PMC:7556614 / 6119-19531 JSONTXT

{"target":"http://pubannotation.org/docs/sourcedb/PMC/sourceid/7556614","sourcedb":"PMC","sourceid":"7556614","source_url":"https://www.ncbi.nlm.nih.gov/pmc/7556614","text":"MATERIALS AND METHODS\n\nMaterials\nAcyclovir (ACV) was acquired from Pharmaniaga Research Centre Sdn. Bhd. (Malaysia). Phospholipon® 90G (lecithin) was bought from Lipoid GmbJ, Mattermannallee, Switzerland. Chitosan, propylene glycol (PG), and polyethylene glycol (PEG-2000, MW: 1900–2200) were purchased from Merck Sdn. Bhd (USA). Dialysis bag-110 (molecular weight cut-off (MWCO): 12,000–14,000 Da) was purchased from HiMedia Laboratories Pvt. Ltd. (India). Rabbit intestinal sac was obtained as a gift from SARSHA Riding Centre. HPLC-grade Acetonitrile was procured from Fisher Scientific Inc. (Malaysia). Water is purified using a MilliQ® Integral Water Purification System with a conductivity of 15 MΩ. Other chemicals were of analytical reagent grade.\n\nExperimental Design\nA 3-factor, 3-level, 17-run Box-Behnken Design (BBD) was adopted to generate polynomial models for the optimisation process of the drug. Three determining factors were the amount of lecithin (X1), chitosan (X2), and PEG 2000 (X3) as shown in Table I. Dependent variables to be optimised were entrapment efficiency (EE%) (Y1), zeta potential (mV) (Y2), and particle size (nm) (Y3). Design-Expert® Software Version12 was employed to establish main effects, interaction effects, and quadratic effects from independent variables, and provide the solution for optimised formulation. The nanoparticle formulations involved, including five centre points, are tabulated in Table II. All experimental results measurement was analysed in triplicate and expressed as mean ± standard deviation and p \u003c 0.05 was considered as significant for the data.\nTable 1 Variables and Their Levels in Box-Behnken Design\nFactors Factor levels\n− 1 (low) 0 (medium) + 1 (high)\nA: lecithin (mg) 100 200 300\nB: chitosan (mg) 10 15 20\nC: PEG 2000 (mg) 20 30 40\nDependent variables Constraints\nY1 = entrapment efficiency (%EE) Maximise\nY2 = particle size (nm) Minimise\nY3 = zeta potential (mV) Maximise\nTable 2 Box-Behnken Experimental Design\nFormulation Independent variable level\nLecithin (X1) Chitosan (X2) PEG 2000 (X3)\nPALN1 0 0 − 1\nPALN2 0 0 + 1\nPALN3 − 1 − 1 0\nPALN4 + 1 + 1 0\nPALN5 − 1 − 1 − 1\nPALN6 − 1 − 1 + 1\nPALN7 + 1 + 1 + 1\nPALN8 0 0 0\nPALN9 0 0 0\nPALN10 0 0 0\nPALN11 + 1 + 1 − 1\nPALN12 0 0 − 1\nPALN13 0 0 + 1\nPALN14 − 1 − 1 0\nPALN15 + 1 + 1 0\nPALN16 0 0 0\nPALN17 0 0 0\n\nPreparation of Acyclovir-Loaded Lecithin-Chitosan Nanoparticles\nALN was prepared by the method reported by Sedef and group with little modification (15). An appropriate amount of lecithin and chitosan was weighed, as shown in Table II. Chitosan was dissolved in 15 mL of 0.1% v/v acetic acid followed by heating to 60°C. Lecithin prepared was dissolved in an organic solvent consisting of 2.5 mL methanol, 2.5 mL chloroform, and 2 mL PG. An amount 15 mg of ACV was added after full solubilisation of lecithin, followed by sonication to solubilise all acyclovir. The lecithin/ACV solution was then added dropwise into chitosan solution under vigorous magnetic stirring. Finally, the nano-suspension was probe sonicated for 4 min.\n\nPEGylation of ALN\nPEGylation was performed by modifying an established method by Arya et al. (23). Prepared PEG 2000 solutions were 200 mg/mL, 300 mg/mL, and 400 mg/mL. One millilitre of the solution was added into nano-suspension formed under Section C. The suspension was agitated at moderate intensity for 30 min, followed by probe sonication with 30% amplitude, pulse on/off the rate of 10 s/5 s, for 2 cycles. The formulated particles were centrifuged and supernatants containing any trace of free ACV were separated for the purification of drug. Final product PEGylated ACV-loaded lecithin/chitosan Nanoparticles (PALNs) were stored for further analysis to study the controlled release of drugs.\n\nOptimisation of Formulation\n\nDrug Entrapment Efficiency Analysis\nAmount of encapsulated ACV in nanoparticles was identified indirectly in high-performance liquid chromatography (HPLC) (24). Agilent 1260 Series HPLC system fitted with a photodiode array detector (DAD), and use Thermo Fisher Syncronis C-18 (5-μm particle size, 150 × 4.60 mm diameter) column. A fixed injection volume of 20 μl per sample, a column temperature of 25°C, and a wavelength of 254 nm were set. The mobile phase used was acetonitrile and pH 6.74 water (10:90, v/v), which was set at an isocratic flow rate of 0.5 ml/min for 10-min run time. Equation (1) showed a calibration curve that fit over the range 100–500 μg/ml with a correlation coefficient of r2 = 0.99990.1 y=117.88803x+43.82506where y represented the peak area in a milli-absorbance unit (mAu) and x represented the ACV concentration (mg/ml).\nIn evaluating the entrapment efficiency (EE), PALNs prepared were stored in − 80°C freezer for 24 h. Then PALNs were thawed and isolated by centrifugation at 48,000×g at 4°C for 20 min. The supernatants consisted of the free drug were:2 EE%=Mtotal–Mfree/Mtotal100%where EE was the entrapment efficiency, and Mtotal and Mfree were a total mass of ACV added and free ACV in supernatant respectively.\n\nZeta Potential Measurement Particle Size and Poly-Dispersity Index\nParticle size, Poly-Dispersity Index (PDI), and zeta potential were determined by DelsaMax PRO Zeta Potential Dynamic Light Scattering Analyzer (Beckman Coulter, USA). To measure the size and zeta potential, 0.1 ml of prepared particles was diluted with 2.0 ml of deionised water loaded in flow cell and scanned for particles. The measurement was done in a 173° scattering angle. The experiment was done with a setting of 25 ± 0.5°C.\n\nCharacterisation of Optimised PALN Formulation\n\nMorphological Analysis\nMorphology study of the nanoparticles was carried out using scanning electron microscopy (SEM) (4). The formulation was lyophilised and further dried using vacuum oven for 30 min and the powder was uniformly spread on the coverslip and further coated with platinum using 208HR High-Resolution Sputter Coater (Ted Pella, INC, CA, USA). Coated sample was then inserted into the specimen chamber and scanned using TM 3030 Plus scanning electron microscope (Hitachi, Tokyo, Japan), at 5 kV.\n\nHigh-Resolution Transmission Electron Microscopy\nThe optimised formulation was visualised by high-resolution transmission electron microscopy (HRTEM). The formulation was diluted with water in ratio of (1:50) and placed on the copper film–coated grid, with a diameter of 3.05 mm and 400 mesh size (Ted Pella, CA, PELCO® 400 Mesh Grids). After placing the particle, they were air dried for 15 min and any access of water was removed using a filter paper. The analysis was done by using a Tecnai G2 20 Twin transmission electron microscope (FEI Company, Oregon, USA).\n\nDifferential Scanning Calorimetry\nThermal properties of optimised formulation were analysed using differential scanning calorimetry (DSC) 214 Polyma differential scanning calorimeter (NETZSCG-Gerätebau GmbH, German). Samples used included lecithin, chitosan, PEG 2000, optimised formulation, pure acyclovir, and a blend of acyclovir, chitosan, lecithin and PEG 2000 (physical mixture, PM). The optimised formulation was dried in SHEL LAB SVAC2-2 vacuum oven (Sheldon Manufacturing Inc., USA) under 35°C and 25 in. Mercury (Hg) before analysis (16). Black alumina was used as a reference. Nitrogen gas purge rate was 20 mL/min. The analysis was conducted with settings shown in Table III. All the scanned samples were analysed in triplicate to plot the data.\nTable 3 DSC Setting for Thermal Analysis of Each Sample\nSample Heating range (°C) Heating rate (°C/min)\nInitial Final\nOptimised formulation 0 320 5\nLecithin 10 100 3\nChitosan 50 320 10\nPEG 2000 0 320 10\nAcyclovir 50 320 10\nPhysical mixture 0 320 10\n\nX-Ray Diffractometry\nThe crystallinity of PALNs synthesised was studied through the sharpness of peak in the XRD spectra by X’Pert3 Powder X-Ray Diffractometer with X’Celerator (Malvern Panalytical, UK). Samples used were lyophilised PALNs, pure ACV, and PM. Samples were radiated with CuK α radiation at 45 kV and 140 mA. 2θ scan range from 0° to 60°, with 3°/min scanning rate, and 0.05° step size were set (17). All the tests were performed in triplicates.\n\nAttenuated Total Reflection-Fourier Transform Infrared Spectrometer\nCompatibility study was conducted using Nicolet iS50 FT-IR Spectrometer (Thermo Fisher Scientific Inc., Waltham, MA). Samples used included pure acyclovir, lecithin, chitosan, PEG 2000, optimised formulation, and PM. Before analysis, optimised formulation was vacuum-dried at 35°C and − 25 in Hg (17). The analysis was done with sixteen scans at scanning range of 600 to 4000 cm−1.\n\nStability Study\nA replicate of optimised PALN was prepared without lyophilisation and assessed for short-term physical stability for 60 days based on ICH guidelines. The sample was stored in 4°C chiller throughout the study period. Nanoparticle size of the sample was measured by using the method as described in section F at an interval of 0th, 15th, 30th, 45th, and 60th days. A p \u003c 0.05 was considered as significant for the obtained data.\n\nIn Vitro Drug Release Profile\nIn vitro drug release profile was conducted on the optimised formulation by employing a dialysis bag method with USP apparatus II. Two sets of dialysis bag (MWCO: 14,000 Da) of suitable length to accommodate the sample were prepared and immersed in 37°C ultrapure water for 30 min for stabilisation. One end of the dialysis bags was tied securely with thread. Then 4 mL of optimised formulation with ACV concentration of 1 mg/mL was injected into each dialysis bag. Another end of the dialysis bags was then tied securely with a thread. The dialysis bags were kept in sink condition in two separated vessels of a RC-8DS dissolution tester (Sinopham Co., Ltd., China) filled with 400 mL of pH 1.2 hydrochloric acid buffer (SGF) and 400 mL of pH 6.4 phosphate buffer saline (SIF) respectively. Dissolution tester was set for 70-rpm rotation and 37°C water bath. One millilitre of sample was taken in triplicate at 0.5 h, 1 h, 2 h, 4 h, 8 h, 12 h, and 24 h. The same volume of fresh medium was re-filled into the corresponding vessel. Samples were analysed with HPLC method as described in section E. Another calibration curve with a smaller range from (1) was generated to ensure the accuracy of ACV concentration derived from peak area. Equation (3) showed a calibration curve that fit over the range of 5 to 50 μg/mL with r2 of 0.99964.3 y=125.32389x+79.70511\nThe drug release profile of optimised formulation was plotted with a cumulative percentage of ACV released against time. Drug release data was fitted into one of the releasing models which were zero order, first order, Higuchi, Korsmeyer-Peppas, and Hixson-Crowell releasing model. A best model kinetic fit describing drug release of the formulation was selected according to the coefficient of determination (r2) that calculated with aid from Microsoft Excel. Equation (4) was used as a formula for the cumulative percentage of drug-released calculation.4 DR%=Mreleased/Mtotal100%where DR was the cumulative percentage of drug released, and Mreleased and Mtotal were mass of acyclovir released and total mass of acyclovir in formulations used respectively.\n\nStudy of Ex vivo Intestinal Permeability\nEx vivo study was performed according to an established method (25) with modification. The study was done using New Zealand white rabbit of age 5–6 months. The rabbit intestine sac was cleaned with cold saline. It was cut into several 9.5-cm-long segments. The intestine segment was tied up securely with thread at one end, followed by the introduction of 2 mL of optimised formulation with 1 mg/mL of ACV concentration into the intestinal opening. The other end was then tied up securely with thread. The rabbit intestine sac containing the formulation was kept in sink condition in a beaker containing 80 mL of pH 7.4 PBS with 1% m/v sodium lauryl sulphate (SLS) at 37°C as releasing medium. The beaker was closed with cover to prevent evaporation of releasing medium. One millilitre of the sample was taken at 0.5 h, 1 h, 2 h, 4 h, 6 h, 8 h, and 10 h in triplicate, and an equal amount of fresh releasing medium was replenished. Samples were analysed with HPLC method as described in section E, and Eq. (3) was used for calculation of ACV concentration in the samples.\nDrug intestinal permeability profile of optimised PALN formulation was plotted with a cumulative amount of ACV permeated into releasing medium against time. Cumulative percentage of drug permeated was calculate with Eq. (5)5 DP%=Mmedium/Mtotal100%where DP was the cumulative percentage of drug permeated and Mmedium was the mass of acyclovir in releasing medium.\nApparent permeability of the formulation as a function of the mucosal surface area was calculated by Eq. (6). In contrast, Eqs. (7) and (8) were used to calculate apparent permeability coefficient and steady-state flux, and Jss of acyclovir in formulation, respectively.6 Apparentpermeabilitymgcm−2=Mmedium/MucosalSurfaceAreacm27 Pappcms−1=dQ/dt1/AC0\nwhere Papp was the apparent permeability coefficient, dQ/dt was the slope for the graph of the cumulative amount of ACV permeated against time in unit of mg/h, A is the surface area of intestinal tissue where the permeation of nanoparticles occurred in the unit of cm2, and C0 gave an initial concentration of ACV in the intestinal sac that was 1 mg/mL (equivalent to 1 mg/cm3).8 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