not allow an efficient grafting of the dendrons due to steric hindrance.
T183	27400-27598	Sentence	denotes	For instance, it has been shown that dendron-functionalized AuNPs showed a size-dependent antiviral activity for influenza virus, where 14 nm particles exhibited a higher efficiency than 2 nm AuNPs.
T184	27599-27775	Sentence	denotes	This has been associated with the low functionalization grade of the small nanoparticles and to the inappropriate spatial distribution of the interacting ligand/receptor pairs.
T185	27776-27881	Sentence	denotes	The development of viral proteomics has profoundly transformed the antiviral and disinfection strategies.
T186	27882-28003	Sentence	denotes	In particular, small molecules and peptides able to target and block the viral biochemical machinery have been developed.
T187	28004-28185	Sentence	denotes	However, despite these efforts into the drug design, many of these molecules suffer from poor biological effect, low concentration in the diseased areas, and undesired side effects.
T188	28186-28328	Sentence	denotes	In this context, AuNPs have been coupled to biologically inactive small molecules to create biologically active multivalent AuNP therapeutics.
T189	28329-28627	Sentence	denotes	A bright example has been reported by Bowman et al., where the authors functionalized AuNPs with SDC-1721, a small membrane fusion inhibitor of HIV.46 The results demonstrated that, while pure SDC-1721 has low activity, functionalized AuNPs are able to inhibit HIV replication at μM concentrations.
T190	28628-28688	Sentence	denotes	Similar results have been reported using targeting peptides.
T191	28689-28904	Sentence	denotes	In particular, it was evidenced that the functionalized AuNPs can sensibly reduce the IC50 up to 2 orders of magnitude compared to pure peptides.47 Preliminary results in vivo confirmed the biosafety of the AuNPs.48
T192	28906-28954	Sentence	denotes	Nanoparticles Generating Reactive Oxygen Species
T193	28955-29145	Sentence	denotes	One of the main advantages of using NPs compared to oxidized metals relies on the slow release of ions and clusters from these particles, leading to an enhancement of the antiviral activity.
T194	29146-29380	Sentence	denotes	Additionally, the use of metal NPs containing Cu or Fe in ionic form catalyzes the generation of radicals via Fenton and Fenton-like reactions oxidizing the capsid proteins and consequently blocking the viral infection at early stage.
T195	29381-30083	Sentence	denotes	For instance, copper ions (derived from sulfates or iodide salts) have been widely used as antiviral agents because of their activity on several kinds of enveloped and non-enveloped viruses including influenza virus,49−51 herpes simplex virus52−54 and hepatitis A virus.55 Their mechanism of action relies on the formation of Cu+ ions (from soluble salts or nanoparticles) that generate hydroxyl radicals.56 The use of metallic copper nanostructures in the form of particles or sheets has shown only a moderate efficiency due to the low concentration and low release of Cu+.56 For these reasons, Cu+ salts, where the copper ions are readily present in their active monocationic form, have been favored.
T196	30084-30610	Sentence	denotes	In particular, CuI nanoparticles (stable at room temperature) have been extensively studied for deactivation of feline calicivirus56 and H1N1 pandemic influenza virus.57 However, the use of copper salts at high concentrations can irreversibly alter reactive oxygen species (ROS) homeostasis of healthy cells, provoking a general toxicity for the organism, limiting their applications to disinfection.56 Nanostructured cuprous and cupric oxides have been also extensively employed as antiviral agents for in vitro applications.
T197	30611-30820	Sentence	denotes	For instance, cuprous oxide nanoparticles (CuONPs) were successfully employed against hepatitis C.58 In particular, it was found that these NPs exerted a favorable antiviral activity with no cytotoxic effects.
T198	30821-30909	Sentence	denotes	CuONPs target the binding and entry step of viral infection to hepatic cells (Figure 5).
T199	30910-31048	Sentence	denotes	Similar results were reported on the use of CuONPs against HSV-1, however without any profound investigation on the antiviral mechanism.59
T200	31049-31191	Sentence	denotes	Figure 5 Huh7.5.1 cells at 72 h post-infection were stained with HCV-positive serum from patients (green signal) and with DAPI (blue signal).
T201	31192-31263	Sentence	denotes	Cuprous oxide NPs (CuONPs) are able to reduce viral infection in vitro.
T202	31264-31305	Sentence	denotes	Reproduced with permission from ref (58).
T203	31306-31334	Sentence	denotes	Copyright 2015 Elsevier B.V.
T204	31335-31555	Sentence	denotes	Alternatively, zinc salts have been successfully used as antimicrobial agents from research up to clinical trials for viral warts.60,61 More recently, ZnO nanoparticles (ZnONPs) were developed for the treatment of HSV-2.
T205	31556-31711	Sentence	denotes	ZnONPs were prepared with a tetrapod morphology.62 The results showed that they can mimic cell surface interacting with the HS present on the viral capsid.
T206	31712-31965	Sentence	denotes	Additionally, these particles have been used for photocatalysis showing to efficiently destroy the viral proteins upon UV irradiation.62 Besides all these interesting examples, in vivo applications are still needed to validate this therapeutic modality.
T207	31966-32072	Sentence	denotes	Due to the generation of high levels of ROS, the toxicity of copper nanoparticles has been widely debated.
T208	32073-32320	Sentence	denotes	The antiviral activity of copper nanoparticles is generally associated with the release of Cu+ ions in solution, thus the leakage of cytotoxic cationic species can be modulated by surface functionalization before in vitro and in vivo applications.
T209	32321-32433	Sentence	denotes	On the other side, the use of nanomaterials generating ROS can find applications in textile and surface coating.
T210	32434-32629	Sentence	denotes	The general broad virucidal efficiency of copper oxide nanoparticles shown for H1N1 pandemic influenza57 should be tested on SARS-Cov-2 and might be used for improving mask protection efficiency.
T211	32631-32651	Sentence	denotes	Carbon Nanomaterials
T212	32652-32796	Sentence	denotes	Due to their diversity, versatility, and tunable surface chemistry, carbon nanomaterials have been attractive for several types of applications.
T213	32797-32939	Sentence	denotes	In particular, the past decade has seen a tremendous raise in the preparation of performant carbon-based nanomaterials in the antiviral field.
T214	32940-33041	Sentence	denotes	Fullerene and its derivatives are the most studied carbon nanomaterials for their virucidal activity.
T215	33042-33175	Sentence	denotes	Due to the lack of solubility of pristine fullerene, functionalization strategies have been developed to prepare water-soluble drugs.
T216	33176-33454	Sentence	denotes	Investigations in the biomedical field evidenced the membranotropic capacity of fullerene derivatives.63 By modulating shape and functions, fullerene derivatives have been shown to possess antiviral properties through inhibition of viral entry and blockage of viral replication.
T217	33455-33542	Sentence	denotes	From these results, the attention has been directed also to other carbon nanomaterials.
T218	33543-33689	Sentence	denotes	In particular, functional carbon dots (CDs) and graphene oxide (GO) have been investigated for their ability to block viral entry into host cells.
T219	33691-33706	Sentence	denotes	Glycofullerenes
T220	33707-33886	Sentence	denotes	The emerging of mortal viruses, like Ebola or Zika, and the lack of suitable treatments led the academic and the industrial communities to look for alternative therapeutic routes.
T221	33887-34050	Sentence	denotes	Most of these pathogens are RNA enveloped viruses, and they share common infection mechanisms that can be targeted for the preparation of wide spectrum antivirals.
T222	34051-34305	Sentence	denotes	The external surface of the envelope of these viruses is covered by glycans that tightly interact with lectin receptors on host cells.64 This strong interaction allows the attachment of the virions to the cells, followed by internalization and infection.
T223	34306-34401	Sentence	denotes	Blocking lectin receptors is a general strategy used to stop viral infection at an early stage.
T224	34402-34652	Sentence	denotes	Fullerenes have been widely investigated as antiviral molecules, drug carriers, or tissue scaffolds.65 Fullerene applications have been recently extended to the design of mannosylated derivatives to block the entry of viral particles into host cells.
T225	34653-34773	Sentence	denotes	Mannose, due to the high affinity with lectin receptors, competes with the virus in the interaction with the host cells.
T226	34774-34948	Sentence	denotes	For example, one of the targets is the inhibition of viral particles through the interaction of mannose with the dendritic cell-specific ICAM-grabbing non-integrin (DC-sign).
T227	34949-35209	Sentence	denotes	DC-sign receptors mediate the interactions between DCs and T cells.66,67 To exploit these characteristics, mannose was combined with fullerene in the design of the so-called glycofullerenes to study their capacity to inhibit Ebola, Dengue, and other pathogens.
T228	35210-35744	Sentence	denotes	For this purpose, different glycofullerenes were synthesized by changing the number of mannose units (from 12 to 36) and the spacers between the fullerene moieties and by varying steric hindrance in order to obtain a library of molecules.66 The synthetic route is composed of three steps based on “click chemistry”: (1) assembly of glycodendrons by Cu(I)-catalyzed azide–alkyne cycloaddition (CuAAC), (2) synthesis of alkyne-substituted Bingel-Hirsch hexakis-adducts, and (3) the coupling between the last two products again by CuAAC.
T229	35745-35876	Sentence	denotes	To increase the number of mannose moieties up to 36, the glycodendron core was changed from malonate to trialkynyl pentaerythritol.
T230	35877-35956	Sentence	denotes	In order to compare the different derivatives, in vitro studies were performed.
T231	35957-36141	Sentence	denotes	Jurkat cells (lymphocyte T CD4 immortalized cells) expressing DC-sign were used to prove the inhibition capacity of the glycofullerenes on viral infection of Ebola (Figure 6, route A).
T232	36142-36449	Sentence	denotes	The study revealed an IC50 in the μM range for the 12 mannose fullerene, a lower efficiency with the 36 mannose fullerene with a short spacer (PEG, with 2 ethylene oxide units), while a nanomolar IC50 was achieved with 36 mannose fullerenes with a longer spacer (PEG, with 3 ethylene oxide units) (Table 3).
T233	36450-36618	Sentence	denotes	This first proof-of-concept study was then expanded, aiming to obtain a better antiviral activity by increasing the valence and inserting longer and flexible spacers.68
T234	36619-36708	Sentence	denotes	Figure 6 Chemical design and general scheme to block the viral entry by glycofullerenes.
T235	36709-37011	Sentence	denotes	Three different shapes of glycofullerenes can act as inhibitor of viral infection: (A) shape composed of monodisperse fullerene bearing mannose, (B) assembly as “sugar balls” of tridecafullerenes exhibiting mannose on the edges, and (C) supramolecular micellar aggregates of fullerenes bearing mannose.
T236	37012-37091	Sentence	denotes	Table 3 Antiviral Activity of Different Fullerenes Functionalized with Mannose
T237	37092-37227	Sentence	denotes	material functional groups core function number of mannose moieties virus number of atoms between fullerene and mannose IC50 ref
T238	37228-37294	Sentence	denotes	glycofullerene monosaccharide malonate 12 Ebola 8 2 μM (66)
T239	37295-37375	Sentence	denotes	glycofullerene monosaccharide trialkynyl pentaerythritol 36 Ebola 22 68 μM
T240	37376-37457	Sentence	denotes	glycofullerene monosaccharide trialkynyl pentaerythritol 36 Ebola 28 0.3 μM
T241	37458-37535	Sentence	denotes	trideca-fullerenesa monosaccharide malonate 120 Ebola 8 20.375 nM (69)
T242	37536-37606	Sentence	denotes	trideca-fullerenesb monosaccharide malonate 120 Ebola 8 0.667 nM
T243	37607-37692	Sentence	denotes	glycofullerene disaccharide trialkynyl pentaerythritol 36 Zika 29 8.35 nM (70)
T244	37693-37708	Sentence	denotes	Dengue 7.71 nM
T245	37709-37793	Sentence	denotes	trideca-fullerenes disaccharide trialkynyl pentaerythritol 120 Zika 29 0.52 nM
T246	37794-37810	Sentence	denotes	Dengue 0.098 nM
T247	37811-37896	Sentence	denotes	trideca-fullerenes disaccharide trialkynyl pentaerythritol 360 Zika 48 0.067 nM
T248	37897-37913	Sentence	denotes	Dengue 0.035 nM
T249	37914-38010	Sentence	denotes	micellar glycofullerenes monosaccharide trialkynyl pentaerythritol 6 Ebola 23 424 nM (72)
T250	38011-38102	Sentence	denotes	micellar glycofullerenes monosaccharide trialkynyl pentaerythritol 12 Ebola 25 196 nM
T251	38103-38163	Sentence	denotes	a Small spacer between core C60 and surrounding fullerenes.
T252	38164-38224	Sentence	denotes	b Large spacer between core C60 and surrounding fullerenes.
T253	38225-38317	Sentence	denotes	Based on these studies, another class of multivalent fullerene dendrimers was then designed.
T254	38318-38461	Sentence	denotes	A fast and controlled synthetic route was developed to achieve giant globular multivalent fullerenes, containing hundreds of functional groups.
T255	38462-38639	Sentence	denotes	The first study was performed with tridecafullerenes containing 120 mannoses.69 The molecular structure is composed of 12 hexakis C60 surrounding a C60 core (Figure 6, route B).
T256	38640-38944	Sentence	denotes	Compared to the previous study, an IC50 3 orders of magnitude lower was measured on the inhibition of Ebola virus (Table 3).66,68 In order to present more carbohydrates at the periphery of the dendrimer, a trialkynyl pentaerythritol derivative allowed to afford a tridecafullerene with 360 carbohydrates.
T257	38945-39367	Sentence	denotes	In this case, the molecule was synthesized with a C60 tridecafullerene bearing α(1,2)mannobioside.70 The use of this disaccharide was already investigated, showing an increase of affinity with DC-sign receptors by a factor of 3–4.71 The synthetic strategy exploited also the use of strain-promoted copper-free cycloaddition of azides to alkynes (SPAAC) for the coupling of the core fullerene to the surrounding fullerenes.
T258	39368-39450	Sentence	denotes	SPAAC allows an easier purification avoiding the removal of cytotoxic copper ions.
T259	39451-39562	Sentence	denotes	The inhibition performance of this molecule was studied in vitro with viral pseudoparticles of Dengue and Zika.
T260	39563-39673	Sentence	denotes	The comparison was made between 360 and 120 disaccharides tridecafullerenes and 36 disaccharide monofullerene.
T261	39674-39807	Sentence	denotes	The results highlighted a picomolar IC50 inhibition on both Zika and Dengue models for the 360 disaccharide glycofullerene (Table 3).
T262	39808-39922	Sentence	denotes	The ability to inhibit other types of viruses allows the use of glycofullerenes as broad spectrum antiviral drugs.
T263	39923-40019	Sentence	denotes	Moreover, the negligible toxicity to other cells proved the biocompatibility of these molecules.
T264	40020-40252	Sentence	denotes	Following an alternative strategy, a supramolecular assembly of monodisperse glycofullerenes, leading to the formation of micelles, was achieved and tested.72 These micelles present a uniform and spherical shape (Figure 6, route C).
T265	40253-40457	Sentence	denotes	The aggregation synthetic route is faster compared to a controlled synthesis of giant glycofullerenes, but it might suffer from low reproducibility and batch-to-batch differences between each formulation.
T266	40458-40721	Sentence	denotes	This self-assembled C60 functionalized with 6 or 12 mannoses exposes a large amount of carbohydrate at the surface, leading to an inhibition of Ebola virus in the nanomolar range (IC50 of 424 nM for six mannoses and 196 nM for 12 mannoses, respectively, Table 3).
T267	40722-40812	Sentence	denotes	Further in vitro studies evidenced again a good biocompatibility of these glycofullerenes.
T268	40813-40950	Sentence	denotes	While there are no in vivo studies yet, these promising results enlarge the panel of molecules in the fight against new emerging viruses.
T269	40951-41081	Sentence	denotes	Functionalized fullerenes have been used for their ability to compete with viral particles through lectin receptors in host cells.
T270	41082-41322	Sentence	denotes	There has been a tremendous advancement in the functionalization of fullerenes leading to the preparation of derivatives with a high amount of mannose, capable to enhance the multivalency effect and thus to increase the therapeutic outcome.
T271	41323-41390	Sentence	denotes	First, glycofullerenes do not have an intrinsic virucidal activity.
T272	41391-41481	Sentence	denotes	They can reduce the infectivity, but they are not able to completely inactivate the virus.
T273	41482-41606	Sentence	denotes	Second, the mechanism of action of glycofullerenes relies on their interaction with host cells and not with viral particles.
T274	41607-41810	Sentence	denotes	Thus, for a therapeutic application, they should be injected at different time points, ensuring that the local concentration is therapeutically relevant to prevent the virus from invading the host cells.
T275	41811-41913	Sentence	denotes	In addition, glycofullerenes can be internalized into host cells losing their viral “shield” activity.
T276	41914-42049	Sentence	denotes	On the other hand, the well-developed surface chemistry of glycofullerenes can be used for other key receptors involved in viral entry.
T277	42050-42249	Sentence	denotes	For instance, in the case of the current SARS-Cov-2 pandemic, a similar click chemistry strategy can be used to anchor ligands recognized by human lung ACE2 receptors and so inhibiting viral entry.73
T278	42251-42277	Sentence	denotes	Other Carbon Nanomaterials
T279	42278-42394	Sentence	denotes	Alongside fullerenes, other carbon nanomaterials (NMs) have been scrutinized for their ability to block viral entry.
T280	42395-42481	Sentence	denotes	CDs and GO are the most known and studied carbon NMs with marked antiviral properties.
T281	42482-42528	Sentence	denotes	CDs are zero-dimensional carbon nanoparticles.
T282	42529-42631	Sentence	denotes	They are generally produced via hydrothermal decomposition of carbon containing “low-cost” precursors.
T283	42632-42792	Sentence	denotes	The use of CDs in the biomedical field has been encouraged by their easy preparation, low toxicity, fluorescence properties, and easy surface functionalization.
T284	42793-43036	Sentence	denotes	Pristine CDs have shown moderate viral blocking activity for HIV infection in vitro.74 This has been associated with the surface of the material rich in carboxylic and hydroxyl groups prone to form noncovalent interaction with viral membranes.
T285	43037-43200	Sentence	denotes	Moreover, due to the complexity of the biological systems these nonspecific interactions could not be so effective in vivo, likely reducing the antiviral efficacy.
T286	43201-43302	Sentence	denotes	Therapeutic targeting molecules can be grafted onto a CD surface to enhance their antiviral activity.
T287	43303-43412	Sentence	denotes	In this context, the design of multifunctional CD platforms can be obtained through two different strategies.
T288	43413-43552	Sentence	denotes	The first consists in a single-step reaction that foresees the insertion of the therapeutic molecule directly into the step of preparation.
T289	43553-43650	Sentence	denotes	Target molecules are decomposed with the other precursors, generating the desired functional CDs.
T290	43651-43758	Sentence	denotes	This protocol is fast and efficient, however the drug loading as well as its activity are hard to estimate.
T291	43759-43889	Sentence	denotes	Indeed, the hydrothermal treatment can alter the chemical structure of the active molecule, thus vanishing its therapeutic effect.
T292	43890-44116	Sentence	denotes	For these reasons, the reaction conditions must be carefully controlled.75 The second method is a two-step reaction and implies the postfunctionalization via amide formation on the surface of the CDs rich in carboxylic groups.
T293	44117-44247	Sentence	denotes	This strategy offers a better chemical control, but the yield and the drug loading may not be quantitative and high, respectively.
T294	44248-44323	Sentence	denotes	Different functionalized CDs were prepared to hamper host cell viral entry.
T295	44324-44463	Sentence	denotes	For instance, benzoxazine (a low water-soluble antiviral agent) was incorporated into the CD structure during their preparation (Figure 7).
T296	44464-44856	Sentence	denotes	The as-prepared CDs showed a broad spectrum viral blocking capacity in vitro for enveloped (e.g., Japanese encephalitis virus, Dengue virus, and Zika virus) and non-enveloped viruses (e.g., porcine parvovirus and adenovirus-associated virus).76 These positive results were explained by the efficient binding and deactivation induced by the multivalent effect of the CDs to the viral particles
T297	44857-44955	Sentence	denotes	Figure 7 Illustration of benzoxazine-functionalized CDs and their broad antiviral entry activity.
T298	44956-44997	Sentence	denotes	Reproduced with permission from ref (76).
T299	44998-45026	Sentence	denotes	Copyright 2019 Elsevier B.V.
T300	45027-45106	Sentence	denotes	Amino-functionalized CDs were also tested for the treatment of human norovirus.
T301	45107-45291	Sentence	denotes	In this study, CDs were functionalized with 2,2′-(ethylenedioxy)bis(ethylamine) (EDA) and 3-ethoxypropylamine (EPA) via amide bond formation.77 These NMs exerted a good viral blockage.
T302	45292-45492	Sentence	denotes	In particular, EPA-functionalized CDs were able to inhibit 100% of viral infection at concentration of 2 μg/mL, while in the case of CDs prepared with the other amines, 80% of inhibition was reported.
T303	45493-45589	Sentence	denotes	These effects have been associated with the higher positive charge of CD-EDA compared to CD-EPA.
T304	45590-45719	Sentence	denotes	Another surface group used for viral targeting is boronic acid (BA), which can bind glycosylated surfaces forming boronic esters.
T305	45720-46220	Sentence	denotes	This strategy was successfully adopted to treat HIV where the boronic groups, linked to different nanoparticles (e.g., silica nanoparticles and nanodiamonds) can target gp120 receptors on the viral envelope inhibiting the infection.78 Another recent study proposed the use of CD functionalized with phenylboronic acid for prevention of HIV infection.74 The functional materials showed good inhibition properties compared to nonfunctionalized CDs by preventing the binding to the target cell in vitro.
T306	46221-46315	Sentence	denotes	Overall, the use of CDs for stopping host cell viral entrance has shown good results in vitro.
T307	46316-46420	Sentence	denotes	However, there is a lack of proofs in vivo limiting their applications to surface disinfection or masks.
T308	46421-46567	Sentence	denotes	In addition, most of the in vitro studies foresee first the contact of the CDs with the viral particles and then their incubation with host cells.
T309	46568-46733	Sentence	denotes	Deeper investigations should be performed adding the NMs at other time points (for instance in infected cells) to understand if the antiviral activity is maintained.
T310	46734-46869	Sentence	denotes	In addition, CDs have been successfully used for photodynamic therapy (generating radicals upon light irradiation) in cancer treatment.
T311	46870-47054	Sentence	denotes	The same approach may be used to combat viral infections, where the antiviral activity induced by the surface modification can be sensibly enhanced by ROS generation under irradiation.
T312	47055-47381	Sentence	denotes	Graphene materials, and in particular GO and reduced GO (rGO), have been used for different biomedical applications including drug delivery, biosensing, and tissue engineering.5 GO platforms have shown also interesting antimicrobial activity.79 Regarding viral infection, GO was used to block the virus entrance in host cells.
T313	47382-47571	Sentence	denotes	GO and rGO can be considered as two-dimensional materials that contain hydrophilic and hydrophobic domains allowing to adsorb many biological molecules including nucleic acids and proteins.
T314	47572-47730	Sentence	denotes	GO showed low interaction with viruses, however its surface functionalization with target molecules can sensibly enhance its affinity for the viral particles.
T315	47731-47985	Sentence	denotes	Additionally, GO can be used as photothermal agent (generation of heat by NIR irradiation) or photodynamic therapy (using visible light irradiation) inactivating the capsids by local thermal shock or by radical formation during irradiation, respectively.
T316	47986-48080	Sentence	denotes	The use of phototherapies may significantly augment the antiviral properties of the materials.
T317	48081-48263	Sentence	denotes	However, we must keep in mind that these therapeutic modalities can be applied only to disinfection, since the radical/heat production may be harmful for the healthy tissues in vivo.
T318	48264-48446	Sentence	denotes	Photodynamic therapy has been successfully exploited using bacteriophage MS2 as a model virus.80 In this study, GO was functionalized with an aptamer recognized by the viral surface.
T319	48447-48608	Sentence	denotes	The results showed that this functionalization is able to enhance the binding efficiency of the MS2 capsids onto the GO surface compared to nonfunctionalized GO.
T320	48609-48775	Sentence	denotes	Subsequently, irradiation in the visible light was able to disinfect the solution, while nonfunctionalized GO showed much less activity due to the lack of adsorption.
T321	48776-49020	Sentence	denotes	Despite these interesting results, this pioneer work remains at an early research stage since the use of high light dose (300 W for 10–140 min) and the lack of material recovery and reuse make its application for surface disinfection difficult.
T322	49021-49080	Sentence	denotes	GO can be also used as a platform to link antiviral agents.
T323	49081-49433	Sentence	denotes	Encouraging results were reported using GO with hypericin for the treatment of a recently appeared duck reovirus.81 More recently, Deokar et al. reported an original rGO-based multifunctional platform for HSV-1 treatment.82 In this work, the authors functionalized the material with organic sulfate groups and iron oxide magnetic nanoparticles (FeNPs).
T324	49434-49531	Sentence	denotes	The rGO functionalized with the sulfate is able to mimic the host cell surface and to bind HSV-1.
T325	49532-49687	Sentence	denotes	Subsequently, the viral particles captured onto the rGO-FeNP surface can be concentrated via magnetic precipitation and destroyed via photothermal therapy.
T326	49688-49800	Sentence	denotes	This approach is highly efficient for disinfection with low energy (1.6 W/cm2 for 7 min) and cost effectiveness.
T327	49801-50036	Sentence	denotes	HS is a common entry receptor in various types of viruses (e.g., herpes viruses, human papillomavirus, Dengue virus).83 The use of organic sulfate-functionalized graphene sheets mimicking HS, like GO and rGO, has been already explored.
T328	50037-50417	Sentence	denotes	However, it is worth noting that these NMs are prone to strongly adsorb proteins in culture environments (coronation), likely inhibiting their antiviral efficacy.84 High loadings of sulfate groups were introduced onto rGO using polyglycerol sulfate.85,86 This approach has been used for inhibition of orthopoxvirus, pseudorabies virus, and African swine fever virus in vitro.85,86
T329	50418-50468	Sentence	denotes	Graphene has also been used as antiviral material.
T330	50469-50737	Sentence	denotes	Polysulfates and fatty amines were grafted onto graphene surface via triazine chemistry for the treatment of herpes simplex virus.87 This strategy promotes the synergy between the electrostatic and hydrophobic interactions, showing incredibly high inhibition efficacy.
T331	50738-50824	Sentence	denotes	Overall, graphene materials have shown a good capacity to block host cell viral entry.
T332	50825-50963	Sentence	denotes	Disinfection with graphene family materials is also promising, offering the possibility to couple high viral binding with phototreatments.
T333	50964-51137	Sentence	denotes	Regarding the GO and rGO activity in cellular environments, different parameters must be considered such as protein coronation, blood circulation time, and activity in vivo.
T334	51138-51245	Sentence	denotes	So far, the use of sulfonic groups introduced via diazonium salt decomposition has been largely privileged.
T335	51246-51437	Sentence	denotes	We take this opportunity to encourage future studies using other targeting groups (e.g., boronic acids) and grafting methods (e.g., epoxide ring opening or hydroxyl esterification reactions).
T336	51439-51474	Sentence	denotes	Mechanical Disruption of the Capsid
T337	51475-51786	Sentence	denotes	The most direct way to suppress viruses and stop the spreading of viral infection is to inactivate them before the attachment to the host cells, by binding to the acceptor proteins.88 One of the most conserved targets of viral attachment ligands is the heparan sulfate proteoglycan (HSPG), previously mentioned.
T338	51787-51983	Sentence	denotes	HSPGs are expressed on the surface of almost all eukaryotic cell types, and many viruses like HIV-1, HSV, human papilloma virus (HPV) exploit HSPGs as the target of their viral attachment ligands.
T339	51984-52153	Sentence	denotes	Bearing in mind this behavior, different studies have used HSPG-mimicking materials to target this type of virus–cell interaction and to achieve broad spectrum efficacy.
T340	52154-52286	Sentence	denotes	For example, in one key study, AuNPs were functionalized with mercaptoethanesulfonate (MES) based on its mimicry of HS (Au-MES NPs).
T341	52287-52504	Sentence	denotes	Au-MES NPs were shown to interfere with viral attachment, viral entry, and cell-to-cell spreading.89 The importance of the polyvalent interactions with the virus makes these NPs a good candidate for antiviral therapy.
T342	52505-52686	Sentence	denotes	However, Au-MES NPs presented virustatic activity, meaning that upon dilution of the NPs, the virus recovers its infectivity due to the reversibility of the cell-virion interaction.
T343	52687-52825	Sentence	denotes	This problem was solved by Stellacci and colleagues who developed NPs coated with mercapto-1-undecanesulfonate (MUS) ligands (Au-MUS NPs).
T344	52826-53244	Sentence	denotes	The long aliphatic and flexible linkers provide stronger associations with the viral particles compared to Au-MES NPs, leading to local distortions and eventually inducing a global deformation and breaking of the capsid that inactivates its contagion irreversibly (Figure 8).90 These Au-MUS NPs were tested against different HSPG-dependent viruses, showing a high viricidal activity over HSV, HPV, and RSV (Figure 9A).
T345	53245-53511	Sentence	denotes	Furthermore, the activity of the Au-MUS NPs was studied in vivo using mice infected with RSV, indicating that the material can prevent pulmonary dissemination of the infection and showing potential use as medically relevant virucidal drugs to fight viral infections.
T346	53512-53793	Sentence	denotes	More recently, the concept was further extended to cyclodextrins modified with mercapto-1-undecanesulfonate, proposing this system as a broad spectrum virucidal macromolecule.91 Besides Au-NPs, a similar mechanism of disruption was studied with other materials like graphene or GO.
T347	53794-54083	Sentence	denotes	In a study, in which the toxicity of graphene was evaluated theoretically, it was also shown that graphene nanosheets can interrupt the hydrophobic protein–protein interaction, which is essential to biological functions.92 This feature was attributed to the hydrophobic nature of graphene.
T348	54084-54227	Sentence	denotes	Thus, it seems energetically favorable for graphene to slide between the interface of two proteins in contact, due to hydrophobic interactions.
T349	54228-54772	Sentence	denotes	In another study inspired by this behavior, the authors performed molecular dynamics simulations of graphene nanosheets in the proximity of the surface of the Ebola viral matrix protein VP40 showing that the nanosheets can break the hydrophobic interactions in VP40, a key protein for the replication and stability of Ebola virus (Figure 8).93 These findings suggest that graphene nanosheets might have potential antiviral activity against Ebola; however, there is a lack of experimental evidence that corroborates this mechanism of disruption.
T350	54773-55229	Sentence	denotes	On the other hand, GO was tested experimentally against Pseudorabies virus and Porcine epidemic diarrhea virus (PEDV), showing significant decrease in the infectivity.94 It was found that the negatively charged surface of GO is important for the adsorption of the virus, whose surface is positively charged, and that GO could directly interact with the viral particles and destroy their structures due to the sharp edges of the material (Figures 8 and 9B).
T351	55230-55296	Sentence	denotes	Figure 8 Mechanical disruption mechanisms of different NMs. Left:
T352	55297-55389	Sentence	denotes	Au-MUS NPs inducing mechanical forces in the virus capsid leading to inactivation.90 Center:
T353	55390-55462	Sentence	denotes	Graphene NSs disrupting VP40 hydrophobic interactions in Ebola.93 Right:
T354	55463-55548	Sentence	denotes	Interaction between negatively charged surface of GO and positively charged capsid.94
T355	55549-55596	Sentence	denotes	Figure 9 Interaction of NMs with viral capsid.
T356	55597-55631	Sentence	denotes	A) Gold NPs acting on HSV-2 virus.
T357	55632-55708	Sentence	denotes	After 90 min, the percentage of destroyed virus was significantly increased.
T358	55709-55720	Sentence	denotes	Scale bars:
T359	55721-55728	Sentence	denotes	100 nm.
T360	55729-55770	Sentence	denotes	Reproduced with permission from ref (90).
T361	55771-55847	Sentence	denotes	Copyright 2018 Springer Nature Limited. (B) GO acting on Pseudorabies virus.
T362	55848-55935	Sentence	denotes	After incubation with GO for 1 h, part of the virus envelope and spikes were destroyed.
T363	55936-55947	Sentence	denotes	Scale bars:
T364	55948-55955	Sentence	denotes	200 nm.
T365	55956-55997	Sentence	denotes	Reproduced with permission from ref (94).
T366	55998-56040	Sentence	denotes	Copyright 2015, American Chemical Society.
T367	56041-56267	Sentence	denotes	The mechanical disruption of the capsid is a peculiar antiviral mechanism associated with some NMs. In particular, the use of specific sulfonates able to mimic heparan sulfate can be also used to target SARS-Cov-2 infection.95
T368	56269-56305	Sentence	denotes	Antiviral Activity Inside Host Cells
T369	56306-56447	Sentence	denotes	Blocking the viral entry, via liquid/surface disinfection or once in the body, is a powerful strategy to hamper early stage viral contagions.
T370	56448-56591	Sentence	denotes	On the other hand, when infections have already spread and reached middle and late stages, alternative pharmacological strategies are required.
T371	56592-56707	Sentence	denotes	The study of the viral pathogenesis and machinery inside host cells has allowed the preparation of different drugs.
T372	56708-56824	Sentence	denotes	Due to the present pandemic, such antiviral drugs are now of top interest in the scientific and medical communities.
T373	56825-57149	Sentence	denotes	So far, few antiviral drugs are clinically available, and their mechanisms of action consist on the inhibition of reverse transcriptase (HIV, hepatitis), DNA inhibition of polymerase (herpes, HIV), inhibition of protease (HIV), blockage of ion channels (influenza), and inhibition of neuramidase (HIV, influenza, hepatitis).
T374	57150-57215	Sentence	denotes	However, these drugs suffer from moderate to severe side effects.
T375	57216-57369	Sentence	denotes	Additionally, rapid mutations in the viral machinery make them resistant to the treatments, making the control and the stop of the infection challenging.
T376	57370-57432	Sentence	denotes	The use of HNMs in drug delivery has shown several advantages.
T377	57433-57499	Sentence	denotes	First, HNMs can increase drug solubility and its circulation time.
T378	57500-57665	Sentence	denotes	Additionally, they can be functionalized with targeting molecules able to direct the drug to the desired organs and so avoiding side effects and reducing the dosage.
T379	57666-57722	Sentence	denotes	More recently, new antiviral mechanisms were discovered.
T380	57723-57907	Sentence	denotes	In particular, it was found that different types of HNMs are able to change the ROS homeostasis in infected host cells, stopping the viral replication and preserving the cell survival.
T381	57908-58034	Sentence	denotes	In this section both drug delivery materials and HNMs with ROS modulation properties will be critically presented (Figure 10).
T382	58035-58120	Sentence	denotes	Figure 10 Illustration of NMs interacting with host cell to prevent viral spreading.
T383	58121-58173	Sentence	denotes	Left: NMs are used for delivery of antiviral agents.
T384	58174-58269	Sentence	denotes	Right: NMs can change the ROS homeostasis slowing down the infection and helping cell survival.
T385	58271-58302	Sentence	denotes	Nanomaterials for Drug Delivery
T386	58303-58373	Sentence	denotes	Different HNMs have been widely tested for drug delivery applications.
T387	58374-58509	Sentence	denotes	The advantages of this strategy are several including enhance drug solubility and the possibility of targeting and multivalent effects.
T388	58510-58689	Sentence	denotes	As a potential broad spectral antiviral agent, AgNPs can prevent the virus from adsorbing to the host cell in the early stage of infection and thus show strong antiviral activity.
T389	58690-58792	Sentence	denotes	After the cells are infected by the virus, there are ways to inhibit cell apoptosis (Figure 2, right).
T390	58793-59111	Sentence	denotes	For example, Lv et al. studied the anti-TGEV activity of AgNPs in swine testicle cells and explored the possible mechanism of AgNP inhibition of TGEV infection-induced apoptosis.18 The results showed that these AgNPs are able to decrease cell apoptosis through the activation of p38/mitochondria-caspase-3 signaling.18
T391	59112-59369	Sentence	denotes	Although the use of AgNPs has shown good antiviral action to further improve the therapeutic effects and reduce both side effects and drug resistance, the way of binding drugs or genes and other therapeutic agents to AgNPs has received particular attention.
T392	59370-59526	Sentence	denotes	It has been observed that AgNPs inhibit the activities of neuraminidase and hemagglutinin, preventing the H1N1 influenza virus from attaching to host cells.
T393	59527-59655	Sentence	denotes	At the same time, the potential molecular mechanisms revealed that caspase-3-mediated apoptosis was inhibited by ROS generation.
T394	59656-59714	Sentence	denotes	AgNPs modified with polyethylenimine (PEI) can bind siRNA.
T395	59715-60020	Sentence	denotes	Ag@PEI@siRNA exhibited superior abilities for enhanced cellular uptake and blocking EV71 virus infection and significantly decreased the apoptotic cell population, which prevented the spread of EV71 virus.37 In addition to drugs and siRNA, neutralizing antibodies in combination with AgNPs were developed.
T396	60021-60175	Sentence	denotes	The results demonstrated that there is an additive effect between the antibody and AgNPs when combined against cell-associated HIV-1 infection in vitro.96
T397	60176-60242	Sentence	denotes	The membranotropic properties of fullerenes were widely exploited.
T398	60243-60586	Sentence	denotes	For example, pristine C60, after accumulation at the cell membrane, can translocate into the cytoplasm by crossing the membrane through multiple energy-dependent pathways despite its hydrophobic character.97 Fullerenes can be made water dispersible using surfactants, sonication or first dissolving them in appropriate organic solvents (DMSO).
T399	60587-60824	Sentence	denotes	The use of fullerenes as inhibitors of viruses started in 1993 with a study focused on HIV infection.98 This work revealed the interaction between C60 derivatives and HIV protease through molecular modeling and experimental verification.
T400	60825-60960	Sentence	denotes	HIV protease (HIV-PR) is involved in the mechanism of replication in the maturation of HIV virion, cleaving newly synthesized proteins.
T401	60961-61229	Sentence	denotes	The active site of HIV protease has a cavity of 10 Å closed to the diameter of C60 cage.99 Molecular docking and experiments on HIV-PR catalytic activity revealed a blockage of the active site of the enzyme by van der Waals interactions leading to an antiviral effect.
T402	61230-61393	Sentence	denotes	The following studies were based on a structure–activity relationship between functionalized fullerenes and HIV-PR with the aim to increase the antiviral activity.
T403	61394-61809	Sentence	denotes	The introduction of pyrrolidinium salts onto C60 was tested against the activity of HIV-1 strain.100 In a second study, C60 bearing two ammonium groups was applied against the activity of HIV-1 and HIV-2 strains.101 The results showed the importance of having two moieties in a precise position on the fullerene cage and the influence of the charge of the different salts sensibly increasing its antiviral activity.
T404	61810-61912	Sentence	denotes	Further investigations using different functional groups (e.g., amino acid derivatives) were explored.
T405	61913-62230	Sentence	denotes	C60 functionalized with aminobutyric acid and aminocaproic acid was able to inhibit HIV viral replication at subnanomolar concentrations.63 In another work, anionic and cationic pyrrolidinium salts and amino acid functionalized fullerene derivatives were used for the inhibition of HIV reverse transcriptase (HIV-RT).
T406	62231-62596	Sentence	denotes	Fullerene compounds were compared to nevirapine, an available drug against HIV-RT, revealing a better inhibition compared to the pure drug.102 The antiviral property of carboxylated fullerenes was confirmed by another study.103 Results obtained in vitro on CEM cell line showed low toxicity and a submicromolar EC50 against HIV-1 and HIV-2 strain viral replication.
T407	62597-62680	Sentence	denotes	Several mechanisms of HIV protease inhibitors have been hypothesized and simulated.
T408	62681-62934	Sentence	denotes	Some studies investigated the action of fullerene derivatives on viral replication cycle and the virus maturation (Figure 11).98,104 For the latter, it was suggested an impairment due to a strong interaction between fullerene and the immature capsid.105
T409	62935-63014	Sentence	denotes	Figure 11 Illustration of fullerene affecting the viral replication mechanism.
T410	63015-63141	Sentence	denotes	Interaction between fullerene and viral protein blocking either the transcription or the translation in the viral replication.
T411	63142-63485	Sentence	denotes	Other RNA viruses share ways of viral replication similar to HIV.106 C60 functionalized with an amino acid derivative was investigated against Hepatitis C RNA polymerase (HCV-RP).102 This essential enzyme for viral replication was inhibited in a submicromolar range, similarly to benzo-1,2,4-thiadiazine, a potent specific inhibitor of HCV-RP.
T412	63486-63776	Sentence	denotes	Another publication highlighted the effect of a C70 poly(carboxylic acid) derivative on different strains of influenza virus (e.g., A, H1N1, H3N2, and B).107 The inhibition was comparable to Tamiflu, rimantadine, ribavirin, and amantadine, but the mechanism of action remains still unclear.
T413	63777-63891	Sentence	denotes	These data emphasize that fullerenes C60 or C70 can be potent universal antiviral drugs for RNA enveloped viruses.
T414	63892-63994	Sentence	denotes	However, clear elucidations of the mechanisms of action and in vivo studies are still a missing point.
T415	63995-64253	Sentence	denotes	Due to their high surface/weight ratio and capacity to pass through cell membranes, carbon nanotubes (CNTs) have been extensively explored for drug and gene delivery applications.108 CNTs have been also successfully used for the delivery of antiviral agents.
T416	64254-64503	Sentence	denotes	Compared to pristine materials, oxidized CNTs (ox-CNTs) showed an inhibitory activity for HIV viruses per se.109 This effect has been associated with the oxygenated groups that increase the hydrophilicity and the colloidal stability of the material.
T417	64504-64692	Sentence	denotes	The antiviral efficiency has been correlated to the ox-CNT interaction with host cells.109 However, it is not clear how and what kind of mechanism blocks the viral machinery in host cells.
T418	64693-64784	Sentence	denotes	Different anchoring strategies have been explored to link antiviral drugs onto CNT surface.
T419	64785-65427	Sentence	denotes	For instance, ox-CNTs were covalently linked to 2-amino-3-nitro-1-(3,5-dimethylbenzyl)-aniline (CHI360) and N-(2-aminophenyl-3-nitro-)-3,5-dimethylbenzenesulfonamide (CHI415), two active non-nucleoside reverse transcriptase inhibitors for HIV treatment.109 Following the covalent conjugation, only a moderate antiviral effect was observed compared to pure drugs, indicating that most probably the NM cell trafficking played a key role on the virucidal activity.109 In another study, ox-CNTs functionalized with cyclodextrin were used for the delivery of acyclovir (a prodrug inhibitor of the viral DNA polymerases) for the treatment of HSV-1.
T420	65428-65729	Sentence	denotes	Preliminary results showed that when acyclovir was delivered via the nanotubes, the viral antireplicative effect was higher than the free drug.110 More recently, a similar approach was applied to herpes virus using cyclodextrin and PEI-functionalized CNTs for co-delivery of cidofovir and plasmid DNA.
T421	65730-66144	Sentence	denotes	However, the antiviral effect of the materials was not explained, and the transfection effect was not satisfactory.111 Functionalized CNTs have been also used for delivery of ribavirin in vivo using grass carp as an animal model for the study of grass carp reovirus. ox-CNTs were first functionalized via amidation with BSA, and then ribavirin was covalently bound to the protein via esterification (Figure 12).112
T422	66145-66246	Sentence	denotes	Figure 12 Schematic procedure of the functionalization of single-walled CNTs (SWCNTs) and ribavirin.
T423	66247-66289	Sentence	denotes	Reproduced with permission from ref (112).
T424	66290-66319	Sentence	denotes	Copyright 2015, Elsevier B.V.
T425	66320-66541	Sentence	denotes	In vivo tests demonstrated that, when ribovirin was shuttled by ox-CNTs, the antiviral efficiency was significantly increased without any evident toxicity and no significant changes in ROS-generating enzymatic activities.
T426	66542-66609	Sentence	denotes	The use of CNTs in drug delivery is however still controversial.113
T427	66610-66699	Sentence	denotes	Graphene-based materials have been limitedly studied as antiviral drug delivery carriers.
T428	66700-66751	Sentence	denotes	Only a few examples can be found in the literature.
T429	66752-67001	Sentence	denotes	Graphene quantum dots (GQDs) were used for drug delivery of CHI360 and CHI415 and tested in vitro against HIV similarly to CNTs.109 Both the prepared GQD-CHI360 and GQD-CHI415 showed a high antiviral activity once into host cells, with low toxicity.
T430	67002-67115	Sentence	denotes	GO has been also used for the delivery of DNAzyme into hepatic cells allowing to block the hepatitis C infection.
T431	67116-67217	Sentence	denotes	This specific DNA single strand is able to recognize the viral mRNA and to silence its expression.114
T432	67218-67292	Sentence	denotes	Overall, carbon NMs proved to be interesting carriers for antiviral drugs.
T433	67293-67393	Sentence	denotes	However, several questions need to be answered before their safe application as antiviral materials.
T434	67394-67639	Sentence	denotes	Different reports have demonstrated that pristine CNTs display relevant toxicity for healthy cells, but by oxidation of the tubes, the side effects can be sensibly reduced.109 In addition, more investigations should be performed on CNT toxicity.
T435	67640-67923	Sentence	denotes	These nanocarriers indeed are not “innocent delivery agents”, but they play a key role in drug internalization pathway and in host cell machinery that might be averse to the expected therapeutic effects.109 So far, CNT application in biological systems has been studied for 20 years.
T436	67924-68047	Sentence	denotes	However, due to their possible toxicity, their real application in clinics seems to be steeper and difficult to achieve.112
T437	68049-68089	Sentence	denotes	Materials Tuning Reactive Oxygen Species
T438	68090-68170	Sentence	denotes	ROS homeostasis in infected cells has been studied for both RNA and DNA viruses.
T439	68171-68685	Sentence	denotes	For instance, it was shown that infection of mice with influenza A decreased the concentration of lung glutathione and the antioxidant vitamin C, providing evidence that the viral infection was associated with oxidative stress in vitro as well as in vivo.115 Similarly, in HIV infection, induced oxidative stress in host T cells and high concentrations of antioxidants are able to slow down the cell-to-cell viral spreading.115 The increase of ROS concentration is a common process in most of the viral infections.
T440	68686-68762	Sentence	denotes	However, the mechanism of radical generation is different from case to case.
T441	68763-68876	Sentence	denotes	Several proofs suggest that modulating ROS homeostasis in infected cells can slow down or block the infection.116
T442	68877-68945	Sentence	denotes	HNMs have been shown to be powerful allies against viral infections.
T443	68946-69067	Sentence	denotes	In particular, metal or metal oxide NMs, once internalized, can regulate the radical production into infected host cells.
T444	69068-69216	Sentence	denotes	In this scenario, the NMs can work following two different mechanisms: (1) enhancing radical activity or (2) quenching ROS inside cell compartments.
T445	69217-69368	Sentence	denotes	In the first case, metal oxide NPs are able to convert superoxide ions into more reactive hydroxyl radical species via Fenton or Fenton-like reactions.
T446	69369-69508	Sentence	denotes	The excess of superoxide ions is able to oxidize the viral proteins and the genetic material and therefore efficiently block the infection.
T447	69509-69687	Sentence	denotes	This approach has been reported using ZnO NPs.117,118 In these studies, ZnO NPs have been successfully applied for the treatment of H1N1 influenza virus and Herpes simplex virus.
T448	69688-69831	Sentence	denotes	The preliminary results showed that PEGylated ZnO particles were able to efficiently reduce the viral infection with IC50 similar to acyclovir.
T449	69832-70023	Sentence	denotes	More importantly, toxicity of ZnO was modulated by functionalization with PEG, which allowed a higher colloidal stability and a more controlled release of Zn2+ ions to catalyze ROS formation.
T450	70024-70229	Sentence	denotes	Indeed, ROS (e.g., superoxide and hydroxyl radicals) produced by the NPs should be highly reactive and should not only damage the exogenous biological molecules but also attack different cell compartments.
T451	70230-70303	Sentence	denotes	Overproduction of ROS may reduce virus spread but also induce cell death.
T452	70304-70483	Sentence	denotes	Interestingly, this approach is applicable only at the early infection stage (after 1 h incubation of the host cells with a virus), but it loses its activity at later time points.
T453	70484-70876	Sentence	denotes	These experimental evidences suggest that materials capable of triggering the formation of ROS are essential in the first phase of the viral replication, most probably inducing the arrest of the viral DNA polymerases, which is active in the first 1–3 h of viral contamination.117,118 This specific mechanism of action restricts ZnO NPs to an application only at the early stage of infections.
T454	70877-71018	Sentence	denotes	However, the same approach with other metals able to induce Fenton or Fenton-like reactions (e.g., Fe, Cu, and Mn) has not been reported yet.
T455	71019-71148	Sentence	denotes	The choice of proper capping agents may allow to control the metal ion release and thus tune the ROS-mediated antiviral activity.
T456	71149-71255	Sentence	denotes	We would like to take an advantage here to suggest the growth of the antiviral research in this direction.
T457	71256-71343	Sentence	denotes	Another successful approach relies on the reduction of ROS concentration in host cells.
T458	71344-71493	Sentence	denotes	ROS scavenging is able to alleviate the toxicity of the infection enhancing cell viability, giving time to start its endogenous antiviral mechanisms.
T459	71494-71567	Sentence	denotes	So, this approach may both block infection and ensure host cell survival.
T460	71568-71665	Sentence	denotes	In this context, selenium NPs (SeNPs) have been extensively studied for their antiviral activity.
T461	71666-71957	Sentence	denotes	The mechanism of action of these NPs relies on the quenching of the radicals into host cells due to the infection, stopping the mitochondria depolarization and the consequent apoptotic cascade.119 Additionally, SeNPs can also adsorb onto the viral capsid sensibly reducing their infectivity.
T462	71958-72065	Sentence	denotes	SeNPs can be prepared via classical mixing of selenium salt precursors in the presence of a reducing agent.
T463	72066-72499	Sentence	denotes	More recently, SeNPs have been instead biosynthesized from Actinobacteria showing good stability and capacity to inhibit Dengue virus in vitro.120 Moreover, SeNPs were used to carry different antiviral drugs including zanamivir,121 oseltamivir,122 amantadine,123 and ribavirin.119 Their functionalization with the desired drug can be easily achieved adding the molecule during their synthesis through the Se ion controlled reduction.
T464	72500-72560	Sentence	denotes	These NMs have been applied for the treatment of H1N1 virus.
T465	72561-72819	Sentence	denotes	Notably, SeNPs with ribavirin (administered via intranasal absorption every 24 h for 3 days) showed that infected mice had much less alveolar collapse and perivascular and peribronchiolar edema, compared to the group challenged with the virus (Figure 13).119
T466	72820-73120	Sentence	denotes	Figure 13 In vivo antiviral efficiency of SeNPs functionalized with ribavirin (Se@RBV). (a) Mice infected by H1N1 virus were treated with physiological saline (Mock), RBV, SeNPs, or Se@RBV. (b) H&E and tunnel staining showing that Se@RBV-treated mice displayed reduced lung damages compared to Mock.
T467	73121-73163	Sentence	denotes	Reproduced with permission from ref (119).
T468	73164-73194	Sentence	denotes	Copyright 2018 Dove Press Ltd.
T469	73195-73340	Sentence	denotes	Due to their efficacy and low toxicity, SeNPs can be considered a useful material for the treatment of other viral diseases including SARS-Cov-2.
T470	73341-73661	Sentence	denotes	As a matter of fact, oxidative stress as well as chronic inflammation may contribute to the aggravation of the Covid-19 symptoms and to the general spread of the infection.124 The use of SeNPs could eventually alleviate the toxicity of infected patients, giving time for the immune system to react against the contagion.
T471	73662-73854	Sentence	denotes	However, the lack of preclinical studies on SeNPs, together the scarce knowledge of their biosafety and long-term toxicity, still remain the main challenges to tackle for clinical translation.
T472	73856-73890	Sentence	denotes	Interaction with the Immune System
T473	73891-74027	Sentence	denotes	The vast majority of the studies show that human survival to viral attack is based on the stimulation and response of our immune system.
T474	74028-74177	Sentence	denotes	When a virus enters and starts infecting tissues, the body reacts and triggers a strong immune response to overcome the pathogen invasion and spread.
T475	74178-74237	Sentence	denotes	Normally, two different immunological reactions take place.
T476	74238-74925	Sentence	denotes	The oxidative stress induced by infection causes the activation of the inflammasome through upregulation of pro-inflammatory cytokines such as IL-1β, pro-IL-18, and NLRP3.122 Excessive upregulation of this mechanism leads to cell damage and eventually to pyroptosis activated by caspases.122 It was also shown that generation of radicals is able to depolarize mitochondria, hence affecting host cell respiration and inducing ROS-mediated apoptosis at the late stage of infection.123 Besides, activation of pro-inflammatory cytokines can alert the immune system blocking the infection (the innate immune response).122 The second immune response is specific (the adaptive immune response).
T477	74926-75061	Sentence	denotes	Immune cells are trained to attack the virus (cellular response), while specific antibodies are produced by B cells (humoral response).
T478	75062-75195	Sentence	denotes	In the last years, HNMs were proved to tune the immune responses, demonstrating to be a possible alternative against viral infection.
T479	75196-75340	Sentence	denotes	In this section the most relevant strategies for the activation of innate and adaptive immune responses using NMs will be described (Figure 14).
T480	75341-75407	Sentence	denotes	Figure 14 Illustration of HNM interaction with the immune system.
T481	75408-75510	Sentence	denotes	Left: HNMs can enhance the production of interferon (IFN) alerting the immune system of the infection.
T482	75511-75517	Sentence	denotes	Right:
T483	75518-75715	Sentence	denotes	Virus-mimicking particles composed of viral proteins and HNMs can induce a strong immunological response activating dendritic cells, T cells, and stimulating B cells to produce specific antibodies.
T484	75717-75739	Sentence	denotes	Innate Immune Response
T485	75740-75843	Sentence	denotes	Innate immune response is the first response that takes place in the presence of any type of infection.
T486	75844-76240	Sentence	denotes	During this early stage, interferon stimulating genes (ISGs) are upregulated in the infected cells.125 This self-defense mechanism slows down the viral replication and alerts sentinel immune cells that start producing proinflammatory cytokines and trigger inflammation.125 However, many viruses are able to escape this complex mechanism, retarding the immune response and spreading the infection.
T487	76241-76319	Sentence	denotes	The interaction of HNMs with the immune system has been more and more studied.
T488	76320-76484	Sentence	denotes	In the case of antiviral HNMs, many examples can be found in the literature where the NMs not only slow down the infection but also tune the innate immune response.
T489	76485-76542	Sentence	denotes	Certain HMNs can display an intrinsic immune stimulation.
T490	76543-76825	Sentence	denotes	We have already mentioned above that in the early stage of infection, AgNPs mainly prevent the virus from entering the host cell through the interaction with the external capsid, but in vitro cellular experiments lack to understand the complex interaction with primary immune cells.
T491	76826-77030	Sentence	denotes	Recent studies have shown that AgNPs can potentially induce the expression of genes involved in innate and adaptive immunity-associated pathways, which are known to play crucial role in immune regulation.
T492	77031-77669	Sentence	denotes	For example, Toll-like receptor 7 can be upregulated by AgNPs after 24 h, by recognizing the single-stranded RNA of the viruses and regulating the antiviral immune response.126 Another study showed that in RSV-infected mice treated with AgNPs, the particles reduced the production of pro-inflammatory TNF-α and IL-6 cytokines, but potentiated the anti-RSV activity of neutrophils in an experimental mouse model.127 However, activation of AgNPs in the reduction of RSV has been noted only when the NM was intranasally inoculated together with the virus, and no results have been reported on the use of AgNPs administrated on infected mice.
T493	77670-77856	Sentence	denotes	In the case of influenza virus infection of lung epithelial cells, it was found that AgNPs targeted infected lung epithelial cells and reduced viral replication, by preventing autophagy.
T494	77857-77972	Sentence	denotes	However, the blockage of the autophagic flux by AgNPs does not inhibit viral replication in already infected cells.
T495	77973-78356	Sentence	denotes	Therefore, AgNPs are more suitable as viral preventive agents due to their pro-inflammatory response rather than drugs.128 More recently, AgNPs were combined with graphene materials and exploited as antiviral material for the treatment of Porcine reproductive and respiratory syndrome virus (PRRSV).129 GO-AgNPs were able to clump the virus diminishing its fusion with cell membrane.
T496	78357-78527	Sentence	denotes	Additionally, once GO-AgNPs were internalized in host cells, they stimulated the ISGs that blocked viral budding and its diffusion to other cells in vitro (Figure 15).129
T497	78528-78618	Sentence	denotes	Figure 15 Scheme of the mechanism of action of GO-AgNPs on activation of innate immunity.
T498	78619-78661	Sentence	denotes	Reproduced with permission from ref (129).
T499	78662-78704	Sentence	denotes	Copyright 2018 American Chemistry Society.
T500	78705-79348	Sentence	denotes	Similarly, CDs used for the treatment of RSV and PRRSV were able to activate the innate immune response via an upregulation of ISGs in vitro.130 Gold nanorods were applied to boost the innate immune response against RSV in vivo.131 Interestingly, it was shown that these nanorods (when intranasally administered with RSV) were not only able to activate the ISGs but also to tune the production of pro- and anti-inflammatory cytokines, resulting in the blocking of the infection with a reduced pulmonary inflammation.131 As drug carriers, HNMs can also affect the internalization pathways, modulate drug efficacy, and the immune cell responses.
T501	79349-79666	Sentence	denotes	For instance, it was found that the isoprinosine immunomodulatory antiviral drug displays a much higher antiviral efficacy in vivo when delivered with CNTs than as a pure drug (in zebrafish larvae food administration), probably due to a better cellular uptake and the anti-inflammatory properties of the nanotubes.132
T502	79667-80115	Sentence	denotes	Fullerenes also exhibit immunomodulation properties through the release of cytokines by bovine alveolar macrophages.133 A study explored the influence of functionalization of C60 with molecules presenting different surface charges like hydroxyl groups and amino acids.134 The study concluded that negative charges upregulated TNF-α up-secretion in RAW 264.7 macrophages, but highly positively or negatively charged surfaces increased cell toxicity.
T503	80116-80218	Sentence	denotes	The upregulation of the cytokine TNF-α is a proof that fullerene can promote cellular immune response.
T504	80219-80368	Sentence	denotes	Despite these preliminary results, the actual mechanisms of interaction between HNMs and the immune system are still at early stage of understanding.
T505	80369-80459	Sentence	denotes	We must consider that the immune response needs to be proportional to the infection grade.
T506	80460-80707	Sentence	denotes	If on one side nanosized immuno-boosters can alert more efficiently the sentinel cells, the use of HNMs that trigger an exaggerated immune response can promote excessive inflammation, damaging healthy cells and promoting uncontrolled side effects.
T507	80708-80927	Sentence	denotes	In the particular context of SARS-Cov-2, the use of AgNPs, fullerenes, or other pro-inflammatory HNMs at the middle and late infection stage may cause an aggravation of the symptoms due to already diffused inflammation.
T508	80928-81370	Sentence	denotes	In particular, most of the studies showed the ability to reduce infection when HNMs were first incubated with the pathogenic virus, thus limiting their potential use in the early stage infection.124 The formulation of HNMs able to both alert the immune system (e.g., upregulating cytokine) and control lung inflammation (e.g., ROS scavenger) even after the early stage infection may be a possible strategy for treatments against SARS viruses.
T509	81372-81396	Sentence	denotes	Adaptive Immune Response
T510	81397-81495	Sentence	denotes	Adaptive immune response is the specific response that the immune system exerts against pathogens.
T511	81496-81959	Sentence	denotes	This mechanism is particularly active toward viral infections where the immune system produces specialized lymphocytes (to fight the virus), called memory B cells (to be effective in case of new infections) and antibodies (corresponding to the humoral response).135 The stimulation of adaptive responses in case of specific infections can be induced artificially through the introduction of attenuated pathogens, stimulating the production of specific antibodies.
T512	81960-82368	Sentence	denotes	This is the principle of vaccination, which is the most common procedure for immunization of large areas of population against many kinds of lethal viruses.135 Besides, the use of viral proteins as antigens in the vaccine formulation leads to neutralizing antibodies, but, due to the low immunogenicity of isolated proteins, does not always stimulate sufficiently the immune system to reach total protection.

T13

0-99

Sentence

denotes

The current emergence caused by SARS-Cov-2 is dramatically changing the everyday life of all of us.

T14

100-232

Sentence

denotes

Due to the high globalization, new viruses can spread all over the world much faster than ever, infecting the communities worldwide.

T15

233-327

Sentence

denotes

The current technologies and measurements are able to sensibly slow down the infection spread.

T16

328-477

Sentence

denotes

However, their cost is tremendously high, impacting the healthcare systems and causing the shutdown of industries and the lockdown of the population.

T17

478-746

Sentence

denotes

The impact of SARS-Cov-2 on the worldwide economy is estimated to be a 2–3% decline, making this the biggest crisis since the world wars.1 A possible vaccine is hopefully expected to come in 1–2 years, originating a buffer period pretty much uncertain for many people.

T18

747-861

Sentence

denotes

Vaccination is the only way known to accelerate the flock immunity without causing further death by this pandemic.

T19

862-1114

Sentence

denotes

Contemporary history has seen the spread of other viral pandemics such as H2N2 flu (1956–1958), H3N3 flu (1968), HIV (peak reached between 2005 and 2012), SARS (2009), while MERS (2012 to now) and Ebola (1975 to now) viruses are in a prepandemic phase.

T20

1115-1242

Sentence

denotes

The continuous colonization of wild nature lands may touch unknown virus reservoirs causing the spread of contagious epidemics.

T21

1243-1577

Sentence

denotes

Due to these facts, there is a clear urgency in the development of viral treatments to avoid the risk of new pandemics.2 In particular, the possibility to have smart antiviral tools able to efficiently disinfect surfaces, block the viral spreading, enhance the survival of infected people, and boost immunization are highly desirable.

T22

1578-1667

Sentence

denotes

In particular, more investments in the next years are expected in the antiviral research.

T23

1668-2283

Sentence

denotes

Hard nanomaterials (HNMs) have been extensively studied for many types of applications including drug delivery, bioimaging, and biosensing.3−5 The use of nanomaterials in biomedical research is highly developed, reaching in some cases clinical approval.6 Despite that, nanomaterials have been mainly developed for cancer therapy, while scarce attention has been spent on their application in viral infections.7 The continuous virology research has more and more increased into viral replication machinery, allowing the preparation and rationalization of more sophisticated vaccine formulations and viral inhibitors.

T24

2284-2420

Sentence

denotes

The use of HNMs may be one of the keys to provide more effective biomedical agents with a wide spectrum of activity in viral pandemics.8

T25

2421-2521

Sentence

denotes

An increasing number of reports describe how HNMs can be successfully applied to block viral spread.

T26

2522-2681

Sentence

denotes

HNMs can be used at different stages of viral infection: blocking viral entry, hampering interaction with infected host cells, and modulating immune responses.

T27

2682-2904

Sentence

denotes

Due to their core composition (e.g., metal oxides, noble metals), HNMs can have an important antiviral activity inactivating some specific proteins of the capsid or dysregulating radical homeostasis in the virus particles.

T28

2905-3067

Sentence

denotes

Additionally, surface functionalization can sensibly increase HNM antiviral activity, enabling the mimicking of host cells or enhancing the targeting efficiency.9

T29

3068-3180

Sentence

denotes

In this review, we will critically analyze different strategies for the application of HNMs as antiviral agents.

T30

3181-3289

Sentence

denotes

The rational and the synthetic strategies will be highlighted and correlated to different relevant examples.

T31

3290-3400

Sentence

denotes

Particular attention will be paid on the mechanisms of antiviral action and on HNM applicability and efficacy.

T32

3401-3564

Sentence

denotes

For the sake of clarity, this review is divided into three parts, namely: blocking viral entry, antiviral activity in host cells, and stimulation of immune system.

T33

3565-3618

Sentence

denotes

We have focused on the different stages of infection.

T34

3619-3803

Sentence

denotes

In the section dedicated to blocking viral entry, the application of HNMs for surface disinfection and inactivation of the virus prior to interaction with host cells will be described.

T35

3804-4023

Sentence

denotes

Subsequently, the interaction of HNMs after internalization into host cells will be addressed, stressing their antiviral delivery features and their activity in viral replication blockage, leading to host cell survival.

T36

4024-4165

Sentence

denotes

Then, activation of immune response induced by HNMs, triggering the innate and the adaptive (e.g., nanovaccines) immunity, will be presented.

T37

4166-4333

Sentence

denotes

The different adopted strategies will be correlated to the nanomaterial core (e.g., composition, size, shape) and surface (e.g., chemistry, surface charge) properties.

T38

4334-4589

Sentence

denotes

Finally, limits (e.g., unknown long-term toxicity), advantages (e.g., high and wide spectrum virucidal activity), and perspectives will be discussed with particular attention to the applications in viral pandemics (e.g., HIV, SARS, and influenza viruses).

T39

4590-4696

Sentence

denotes

This review is addressed to material and biomaterials scientists who are interested in antiviral research.

T40

4697-4861

Sentence

denotes

We acknowledge that application of HNMs as antiviral agents is still in the early stages; however, we believe that the research on this topic is going to grow soon.

T41

4862-4995

Sentence

denotes

Thus, with this contribution we genuinely hope to inspire researchers in the preparation of smart and efficient HNM antiviral agents.

T42

4997-5017

Sentence

denotes

Blocking Viral Entry

T43

5018-5098

Sentence

denotes

The last decades have been characterized by increasing investigation on viruses.

T44

5099-5277

Sentence

denotes

In particular, their surface charge, protein composition, and host cell entry mechanism have been elucidated, allowing to formulate the first-generation wide spectrum antivirals.

T45

5278-5397

Sentence

denotes

Blocking the viral entry is one of the most common known antimicrobial procedure to stop infections at the early stage.

T46

5398-5524

Sentence

denotes

In this context, antiviral materials have been used for surface disinfection or for epidemic limitation in humans and animals.

T47

5525-5690

Sentence

denotes

Due to their high surface to volume ratio, composition, and tunable surface chemistry, HNMs are now more and more studied as powerful agents in blocking viral entry.

T48

5691-6024

Sentence

denotes

As for other biological interactions, the attachment and entry of viruses into host cells are mediated by multivalent interactions between the surface of the virus and cell surface receptors.10 Nanomaterials can display multivalency that makes them able to compete with the host cells on virus attachment, limiting their infectivity.

T49

6025-6110

Sentence

denotes

The mechanism of antiviral actions relies on the inactivation of the capsid proteins.

T50

6111-6310

Sentence

denotes

As a matter of fact, HNMs have been used for: (1) blocking target proteins for viral entry, (2) capsid protein oxidation, (3) mimicking cell surface, and (4) mechanical rupture of viruses (Figure 1).

T51

6311-6463

Sentence

denotes

These strategies target proteins and mechanisms of entry common in most of the viruses, thus allowing the preparation of wide spectrum antiviral agents.

T52

6464-6571

Sentence

denotes

In this section, the application of different HNMs as powerful inhibitors of viral entry will be discussed.

T53

6572-6741

Sentence

denotes

Figure 1 Illustration of the main mechanisms of blocking virus entry into host cells: capsid denaturation, mimicking cell surface, and mechanical breaking of the virus.

T54

6743-6762

Sentence

denotes

Noble Nanoparticles

T55

6763-6943

Sentence

denotes

Noble nanoparticles (NPs), made of gold and silver, are attractive as antiviral agents for their surface functionalization versatility and their capacity to cleave disulfide bonds.

T56

6944-7030

Sentence

denotes

Their use in disinfection has been extensively studied for different types of viruses.

T57

7031-7187

Sentence

denotes

The morphology and the size of the NPs play a crucial role in their ability to efficiently interact with the capsids and in their toxicity for the organism.

T58

7188-7314

Sentence

denotes

These nanomaterials are characterized by a very large specific surface area (inversely proportional to the particle diameter).

T59

7315-7473

Sentence

denotes

As the particle size becomes smaller and smaller, the percentage of surface atoms increases, creating many unsaturated bonds due to lack of neighboring atoms.

T60

7474-7553

Sentence

denotes

As a consequence, AgNPs and AuNPs have unstable atoms with high surface energy.

T61

7554-7666

Sentence

denotes

This kind of structure provides a lot of contact adsorption sites and reaction points for further modifications.

T62

7667-7772

Sentence

denotes

These chemical features allow to easily combine surface NP atoms with other atoms through chemical bonds.

T63

7773-7904

Sentence

denotes

Besides the composition of the metal core, several studies have pointed out the importance of the control of the surface chemistry.

T64

7905-8054

Sentence

denotes

The surface groups can: (1) stabilize NPs in the biological media, (2) insert targeting agents, and (3) enhance the circulation time inside the body.

T65

8055-8234

Sentence

denotes

Antiviral efficiency can be also enhanced by the multivalency effect, where highly branched ligands are used to locally augment the local concentration of the targeting molecules.

T66

8235-8386

Sentence

denotes

In this section the main strategies and results for silver (AgNPs) and gold (AuNPs) nanoparticles in blocking viral entry will be critically discussed.

T67

8388-8408

Sentence

denotes

Silver Nanoparticles

T68

8409-8540

Sentence

denotes

Many studies have shown that naked AgNPs have a good effect on the control and prevention of a variety of viral diseases (Table 1).

T69

8541-8605

Sentence

denotes

However, the antiviral mechanism of nanosilver is still unclear.

T70

8606-8671

Sentence

denotes

The antiviral action is associated with the following mechanisms:

T71

8672-8852

Sentence

denotes

Nanosilver can prevent the virus from entering the host cells and inhibit the virus from binding to the cell receptor, thereby stopping the virus from infecting the targeted cells.

T72

8853-9000

Sentence

denotes

AgNPs may be able to bind the viral surface protein and inhibit the interaction between the virus and the cell membrane receptors (Figure 2, left).

T73

9001-9225

Sentence

denotes

However, it has been also reported that AgNPs can inactivate the virus through denaturation of surface proteins containing cysteine and methionine residues present on the viral capsid, in a similar way reported for bacteria.

T74

9226-9673

Sentence

denotes

For example, AgNPs smaller than 10 nm were shown to interact with the sulfur-bearing residues of gp120 glycoprotein knobs distributed on the lipid membrane of HIV-1 virus, preventing the virus from binding to CD4 receptor site on the host cells, thus inhibiting the viral infection.11 By means of a viral adsorption assay, it was shown that the AgNP mechanism of anti-HIV action is based on the inhibition of the initial stages of the HIV-1 cycle.

T75

9674-9926

Sentence

denotes

To demonstrate that the antiviral effect of AgNPs is due to the particle structure rather than to silver ions present in solution, the antiviral activity of silver sulfadiazine (AgSD) and silver nitrate (known antibacterial silver salts) was evaluated.

T76

9927-10317

Sentence

denotes

Both salts showed a much lower therapeutic index than AgNPs in vitro, indicating that silver ions themselves are less efficient.12 These results point out that the antiviral efficacy is not only related to the dose of Ag+ ions present in solution but is also regulated by different other parameters (e.g., size, charge, and surface functionalization) associated with the nanosize dimension.

T77

10318-10667

Sentence

denotes

For instance, in the case of Herpesviridae and Paramyxoviridae viruses (both enveloped viruses with embedded viral-encoded glycoproteins), AgNPs can effectively reduce their infectivity, by blocking the interaction between the viral particles and the host cells with an antiviral activity strictly dependent on the size and ζ potential of the AgNPs.

T78

10668-10766

Sentence

denotes

As a general observation, it was reported that smaller nanoparticles have better antiviral effect.

T79

10767-11163

Sentence

denotes

This effect was associated with the increase of the surface area, where smaller-sized AgNPs could bind more efficiently to the viral particles exerting a higher antiviral activity.13 Another study reported the impairment of Peste des petits ruminants virus (PPRV) replication after incubating infectious viral particles with AgNPs, which did not exhibit any virucidal effect even up to 900 μg/mL.

T80

11164-11300

Sentence

denotes

This result suggested that the anti-PPRV activity of the AgNPs is due to the inhibitory effect on viral replication in the target cells.

T81

11301-11407

Sentence

denotes

AgNPs do not prevent the binding of PPRV to host cells, but inhibit the entry of viruses into these cells.

T82

11408-11576

Sentence

denotes

AgNPs can also interact with the surface and core of PPRV, but this interaction cannot kill the virus directly.12 The same results were then confirmed on other viruses.

T83

11577-11687

Sentence

denotes

AgNPs with a diameter of 25 nm inhibited Vaccinia virus replication by preventing viral entry into host cells.

T84

11688-11854

Sentence

denotes

However, AgNPs cannot prevent the virus from adsorbing onto the cells, and this virus is still infectious, indicating that AgNPs lack a direct virus-killing effect.13

T85

11855-12290

Sentence

denotes

Figure 2 Potential antiviral mechanism of AgNPs. (1) AgNPs interact with viral envelope and/or viral surface proteins; (2) AgNPs interact with cell membranes and block viral penetration; (3) AgNPs block cellular pathways of viral entry; (4) AgNPs interact with viral genome; (5) AgNPs interact with viral factors necessary for viral replication; and (6) AgNPs interact with cellular factors necessary for productive viral replication.

T86

12291-12332

Sentence

denotes

Reproduced with permission from ref (14).

T87

12333-12367

Sentence

denotes

T88

12368-12432

Sentence

denotes

Table 1 Antiviral AgNPs and Their Possible Mechanisms of Action

T89

12433-12504

Sentence

denotes

virus shape size (nm) active concentration mechanism of action ref

T90

12505-12567

Sentence

denotes

HIV-1 spherical 1–10 25 μg/mL interaction with gp120 (11)

T91

12568-12629

Sentence

denotes

HIV-1 IIIB – 30–50 440 μg/mL interaction with gp120 (19)

T92

12630-12755

Sentence

denotes

HSV-1, HSV-2, and HPIV-3 – 20–50 not available possible interaction directly with the viral envelope or its protein (20)

T93

12756-12863

Sentence

denotes

Adenovirus type 3 spherical 5–18 25 μg/mL direct destruction of virus particles and DNA structure (15)

T94

12864-13025

Sentence

denotes

H1N1 influenza A virus spherical 5–20 12.5 μg/mL inhibition of respiratory enzymes and electron transport components and interference with DNA function (21)

T95

13026-13133

Sentence

denotes

HBV spherical 10–50 5 μM interaction with double-stranded DNA and/or binding with viral particles (16)

T96

13134-13214

Sentence

denotes

PPRV spherical 5–30 11.1 μg/mL interaction with virus surface and core (12)

T97

13215-13305

Sentence

denotes

Vaccinia virus spherical 25 not available preventing viral entry into host cells (13)

T98

13306-13406

Sentence

denotes

Monkey pox virus (MPV) – 10–80 12.5 μg/mL blocking virus-host cell binding and penetration (22)

T99

13407-13499

Sentence

denotes

Tacaribe virus (TCRV) – 5–10 25 μg/mL inactivation of virus particles before entry (23)

T100

13500-13611

Sentence

denotes

Poliovirus spherical 4–9 3.1 ppm preventing viral particles from binding to the receptors of RD cells (24)

T101

13612-13725

Sentence

denotes

TGEV spherical <20 12.5 μg/mL direct interaction with TGEV surface protein, such as TGEV S glycoprotein (18)

T102

13726-13745

Sentence

denotes

linear 60000–80000

T103

13746-13943

Sentence

denotes

linear 20000–30000 Alternatively, nanosilver can be combined with viral nucleic acids to change the capsid structure, affect the replication of viral genetic material, and make the virus inactive.

T104

13944-14379

Sentence

denotes

For example, TEM analyses have shown that NPs can cause a change of the structure of the Ad3 virus from a hexahedral shape to an irregular shape, destroying its fibers and capsid proteins, leading to inhibition of the virus from binding to the host cells and destroying the DNA structure, preventing adenoviral infection.15 Nanosilver can also bind directly to the double-stranded DNA of hepatitis B virus to inhibit its replication.16

T105

14380-14497

Sentence

denotes

In other studies, it has been demonstrated that silver ions released from nanosilver can directly damage the viruses.

T106

14498-14567

Sentence

denotes

Based in this property, an interesting application has been proposed.

T107

14568-14696

Sentence

denotes

AgNPs were used as a coating on polyurethane condoms, effectively inhibiting the activity of HIV and herpes simplex virus (HSV).

T108

14697-14857

Sentence

denotes

The hypothesized mechanism is that silver ions are transferred directly from oxidized NPs to biological targets, such as viral membrane proteins gp120 and gp41.

T109

14858-14981

Sentence

denotes

In addition, a small amount of silver ion is also released from the coated contraceptives to improve the antiviral level.17

T110

14982-15208

Sentence

denotes

Although the studies on naked AgNPs to reduce viral infectivity have shown their potential as broad-spectrum antiviral agents, the understanding of the specific antiviral action mechanism still needs to be elucidated in depth.

T111

15209-15494

Sentence

denotes

Many studies have shown that the antiviral performance of naked AgNPs is related to their size, and smaller nanoparticles have better antiviral activities.16 In addition to particle size, the antiviral action of AgNP morphology has also attracted interest to fight against coronavirus.

T112

15495-15690

Sentence

denotes

AgNPs and two types of silver nanowires were able to significantly cause an inhibitory effect on coronavirus transmissible gastroenteritis (TGEV)-induced host cell infection and TGEV replication.

T113

15691-15852

Sentence

denotes

The mechanism is likely based on a direct interaction of AgNPs with TGEV surface proteins (e.g., TGEV glycoproteins) to inhibit the beginning of viral infection.

T114

15853-16029

Sentence

denotes

It is possible that AgNPs and Ag nanowires alter the structure of some surface proteins of TGEV and then inhibit their recognition and adhesion to the cellular receptor pAPN.18

T115

16030-16235

Sentence

denotes

Although the potential of AgNPs as antiviral agents has been commonly recognized, unfortunately, their wide biological applications are limited by the risks of self-aggregation and environmental pollution.

T116

16236-16466

Sentence

denotes

Silver ions can be released from the surface of AgNPs and potentially pollute the environment, and their agglomeration into bulkier particles or fibers may change their biological characteristics, diminishing the antiviral effect.

T117

16467-16721

Sentence

denotes

In several cases, it has been reported that naked AgNPs may affect human health.25 Therefore, research and development of AgNPs whose surface is modified or stabilized by protecting molecular layers is an urgent need to overcome these problems (Table 2).

T118

16722-16802

Sentence

denotes

Poly(N-vinyl-2-pyrrolidone) (PVP) is the most commonly used stabilizer of AgNPs.

T119

16803-17408

Sentence

denotes

The PVP-coated AgNPs are able to inhibit the activities of HIV-1, herpes simplex 2 virus (HSV-2), and respiratory syncytial virus (RSV).11,26,27 But compared to foamy carbon, small-sized PVP and BSA-coated AgNPs showed poor antiviral activity to the HIV-1 virus.11 For RSV, PVP-coated AgNPs have a specific binding capacity to the viral surface, evidencing a regular spatial arrangement and a clear interaction with G-protein.26 In addition, to improve the stability of AgNPs, their surface modification with antiviral drugs was proved to reduce the drug resistance caused by the drugs administered alone.

T120

17409-17506

Sentence

denotes

Tannic acid-modified AgNPs showed good antiviral effects on HSV-2 infection in vitro and in vivo.

T121

17507-17600

Sentence

denotes

The viral infection was inhibited only when these NPs directly interacted with HSV-2 virions.

T122

17601-17687

Sentence

denotes

Indeed, the pretreatment of host cells with such AgNPs did inhibit the entry of HSV-2.

T123

17688-17925

Sentence

denotes

Due to the high affinity of tannins to proteins and sugars, tannic acid can bind glycoproteins on the surface of viruses to make them inert, impairing glycoprotein function and preventing viruses from attaching and entering host cells.28

T124

17926-18001

Sentence

denotes

Table 2 Surface-Modified Antiviral AgNPs and Possible Mechanisms of Action

T125

18002-18060

Sentence

denotes

virus shape size (nm) coating mechanism of action ref

T126

18061-18140

Sentence

denotes

HIV-1 spherical 1–10 foamy carbon, PVP and BSA interaction with gp120 (11)

T127

18141-18263

Sentence

denotes

RSV spherical – PVP, BSA, and recombinant F protein (RF 412) interaction with the G-protein on the virus surface (26)

T128

18264-18382

Sentence

denotes

H1N1 influenza virus spherical 2–5 Oseltamivir inhibition of the activity of neuraminidase and hemagglutinin (34)

T129

18383-18399

Sentence

denotes

Amantadine (35)

T130

18400-18415

Sentence

denotes

Zanamivir (36)

T131

18416-18481

Sentence

denotes

inhibition of accumulation of reactive oxygen species (ROS)

T132

18482-18597

Sentence

denotes

HAV, NoV and CoxB4 spherical – polyphosphonium-oligochitosans preventing viral attachment and penetration (29)

T133

18598-18691

Sentence

denotes

MPV spherical 10–80 polysaccharide blocking virus-host cell binding and penetration (22)

T134

18692-18781

Sentence

denotes

TCRV spherical 10 polysaccharide inactivation of virus particles prior to entry (23)

T135

18782-18886

Sentence

denotes

HSV-1 spherical 4 mercaptoethanesulfonate competition for the binding of the virus to the cell (30)

T136

18887-19024

Sentence

denotes

Enterovirus 71 (EV71) spherical 2–5 PEI and antiviral siRNA inhibition of the accumulation of ROS and activation of AKT and p53 (37)

T137

19025-19221

Sentence

denotes

HSV-2 spherical 13, 33, 46 tannic acid direct interaction and blocking of virus attachment, penetration and spread (28) The surface modification can also exert a synergistic antiviral effect.

T138

19222-19360

Sentence

denotes

AgNPs decorated with polyphosphonium-oligochitosan (PQPOC) exhibited moderate to excellent antiviral activity against HAV, NoV, and CoxB4.

T139

19361-19470

Sentence

denotes

In addition, AgNPs could interact with the virion glycoproteins and prevent viral attachment and penetration.

T140

19471-19712

Sentence

denotes

PQPOC can also serve as an effective virus inhibitor by blocking the interaction of the targeted virus with the host through the electrostatic interaction between the cationic polymers and the negatively charged binding sites of the virus.29

T141

19713-19809

Sentence

denotes

Surface-modified AgNPs can also prevent viral infection by competitive adsorption on host cells.

T142

19810-19986

Sentence

denotes

The process of infection of cells by herpes simplex virus type 1 (HSV-1) involves the interaction between viral envelope glycoproteins and heparan sulfate (HS) on cell surface.

T143

19987-20189

Sentence

denotes

Therefore, researchers designed AgNPs capped with mercaptoethanesulfonate (Ag-MES) to compete with the cellular HS through the sulfonate end groups, thereby blocking the virus from entering the cells.30

T144

20190-20696

Sentence

denotes

A few years ago, it was shown that curcumin could prevent the replication and the budding of RSV,31 but the disadvantage of poor solubility and low bioavailability limited its clinical application.32 Curcumin was used as a reducing and capping agent to prepare stable curcumin AgNPs (cAgNPs) under physiological conditions. cAgNPs could reduce cytopathic effects induced by RSV and showed efficient antiviral activity against infection by directly inactivating the virus prior to entry into the host cells.

T145

20697-20782

Sentence

denotes

Its antiviral effect was higher than curcumin alone or unmodified AgNPs (Figure 3).33

T146

20783-20916

Sentence

denotes

Figure 3 Schematic representation of the synthesis of cAgNPs (A) and a proposed inhibition mode of cAgNPs against RSV infection (B).

T147

20917-21095

Sentence

denotes

The inhibition mode of (B) shows that cAgNPs can reduce the binding ability of virus with the binding centers on the surface of cells (b) as compared to those without cAgNPs (a).

T148

21096-21137

Sentence

denotes

Reproduced with permission from ref (33).

T149

21138-21177

Sentence

denotes

T150

21178-21335

Sentence

denotes

Alternatively, Zhu et al. prepared AgNPs surface-modified with oseltamivir, amantadine, and zanamivir (Ag@OTV,34 Ag@AM,35 and Ag@ZNV36), by chemical methods.

T151

21336-21456

Sentence

denotes

The results showed that these nanoparticles can directly interact with the virions, resulting in viral function damages.

T152

21457-21540

Sentence

denotes

Overall different studies have reported the capacity of AgNPs to block viral entry.

T153

21541-21739

Sentence

denotes

However, there is not a concerted antiviral mechanism, but their activity differs from case to case, based on viral particle adsorption, capsid structure alteration, or surface protein denaturation.

T154

21740-21970

Sentence

denotes

For AgNPs, the antiviral activity can be associated with different parameters including size, shape, surface charge, and functionalization but also to the topical release of silver ions able to disturb the viral cycle replication.

T155

21971-22148

Sentence

denotes

As described before, bare AgNPs can be used as disinfectant agents, however their use in biological media is limited by their low colloidal stability and potential cytotoxicity.

T156

22149-22333

Sentence

denotes

Surface functionalization can alleviate cytotoxicity, but it can also mask the nanoparticle surface, reducing their affinity for viral particles, thus reducing AgNP antiviral activity.

T157

22334-22459

Sentence

denotes

For these reasons, AgNPs at the moment could find application mainly for surface disinfection and for topical administration.

T158

22460-22550

Sentence

denotes

Further studies are needed to prepare safer AgNP formulations for systemic administration.

T159

22551-22804

Sentence

denotes

In particular, the clarification of the antiviral mechanisms and the use of surface functional groups able to stabilize AgNPs in biological fluids without affecting their prominent antiviral activity are probably the most important challenges to tackle.

T160

22806-22824

Sentence

denotes

Gold Nanoparticles

T161

22825-23055

Sentence

denotes

Compared to AgNPs, AuNPs exhibit reduced toxicity on healthy cells, making them more attractive for in vivo and clinical applications.38 Indeed, AuNPs have been successfully tested as inhibitors of viral entry into the host cells.

T162

23056-23253

Sentence

denotes

AuNPs interact with hemagglutinin (HA), where Au is able to oxidize the disulfide bond of this glycoprotein causing its inactivation, thus impeding the membrane fusion of the virus with host cells.

T163

23254-23715

Sentence

denotes

Targeting HA has emerged as an alternative strategy to the actual therapies (e.g., matrix protein 2 and neuramidase), especially to pandemic viruses that show an accelerated mutation speed of their surface proteins, hence a resistance to conventional treatments increasing their infectivity and mortality.38 This strategy has been applied to influenza (e.g., H1N1, HCV) and herpes viruses.39−44 The activity of AuNPs is proportional to the surface area exposed.

T164

23716-23833

Sentence

denotes

As a consequence, the size and the morphology of these metal NPs play a substantial role in their antiviral activity.

T165

23834-24152

Sentence

denotes

Recently, Kim et al. have reported that porous AuNPs are able to inhibit influenza A infection more efficiently than nonporous AuNPs.39 This effect has been associated with the higher surface area of the porous material that favors their interaction with capsids and thus increases their antiviral activity (Figure 4).

T166

24153-24262

Sentence

denotes

Figure 4 Schematic illustration of inactivation of influenza A virus (IAV) treated with porous AuNP (PoGNP).

T167

24263-24339

Sentence

denotes

PoGNP interacts with IAV surface proteins and cleaves their disulfide bonds.

T168

24340-24395

Sentence

denotes

Inactivated viruses exhibit lower infectivity to cells.

T169

24396-24476

Sentence

denotes

Reproduced with permission under a Creative Commons CC-BY license from ref (39).

T170

24477-24529

Sentence

denotes

T171

24530-24671

Sentence

denotes

Besides the per se antiviral activity, AuNP surface modifications have been developed in order to enhance their overall therapeutic benefits.

T172

24672-24790

Sentence

denotes

The engineering of tailored AuNPs with selected ligands has allowed the preparation of efficient antiviral nanoagents.

T173

24791-24921

Sentence

denotes

The target ligands can be introduced directly during the particle synthesis via ligand exchange reactions or ligand modifications.

T174

24922-25409

Sentence

denotes

For instance, direct reduction of gold ions in the presence of gallic acid produced homogeneous AuNPs able to sensibly reduce herpes simplex virus infection in vitro.40 Compared to free ligand NPs, functionalized AuNPs benefit from the multivalency effect and higher circulation times, decreasing the needed therapeutic concentrations.39 Functionalized AuNPs can present organic groups that mimic host cell surfaces or other specific molecular patterns that selectively target the virus.

T175

25410-25532

Sentence

denotes

Normally, negative charges are used to mimic cell surfaces and favor the interaction between the particles and the capsid.

T176

25533-26024

Sentence

denotes

In particular, sulfonates and organic sulfates have been used for their capacity to attract the virus via capsid protein interaction and block the HA activity.41 AuNPs functionalized with sulfonates showed an increasing inhibition of influenza A compared to the nanoparticles capped with succinic acid.42 This study also demonstrated that there is not a correlation between the negative charge and the antiviral activity, but instead the inhibition depends mainly on the organic groups used.

T177

26025-26213

Sentence

denotes

Thiol-capped AuNPs also displayed powerful inactivation of bovine viral diarrhea virus in vitro.43 Multivalency has been exploited in more complex systems using dendrons as capping agents.

T178

26214-26396

Sentence

denotes

This strategy allows to generate higher concentrations of the target ligand in close proximity to the AuNPs and to increase the binding efficiency of the nanoparticles to the capsid.

T179

26397-26513

Sentence

denotes

The driving force of the antiviral efficiency relies on the concentration of the targeting agent onto the particles.

T180

26514-26691

Sentence

denotes

Sulfonated dendrons were grafted to AuNPs via a sulfide bond and tested for HIV inhibition.44 The results showed that the decorated AuNPs exerted a higher affinity to the virus.

T181

26692-26932

Sentence

denotes

Additionally, comparing AuNPs functionalized with different generation dendrons, those with a third generation displayed the highest inhibition performance with an IC50 below 0.1 μmol/mL, thus making them attractive for in vivo translation.

T182

26933-27399

Sentence

denotes

It is worth noting that the inhibition efficiency is strictly dependent on the available sulfonate groups present on the surface of the NPs, making crucial a thorough characterization of the material.44 The size of the AuNPs clearly plays an important role in the concentration of targeting ligands exposed per particle.45 Indeed, too big NPs have a limited surface area, while too small would not allow an efficient grafting of the dendrons due to steric hindrance.

T183

27400-27598

Sentence

denotes

For instance, it has been shown that dendron-functionalized AuNPs showed a size-dependent antiviral activity for influenza virus, where 14 nm particles exhibited a higher efficiency than 2 nm AuNPs.

T184

27599-27775

Sentence

denotes

This has been associated with the low functionalization grade of the small nanoparticles and to the inappropriate spatial distribution of the interacting ligand/receptor pairs.

T185

27776-27881

Sentence

denotes

The development of viral proteomics has profoundly transformed the antiviral and disinfection strategies.

T186

27882-28003

Sentence

denotes

In particular, small molecules and peptides able to target and block the viral biochemical machinery have been developed.

T187

28004-28185

Sentence

denotes

However, despite these efforts into the drug design, many of these molecules suffer from poor biological effect, low concentration in the diseased areas, and undesired side effects.

T188

28186-28328

Sentence

denotes

In this context, AuNPs have been coupled to biologically inactive small molecules to create biologically active multivalent AuNP therapeutics.

T189

28329-28627

Sentence

denotes

A bright example has been reported by Bowman et al., where the authors functionalized AuNPs with SDC-1721, a small membrane fusion inhibitor of HIV.46 The results demonstrated that, while pure SDC-1721 has low activity, functionalized AuNPs are able to inhibit HIV replication at μM concentrations.

T190

28628-28688

Sentence

denotes

Similar results have been reported using targeting peptides.

T191

28689-28904

Sentence

denotes

In particular, it was evidenced that the functionalized AuNPs can sensibly reduce the IC50 up to 2 orders of magnitude compared to pure peptides.47 Preliminary results in vivo confirmed the biosafety of the AuNPs.48

T192

28906-28954

Sentence

denotes

Nanoparticles Generating Reactive Oxygen Species

T193

28955-29145

Sentence

denotes

One of the main advantages of using NPs compared to oxidized metals relies on the slow release of ions and clusters from these particles, leading to an enhancement of the antiviral activity.

T194

29146-29380

Sentence

denotes

Additionally, the use of metal NPs containing Cu or Fe in ionic form catalyzes the generation of radicals via Fenton and Fenton-like reactions oxidizing the capsid proteins and consequently blocking the viral infection at early stage.

T195

29381-30083

Sentence

denotes

For instance, copper ions (derived from sulfates or iodide salts) have been widely used as antiviral agents because of their activity on several kinds of enveloped and non-enveloped viruses including influenza virus,49−51 herpes simplex virus52−54 and hepatitis A virus.55 Their mechanism of action relies on the formation of Cu+ ions (from soluble salts or nanoparticles) that generate hydroxyl radicals.56 The use of metallic copper nanostructures in the form of particles or sheets has shown only a moderate efficiency due to the low concentration and low release of Cu+.56 For these reasons, Cu+ salts, where the copper ions are readily present in their active monocationic form, have been favored.

T196

30084-30610

Sentence

denotes

In particular, CuI nanoparticles (stable at room temperature) have been extensively studied for deactivation of feline calicivirus56 and H1N1 pandemic influenza virus.57 However, the use of copper salts at high concentrations can irreversibly alter reactive oxygen species (ROS) homeostasis of healthy cells, provoking a general toxicity for the organism, limiting their applications to disinfection.56 Nanostructured cuprous and cupric oxides have been also extensively employed as antiviral agents for in vitro applications.

T197

30611-30820

Sentence

denotes

For instance, cuprous oxide nanoparticles (CuONPs) were successfully employed against hepatitis C.58 In particular, it was found that these NPs exerted a favorable antiviral activity with no cytotoxic effects.

T198

30821-30909

Sentence

denotes

CuONPs target the binding and entry step of viral infection to hepatic cells (Figure 5).

T199

30910-31048

Sentence

denotes

Similar results were reported on the use of CuONPs against HSV-1, however without any profound investigation on the antiviral mechanism.59

T200

31049-31191

Sentence

denotes

Figure 5 Huh7.5.1 cells at 72 h post-infection were stained with HCV-positive serum from patients (green signal) and with DAPI (blue signal).

T201

31192-31263

Sentence

denotes

Cuprous oxide NPs (CuONPs) are able to reduce viral infection in vitro.

T202

31264-31305

Sentence

denotes

Reproduced with permission from ref (58).

T203

31306-31334

Sentence

denotes

T204

31335-31555

Sentence

denotes

Alternatively, zinc salts have been successfully used as antimicrobial agents from research up to clinical trials for viral warts.60,61 More recently, ZnO nanoparticles (ZnONPs) were developed for the treatment of HSV-2.

T205

31556-31711

Sentence

denotes

ZnONPs were prepared with a tetrapod morphology.62 The results showed that they can mimic cell surface interacting with the HS present on the viral capsid.

T206

31712-31965

Sentence

denotes

Additionally, these particles have been used for photocatalysis showing to efficiently destroy the viral proteins upon UV irradiation.62 Besides all these interesting examples, in vivo applications are still needed to validate this therapeutic modality.

T207

31966-32072

Sentence

denotes

Due to the generation of high levels of ROS, the toxicity of copper nanoparticles has been widely debated.

T208

32073-32320

Sentence

denotes

The antiviral activity of copper nanoparticles is generally associated with the release of Cu+ ions in solution, thus the leakage of cytotoxic cationic species can be modulated by surface functionalization before in vitro and in vivo applications.

T209

32321-32433

Sentence

denotes

On the other side, the use of nanomaterials generating ROS can find applications in textile and surface coating.

T210

32434-32629

Sentence

denotes

The general broad virucidal efficiency of copper oxide nanoparticles shown for H1N1 pandemic influenza57 should be tested on SARS-Cov-2 and might be used for improving mask protection efficiency.

T211

32631-32651

Sentence

denotes

Carbon Nanomaterials

T212

32652-32796

Sentence

denotes

Due to their diversity, versatility, and tunable surface chemistry, carbon nanomaterials have been attractive for several types of applications.

T213

32797-32939

Sentence

denotes

In particular, the past decade has seen a tremendous raise in the preparation of performant carbon-based nanomaterials in the antiviral field.

T214

32940-33041

Sentence

denotes

Fullerene and its derivatives are the most studied carbon nanomaterials for their virucidal activity.

T215

33042-33175

Sentence

denotes

Due to the lack of solubility of pristine fullerene, functionalization strategies have been developed to prepare water-soluble drugs.

T216

33176-33454

Sentence

denotes

Investigations in the biomedical field evidenced the membranotropic capacity of fullerene derivatives.63 By modulating shape and functions, fullerene derivatives have been shown to possess antiviral properties through inhibition of viral entry and blockage of viral replication.

T217

33455-33542

Sentence

denotes

From these results, the attention has been directed also to other carbon nanomaterials.

T218

33543-33689

Sentence

denotes

In particular, functional carbon dots (CDs) and graphene oxide (GO) have been investigated for their ability to block viral entry into host cells.

T219

33691-33706

Sentence

denotes

Glycofullerenes

T220

33707-33886

Sentence

denotes

The emerging of mortal viruses, like Ebola or Zika, and the lack of suitable treatments led the academic and the industrial communities to look for alternative therapeutic routes.

T221

33887-34050

Sentence

denotes

Most of these pathogens are RNA enveloped viruses, and they share common infection mechanisms that can be targeted for the preparation of wide spectrum antivirals.

T222

34051-34305

Sentence

denotes

The external surface of the envelope of these viruses is covered by glycans that tightly interact with lectin receptors on host cells.64 This strong interaction allows the attachment of the virions to the cells, followed by internalization and infection.

T223

34306-34401

Sentence

denotes

Blocking lectin receptors is a general strategy used to stop viral infection at an early stage.

T224

34402-34652

Sentence

denotes

Fullerenes have been widely investigated as antiviral molecules, drug carriers, or tissue scaffolds.65 Fullerene applications have been recently extended to the design of mannosylated derivatives to block the entry of viral particles into host cells.

T225

34653-34773

Sentence

denotes

Mannose, due to the high affinity with lectin receptors, competes with the virus in the interaction with the host cells.

T226

34774-34948

Sentence

denotes

For example, one of the targets is the inhibition of viral particles through the interaction of mannose with the dendritic cell-specific ICAM-grabbing non-integrin (DC-sign).

T227

34949-35209

Sentence

denotes

DC-sign receptors mediate the interactions between DCs and T cells.66,67 To exploit these characteristics, mannose was combined with fullerene in the design of the so-called glycofullerenes to study their capacity to inhibit Ebola, Dengue, and other pathogens.

T228

35210-35744

Sentence

denotes

For this purpose, different glycofullerenes were synthesized by changing the number of mannose units (from 12 to 36) and the spacers between the fullerene moieties and by varying steric hindrance in order to obtain a library of molecules.66 The synthetic route is composed of three steps based on “click chemistry”: (1) assembly of glycodendrons by Cu(I)-catalyzed azide–alkyne cycloaddition (CuAAC), (2) synthesis of alkyne-substituted Bingel-Hirsch hexakis-adducts, and (3) the coupling between the last two products again by CuAAC.

T229

35745-35876

Sentence

denotes

To increase the number of mannose moieties up to 36, the glycodendron core was changed from malonate to trialkynyl pentaerythritol.

T230

35877-35956

Sentence

denotes

In order to compare the different derivatives, in vitro studies were performed.

T231

35957-36141

Sentence

denotes

Jurkat cells (lymphocyte T CD4 immortalized cells) expressing DC-sign were used to prove the inhibition capacity of the glycofullerenes on viral infection of Ebola (Figure 6, route A).

T232

36142-36449

Sentence

denotes

The study revealed an IC50 in the μM range for the 12 mannose fullerene, a lower efficiency with the 36 mannose fullerene with a short spacer (PEG, with 2 ethylene oxide units), while a nanomolar IC50 was achieved with 36 mannose fullerenes with a longer spacer (PEG, with 3 ethylene oxide units) (Table 3).

T233

36450-36618

Sentence

denotes

This first proof-of-concept study was then expanded, aiming to obtain a better antiviral activity by increasing the valence and inserting longer and flexible spacers.68

T234

36619-36708

Sentence

denotes

Figure 6 Chemical design and general scheme to block the viral entry by glycofullerenes.

T235

36709-37011

Sentence

denotes

Three different shapes of glycofullerenes can act as inhibitor of viral infection: (A) shape composed of monodisperse fullerene bearing mannose, (B) assembly as “sugar balls” of tridecafullerenes exhibiting mannose on the edges, and (C) supramolecular micellar aggregates of fullerenes bearing mannose.

T236

37012-37091

Sentence

denotes

Table 3 Antiviral Activity of Different Fullerenes Functionalized with Mannose

T237

37092-37227

Sentence

denotes

material functional groups core function number of mannose moieties virus number of atoms between fullerene and mannose IC50 ref

T238

37228-37294

Sentence

denotes

glycofullerene monosaccharide malonate 12 Ebola 8 2 μM (66)

T239

37295-37375

Sentence

denotes

glycofullerene monosaccharide trialkynyl pentaerythritol 36 Ebola 22 68 μM

T240

37376-37457

Sentence

denotes

glycofullerene monosaccharide trialkynyl pentaerythritol 36 Ebola 28 0.3 μM

T241

37458-37535

Sentence

denotes

trideca-fullerenesa monosaccharide malonate 120 Ebola 8 20.375 nM (69)

T242

37536-37606

Sentence

denotes

trideca-fullerenesb monosaccharide malonate 120 Ebola 8 0.667 nM

T243

37607-37692

Sentence

denotes

glycofullerene disaccharide trialkynyl pentaerythritol 36 Zika 29 8.35 nM (70)

T244

37693-37708

Sentence

denotes

Dengue 7.71 nM

T245

37709-37793

Sentence

denotes

trideca-fullerenes disaccharide trialkynyl pentaerythritol 120 Zika 29 0.52 nM

T246

37794-37810

Sentence

denotes

Dengue 0.098 nM

T247

37811-37896

Sentence

denotes

trideca-fullerenes disaccharide trialkynyl pentaerythritol 360 Zika 48 0.067 nM

T248

37897-37913

Sentence

denotes

Dengue 0.035 nM

T249

37914-38010

Sentence

denotes

micellar glycofullerenes monosaccharide trialkynyl pentaerythritol 6 Ebola 23 424 nM (72)

T250

38011-38102

Sentence

denotes

micellar glycofullerenes monosaccharide trialkynyl pentaerythritol 12 Ebola 25 196 nM

T251

38103-38163

Sentence

denotes

a Small spacer between core C60 and surrounding fullerenes.

T252

38164-38224

Sentence

denotes

b Large spacer between core C60 and surrounding fullerenes.

T253

38225-38317

Sentence

denotes

Based on these studies, another class of multivalent fullerene dendrimers was then designed.

T254

38318-38461

Sentence

denotes

A fast and controlled synthetic route was developed to achieve giant globular multivalent fullerenes, containing hundreds of functional groups.

T255

38462-38639

Sentence

denotes

The first study was performed with tridecafullerenes containing 120 mannoses.69 The molecular structure is composed of 12 hexakis C60 surrounding a C60 core (Figure 6, route B).

T256

38640-38944

Sentence

denotes

Compared to the previous study, an IC50 3 orders of magnitude lower was measured on the inhibition of Ebola virus (Table 3).66,68 In order to present more carbohydrates at the periphery of the dendrimer, a trialkynyl pentaerythritol derivative allowed to afford a tridecafullerene with 360 carbohydrates.

T257

38945-39367

Sentence

denotes

In this case, the molecule was synthesized with a C60 tridecafullerene bearing α(1,2)mannobioside.70 The use of this disaccharide was already investigated, showing an increase of affinity with DC-sign receptors by a factor of 3–4.71 The synthetic strategy exploited also the use of strain-promoted copper-free cycloaddition of azides to alkynes (SPAAC) for the coupling of the core fullerene to the surrounding fullerenes.

T258

39368-39450

Sentence

denotes

SPAAC allows an easier purification avoiding the removal of cytotoxic copper ions.

T259

39451-39562

Sentence

denotes

The inhibition performance of this molecule was studied in vitro with viral pseudoparticles of Dengue and Zika.

T260

39563-39673

Sentence

denotes

The comparison was made between 360 and 120 disaccharides tridecafullerenes and 36 disaccharide monofullerene.

T261

39674-39807

Sentence

denotes

The results highlighted a picomolar IC50 inhibition on both Zika and Dengue models for the 360 disaccharide glycofullerene (Table 3).

T262

39808-39922

Sentence

denotes

The ability to inhibit other types of viruses allows the use of glycofullerenes as broad spectrum antiviral drugs.

T263

39923-40019

Sentence

denotes

Moreover, the negligible toxicity to other cells proved the biocompatibility of these molecules.

T264

40020-40252

Sentence

denotes

Following an alternative strategy, a supramolecular assembly of monodisperse glycofullerenes, leading to the formation of micelles, was achieved and tested.72 These micelles present a uniform and spherical shape (Figure 6, route C).

T265

40253-40457

Sentence

denotes

The aggregation synthetic route is faster compared to a controlled synthesis of giant glycofullerenes, but it might suffer from low reproducibility and batch-to-batch differences between each formulation.

T266

40458-40721

Sentence

denotes

This self-assembled C60 functionalized with 6 or 12 mannoses exposes a large amount of carbohydrate at the surface, leading to an inhibition of Ebola virus in the nanomolar range (IC50 of 424 nM for six mannoses and 196 nM for 12 mannoses, respectively, Table 3).

T267

40722-40812

Sentence

denotes

Further in vitro studies evidenced again a good biocompatibility of these glycofullerenes.

T268

40813-40950

Sentence

denotes

While there are no in vivo studies yet, these promising results enlarge the panel of molecules in the fight against new emerging viruses.

T269

40951-41081

Sentence

denotes

Functionalized fullerenes have been used for their ability to compete with viral particles through lectin receptors in host cells.

T270

41082-41322

Sentence

denotes

There has been a tremendous advancement in the functionalization of fullerenes leading to the preparation of derivatives with a high amount of mannose, capable to enhance the multivalency effect and thus to increase the therapeutic outcome.

T271

41323-41390

Sentence

denotes

First, glycofullerenes do not have an intrinsic virucidal activity.

T272

41391-41481

Sentence

denotes

They can reduce the infectivity, but they are not able to completely inactivate the virus.

T273

41482-41606

Sentence

denotes

Second, the mechanism of action of glycofullerenes relies on their interaction with host cells and not with viral particles.

T274

41607-41810

Sentence

denotes

Thus, for a therapeutic application, they should be injected at different time points, ensuring that the local concentration is therapeutically relevant to prevent the virus from invading the host cells.

T275

41811-41913

Sentence

denotes

In addition, glycofullerenes can be internalized into host cells losing their viral “shield” activity.

T276

41914-42049

Sentence

denotes

On the other hand, the well-developed surface chemistry of glycofullerenes can be used for other key receptors involved in viral entry.

T277

42050-42249

Sentence

denotes

For instance, in the case of the current SARS-Cov-2 pandemic, a similar click chemistry strategy can be used to anchor ligands recognized by human lung ACE2 receptors and so inhibiting viral entry.73

T278

42251-42277

Sentence

denotes

Other Carbon Nanomaterials

T279

42278-42394

Sentence

denotes

Alongside fullerenes, other carbon nanomaterials (NMs) have been scrutinized for their ability to block viral entry.

T280

42395-42481

Sentence

denotes

CDs and GO are the most known and studied carbon NMs with marked antiviral properties.

T281

42482-42528

Sentence

denotes

CDs are zero-dimensional carbon nanoparticles.

T282

42529-42631

Sentence

denotes

They are generally produced via hydrothermal decomposition of carbon containing “low-cost” precursors.

T283

42632-42792

Sentence

denotes

The use of CDs in the biomedical field has been encouraged by their easy preparation, low toxicity, fluorescence properties, and easy surface functionalization.

T284

42793-43036

Sentence

denotes

Pristine CDs have shown moderate viral blocking activity for HIV infection in vitro.74 This has been associated with the surface of the material rich in carboxylic and hydroxyl groups prone to form noncovalent interaction with viral membranes.

T285

43037-43200

Sentence

denotes

Moreover, due to the complexity of the biological systems these nonspecific interactions could not be so effective in vivo, likely reducing the antiviral efficacy.

T286

43201-43302

Sentence

denotes

Therapeutic targeting molecules can be grafted onto a CD surface to enhance their antiviral activity.

T287

43303-43412

Sentence

denotes

In this context, the design of multifunctional CD platforms can be obtained through two different strategies.

T288

43413-43552

Sentence

denotes

The first consists in a single-step reaction that foresees the insertion of the therapeutic molecule directly into the step of preparation.

T289

43553-43650

Sentence

denotes

Target molecules are decomposed with the other precursors, generating the desired functional CDs.

T290

43651-43758

Sentence

denotes

This protocol is fast and efficient, however the drug loading as well as its activity are hard to estimate.

T291

43759-43889

Sentence

denotes

Indeed, the hydrothermal treatment can alter the chemical structure of the active molecule, thus vanishing its therapeutic effect.

T292

43890-44116

Sentence

denotes

For these reasons, the reaction conditions must be carefully controlled.75 The second method is a two-step reaction and implies the postfunctionalization via amide formation on the surface of the CDs rich in carboxylic groups.

T293

44117-44247

Sentence

denotes

This strategy offers a better chemical control, but the yield and the drug loading may not be quantitative and high, respectively.

T294

44248-44323

Sentence

denotes

Different functionalized CDs were prepared to hamper host cell viral entry.

T295

44324-44463

Sentence

denotes

For instance, benzoxazine (a low water-soluble antiviral agent) was incorporated into the CD structure during their preparation (Figure 7).

T296

44464-44856

Sentence

denotes

The as-prepared CDs showed a broad spectrum viral blocking capacity in vitro for enveloped (e.g., Japanese encephalitis virus, Dengue virus, and Zika virus) and non-enveloped viruses (e.g., porcine parvovirus and adenovirus-associated virus).76 These positive results were explained by the efficient binding and deactivation induced by the multivalent effect of the CDs to the viral particles

T297

44857-44955

Sentence

denotes

Figure 7 Illustration of benzoxazine-functionalized CDs and their broad antiviral entry activity.

T298

44956-44997

Sentence

denotes

Reproduced with permission from ref (76).

T299

44998-45026

Sentence

denotes

T300

45027-45106

Sentence

denotes

Amino-functionalized CDs were also tested for the treatment of human norovirus.

T301

45107-45291

Sentence

denotes

In this study, CDs were functionalized with 2,2′-(ethylenedioxy)bis(ethylamine) (EDA) and 3-ethoxypropylamine (EPA) via amide bond formation.77 These NMs exerted a good viral blockage.

T302

45292-45492

Sentence

denotes

In particular, EPA-functionalized CDs were able to inhibit 100% of viral infection at concentration of 2 μg/mL, while in the case of CDs prepared with the other amines, 80% of inhibition was reported.

T303

45493-45589

Sentence

denotes

These effects have been associated with the higher positive charge of CD-EDA compared to CD-EPA.

T304

45590-45719

Sentence

denotes

Another surface group used for viral targeting is boronic acid (BA), which can bind glycosylated surfaces forming boronic esters.

T305

45720-46220

Sentence

denotes

This strategy was successfully adopted to treat HIV where the boronic groups, linked to different nanoparticles (e.g., silica nanoparticles and nanodiamonds) can target gp120 receptors on the viral envelope inhibiting the infection.78 Another recent study proposed the use of CD functionalized with phenylboronic acid for prevention of HIV infection.74 The functional materials showed good inhibition properties compared to nonfunctionalized CDs by preventing the binding to the target cell in vitro.

T306

46221-46315

Sentence

denotes

Overall, the use of CDs for stopping host cell viral entrance has shown good results in vitro.

T307

46316-46420

Sentence

denotes

However, there is a lack of proofs in vivo limiting their applications to surface disinfection or masks.

T308

46421-46567

Sentence

denotes

In addition, most of the in vitro studies foresee first the contact of the CDs with the viral particles and then their incubation with host cells.

T309

46568-46733

Sentence

denotes

Deeper investigations should be performed adding the NMs at other time points (for instance in infected cells) to understand if the antiviral activity is maintained.

T310

46734-46869

Sentence

denotes

In addition, CDs have been successfully used for photodynamic therapy (generating radicals upon light irradiation) in cancer treatment.

T311

46870-47054

Sentence

denotes

The same approach may be used to combat viral infections, where the antiviral activity induced by the surface modification can be sensibly enhanced by ROS generation under irradiation.

T312

47055-47381

Sentence

denotes

Graphene materials, and in particular GO and reduced GO (rGO), have been used for different biomedical applications including drug delivery, biosensing, and tissue engineering.5 GO platforms have shown also interesting antimicrobial activity.79 Regarding viral infection, GO was used to block the virus entrance in host cells.

T313

47382-47571

Sentence

denotes

GO and rGO can be considered as two-dimensional materials that contain hydrophilic and hydrophobic domains allowing to adsorb many biological molecules including nucleic acids and proteins.

T314

47572-47730

Sentence

denotes

GO showed low interaction with viruses, however its surface functionalization with target molecules can sensibly enhance its affinity for the viral particles.

T315

47731-47985

Sentence

denotes

Additionally, GO can be used as photothermal agent (generation of heat by NIR irradiation) or photodynamic therapy (using visible light irradiation) inactivating the capsids by local thermal shock or by radical formation during irradiation, respectively.

T316

47986-48080

Sentence

denotes

The use of phototherapies may significantly augment the antiviral properties of the materials.

T317

48081-48263

Sentence

denotes

However, we must keep in mind that these therapeutic modalities can be applied only to disinfection, since the radical/heat production may be harmful for the healthy tissues in vivo.

T318

48264-48446

Sentence

denotes

Photodynamic therapy has been successfully exploited using bacteriophage MS2 as a model virus.80 In this study, GO was functionalized with an aptamer recognized by the viral surface.

T319

48447-48608

Sentence

denotes

The results showed that this functionalization is able to enhance the binding efficiency of the MS2 capsids onto the GO surface compared to nonfunctionalized GO.

T320

48609-48775

Sentence

denotes

Subsequently, irradiation in the visible light was able to disinfect the solution, while nonfunctionalized GO showed much less activity due to the lack of adsorption.

T321

48776-49020

Sentence

denotes

Despite these interesting results, this pioneer work remains at an early research stage since the use of high light dose (300 W for 10–140 min) and the lack of material recovery and reuse make its application for surface disinfection difficult.

T322

49021-49080

Sentence

denotes

GO can be also used as a platform to link antiviral agents.

T323

49081-49433

Sentence

denotes

Encouraging results were reported using GO with hypericin for the treatment of a recently appeared duck reovirus.81 More recently, Deokar et al. reported an original rGO-based multifunctional platform for HSV-1 treatment.82 In this work, the authors functionalized the material with organic sulfate groups and iron oxide magnetic nanoparticles (FeNPs).

T324

49434-49531

Sentence

denotes

The rGO functionalized with the sulfate is able to mimic the host cell surface and to bind HSV-1.

T325

49532-49687

Sentence

denotes

Subsequently, the viral particles captured onto the rGO-FeNP surface can be concentrated via magnetic precipitation and destroyed via photothermal therapy.

T326

49688-49800

Sentence

denotes

This approach is highly efficient for disinfection with low energy (1.6 W/cm2 for 7 min) and cost effectiveness.

T327

49801-50036

Sentence

denotes

HS is a common entry receptor in various types of viruses (e.g., herpes viruses, human papillomavirus, Dengue virus).83 The use of organic sulfate-functionalized graphene sheets mimicking HS, like GO and rGO, has been already explored.

T328

50037-50417

Sentence

denotes

However, it is worth noting that these NMs are prone to strongly adsorb proteins in culture environments (coronation), likely inhibiting their antiviral efficacy.84 High loadings of sulfate groups were introduced onto rGO using polyglycerol sulfate.85,86 This approach has been used for inhibition of orthopoxvirus, pseudorabies virus, and African swine fever virus in vitro.85,86

T329

50418-50468

Sentence

denotes

Graphene has also been used as antiviral material.

T330

50469-50737

Sentence

denotes

Polysulfates and fatty amines were grafted onto graphene surface via triazine chemistry for the treatment of herpes simplex virus.87 This strategy promotes the synergy between the electrostatic and hydrophobic interactions, showing incredibly high inhibition efficacy.

T331

50738-50824

Sentence

denotes

Overall, graphene materials have shown a good capacity to block host cell viral entry.

T332

50825-50963

Sentence

denotes

Disinfection with graphene family materials is also promising, offering the possibility to couple high viral binding with phototreatments.

T333

50964-51137

Sentence

denotes

Regarding the GO and rGO activity in cellular environments, different parameters must be considered such as protein coronation, blood circulation time, and activity in vivo.

T334

51138-51245

Sentence

denotes

So far, the use of sulfonic groups introduced via diazonium salt decomposition has been largely privileged.

T335

51246-51437

Sentence

denotes

We take this opportunity to encourage future studies using other targeting groups (e.g., boronic acids) and grafting methods (e.g., epoxide ring opening or hydroxyl esterification reactions).

T336

51439-51474

Sentence

denotes

Mechanical Disruption of the Capsid

T337

51475-51786

Sentence

denotes

The most direct way to suppress viruses and stop the spreading of viral infection is to inactivate them before the attachment to the host cells, by binding to the acceptor proteins.88 One of the most conserved targets of viral attachment ligands is the heparan sulfate proteoglycan (HSPG), previously mentioned.

T338

51787-51983

Sentence

denotes

HSPGs are expressed on the surface of almost all eukaryotic cell types, and many viruses like HIV-1, HSV, human papilloma virus (HPV) exploit HSPGs as the target of their viral attachment ligands.

T339

51984-52153

Sentence

denotes

Bearing in mind this behavior, different studies have used HSPG-mimicking materials to target this type of virus–cell interaction and to achieve broad spectrum efficacy.

T340

52154-52286

Sentence

denotes

For example, in one key study, AuNPs were functionalized with mercaptoethanesulfonate (MES) based on its mimicry of HS (Au-MES NPs).

T341

52287-52504

Sentence

denotes

Au-MES NPs were shown to interfere with viral attachment, viral entry, and cell-to-cell spreading.89 The importance of the polyvalent interactions with the virus makes these NPs a good candidate for antiviral therapy.

T342

52505-52686

Sentence

denotes

However, Au-MES NPs presented virustatic activity, meaning that upon dilution of the NPs, the virus recovers its infectivity due to the reversibility of the cell-virion interaction.

T343

52687-52825

Sentence

denotes

This problem was solved by Stellacci and colleagues who developed NPs coated with mercapto-1-undecanesulfonate (MUS) ligands (Au-MUS NPs).

T344

52826-53244

Sentence

denotes

The long aliphatic and flexible linkers provide stronger associations with the viral particles compared to Au-MES NPs, leading to local distortions and eventually inducing a global deformation and breaking of the capsid that inactivates its contagion irreversibly (Figure 8).90 These Au-MUS NPs were tested against different HSPG-dependent viruses, showing a high viricidal activity over HSV, HPV, and RSV (Figure 9A).

T345

53245-53511

Sentence

denotes

Furthermore, the activity of the Au-MUS NPs was studied in vivo using mice infected with RSV, indicating that the material can prevent pulmonary dissemination of the infection and showing potential use as medically relevant virucidal drugs to fight viral infections.

T346

53512-53793

Sentence

denotes

More recently, the concept was further extended to cyclodextrins modified with mercapto-1-undecanesulfonate, proposing this system as a broad spectrum virucidal macromolecule.91 Besides Au-NPs, a similar mechanism of disruption was studied with other materials like graphene or GO.

T347

53794-54083

Sentence

denotes

In a study, in which the toxicity of graphene was evaluated theoretically, it was also shown that graphene nanosheets can interrupt the hydrophobic protein–protein interaction, which is essential to biological functions.92 This feature was attributed to the hydrophobic nature of graphene.

T348

54084-54227

Sentence

denotes

Thus, it seems energetically favorable for graphene to slide between the interface of two proteins in contact, due to hydrophobic interactions.

T349

54228-54772

Sentence

denotes

In another study inspired by this behavior, the authors performed molecular dynamics simulations of graphene nanosheets in the proximity of the surface of the Ebola viral matrix protein VP40 showing that the nanosheets can break the hydrophobic interactions in VP40, a key protein for the replication and stability of Ebola virus (Figure 8).93 These findings suggest that graphene nanosheets might have potential antiviral activity against Ebola; however, there is a lack of experimental evidence that corroborates this mechanism of disruption.

T350

54773-55229

Sentence

denotes

On the other hand, GO was tested experimentally against Pseudorabies virus and Porcine epidemic diarrhea virus (PEDV), showing significant decrease in the infectivity.94 It was found that the negatively charged surface of GO is important for the adsorption of the virus, whose surface is positively charged, and that GO could directly interact with the viral particles and destroy their structures due to the sharp edges of the material (Figures 8 and 9B).

T351

55230-55296

Sentence

denotes

Figure 8 Mechanical disruption mechanisms of different NMs. Left:

T352

55297-55389

Sentence

denotes

Au-MUS NPs inducing mechanical forces in the virus capsid leading to inactivation.90 Center:

T353

55390-55462

Sentence

denotes

Graphene NSs disrupting VP40 hydrophobic interactions in Ebola.93 Right:

T354

55463-55548

Sentence

denotes

Interaction between negatively charged surface of GO and positively charged capsid.94

T355

55549-55596

Sentence

denotes

Figure 9 Interaction of NMs with viral capsid.

T356

55597-55631

Sentence

denotes

A) Gold NPs acting on HSV-2 virus.

T357

55632-55708

Sentence

denotes

After 90 min, the percentage of destroyed virus was significantly increased.

T358

55709-55720

Sentence

denotes

Scale bars:

T359

55721-55728

Sentence

denotes

100 nm.

T360

55729-55770

Sentence

denotes

Reproduced with permission from ref (90).

T361

55771-55847

Sentence

denotes

T362

55848-55935

Sentence

denotes

After incubation with GO for 1 h, part of the virus envelope and spikes were destroyed.

T363

55936-55947

Sentence

denotes

Scale bars:

T364

55948-55955

Sentence

denotes

200 nm.

T365

55956-55997

Sentence

denotes

Reproduced with permission from ref (94).

T366

55998-56040

Sentence

denotes

T367

56041-56267

Sentence

denotes

The mechanical disruption of the capsid is a peculiar antiviral mechanism associated with some NMs. In particular, the use of specific sulfonates able to mimic heparan sulfate can be also used to target SARS-Cov-2 infection.95

T368

56269-56305

Sentence

denotes

Antiviral Activity Inside Host Cells

T369

56306-56447

Sentence

denotes

Blocking the viral entry, via liquid/surface disinfection or once in the body, is a powerful strategy to hamper early stage viral contagions.

T370

56448-56591

Sentence

denotes

On the other hand, when infections have already spread and reached middle and late stages, alternative pharmacological strategies are required.

T371

56592-56707

Sentence

denotes

The study of the viral pathogenesis and machinery inside host cells has allowed the preparation of different drugs.

T372

56708-56824

Sentence

denotes

Due to the present pandemic, such antiviral drugs are now of top interest in the scientific and medical communities.

T373

56825-57149

Sentence

denotes

So far, few antiviral drugs are clinically available, and their mechanisms of action consist on the inhibition of reverse transcriptase (HIV, hepatitis), DNA inhibition of polymerase (herpes, HIV), inhibition of protease (HIV), blockage of ion channels (influenza), and inhibition of neuramidase (HIV, influenza, hepatitis).

T374

57150-57215

Sentence

denotes

However, these drugs suffer from moderate to severe side effects.

T375

57216-57369

Sentence

denotes

Additionally, rapid mutations in the viral machinery make them resistant to the treatments, making the control and the stop of the infection challenging.

T376

57370-57432

Sentence

denotes

The use of HNMs in drug delivery has shown several advantages.

T377

57433-57499

Sentence

denotes

First, HNMs can increase drug solubility and its circulation time.

T378

57500-57665

Sentence

denotes

Additionally, they can be functionalized with targeting molecules able to direct the drug to the desired organs and so avoiding side effects and reducing the dosage.

T379

57666-57722

Sentence

denotes

More recently, new antiviral mechanisms were discovered.

T380

57723-57907

Sentence

denotes

In particular, it was found that different types of HNMs are able to change the ROS homeostasis in infected host cells, stopping the viral replication and preserving the cell survival.

T381

57908-58034

Sentence

denotes

In this section both drug delivery materials and HNMs with ROS modulation properties will be critically presented (Figure 10).

T382

58035-58120

Sentence

denotes

Figure 10 Illustration of NMs interacting with host cell to prevent viral spreading.

T383

58121-58173

Sentence

denotes

Left: NMs are used for delivery of antiviral agents.

T384

58174-58269

Sentence

denotes

Right: NMs can change the ROS homeostasis slowing down the infection and helping cell survival.

T385

58271-58302

Sentence

denotes

Nanomaterials for Drug Delivery

T386

58303-58373

Sentence

denotes

Different HNMs have been widely tested for drug delivery applications.

T387

58374-58509

Sentence

denotes

The advantages of this strategy are several including enhance drug solubility and the possibility of targeting and multivalent effects.

T388

58510-58689

Sentence

denotes

As a potential broad spectral antiviral agent, AgNPs can prevent the virus from adsorbing to the host cell in the early stage of infection and thus show strong antiviral activity.

T389

58690-58792

Sentence

denotes

After the cells are infected by the virus, there are ways to inhibit cell apoptosis (Figure 2, right).

T390

58793-59111

Sentence

denotes

For example, Lv et al. studied the anti-TGEV activity of AgNPs in swine testicle cells and explored the possible mechanism of AgNP inhibition of TGEV infection-induced apoptosis.18 The results showed that these AgNPs are able to decrease cell apoptosis through the activation of p38/mitochondria-caspase-3 signaling.18

T391

59112-59369

Sentence

denotes

Although the use of AgNPs has shown good antiviral action to further improve the therapeutic effects and reduce both side effects and drug resistance, the way of binding drugs or genes and other therapeutic agents to AgNPs has received particular attention.

T392

59370-59526

Sentence

denotes

It has been observed that AgNPs inhibit the activities of neuraminidase and hemagglutinin, preventing the H1N1 influenza virus from attaching to host cells.

T393

59527-59655

Sentence

denotes

At the same time, the potential molecular mechanisms revealed that caspase-3-mediated apoptosis was inhibited by ROS generation.

T394

59656-59714

Sentence

denotes

AgNPs modified with polyethylenimine (PEI) can bind siRNA.

T395

59715-60020

Sentence

denotes

Ag@PEI@siRNA exhibited superior abilities for enhanced cellular uptake and blocking EV71 virus infection and significantly decreased the apoptotic cell population, which prevented the spread of EV71 virus.37 In addition to drugs and siRNA, neutralizing antibodies in combination with AgNPs were developed.

T396

60021-60175

Sentence

denotes

The results demonstrated that there is an additive effect between the antibody and AgNPs when combined against cell-associated HIV-1 infection in vitro.96

T397

60176-60242

Sentence

denotes

The membranotropic properties of fullerenes were widely exploited.

T398

60243-60586

Sentence

denotes

For example, pristine C60, after accumulation at the cell membrane, can translocate into the cytoplasm by crossing the membrane through multiple energy-dependent pathways despite its hydrophobic character.97 Fullerenes can be made water dispersible using surfactants, sonication or first dissolving them in appropriate organic solvents (DMSO).

T399

60587-60824

Sentence

denotes

The use of fullerenes as inhibitors of viruses started in 1993 with a study focused on HIV infection.98 This work revealed the interaction between C60 derivatives and HIV protease through molecular modeling and experimental verification.

T400

60825-60960

Sentence

denotes

HIV protease (HIV-PR) is involved in the mechanism of replication in the maturation of HIV virion, cleaving newly synthesized proteins.

T401

60961-61229

Sentence

denotes

The active site of HIV protease has a cavity of 10 Å closed to the diameter of C60 cage.99 Molecular docking and experiments on HIV-PR catalytic activity revealed a blockage of the active site of the enzyme by van der Waals interactions leading to an antiviral effect.

T402

61230-61393

Sentence

denotes

The following studies were based on a structure–activity relationship between functionalized fullerenes and HIV-PR with the aim to increase the antiviral activity.

T403

61394-61809

Sentence

denotes

The introduction of pyrrolidinium salts onto C60 was tested against the activity of HIV-1 strain.100 In a second study, C60 bearing two ammonium groups was applied against the activity of HIV-1 and HIV-2 strains.101 The results showed the importance of having two moieties in a precise position on the fullerene cage and the influence of the charge of the different salts sensibly increasing its antiviral activity.

T404

61810-61912

Sentence

denotes

Further investigations using different functional groups (e.g., amino acid derivatives) were explored.

T405

61913-62230

Sentence

denotes

C60 functionalized with aminobutyric acid and aminocaproic acid was able to inhibit HIV viral replication at subnanomolar concentrations.63 In another work, anionic and cationic pyrrolidinium salts and amino acid functionalized fullerene derivatives were used for the inhibition of HIV reverse transcriptase (HIV-RT).

T406

62231-62596

Sentence

denotes

Fullerene compounds were compared to nevirapine, an available drug against HIV-RT, revealing a better inhibition compared to the pure drug.102 The antiviral property of carboxylated fullerenes was confirmed by another study.103 Results obtained in vitro on CEM cell line showed low toxicity and a submicromolar EC50 against HIV-1 and HIV-2 strain viral replication.

T407

62597-62680

Sentence

denotes

Several mechanisms of HIV protease inhibitors have been hypothesized and simulated.

T408

62681-62934

Sentence

denotes

Some studies investigated the action of fullerene derivatives on viral replication cycle and the virus maturation (Figure 11).98,104 For the latter, it was suggested an impairment due to a strong interaction between fullerene and the immature capsid.105

T409

62935-63014

Sentence

denotes

Figure 11 Illustration of fullerene affecting the viral replication mechanism.

T410

63015-63141

Sentence

denotes

Interaction between fullerene and viral protein blocking either the transcription or the translation in the viral replication.

T411

63142-63485

Sentence

denotes

Other RNA viruses share ways of viral replication similar to HIV.106 C60 functionalized with an amino acid derivative was investigated against Hepatitis C RNA polymerase (HCV-RP).102 This essential enzyme for viral replication was inhibited in a submicromolar range, similarly to benzo-1,2,4-thiadiazine, a potent specific inhibitor of HCV-RP.

T412

63486-63776

Sentence

denotes

Another publication highlighted the effect of a C70 poly(carboxylic acid) derivative on different strains of influenza virus (e.g., A, H1N1, H3N2, and B).107 The inhibition was comparable to Tamiflu, rimantadine, ribavirin, and amantadine, but the mechanism of action remains still unclear.

T413

63777-63891

Sentence

denotes

These data emphasize that fullerenes C60 or C70 can be potent universal antiviral drugs for RNA enveloped viruses.

T414

63892-63994

Sentence

denotes

However, clear elucidations of the mechanisms of action and in vivo studies are still a missing point.

T415

63995-64253

Sentence

denotes

Due to their high surface/weight ratio and capacity to pass through cell membranes, carbon nanotubes (CNTs) have been extensively explored for drug and gene delivery applications.108 CNTs have been also successfully used for the delivery of antiviral agents.

T416

64254-64503

Sentence

denotes

Compared to pristine materials, oxidized CNTs (ox-CNTs) showed an inhibitory activity for HIV viruses per se.109 This effect has been associated with the oxygenated groups that increase the hydrophilicity and the colloidal stability of the material.

T417

64504-64692

Sentence

denotes

The antiviral efficiency has been correlated to the ox-CNT interaction with host cells.109 However, it is not clear how and what kind of mechanism blocks the viral machinery in host cells.

T418

64693-64784

Sentence

denotes

Different anchoring strategies have been explored to link antiviral drugs onto CNT surface.

T419

64785-65427

Sentence

denotes

For instance, ox-CNTs were covalently linked to 2-amino-3-nitro-1-(3,5-dimethylbenzyl)-aniline (CHI360) and N-(2-aminophenyl-3-nitro-)-3,5-dimethylbenzenesulfonamide (CHI415), two active non-nucleoside reverse transcriptase inhibitors for HIV treatment.109 Following the covalent conjugation, only a moderate antiviral effect was observed compared to pure drugs, indicating that most probably the NM cell trafficking played a key role on the virucidal activity.109 In another study, ox-CNTs functionalized with cyclodextrin were used for the delivery of acyclovir (a prodrug inhibitor of the viral DNA polymerases) for the treatment of HSV-1.

T420

65428-65729

Sentence

denotes

Preliminary results showed that when acyclovir was delivered via the nanotubes, the viral antireplicative effect was higher than the free drug.110 More recently, a similar approach was applied to herpes virus using cyclodextrin and PEI-functionalized CNTs for co-delivery of cidofovir and plasmid DNA.

T421

65730-66144

Sentence

denotes

However, the antiviral effect of the materials was not explained, and the transfection effect was not satisfactory.111 Functionalized CNTs have been also used for delivery of ribavirin in vivo using grass carp as an animal model for the study of grass carp reovirus. ox-CNTs were first functionalized via amidation with BSA, and then ribavirin was covalently bound to the protein via esterification (Figure 12).112

T422

66145-66246

Sentence

denotes

Figure 12 Schematic procedure of the functionalization of single-walled CNTs (SWCNTs) and ribavirin.

T423

66247-66289

Sentence

denotes

Reproduced with permission from ref (112).

T424

66290-66319

Sentence

denotes

T425

66320-66541

Sentence

denotes

In vivo tests demonstrated that, when ribovirin was shuttled by ox-CNTs, the antiviral efficiency was significantly increased without any evident toxicity and no significant changes in ROS-generating enzymatic activities.

T426

66542-66609

Sentence

denotes

The use of CNTs in drug delivery is however still controversial.113

T427

66610-66699

Sentence

denotes

Graphene-based materials have been limitedly studied as antiviral drug delivery carriers.

T428

66700-66751

Sentence

denotes

Only a few examples can be found in the literature.

T429

66752-67001

Sentence

denotes

Graphene quantum dots (GQDs) were used for drug delivery of CHI360 and CHI415 and tested in vitro against HIV similarly to CNTs.109 Both the prepared GQD-CHI360 and GQD-CHI415 showed a high antiviral activity once into host cells, with low toxicity.

T430

67002-67115

Sentence

denotes

GO has been also used for the delivery of DNAzyme into hepatic cells allowing to block the hepatitis C infection.

T431

67116-67217

Sentence

denotes

This specific DNA single strand is able to recognize the viral mRNA and to silence its expression.114

T432

67218-67292

Sentence

denotes

Overall, carbon NMs proved to be interesting carriers for antiviral drugs.

T433

67293-67393

Sentence

denotes

However, several questions need to be answered before their safe application as antiviral materials.

T434

67394-67639

Sentence

denotes

Different reports have demonstrated that pristine CNTs display relevant toxicity for healthy cells, but by oxidation of the tubes, the side effects can be sensibly reduced.109 In addition, more investigations should be performed on CNT toxicity.

T435

67640-67923

Sentence

denotes

These nanocarriers indeed are not “innocent delivery agents”, but they play a key role in drug internalization pathway and in host cell machinery that might be averse to the expected therapeutic effects.109 So far, CNT application in biological systems has been studied for 20 years.

T436

67924-68047

Sentence

denotes

However, due to their possible toxicity, their real application in clinics seems to be steeper and difficult to achieve.112

T437

68049-68089

Sentence

denotes

Materials Tuning Reactive Oxygen Species

T438

68090-68170

Sentence

denotes

ROS homeostasis in infected cells has been studied for both RNA and DNA viruses.

T439

68171-68685

Sentence

denotes

For instance, it was shown that infection of mice with influenza A decreased the concentration of lung glutathione and the antioxidant vitamin C, providing evidence that the viral infection was associated with oxidative stress in vitro as well as in vivo.115 Similarly, in HIV infection, induced oxidative stress in host T cells and high concentrations of antioxidants are able to slow down the cell-to-cell viral spreading.115 The increase of ROS concentration is a common process in most of the viral infections.

T440

68686-68762

Sentence

denotes

However, the mechanism of radical generation is different from case to case.

T441

68763-68876

Sentence

denotes

Several proofs suggest that modulating ROS homeostasis in infected cells can slow down or block the infection.116

T442

68877-68945

Sentence

denotes

HNMs have been shown to be powerful allies against viral infections.

T443

68946-69067

Sentence

denotes

In particular, metal or metal oxide NMs, once internalized, can regulate the radical production into infected host cells.

T444

69068-69216

Sentence

denotes

In this scenario, the NMs can work following two different mechanisms: (1) enhancing radical activity or (2) quenching ROS inside cell compartments.

T445

69217-69368

Sentence

denotes

In the first case, metal oxide NPs are able to convert superoxide ions into more reactive hydroxyl radical species via Fenton or Fenton-like reactions.

T446

69369-69508

Sentence

denotes

The excess of superoxide ions is able to oxidize the viral proteins and the genetic material and therefore efficiently block the infection.

T447

69509-69687

Sentence

denotes

This approach has been reported using ZnO NPs.117,118 In these studies, ZnO NPs have been successfully applied for the treatment of H1N1 influenza virus and Herpes simplex virus.

T448

69688-69831

Sentence

denotes

The preliminary results showed that PEGylated ZnO particles were able to efficiently reduce the viral infection with IC50 similar to acyclovir.

T449

69832-70023

Sentence

denotes

More importantly, toxicity of ZnO was modulated by functionalization with PEG, which allowed a higher colloidal stability and a more controlled release of Zn2+ ions to catalyze ROS formation.

T450

70024-70229

Sentence

denotes

Indeed, ROS (e.g., superoxide and hydroxyl radicals) produced by the NPs should be highly reactive and should not only damage the exogenous biological molecules but also attack different cell compartments.

T451

70230-70303

Sentence

denotes

Overproduction of ROS may reduce virus spread but also induce cell death.

T452

70304-70483

Sentence

denotes

Interestingly, this approach is applicable only at the early infection stage (after 1 h incubation of the host cells with a virus), but it loses its activity at later time points.

T453

70484-70876

Sentence

denotes

These experimental evidences suggest that materials capable of triggering the formation of ROS are essential in the first phase of the viral replication, most probably inducing the arrest of the viral DNA polymerases, which is active in the first 1–3 h of viral contamination.117,118 This specific mechanism of action restricts ZnO NPs to an application only at the early stage of infections.

T454

70877-71018

Sentence

denotes

However, the same approach with other metals able to induce Fenton or Fenton-like reactions (e.g., Fe, Cu, and Mn) has not been reported yet.

T455

71019-71148

Sentence

denotes

The choice of proper capping agents may allow to control the metal ion release and thus tune the ROS-mediated antiviral activity.

T456

71149-71255

Sentence

denotes

We would like to take an advantage here to suggest the growth of the antiviral research in this direction.

T457

71256-71343

Sentence

denotes

Another successful approach relies on the reduction of ROS concentration in host cells.

T458

71344-71493

Sentence

denotes

ROS scavenging is able to alleviate the toxicity of the infection enhancing cell viability, giving time to start its endogenous antiviral mechanisms.

T459

71494-71567

Sentence

denotes

So, this approach may both block infection and ensure host cell survival.

T460

71568-71665

Sentence

denotes

In this context, selenium NPs (SeNPs) have been extensively studied for their antiviral activity.

T461

71666-71957

Sentence

denotes

The mechanism of action of these NPs relies on the quenching of the radicals into host cells due to the infection, stopping the mitochondria depolarization and the consequent apoptotic cascade.119 Additionally, SeNPs can also adsorb onto the viral capsid sensibly reducing their infectivity.

T462

71958-72065

Sentence

denotes

SeNPs can be prepared via classical mixing of selenium salt precursors in the presence of a reducing agent.

T463

72066-72499

Sentence

denotes

More recently, SeNPs have been instead biosynthesized from Actinobacteria showing good stability and capacity to inhibit Dengue virus in vitro.120 Moreover, SeNPs were used to carry different antiviral drugs including zanamivir,121 oseltamivir,122 amantadine,123 and ribavirin.119 Their functionalization with the desired drug can be easily achieved adding the molecule during their synthesis through the Se ion controlled reduction.

T464

72500-72560

Sentence

denotes

These NMs have been applied for the treatment of H1N1 virus.

T465

72561-72819

Sentence

denotes

Notably, SeNPs with ribavirin (administered via intranasal absorption every 24 h for 3 days) showed that infected mice had much less alveolar collapse and perivascular and peribronchiolar edema, compared to the group challenged with the virus (Figure 13).119

T466

72820-73120

Sentence

denotes

Figure 13 In vivo antiviral efficiency of SeNPs functionalized with ribavirin (Se@RBV). (a) Mice infected by H1N1 virus were treated with physiological saline (Mock), RBV, SeNPs, or Se@RBV. (b) H&E and tunnel staining showing that Se@RBV-treated mice displayed reduced lung damages compared to Mock.

T467

73121-73163

Sentence

denotes

Reproduced with permission from ref (119).

T468

73164-73194

Sentence

denotes

T469

73195-73340

Sentence

denotes

Due to their efficacy and low toxicity, SeNPs can be considered a useful material for the treatment of other viral diseases including SARS-Cov-2.

T470

73341-73661

Sentence

denotes

As a matter of fact, oxidative stress as well as chronic inflammation may contribute to the aggravation of the Covid-19 symptoms and to the general spread of the infection.124 The use of SeNPs could eventually alleviate the toxicity of infected patients, giving time for the immune system to react against the contagion.

T471

73662-73854

Sentence

denotes

However, the lack of preclinical studies on SeNPs, together the scarce knowledge of their biosafety and long-term toxicity, still remain the main challenges to tackle for clinical translation.

T472

73856-73890

Sentence

denotes

Interaction with the Immune System

T473

73891-74027

Sentence

denotes

The vast majority of the studies show that human survival to viral attack is based on the stimulation and response of our immune system.

T474

74028-74177

Sentence

denotes

When a virus enters and starts infecting tissues, the body reacts and triggers a strong immune response to overcome the pathogen invasion and spread.

T475

74178-74237

Sentence

denotes

Normally, two different immunological reactions take place.

T476

74238-74925

Sentence

denotes

The oxidative stress induced by infection causes the activation of the inflammasome through upregulation of pro-inflammatory cytokines such as IL-1β, pro-IL-18, and NLRP3.122 Excessive upregulation of this mechanism leads to cell damage and eventually to pyroptosis activated by caspases.122 It was also shown that generation of radicals is able to depolarize mitochondria, hence affecting host cell respiration and inducing ROS-mediated apoptosis at the late stage of infection.123 Besides, activation of pro-inflammatory cytokines can alert the immune system blocking the infection (the innate immune response).122 The second immune response is specific (the adaptive immune response).

T477

74926-75061

Sentence

denotes

Immune cells are trained to attack the virus (cellular response), while specific antibodies are produced by B cells (humoral response).

T478

75062-75195

Sentence

denotes

In the last years, HNMs were proved to tune the immune responses, demonstrating to be a possible alternative against viral infection.

T479

75196-75340

Sentence

denotes

In this section the most relevant strategies for the activation of innate and adaptive immune responses using NMs will be described (Figure 14).

T480

75341-75407

Sentence

denotes

Figure 14 Illustration of HNM interaction with the immune system.

T481

75408-75510

Sentence

denotes

Left: HNMs can enhance the production of interferon (IFN) alerting the immune system of the infection.

T482

75511-75517

Sentence

denotes

Right:

T483

75518-75715

Sentence

denotes

Virus-mimicking particles composed of viral proteins and HNMs can induce a strong immunological response activating dendritic cells, T cells, and stimulating B cells to produce specific antibodies.

T484

75717-75739

Sentence

denotes

Innate Immune Response

T485

75740-75843

Sentence

denotes

Innate immune response is the first response that takes place in the presence of any type of infection.

T486

75844-76240

Sentence

denotes

During this early stage, interferon stimulating genes (ISGs) are upregulated in the infected cells.125 This self-defense mechanism slows down the viral replication and alerts sentinel immune cells that start producing proinflammatory cytokines and trigger inflammation.125 However, many viruses are able to escape this complex mechanism, retarding the immune response and spreading the infection.

T487

76241-76319

Sentence

denotes

The interaction of HNMs with the immune system has been more and more studied.

T488

76320-76484

Sentence

denotes

In the case of antiviral HNMs, many examples can be found in the literature where the NMs not only slow down the infection but also tune the innate immune response.

T489

76485-76542

Sentence

denotes

Certain HMNs can display an intrinsic immune stimulation.

T490

76543-76825

Sentence

denotes

We have already mentioned above that in the early stage of infection, AgNPs mainly prevent the virus from entering the host cell through the interaction with the external capsid, but in vitro cellular experiments lack to understand the complex interaction with primary immune cells.

T491

76826-77030

Sentence

denotes

Recent studies have shown that AgNPs can potentially induce the expression of genes involved in innate and adaptive immunity-associated pathways, which are known to play crucial role in immune regulation.

T492

77031-77669

Sentence

denotes

For example, Toll-like receptor 7 can be upregulated by AgNPs after 24 h, by recognizing the single-stranded RNA of the viruses and regulating the antiviral immune response.126 Another study showed that in RSV-infected mice treated with AgNPs, the particles reduced the production of pro-inflammatory TNF-α and IL-6 cytokines, but potentiated the anti-RSV activity of neutrophils in an experimental mouse model.127 However, activation of AgNPs in the reduction of RSV has been noted only when the NM was intranasally inoculated together with the virus, and no results have been reported on the use of AgNPs administrated on infected mice.

T493

77670-77856

Sentence

denotes

In the case of influenza virus infection of lung epithelial cells, it was found that AgNPs targeted infected lung epithelial cells and reduced viral replication, by preventing autophagy.

T494

77857-77972

Sentence

denotes

However, the blockage of the autophagic flux by AgNPs does not inhibit viral replication in already infected cells.

T495

77973-78356

Sentence

denotes

Therefore, AgNPs are more suitable as viral preventive agents due to their pro-inflammatory response rather than drugs.128 More recently, AgNPs were combined with graphene materials and exploited as antiviral material for the treatment of Porcine reproductive and respiratory syndrome virus (PRRSV).129 GO-AgNPs were able to clump the virus diminishing its fusion with cell membrane.

T496

78357-78527

Sentence

denotes

Additionally, once GO-AgNPs were internalized in host cells, they stimulated the ISGs that blocked viral budding and its diffusion to other cells in vitro (Figure 15).129

T497

78528-78618

Sentence

denotes

Figure 15 Scheme of the mechanism of action of GO-AgNPs on activation of innate immunity.

T498

78619-78661

Sentence

denotes

Reproduced with permission from ref (129).

T499

78662-78704

Sentence

denotes

T500

78705-79348

Sentence

denotes

Similarly, CDs used for the treatment of RSV and PRRSV were able to activate the innate immune response via an upregulation of ISGs in vitro.130 Gold nanorods were applied to boost the innate immune response against RSV in vivo.131 Interestingly, it was shown that these nanorods (when intranasally administered with RSV) were not only able to activate the ISGs but also to tune the production of pro- and anti-inflammatory cytokines, resulting in the blocking of the infection with a reduced pulmonary inflammation.131 As drug carriers, HNMs can also affect the internalization pathways, modulate drug efficacy, and the immune cell responses.

T501

79349-79666

Sentence

denotes

For instance, it was found that the isoprinosine immunomodulatory antiviral drug displays a much higher antiviral efficacy in vivo when delivered with CNTs than as a pure drug (in zebrafish larvae food administration), probably due to a better cellular uptake and the anti-inflammatory properties of the nanotubes.132

T502

79667-80115

Sentence

denotes

Fullerenes also exhibit immunomodulation properties through the release of cytokines by bovine alveolar macrophages.133 A study explored the influence of functionalization of C60 with molecules presenting different surface charges like hydroxyl groups and amino acids.134 The study concluded that negative charges upregulated TNF-α up-secretion in RAW 264.7 macrophages, but highly positively or negatively charged surfaces increased cell toxicity.

T503

80116-80218

Sentence

denotes

The upregulation of the cytokine TNF-α is a proof that fullerene can promote cellular immune response.

T504

80219-80368

Sentence

denotes

Despite these preliminary results, the actual mechanisms of interaction between HNMs and the immune system are still at early stage of understanding.

T505

80369-80459

Sentence

denotes

We must consider that the immune response needs to be proportional to the infection grade.

T506

80460-80707

Sentence

denotes

If on one side nanosized immuno-boosters can alert more efficiently the sentinel cells, the use of HNMs that trigger an exaggerated immune response can promote excessive inflammation, damaging healthy cells and promoting uncontrolled side effects.

T507

80708-80927

Sentence

denotes

In the particular context of SARS-Cov-2, the use of AgNPs, fullerenes, or other pro-inflammatory HNMs at the middle and late infection stage may cause an aggravation of the symptoms due to already diffused inflammation.

T508

80928-81370

Sentence

denotes

In particular, most of the studies showed the ability to reduce infection when HNMs were first incubated with the pathogenic virus, thus limiting their potential use in the early stage infection.124 The formulation of HNMs able to both alert the immune system (e.g., upregulating cytokine) and control lung inflammation (e.g., ROS scavenger) even after the early stage infection may be a possible strategy for treatments against SARS viruses.

T509

81372-81396

Sentence

denotes

Adaptive Immune Response

T510

81397-81495

Sentence

denotes

Adaptive immune response is the specific response that the immune system exerts against pathogens.

T511

81496-81959

Sentence

denotes

This mechanism is particularly active toward viral infections where the immune system produces specialized lymphocytes (to fight the virus), called memory B cells (to be effective in case of new infections) and antibodies (corresponding to the humoral response).135 The stimulation of adaptive responses in case of specific infections can be induced artificially through the introduction of attenuated pathogens, stimulating the production of specific antibodies.

T512

81960-82368

Sentence

denotes

This is the principle of vaccination, which is the most common procedure for immunization of large areas of population against many kinds of lethal viruses.135 Besides, the use of viral proteins as antigens in the vaccine formulation leads to neutralizing antibodies, but, due to the low immunogenicity of isolated proteins, does not always stimulate sufficiently the immune system to reach total protection.

T513

82369-82472

Sentence

denotes

More recently, nanotechnology has been applied to develop more efficient vaccines (e.g., nanovaccines).

T514

82473-82803

Sentence

denotes

The use of nanostructures with a size similar to virus (virus-like nanoparticles) sensibly enhances the response helping to reach immunity.135 HNMs can adsorb viral particles and present them to the immune system.136,137 This method of vaccination has been successfully applied in vivo for the challenge of herpes simplex 2 virus.

T515

82804-82905

Sentence

denotes

HSV-2 starts its spreading in vaginal tissues and then diffuses to the neurons causing death in mice.

T516

82906-83102

Sentence

denotes

ZnO NPs (teardrop morphology), after vaginal inoculation with HSV-2, are able not only to prevent viral cell adhesion but also to expose viral antigens to T cells and DCs, leading to immunization.

T517

83103-83187

Sentence

denotes

The preclinical trials against HSV-2 showed a survival to infection higher than 90%.

T518

83188-83350

Sentence

denotes

This approach highlights the possibility to couple cell mimicking NMs to other co-adjuvants for the formulation of large spectrum nanovaccines (Figure 16).136,137

T519

83351-83706

Sentence

denotes

Figure 16 (A) Scanning electron microscopy images of ZnO tetrapod nanoparticles (ZOTEN) synthesized by flame transport synthesis. (B) Mice were challenged intravaginally with HSV-2 333 with or without ZOTEN. (C) To monitor progression of infection, mice were observed daily for the development of lesions around the vaginal opening, and base of the tail.

T520

83707-83774

Sentence

denotes

Representative images from three independent experiments are shown.

T521

83775-83817

Sentence

denotes

Reproduced with permission from ref (137).

T522

83818-83880

Sentence

denotes

T523

83881-83973

Sentence

denotes

HNMs have been also applied to the delivery of antigens, exposing them to the immune system.

T524

83974-84603

Sentence

denotes

Fullerenes were found as suitable carriers for the delivery of drugs or nucleic acids.138 Functionalized fullerene can also self-assemble into virus-sized NPs.139 Investigated as vaccines in cancer immune therapy,140 polyhydroxy fullerenes (called fullerenols) display interesting properties for antiviral therapy, based on their capacity to self-assemble into virus-like particles (VLPs) and so to enhance the immunogenicity of the antigens.141 The great advantage of this strategy relies on the easy encapsulation process during the self-assembly making fullerenols versatile for the formulation of different kinds of vaccines.

T525

84604-85027

Sentence

denotes

These VLPs were investigated against HIV-1141 and hepatitis C viruses.142 Compared to conventional protein- or peptide-based vaccines intended to induce antigen-specific adaptive immune responses, DNA vaccines are more stable, cost-effective, easy to manufacture, and safe in handling.143 However, DNA vaccines have the disadvantage of being poorly immunogenic.144 Fullerenol VLPs allow to avoid the use of other adjuvants.

T526

85028-85165

Sentence

denotes

In the case of a vaccine against HIV-1, fullerenol VLPs penetrated easily into the cells resulting in an enhancement of DNA transfection.

T527

85166-85396

Sentence

denotes

This was proved in a study using fullerenol encapsulating DNA encoding the HIV-1 envelope protein gp145 (Figure 17).141In vitro assays were performed in human embryonic kidney cells line (HEK293) showing good transfection ability.

T528

85397-85590

Sentence

denotes

Following various immunization routes (e.g., activation of Toll-like receptor signaling or effector memory T cell immune response), fullerenol VLPs can induce an innate and a cellular immunity.

T529

85591-85763

Sentence

denotes

A similar study was performed for hepatitis C using the HCV recombinant protein as antigen,142 confirming the potential efficacy of using fullerenols as antiviral vaccines.

T530

85764-85835

Sentence

denotes

Nevertheless, these results require further mechanistic investigations.

T531

85836-86133

Sentence

denotes

Indeed, in vitro studies also evidenced a suppressive effect of acquired immune response of C60 pyrrolidine tris-acid and fullerenol C60(OH)36.145 The fullerenol had a dose-dependent effect on T cell receptor-mediated activation and antibody production by B cells under anti-CD40/IL-4 stimulation.

T532

86134-86184

Sentence

denotes

However, the molecular mechanism is still unknown.

T533

86185-86814

Sentence

denotes

Figure 17 Use of fullerenol as co-adjuvant for vaccination. (a) The structure of fullerenol, red balls represent O and white for H on the fullerene surface, and green balls represent C atoms. (b) The schematic diagram of HIV Env plasmid DNA encapsulated during the self-assembly of fullerenol. (c) TEM image of Env entrapped by fullerenol. (d) Compared to naked Env immunization group, IFN-γ production (immunospot) was significantly enhanced when mice were immunized with the formulation via various immunization routes, including intradermal (i.d.), intramuscular (i.m.), subcutaneous (s.c.), and intranasal (i.n.) injections.

T534

86815-86891

Sentence

denotes

Fullerenol could decrease the antigen dosage (e) and immunization times (f).

T535

86892-86934

Sentence

denotes

Reproduced with permission from ref (141).

T536

86935-86973

Sentence

denotes

T537

86974-87412

Sentence

denotes

Other HNMs including AuNPs (two subcutaneous injections in guinea pigs) and nanodiamonds (three subcutaneous injections in Balb/C mice) were explored as carriers of viral proteins for immunization of swine transmissible gastroenteritis virus and H7N9 influenza, respectively, with good preliminary results.146,147 In these cases, the vaccine formulation relies on the adsorption of the viral antigen onto the surface of the nanoparticles.

T538

87413-87474

Sentence

denotes

However, an effective vaccination depends on several factors.

T539

87475-87547

Sentence

denotes

First of all, the size of the NPs plays a key role on the immune system.

T540

87548-87781

Sentence

denotes

For instance, size-dependent vaccination efficacy has been reported in mice immunization against the foot-and-mouth disease virus (intraperitoneal and subcutaneous injection, every 7 days for 7 weeks) using AuNPs as antigen carriers.

T541

87782-88003

Sentence

denotes

In this study, the most effective activity to stimulate the immune system was exerted by particles with a diameter in the range of 8 nm.148 Both smaller or bigger particles evidenced a drop-off of the immunization effect.

T542

88004-88173

Sentence

denotes

This aspect cannot be ascribed to the antigen concentration, but must be associated only to the nanoparticle size; however, the mechanism of interaction remains unknown.

T543

88174-88266

Sentence

denotes

The selected antigen plays also a crucial role in the preparation of wide spectrum vaccines.

T544

88267-88405

Sentence

denotes

For instance, in the case of influenza, two major membrane glycoproteins, hemagglutinin and neuraminidase, are generally used as antigens.

T545

88406-88621

Sentence

denotes

However, the antibodies produced by this vaccination strategy are selective to the dominant epitope which has a low effectiveness or is totally ineffective against other epitopes or other kinds of influenza viruses.

T546

88622-88828

Sentence

denotes

M2 (a viral protein responsible for the budding and scission of the influenza virus) is commonly expressed in different types of influenza viruses with a high rate of conservation but with low antigenicity.

T547

88829-88959

Sentence

denotes

It has been shown that AuNPs functionalized with M2e protein have a high immunization capacity in comparison to the antigen alone.

T548

88960-89294

Sentence

denotes

Mice immunized with AuNPs (two intranasal injections), and then challenged, showed a survival rate higher than 90% to California-H1N1pdm, Victoria-H3N2, and Vietnam-H5N1 infections.149 This strategy shows that HNMs can be used to boost the immune response of low immunogenic molecules, providing a wide spectrum vaccination potential.

T549

89295-89527

Sentence

denotes

Unfortunately, it has not been determined if the budding process in SARS-Cov-2 is mediated by viral proteins or via the host cell’s endosomal sorting complex, thus more research on the SARS-Cov-2 viral machinery is highly desirable.

T550

89528-89742

Sentence

denotes

All these approaches are based on the capacity of the nanoparticles to adsorb the antigens and expose them to the immune system in the appropriate conformation to produce the neutralizing and protective antibodies.

T551

89743-89801

Sentence

denotes

However, the adsorption is not an easy process to control.

T552

89802-90052

Sentence

denotes

For instance, AuNPs have been ineffective in the immunization against SARS-Cov, when S viral proteins were used as antigens.150 In particular, the immunization with the protein alone was more efficient than when it was adsorbed onto the AuNP surface.

T553

90053-90207

Sentence

denotes

This failure was associated with a conformational change or denaturation of the antigen, which did not lead to the production of the specific antibody.150

T554

90208-90931

Sentence

denotes

Covalent chemistry strategies offer a higher control on the antigen quantification with higher reproducibility and possibility to bind different groups onto the surface of HNMs. For example, calcium phosphate nanoparticles (CaPNPs) were successfully covalently functionalized with Hen Egg lysozyme as model antigen showing an immunization 100 times higher in vivo compared to antigens alone using (one subdermal injection in mice).151 A similar approach was used with iron oxide NPs using mannose (to target DCs) and hepatitis B antigen showing good immunological activity in vitro (two subdermal injections in mice at 14 days distance).152 More recently, other types of vaccination strategies have been applied using HNMs.

T555

90932-91011

Sentence

denotes

A smart example has been reported using multifunctional CaPNPs on herpes virus.

T556

91012-91162

Sentence

denotes

In this study, CaPNPs have been covalently functionalized with alum/MPL as the adjuvant and two peptides as antigens selected via reverse vaccination.

T557

91163-91460

Sentence

denotes

The NM stimulated the immune system generating highly efficient antibodies able to block cell-to-cell infection of herpes virus in vivo (three intramuscular injections in mice every 14 days), increasing the survival rate of immunized mice to 100% against the controls (20% survival, Figure 18).153

T558

91461-91506

Sentence

denotes

Figure 18 Scheme of CaPNPs used for vaccine.

T559

91507-91512

Sentence

denotes

Left:

T560

91513-91593

Sentence

denotes

CaPNPs functionalized with CpGm adjuvant and two different peptides as antigens.

T561

91594-91604

Sentence

denotes

Top right:

T562

91605-91667

Sentence

denotes

CaPNPs were able to reduce cell-to-cell virus spread in vitro.

T563

91668-91681

Sentence

denotes

Bottom right:

T564

91682-91740

Sentence

denotes

CaP nanovaccines were able to immunize mice against HSV-1.

T565

91741-91783

Sentence

denotes

Reproduced with permission from ref (153).

T566

91784-91812

Sentence

denotes

T567

91814-91838

Sentence

denotes

Summary and Perspectives

T568

91839-91940

Sentence

denotes

The recent history has shown the spread of different viral pandemics such as H1N1 flu, HIV, and SARS.

T569

91941-92103

Sentence

denotes

Nowadays, the SARS-Cov-2 pandemic global lockdown has profoundly changed the daily life of most humans, causing uncertainty in short and middle time perspectives.

T570

92104-92269

Sentence

denotes

In this context, the scientific community has responded to protect the population by studying new vaccines and disinfection methods to be applied in the near future.

T571

92270-92434

Sentence

denotes

Despite this tough work, a SARS-Cov-2 vaccination will hopefully be available in 1–2 years, making this epidemic transient period gloomy with increased instability.

T572

92435-92559

Sentence

denotes

The study of more effective vaccines and the production of a wide range antiviral agents is nowadays an extremely hot topic.

T573

92560-92666

Sentence

denotes

HNMs comprise a family of materials that share a nanostructured hard core and a tunable surface chemistry.

T574

92667-92759

Sentence

denotes

In this contribution, we have methodically reviewed different HNMs for antiviral properties.

T575

92760-92927

Sentence

denotes

HNMs can have antiviral properties per se, blocking the viral replication and diffusion, or their antiviral properties can be tailored, playing with surface chemistry.

T576

92928-93006

Sentence

denotes

HNMs can be used to block viral entry and arrest infection at the early stage.

T577

93007-93258

Sentence

denotes

The mechanisms rely on different actions including breaking of capsid disulfide bonds (e.g., noble metal nanoparticles), capsid oxidation (e.g., CuONPs), mimicking of cell surface (e.g., carbon NMs), or mechanical disruption (e.g., AuNPs or graphene).

T578

93259-93382

Sentence

denotes

Surface functionalization additionally confers a higher specificity and pharmacological activity toward the targeted virus.

T579

93383-93688

Sentence

denotes

In particular, the high local concentration of ligands on functionalized HNM surface imparts a high multivalent effect, enhancing the viral trapping efficiency of NMs. Interestingly, HNMs that show an intrinsic antiviral activity can further enhance their antiviral efficacy via surface functionalization.

T580

93689-93867

Sentence

denotes

Some antiviral HNMs are good photosensitizers (e.g., CuONPs) or exert photothermal activity (e.g., carbon NMs), thus their antiviral activity can be trigged by light stimulation.

T581

93868-94004

Sentence

denotes

More importantly, most of the settled strategies target common viral entry mechanisms and can be adopted to fight a wide viral spectrum.

T582

94005-94090

Sentence

denotes

HNMs have been also applied as antiviral agents by their interaction with host cells.

T583

94091-94223

Sentence

denotes

HNMs are able to block viral replication machinery in host cells (e.g., CNTs and fullerenes), inhibiting endogenous enzyme activity.

T584

94224-94383

Sentence

denotes

Additionally, HNMs can be explored for the delivery of antiviral molecules, showing a better antiviral activity and reducing side effects in vitro and in vivo.

T585

94384-94517

Sentence

denotes

Some HNMs can also regulate the ROS homeostasis of host cells, reduce apoptosis, and enhance host cell survival during the infection.

T586

94518-94573

Sentence

denotes

Finally, we have reviewed the role of HNMs on immunity.

T587

94574-94695

Sentence

denotes

In particular, HNMs can stimulate the innate immune response, mainly inducing overexpression of interferon and cytokines.

T588

94696-94787

Sentence

denotes

This effect can alert sentinel cells and generally warn the immune system of the infection.

T589

94788-94885

Sentence

denotes

HNMs can be also used for the activation of the adaptive immune response, foreseeing vaccination.

T590

94886-95023

Sentence

denotes

Due to the similar size of a virus, functionalized HNMs with antiviral molecules (virus-like particles) can enhance their immunogenicity.

T591

95024-95108

Sentence

denotes

Certainly a huge effort should be done for translation of the research into clinics.

T592

95109-95189

Sentence

denotes

Indeed, HNM applications as antivirals are still in the early phase of research.

T593

95190-95256

Sentence

denotes

Several challenges still need to be tackled before their safe use.

T594

95257-95338

Sentence

denotes

At the current stage, some HNMs have been approved only for surface disinfection.

T595

95339-95507

Sentence

denotes

For instance, CuNPs have been used in filters for the preparation of highly efficient broad spectrum antiviral masks.50 Some HNMs have been already clinically approved.

T596

95508-95843

Sentence

denotes

For example, FeNPs were approved for imaging and as a drug to treat iron deficiency anemia in adult patients with chronic kidney disease, while AuNPs are in clinical trials for the treatment of prostate cancer (photothermal therapy).154,155 However, at the moment no clinical trials are running for the use of HNMs as antiviral agents.

T597

95844-95935

Sentence

denotes

In fact, there are still several concerns on the applications of HNMs in drug formulations.

T598

95936-96074

Sentence

denotes

Compared to molecules, where mainly concentration and exposure routes are concerned, solving HNM toxicity issues is much more complicated.

T599

96075-96203

Sentence

denotes

Composition, size, shape, and surface functionalization must be considered to respond to the requirement for safety regulations.

T600

96204-96287

Sentence

denotes

Additionally, interaction on HNMs with the immune system must be better elucidated.

T601

96288-96763

Sentence

denotes

In particular, the activation of the immune system and complement activation-related pseudoallergy must be taken into account.156 For instance, ferumoxytol (a FeNP-based drug) has been reported to generate severe anaphylactic reactions in humans, 18 of which were fatal.156 This is due to the possible interaction of HNMs with mast cells, provoking their degranulation/activation and release of histamine even at the first exposure (pseudoallergic-mediated hypersensitivity).

T602

96764-97101

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Besides, the mechanism of activation of these cells is still unknown, although it was found that it depends on the HNM composition, size, and surface chemistry and on the corona formation.156 On the other hand, it has been demonstrated that HNMs can travel to the draining lymph nodes, targeting resident dendritic cells and macrophages.

T603

97102-97496

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Therefore, they are able to interact with antigen presenting cells to stimulate innate and adaptive immune responses.156 We believe that a joint venture of different chemists, materials scientists, virologists, toxicologists, and medical doctors can push forward the preparation and safe application of HNMs in this field, hoping to prevent and eventually block the rise of new viral pandemics.

PMC:7376974 / 1497-98993 JSON TXT 12 Projects

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PMC:7376974 / 1497-98993 JSONTXT 12 Projects

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PMC:7376974 / 1497-98993 JSON TXT 12 Projects