Optimisation of Formulation
The Box-Behnken design with 3 × 3 factorial design generated 17 trail with 5 centre points runs for the preparation of PEGylated lipid polymeric nanoparticles (PALNs) as shown in Table II. The optimisation data shows that the concentration of independent factors lecithin (A), chitosan (B), and PEG 2000 (C) exerts an individual negative and positive effect on the dependent factors i.e. entrapment efficiency (Y1), particle size (Y2), and zeta potential (Y3). The best fitted equation was selected as quadratic for all the 3 dependent factors. The effect was shown through 3D response surface depicting the interaction between independent variables and dependent factors as shown in Fig. 2. Before the BBD development, some preliminary studies were conducted using the ratio of the lecithin and chitosan was selected. The aim was to get the maximum entrapment efficiency and minimum range for particle size. The probe sonication was selected at 4 min, as in our preliminary studies, it was concluded that high sonication with leaded to breakage in the particle and 1-–2-min sonication produced particles with large diameters.
Fig. 2 Graphical presentation of a drug entrapment efficiency, b zeta potential, and c particle size and PDI PALNs formulation

Drug Entrapment Efficiency
The EE is shown in Table IV and Fig. 2 a represented the graphical presentation of EE for all formulations. The results obtained for the drug EE ranged from 68 to 87%. The use of sonication for solubilisation of ACV in organic solvent allowed it to be correctly attached to lecithin and get encapsulated into nanoparticles formed.
Table 4 Observed and predicted value of encapsulation efficiency (Y1), particle size (Y2) and zeta potential (Y3) of formulations in the Box-Behnken design
Sample EE, % (Y1) Zeta potential, mV (Y2) Particle size, nm (Y3) PDI
Observed Predicted Observed Predicted Observed Predicted
PALN1 83.81 ± 1.93 84.84 37.70 ± 1.16 37.19 187.7 ± 3.75 186.09 0.238 ± 0.01
PALN2 82.15 ± 6.26 81.07 48.33 ± 1.93 50.39 264.8 ± 12.21 254.17 0.429 ± 0.12
PALN3 77.28 ± 0.58 78.00 43.73 ± 5.78 44.00 177.7 ± 5.23 180.51 0.216 ± 0.01
PALN4 83.44 ± 2.47 82.77 35.03 ± 1.76 33.20 231.7 ± 6.32 241.17 0.228 ± 0.02
PALN5 74.17 ± 6.10 72.42 31.37 ± 1.48 31.60 187.9 ± 9.56 186.76 0.243 ± 0.01
PALN6 68.45 ± 1.79 68.81 50.47 ± 1.65 48.13 186.8 ± 8.98 194.69 0.232 ± 0.02
PALN7 70.17 ± 9.14 71.92 42.07 ± 1.21 41.83 254.8 ± 15.45 256.00 0.237 ± 0.00
PALN8 84.72 ± 1.90 85.44 25.47 ± 1.33 28.09 156.4 ± 8.98 162.83 0.314 ± 0.01
PALN9 86.67 ± 3.27 85.44 31.17 ± 1.33 28.09 178.7 ± 10.54 162.87 0.179 ± 0.03
PALN10 84.67 ± 3.56 85.44 27.63 ± 2.94 28.09 166.1 ± 3.65 162.87 0.244 ± 0.01
PALN11 83.97 ± 1.52 83.61 21.30 ± 2.48 23.63 198.1 ± 4.78 190.29 0.167 ± 0.02
PALN12 83.57 ± 2.03 84.64 20.77 ± 5.59 18.70 240.2 ± 4.56 250.82 0.208 ± 0.03
PALN13 74.13 ± 4.69 73.10 39.73 ± 1.60 40.24 254.7 ± 16.87 256.30 0.347 ± 0.07
PALN14 70.86 ± 3.61 71.53 24.20 ± 1.56 26.02 251.5 ± 24.56 242.08 0.360 ± 0.07
PALN15 81.77 ± 6.41 81.06 22.83 ± 2.28 22.55 249.2 ± 16.87 246.42 0.344 ± 0.03
PALN16 86.09 ± 2.93 85.44 28.49 ± 1.76 28.10 157.5 ± 3.45 162.83 0.263 ± 0.01
PALN17 85.06 ± 2.88 85.44 27.69 ± 1.97 28.10 155.3 ± 7.65 162.82 0.228 ± 0.02
Design-Expert software generated Eq. (9) that expressed the effect from independent factors, which were the amount of lecithin (X1), amount of chitosan (X2), and amount of PEG 2000 (X3) on drug entrapment efficiency of the formulation.9 Y1=85.44+3.57X1–3.83X2–2.04X3–2.02X1X2+1.19X1X3–1.95X2X3–6.91X12–4.34X22–0.19X32
A positive value for coefficient indicates the increment in the factor results in an appropriate improvement in the response and vice versa. The equation with adjusted R2 of 0.9398 indicates a good fit. Interaction between the factors affecting drug entrapment efficiency was as shown in Fig. 3 a–c; it is to be noted that the X1 and X2 in the figures indicate the X-axis instead of the independent factors.
Fig. 3 Three-dimensional (3-D) response surface plot showing effect of the independent factors on the depended responses EE%, particle size, and zeta potential
Figure 3 a showed the increment of drug entrapment efficiency by increasing lecithin amount and decreasing chitosan. It is also observed that drug entrapment efficiency reduced at level 1 of lecithin, as micelle formed when lecithin concentration exceeds critical micelle concentration which could affect the encapsulation process. Lowering the amount of lecithin also led to poor encapsulation efficiency as less available hydrophilic heads to be attracted by ACV (26). Increment in chitosan concentration amount improved self-assembling process between chitosan and lecithin, which also resulted in weakly attraction between ACV and lecithin and ACV will not be entrapped. It is noticed that the interaction effects between lecithin and chitosan have the most substantial impact on entrapment efficiency.
Figure 3 b and c showed the interaction between PEG 2000 as well as between chitosan and lecithin on entrapment efficiency was weaker when compared with lecithin and chitosan. EE decreases slightly with an increment of PEG 2000 amount, which might be due to the interaction between the PEG 2000 and chitosan, as it affects the electrostatic interaction between lecithin and chitosan, causing the tight junction to be disrupted upon mixing, resulting in drug leaking. The disruption was attributed to the ability of PEG 2000 to enter the loosely packed structure (31). A suitable amount of lecithin (X1) should be used in pair with sufficient but not excessive amount of chitosan (X2), and less PEG 2000 (X3) for high EE.

Zeta Potential Measurements
Zeta potential for all formulations was determined and tabulated in Table IV as observed values. Figure 2 b showed the graphical presentation of zeta potential measurement for 17 formulations. The zeta potential ranged from + 20.77 ± 5.59 to + 50.47 47 ± 1.65 mV. The zeta potential was mainly determined by lecithin and chitosan used. However, PEG 2000 as a polymer grafting on the lecithin-chitosan core-shell structure might be impacting the stability of the nanoparticle. The values can be used as an indicator that all the prepared hybrid nanoparticle formulations were highly stable. Using Design-Expert software, the relationship between the used amount of lecithin, chitosan, and PEG 2000 on zeta potential can be explained using Eq. (10) with a high R2 value of 0.9294.10 Y2=28.09–3.57X1+8.68X2–7.16X3+0.42X1X2+1.83X1X3+2.08X2X3+1.51X12+6.7X22+1.84X32
Figure 3 d showed positive linear relationship between zeta potential and amount of chitosan used for developing the nanoparticles. The relationship was predictable since chitosan dissolves in an aqueous solvent, exhibiting net positive charges due to the protonated amine group (30). When the amount of lecithin used decreases, the zeta potential increases, proving that negatively charged lecithin had reacted with positively charged chitosan, producing a negative effect on zeta potential. The same trend can be seen in Fig. 3 e, where PEG 2000 with a surface charge of − 2 to − 7 mV also had a negative effect on zeta potential, but to a lower extent to that of lecithin (32). It can be seen that chitosan was capable of producing formulation with zeta potential greater than 50 mV. Still, the use of PEG 2000 and lecithin, or to be exact, the electrostatic interaction between chitosan and other components, causes the reduction of zeta potential. Figure 3 f further supported the point that lecithin and PEG 2000 were responsible for zeta potential decrement. When a higher proportion of PEG2000 (40 mg) and lecithin (300 mg) was used, the zeta potential significantly decreases in the nanoparticles.

Particle Size and PDI Analysis
Particle size and PDI obtained from all 17 formulations were recorded in Table IV as observed values and illustrated in Fig. 2 c. The particle size ranged from 155.31 ± 6.87 to 264.82 ± 4.33 nm, with PDI ranged from 0.179 ± 0.03 to 0.429 ± 0.12. In overall, the results were accepted as it is capable of exhibiting enhanced permeability and retention (EPR) effect. Equation (11) describes the effect of three independent factors on particle size, with high r2 value of 0.9162 to prove that the equation is reliable.11 Y2=162.83+16.23X1+18.39X2+16.72X3+14.47X1X2–14.07X1X3–15.65X2X3+17.39X12+26.7X22+47.32X32
We observed that particle size was significantly impacted by amount of lecithin, chitosan, and PEG 2000 used, as shown in Fig. 3 g–i.
The particle size of PALN increased with the amount of lecithin and chitosan used in the nanoparticles. As more lecithin and chitosan were used, more drugs attracted and entrapped in the core, forming bulkier nanoparticle. The nanoparticles formed showed a size smaller than 200 nm, as a proven larger proportion of smaller nanoparticle size area in Fig. 4. To decrease the amount of chitosan injudiciously was not preferred as it might be insufficient to form a complete spherical structure with lecithin, which was bulkier with attached drug. Thus, lecithin to chitosan ratio must be adjust to ensure complete self-assembling process as it leads to rigid spherical nanoparticle occupying less space. On the other hand, increment in PEG 2000 thickens the layer on PALN, resulting in size increment (32). It is also observed that without sufficient PEG 2000, the drug residence time in systemic blood circulation might not be able to be significantly prolonged for nanoparticles.
Fig. 4 SEM image of PALN1 at a × 12,000, b× 30,000, and HRTEM image of PALNs at c× 7800

Statistical Analysis of Experimental Data by Design-Expert Software
Table V summarised quadratic equation used in describing the effect of three independent variables on three dependent variables. All factor with (p value< 0.05) were considered as significant. The expected values for Y1, Y2, and Y3 was shown in Table IV. Due to negligible difference between observed and predicted value, a quadratic Eqs. (9), (10), and (11) can serve as a good indicator of behaviour and interaction relationship between the independent factors.
Table 5 Statistical Analysis Results of Entrapment Efficiency, Particle Size, and Zeta Potential
Parameter Drug EE (%) Particle size (nm) Zeta potential (mV)
Coefficient p value Coefficient p value Coefficient p value
Intercept 85.4405 < 0.0001* 162.826 < 0.0001* 28.0896 < 0.0001*
X1 3.5735 0.0003* 16.2333 0.0054* − 3.5675 0.0050*
X2 − 3.8271 0.0002* 18.3916 0.0028* 8.6821 < 0.0001*
X3 − 2.0429 0.0064* 16.7166 0.0047* − 7.1571 < 0.0001*
X1X2 − 2.0193 0.0315* 14.4667 0.0410* 0.4175 0.7481
X1X3 1.1877 0.1588 − 14.0668 0.0454* 1.8325 0.1860
X2X3 − 1.9454 0.0363* − 15.65 0.0305* 2.0833 0.1395
X12 − 6.9129 < 0.0001* 17.3868 0.0178* 1.5143 0.2538
X22 − 4.3391 0.0006* 26.7035 0.0021* 6.6986 0.0009*
X32 − 0.1888 0.8044 47.3201 < 0.0001* 1.8436 0.1739
*p value less than 0.05
The measurements considering for all obtained and software’s recommendation, formulation PALN1 with high EE of 83.81 ± 1.93%, the particle size of 187.7 ± 3.75 nm, and PDI of 0.238 ± 0.01. The values of PALN1 were selected due to its higher EE and optimum size. The zeta potential of + 37.70 ± 1.16 mV of PALN1 was found to be very stable. The ratio of lecithin to chitosan for the optimised formulation is exactly 20:1, which was known to be the perfect proportion of both materials to get a small-sized nanoparticle with spherical shape (33).