Enhancement of Liver Targetability through Statistical Optimization and Surface Modification of Biodegradable Nanocapsules Loaded with Lamivudine

The intention of the current work was to develop and optimize the formulation of biodegradable polymeric nanocapsules for lamivudine (LMV) in order to obtain desired physical characteristics so as to have improved liver targetability. Nanocapsules were prepared in this study as aqueous-core nanocapsules (ACNs) with poly(lactide-co-glycolide) using a modified multiple emulsion technique. LMV was taken as a model drug to investigate the potential of ACNs developed in this work in achieving the liver targetability. Three formulations factors were chosen and 33 factorial design was adopted. The selected formulation factors were optimized statistically so as to have the anticipated characteristics of the ACNs viz. maximum entrapment efficiency, minimum particle size, and less drug release rate constant. The optimized LMV-ACNs were found to have 71.54 ± 1.93% of entrapment efficiency and 288.36 ± 2.53 nm of particle size with zeta potential of −24.7 ± 1.2 mV and 0.095 ± 0.006 h−1 of release rate constant. This optimized formulation was subjected to surface modification by treating with sodium lauryl sulphate (SLS), which increased the zeta potential to a maximum of −41.6 ± 1.3 mV at a 6 mM concentration of SLS. The results of in vivo pharmacokinetics from blood and liver tissues indicated that hepatic bioavailability of LMV was increased from 13.78 ± 3.48 μg/mL ∗ h for LMV solution to 32.94 ± 5.12 μg/mL ∗ h for the optimized LMV-ACNs and to 54.91 ± 6.68 μg/mL ∗ h for the surface-modified LMV-ACNs.


Introduction
Lamivudine (LMV) is an antiviral drug of class nucleoside reverse transcriptase inhibitors, used in the hepatitis B treatment.Hepatocytes are the major target for the livertropic viruses such as hepatitis B virus (HBV), and these cells are the site of replication for HBV [1,2].Hence, any anti-HBV drug like LMV needs to be developed into a dosage form that can deliver the drug predominantly to the liver tissue so as to achieve the highest therapeutic beneft with fewer side efects.
Colloidal drug delivery systems are capable of delivering the loaded drug at the target site of action, thus improving the therapeutic efcacy and reducing the side efects of the drug [3].Polymeric nanoparticles are physically more stable and fexible for modifcation.Tese can be modifed into having a wide range of surface properties among the different systems viz.solid lipid nanoparticles, niosomes, and liposomes [4,5].Biodegradable polymers like poly (lactideco-glycolide) (PLGA) are less toxic and digestible in the body fuids.Hence, these are most widely employed currently for nanoparticle preparation [6].Liver targeting can be achieved by broadly two types of approaches viz.active targeting and passive targeting approaches.Active targeting approaches need liver-specifc ligands on the nanoparticle surface otherwise targeting cannot be achieved.Targeting ligands for cell-specifc receptors on the liver have to be chosen selectively to guide active targeting of the nanoparticles.Few such receptors on the liver for example are asialoglycoprotein receptor (ASGP-R) on the hepatocytes, galactose receptors on the Kupfer cells, uroplasminogen receptors on the hepatic stellate cells, hyaluronan fbronectin, and denatured collagen receptors on the sinusoidal endothelial cells.Tough few such ligands such as lactobionic acid, glycyrrhetinic acid, and asialofetuin are identifed and reported, still their reliability and reproducibility are yet to be studied [7].Targeting by external triggering (like applying an external magnetic feld or sound waves) demands sophisticated facility, and also, the patient is needed to be hospitalized [8][9][10].Targeted drug delivery by biochemical triggering/impulses may also be possible, but this limits the selection of carrier systems or polymers Taking into account these difculties and limitations, liver targeting through passive targeting approaches can possibly be simple yet reliable.
Passive targeting of the liver can be achieved by making use of the characteristics of the liver, particularly its endocytosis property.Te fate of nanoparticles upon intravenous administration largely depends on their physical characteristics.Tose characteristics include particle size and surface properties such as hydrophobicity and zeta potential [11].Nanoparticles with particle sizes below 200 nm can escape phagocytosis by the reticuloendothelial system (RES)/macrophages and can have high circulatory time in the blood.On the other hand, nanoparticles of size just above 200 nm with a hydrophobic surface and high negative zeta potential are readily phagocytized by RES and delivered into RES-rich organs like the liver [12,13].Modifcation of surface hydrophobicity, charge, and particle size can be achieved by carefully controlling the formulation of nanoparticles.Hence, in this work, passive targeting of the liver through modifcation of size and surface properties was opted.
Te major aim of this work was to develop aqueous-core nanocapsules (ACNs) to achieve high drug entrapment as well as liver targetability of the loaded drug.Few literature reports suggested that ACNs, a novel form of polymeric nanoparticles, have high potential in loading hydrophilic drugs to a greater extent.Vignaud et al. developed ACNs for a high water-soluble drug, doxorubicin HCl.Trough these ACNs, the authors could obtain the entrapment efciency of the doxorubicin HCl to a maximum of 80% [14].Cosco et al. developed ACNs with PLA for loading a high water-soluble drug, gemcitabine HCL.Tese authors also reported that the ACNs were efcient in achieving higher loading of watersoluble drugs [15].Deng et al. also described the efciency of polymeric ACNs in improving the loading of high watersoluble drugs in their review [16].LMV, considering its physicochemical properties and therapeutic action in the liver tissue, was taken as a model drug to study the ability of the ACNs to deliver the drug to the liver tissue.Te ACNs for LMV were prepared through the W/O/W emulsifcation method using PLGA RG503H, a hydrophobic and biodegradable polymer.Te development of ACNs was performed through the design of experiments (DoE) approach.DoE is a statistical tool whose application is now mandated by the regulatory bodies in pharmaceutical/biotechnological industries.Under the DoE, the preparation of ACNs was designed as a 3 3 factorial design of response surface methodology.Tree formulation parameters were taken as the critical material attributes (CMAs) viz.concentrations of the PLGA and the surfactant in secondary emulsion, and also the nature of the external water phase.Te prepared ACNs were subjected to thermal analysis to know the physical state of LMV in the ACNs.Particle size (PS) and surface charge were analyzed by Zetasizer.Morphology of the surface of the ACNs was investigated by transmission electron microscopy (TEM).Also, entrapment efciency (EE) and in vitro drug release studies by the dialysis bag method were performed.To optimize the formulation of ACNs, the responses taken were EE, PS, and drug release rate constant (k).Formulation of the ACNs was optimized by the desirability functions approach, a statistical approach, with the target of reduced PS (to have more difusivity into tissues including the target liver tissue), high EE (to reduce the weight of formulation per dose), and less drug release rate constant (to get a prolonged duration of action).Later, the optimized formulation of LMV-ACNs was subjected to surface modifcation upon treatment with sodium lauryl sulphate, an anionic surfactant to enhance the negative zeta potential of LMV-ACNs.Tese formulations were studied for in vivo pharmacokinetic studies in rat models to know the hepatic distribution and hepatic bioavailability of the optimized and then surface-modifed LMV-ACNs.Te fnal formulation was also studied for its cytotoxicity.Similar work was reported by Srikar and Rani [17] by taking tenofovir as the model drug to develop ACNs with the objective of only formulation optimization.However, in the current work, the optimized formulation of the LMV-ACNs was subjected to in vivo pharmacokinetic studies so as to justify its liver targeting potential.

Fourier Transform Infrared Spectroscopy (FTIR).
Te compatibility of LMV with PLGA, Pluronic F-68, and SLS was tested using FTIR (Alpha, Bruker).Pure LMV and 2 Advances in Pharmacological and Pharmaceutical Sciences physical mixtures (100 mg of LMV with 100 mg of each of the above excipients) were prepared as pellets in a hydraulic press after thoroughly mixing with potassium bromide.Tese pellets were individually exposed to scanning in wavelength regions from 4000 cm −1 to 400 cm −1 [18], and spectra were recorded.

Preparation of LMV-ACNs
2.3.1.Experimental Design.In the present work, LMV-ACNs were developed by modifed multiple emulsifcation (W/O/W).Te three most signifcant formulation parameters were selected based on the knowledge from previous literature, and each was taken at three levels.Te ranges of the independent variables were taken based on several preliminary trials.Tese trials were performed with varying levels of independent factors.Te levels for optimization were fnalized as the minimum and maximum levels at which nanoparticles were yielded with sufcient suitability to perform characterization studies.Tese were concentration of polymer out of total weight of nanocapsules (factor A: PLGA RG503H-50%, 62.5% and 75% w/w), the concentration of Pluronic F-68 in the outer aqueous phase (factor B: 0%, 0.25% and 0.5% w/v), and the proportion of glycerol in the outermost water phase (factor C: 0%, 25% and 50% v/v).With Design-Expert v8.0 software, a 3 3 fullfactorial design was created for the experiment.Tere are a total of 28 runs because each combination of the factor levels was treated as a single block with a single center point.Te PS, EE, and k were chosen as response variables.Table 1 provides a description of the components and the matching levels that were taken, and Table 2 provides information on the experimental runs.

Preparation of LMV-ACNs.
Multiple emulsifcation method as reported by Cruz et al. [19] with some modifcations was employed to develop LMV-ACNs.Te same method is employed for the development of the ACNs of tenofovir in our earlier work [17] was employed here with the change of drug into LMV.Te inner aqueous phase (W1) was made by solubilizing LMV in the solvent mixture of methanol and water taken at a 1 : 4 ratio.Te polymer was solubilized in chloroform to constitute the organic phase (O).Two mL of the W1 was added drop by drop into 10 mL of the O, which was maintained kept mixing at 12000 rpm and continued for 30 minutes to develop the primary emulsion of w/o type.Ten, immediately, this emulsion dropped slowly into 20 ml of external phase (glycerol and Pluronic F-68 concentrations were as shown in the table) (W2) under continuous mixing to yield multiple emulsions of W1/O/W2.Mixing was continued until chloroform from the middle organic phase was evaporated to yield PLGA nanocapsules with an aqueous core containing LMV (hence, named LMV-ACNs).Ten, the LMV-ACNs were recovered as a pellet from the nanosuspension by centrifugation at 8,000 rpm and 4 °C for 30 min (Sorvall ST 8R, TermoFisher Scientifc).Te pellet of LMV-ACNs was dispersed in fresh distilled water containing mannitol as cryoprotectant and lyophilized (FDB-5502, Operon) for 24 hours to obtain powdered LMV-ACNs.

Surface Modifcation.
Te optimized formulation of LMV-ACNs was subjected to surface modifcation with the objective of enhancing the negative zeta potential.For this, a sequence of aqueous solutions of SLS was prepared in order to obtain 0.5, 1.0, 2.0, 4.0, 6.0, and 8.0 mM concentrations.Te optimized formulation of LMV-ACNs of weight 0.2 g was added separately into 10 mL of each of the above SLS solutions.Tese mixtures were mechanically stirred (RQ-5 Plus, Remi) at 1000 rpm for about two hours at room temperature and then studied for their zeta potential [20,21].

Diferential Scanning Calorimetry (DSC)
. By using the DSC (DSC-60, Shimadzu) on the pure drug, pure polymer, and optimized LMV-ACNs, the physical states of LMV and PLGA in the manufactured ACNs were examined [22].Te samples were prepared as per the process reported [17], and the DSC was carried out by increasing the temperature in a nitrogen atmosphere from 20 to 250 degrees Celsius at a speed of 10 °C/min.Te spectra were then recorded.

TEM Analysis.
Te surface of the LMV-ACNs was examined employing a transmission electron microscope (Tecnai G2-30, FEI, Netherlands).Te optimized formulation of LMV-ACNs was dispersed in water to sufcient dilution, and a drop of it was attached to the carbonated copper grid and waited till dried.Ten, this was viewed under the microscope, and photomicrographs were taken [23].

Particle Size and Zeta Potential.
Based on the dynamic light scattering (DLS) concept, ZetaSizer Nano ZS90 (Malvern Instruments, UK) [24] evaluated the sizes and zeta potentials of the produced LMV-ACNs.Te analysis was carried out with a fxed scattering angle of 90 °and a temperature of 25 °C.After properly diluting each sample with distilled water, measurements were made in triplicate for each.

Entrapment Efciency.
After preparation, the obtained LMV-ACNs nanosuspension was centrifuged at 8,000 rpm and 4 °C for a period of 30 min.Te supernatant was collected and subjected to a spectrophotometry (Evolution 201, TermoFisher Scientifc) assay to estimate the amount of LMV that remained unentrapped into ACNs at a maximum wavelength (λ max ) of 271 nm.From this value, the LMV entrapped can be obtained by subtracting the obtained unentrapped amount from the initial amount of LMV taken [14,25].Te EE was quantifed by using the following formula: Advances in Pharmacological and Pharmaceutical Sciences EE(%) � Amount of LMV taken − Amount of unentrapped LMV Amount of LMV taken × 100. (1)
One dose equivalent LMV-ACNs were dispersed in a small amount of water and transferred into the dialysis bag. 100 mL of 0.1N HCl as bufer medium was taken in a beaker and was kept for continuous stirring at 100 rpm on a magnetic stirrer (1MLH, Remi).Te dialysis bag containing the ACNs was immersed in the beaker containing the medium.Samples of two mL from the medium were taken at prefxed time points for a total period of 24 h.After every sampling, two mL of fresh bufer was substituted into the beaker.Te samples were quantifed by measuring absorbance at 271 nm in a spectrophotometer to quantify the amount of LMV released from the ACNs.
2.9.Design Validation and Optimization.Design-Expert software was used to perform the DoE validation.All the formulations of LMV-ACNs, obtained by performing the runs according to the model, were estimated for the selected response variables.Te obtained values of these response variables were analyzed statistically by the response surface polynomial quadratic model.Plots of predicted versus actual To meet the objectives for each of the three responses of the LMV-ACNs, the optimization of the design's chosen formulation elements was carried out.Te goals were set as reduced PS (to have more difusivity into tissues, including the target liver tissue), high EE (to reduce the weight of formulation per dose), and less k (to get a prolonged duration of action) [28,29].

In Vitro Cytotoxicity Studies.
Te MTT test was used to assess the toxicity of LMV-ACNs on HeLa cell lines that were procured from NCCS, Pune [30].Tis test was carried out similarly as we reported earlier [17].
2.11.In Vivo Pharmacokinetic Studies.Male Wistar rats having 236−261 g of body weight were chosen for the in vivo biodistribution and pharmacokinetic investigations.Te rats were maintained in an animal house at 22 ± 0.5 °C temperature with 50 ± 5% RH.Te study protocol was studied and accepted by the Institute Animal Ethics Committee (IAEC) of the University College of Pharmaceutical Sciences, Acharya Nagarjuna University, Guntur (IAEC No.: ANUCPS/IAEC/AH/P/20/2015). Te rats were maintained for overnight fasting with allowance to take water only until four hours after dosing.
All 24 animals were separated into four groups, containing six animals in every group.Te groups were labelled as L1: control; L2: aqueous LMV solution; L3: optimized LMV-ACNs; and L4: surface-modifed LMV-ACNs.Except for the control group, all the rats in the remaining three groups were administered with the respective formulation at the same LMV equivalent dose of 7.6 mg/kg [31].Te dose was adjusted to 0.4 mL and was given through the saphenous vein of one leg.Blood samples were taken from the lateral saphenous vein of the second leg at 0.5, 1.0, 2.0, 4.0, 8.0, 12.0, 18.0, and 24.0 h after dosing.After every time point, three animals from each group were sacrifced and the liver was isolated.Te animals were exposed to CO 2 for anesthetization.Ten, the anesthetized rats were euthanized carefully by cervical dislocation [32].Te isolated liver was homogenized in an isotonic phosphate bufer of pH 7.4.Te tissue was transferred into 10 mL of the bufer in a glass homogenizing cup supplied with the glass-tefon tissue homogenizer (Remi, RQ-127 A/D).Te tissue was homogenized for 2 min.at 8000 rpm [33].Ten, the obtained homogenate was subjected to LMV extraction.

Preparation of Biological Samples.
Liquid-liquid extraction technique was employed to extract the LMV from the biological samples [34].Plasma was taken by centrifuging the blood samples at 8000 rpm for 15 min.at 4 °C.100 μL of the plasma sample or liver homogenate, 10 μL of IS solution (Nelfnavir 50 μg/mL) were mixed in a vortex mixer (CM-101 Plus, Remi) for 20 sec., and 1.5 mL of acetonitrile (ACN) was added and again mixed for 15 min.Te supernatant was separated and dried on a constant temperature water bath until the complete evaporation of ACN.Te dried residue was diluted with the mobile phase.Tese samples were stored at −25 °C until analysis using highperformance liquid chromatography (HPLC).

Sample Analysis.
LMV in the biological samples was estimated by a modifed and validated HPLC (Infnity II LC System, Agilent) method reported by AV Singh et al.Nelfnavir at 50 μg/mL was added as the internal standard (IS).Te mobile phase was composed of 0.25% triethylamine bufer (pH 3.0) and ACN at a 70 : 30 ratio with a fow rate of 1 mL/min.20 μL of the sample was administered into the column (Poroshell 120 EC-C 18; 4.6 × 100 mm) and ran the system for 5 min.Te LMV was detected at 258 nm using a PDA detector.
2.12.Statistical Analysis.All the statistical analysis including the ANOVA was performed using the Design-Expert software.Te statistical signifcance was conveyed at p < 0.05.All the experimental results were presented as mean-± standard deviation of the three observations.

FT-IR.
Te spectra of pure LMV and its physical mixtures with the selected excipients are shown in Figure 1. Figure 1(a) of pure LMV exhibited peaks at 3326.53, 1652.46,1286.25, and 1159.79 cm −1 corresponding to the characteristic groups of LMV viz.amino group stretch, carbonyl of cysteine ring, asymmetrical oxathiolane C-O-C stretching, and symmetrical oxathiolane C-O-C stretching, respectively [35].Spectra (Figures 1(b)-1(d)) of the LMV mixtures with the taken polymer and surfactants also exhibited the above characteristic peaks at the matching wave numbers as those of pure LMV.Hence, there was no incompatibility aroused between LMV and the excipients, and these excipients could be used in the development of formulations for LMV.

TEM.
Te surface morphology of the LMV-ACNs was studied by TEM, and the photographs are presented in Figure 2. Tese illustrated that the prepared ACNs were almost spherical and their surface was smooth and uniform without any dents or protrusions.

DSC.
During the preparation of the ACNs, LMV was taken as an aqueous solution.Hence, the state of the LMV in the developed ACNs had to be investigated.For this purpose, DSC was performed for the pure LMV, pure PLGA, and the LMV-ACNs, and the obtained spectra are illustrated in Figure 3. Te pure LMV spectrum showed an endotherm sharply near 180 °C which corresponded to the melting point of LMV, and this confrmed that the pure LMV was in the crystalline state.However, the spectrum of the LMV-ACNs did not indicate any such endotherm.Tis result designated that the crystalline LMV might be either in the molecular Advances in Pharmacological and Pharmaceutical Sciences  ( From the equations, it was deduced that all three of the selected factors had a positive efect on EE; factor A had a positive efect, while factors B and C had a negative efect on the PS; it was deduced that all three of the factors had a negative efect on k.Te parts that follow go into further detail about these infuences.

Entrapment Efciency.
Te EE values were obtained in the range of 21.59-73.41%(as shown in Table 2).Te infuences of the factors on EE are shown in Figures 4(a It was very visible how the concentration of the polymer afected the EE, which rose with the level of factor A. Tis could be as a result of the drug being bound more frmly by a high concentration of polymer, which would reduce drug leakage.Increases in viscosity brought about by increased polymer concentrations also reduced drug difusion from ACNs, increasing EE [37].Additionally, at greater polymer levels, the reduction in surface area and increase in path length caused by the larger particles limited the drug's escape through difusion out of the nanocapsules, increasing the EE [38].Te efect of factor B was intriguing since the EE rose when it was increased from 0.0% to 0.25%, but it decreased when it was increased to 0.5%.Te frst increase might have resulted from the surfactant stabilizing the emulsion and preventing the drug leakage.However, at a further rise in surfactant concentration, the drug would difuse out of the nanocapsules and become micellarly soluble in the external aqueous phase [39].Te rise in viscosity and density of the exterior phase of the secondary emulsion upon raising the level of factor C may be responsible for the increase in EE Advances in Pharmacological and Pharmaceutical Sciences that follows an increase in glycerol content.According to the Stokes-Einstein equation [40], viscosity can decrease diffusion, which means that less amount of drug would leak into the external phase and boost EE.Table 3 displays the signifcant results for each of the three factors at p < 0.05.
It was discovered that the highest EE was just 74.31%.Tis may be because the LMV has a high water solubility, which could lead to some leakage.However, in the case of LMV and other comparable highly water-soluble drugs, these EE values for the ACNs produced with PLGA were   8 Advances in Pharmacological and Pharmaceutical Sciences found to be higher than those reported by other authors using alternative methodologies [41][42][43].Tis shows that the water-soluble drug could be efectively loaded into nanocapsules using both the PLGA polymer and the ACNs approach used in this work.

Particle Size.
All formulations of LMV-ACNs were found to have PS ranging from 231.3 to 341.2 nm (Table 2).Contour plots illustrating the impact of the factors on PS are displayed in Figures 4(c) and 4(d).PS increased as the level of factor A increased.Tis might be as a result of the polymer depositing on the core material's surface after the solvent is removed [44].Higher polymer levels may, therefore, cause more polymers to settle around the globules in the dispersed phase, resulting in larger particles.At higher viscosities, shearing efectiveness may also be reduced, potentially resulting in the creation of big particles [45].It was discovered that the surfactant-containing nanoparticles had smaller particles than the surfactant-free ones.Tis could be as a result of surfactant's capacity to reduce interfacial free energy, which could lead to the formation of a more stable fner emulsion.Tese fndings followed a similar pattern to those reported by Krishnamachari et al. [46] and Gupta et al. [47].When water was used as the secondary emulsion's external phase, higher particle sizes were seen than when aqueous glycerol was used at 25% and 50% v/v.Te glycerol concentration-induced increase in the outer phase's viscosity would prevent the globules from the primary emulsion from aggregating.As can be seen in Table 3, every component was determined to have a signifcant impact on particle size at p < 0.05.Advances in Pharmacological and Pharmaceutical Sciences 3.7.Drug Release Studies.Figures 4(e) and 4(f ) illustrate how three factors afect the response k.Te generated ACNs' drug release was shown to be signifcantly impacted by factor A, with a drop in k observed with an increase in polymer content.Tis could be because as the level of factor A increases, the k decreases due to an increase in particle size.
Te difusion path length would increase with increasing particle size, which could lead to a reduction in the drug release rate.Te impact of the factor B was noteworthy, as evidenced by the observation of reduced drug release with an increase in concentration.Te primary emulsion's outer volatile organic phase (chloroform) may interact more with 10 Advances in Pharmacological and Pharmaceutical Sciences the external aqueous phase as surfactant concentration rises, slowing the rate of evaporation.As a result, the ACNs with a stifer membrane were produced, and the drug release rate was reduced [39,48].Factor C also had an impact, as an increase in glycerol concentration was accompanied by a decrease in k value.Tis may be the result of the viscosity increasing at higher glycerol concentrations, which may have slowed the evaporation of chloroform.Te polymer membrane of the nanocapsules produced by delayed evaporation rates may have been stifer and less porous [48], which may have slowed the pace at which drugs are released from them.All three factors' efects were determined to be signifcant at p < 0.05 (Table 3).All LMV-ACN formulations were shown to ft the frstorder kinetics of drug release, as shown by the zero-and frst-order kinetic plots.Te non-Fickian difusion mechanism of drug release was determined using Higuchi's and Korsmeyer-Peppas plots.

Design Validation.
Te adjusted and predicted R 2 values for each response variable were found to be close, with a diference between them of less than 0.2, suggesting that this may be explored for optimization or design space development.Table 3 displays the results of the ANOVA.Each response's model F value indicates that the model was signifcant at p < 0.05.Tis suggests that the response surface quadratic model was suitable for elucidating the impact of the factors on the responses.Furthermore, supporting this was the negligible lack-of-ft values.Tese fndings signifed that this could be extended to further optimization.
3.9.Optimization.Out of all the alternatives the software ofered for the set desire, one solution (as illustrated in Figure 5) had the highest desirability of 0.888 at a combination of factors of 75% w/w of the factor A, 0.45% w/v of the factor B, and 50% v/v of the factor C. At this combination, the values of the response factors are, as indicated by the software, 70.69% of EE, 294.99 nm of PS, and 0.099 h −1 of k.Using this ideal combination of the factors, a new formulation of LMV-ACNs was created and the characterization tests were carried out for the response variables.Te results showed that the EE was 71.54%, the PS was 288.4 nm with a polydisperse index (PDI) of 0.245, the zeta potential was −24.7 mV, and the k was 0.095 h −1 .Tese outcomes and the ones provided by the design showed a strong correlation.As a result, the optimal formulation of LMV-ACNs was thought to be this combination of the three factor levels.

Surface Modifcation.
Te zeta potential of the optimized LMV-ACNs was −24.7 mV.Tis negative zeta potential might be due to the polymer PLGA RG503H which contains free carboxylic acid groups as the end groups on it [49,50].Tis high negative zeta potential is an advantage as it can control the aggregation of ACNs in the dispersion and can improve the physical stability.
Te literature states that nanoparticles with high negative zeta potential can be easily opsonized and cleared from the blood to reach RES-rich organs such as the liver [51].So, the optimized ACNs of LMV were further treated with SLS in order to increase their negative zeta potential.After treatment with SLS, the zeta potential of LMV-ACNs was observed to be increased to a maximum zeta potential of −41.6 mV at a 6 mM concentration of SLS.Hence, with this zeta potential, the ACNs were assumed to have more targetability to reach the liver, which had to be confrmed by in vivo biodistribution studies.3.12.In Vivo Biodistribution/Bioavailability Studies.In vivo biodistribution/bioavailability studies were performed for pure LMV solution and optimized LMV-ACNs and surfacemodifed LMV-ACNs in rats.From the HPLC results, the LMV was found to be eluted at 2.38 min.and the Nelfnavir (IS) was eluted at 3.57 min.Te ratios of the peak areas of the LMV and the IS were equated against the previously developed calibration curve to quantify the plasma concentration of the LMV.Te obtained data of plasma and liver drug concentrations are illustrated in Figures 7(a) and 7(b), respectively.Tese data were subjected to noncompartmental analysis to fnd various pharmacokinetic properties so as to understand the impact of formulation of ACNs and their surface modifcation on plasma and the hepatic bioavailability of LMV.Te results are shown in Table 4.In plasma, steadystate volume of distribution (V ss ) and elimination half-life (t 1/

In Vitro Cytotoxicity
2 ) increased when formulating LMV into ACNs which indicates that LMV distributes more into the body as the nanoparticles can difuse into various tissues.Whereas for the data obtained from the liver tissue, the V ss was found to decrease from 0.58 L/kg of LMV solution to 0.23 L/kg of LMV-ACNs.Tis indicated that more concentration of the drug was confned in the liver since only fewer amounts were distributed out of the liver.Tis could be attributed to the surface hydrophobicity [53,54] and negative zeta potential of the LMV-ACNs as they were made of PLGA that might induce their phagocytosis and make them more available to the liver [12,50].Tis could also be further justifed by the observed hepatic AUC, which increased to 32.94 μg/mL * h for the LMV-ACNs compared to 13.78 μg/mL * h for pure LMV solution.So, it is evident that LMV-ACNs prepared from PLGA make the LMV more available in the liver where it is actually needed.Still, a further signifcant (p < 0.05) increase in hepatic AUC (66.7%) and decrease in V d (64.3%) were observed upon surface modifcation of LMV-ACNs with SLS.Tis might be due to the increased negative zeta potential which could improve the phagocytosis of nanoparticles into RES-rich organs such as the liver.Tis was further supported by a signifcant increase (p < 0.05) in hepatic mean residence time (MRT) (34.7%) and a decrease in clearance (19.7%) upon surface modifcation of LMV-ACNs.
Tese observed results are justifed by the work reported by Nag et al. [55] regarding the passive targeting ability of the PLGA nanoparticles.Tese authors developed PLGA nanoparticles for loading tannic acid (TA) and vitamin E for the treatment of alcoholic liver damage (ALD).Te prepared nanoparticles were found to have a zeta potential of −21.2 mV.Te in vivo and histopathology results revealed that recovery of the liver was highest in the animals treated with the PLGA-TA-E nanoparticles than those treated with plain TA and plain vitamin E. Te authors ascribed this result to the improved delivery of the TA and vitamin E to the liver by the nanoparticles owing to their hydrophobic nature and surface charge.In one more study reported by Zhang et al. [56], Cholesterol-based nanoparticles were developed for loading miRNA to treat ALD.Besides, the miRNA was paired with polyethyleneimine (PEI) and made into nanoparticles.In vivo biodistribution and histopathology studies revealed greater accumulation of the RNA from the cholesterol-based nanoparticles than the PEIpaired RNA.Te authors attributed this observation to the surface hydrophobicity of the nanoparticles owing to the presence of cholesterol.Few other similar reports regarding the passive liver targeting ability of nanoparticles are reviewed by Warner et al. [57].
Tese recent literature reports suggested that the proposed mechanism of liver targeting in this work is justifed.Besides the charge, the surface hydrophobicity of the PLGA nanoparticles to a greater is responsible for the passive liver targeting.Opsonins in the blood easily detect and attack the hydrophobic particles that are administered into the blood through the IV route.Te opsonized nanoparticles can be readily engulfed by the reticuloendothelial system (RES) rich organs such as liver and spleen [58,59].Tis mechanism of phagocytosis is even more prominent for the nanoparticles with a negative charge and with a size of above 200 nm [57].Tese possible mechanisms could be responsible for the greater accumulation of the LMV from the PLGA nanoparticles developed in this work.

Conclusion
Te current research work was executed out with the objective of achieving liver targetability by developing biodegradable nanocapsules for delivering LMV.Te experiment was designed successfully with 3 3 full-factorial design.All three selected formulation parameters were observed to have a quadratic efect on the three responses.Tis further proceeded to graphical optimization.Te optimized formulation was obtained as ACNs containing PLGA at 75% w/w, Pluronic-F68 at 0.45% w/v, and glycerol in the external phase at 50.00% v/v.Te optimized ACNs were further coated with SLS so as to increase the zeta potential.Te in vivo biodistribution studies indicated that the optimized ACNs increased the hepatic bioavailability of LMV by 139% when compared to pure LMV.Further, surface modifcation of the optimized ACNs resulted in an increase in the hepatic bioavailability of LMV by 66.7% when compared to the optimized ACNs.Tese fndings designated that the hepatic targetability was accomplished by Advances in Pharmacological and Pharmaceutical Sciences developing the ACNs and was further increased by surface modifcation, thus demonstrating the successful achievement of the study objectives.Hence, this ACN formulation can be extended to drugs like LMV in achieving liver-specifc delivery to enhance their therapeutic outcomes.
) and 4(b) for LMV-ACNs in the form of contour plots.

Figure 4 :
Figure 4: Contour plots showing (a) efect of the factors A and B on entrapment efciency, (b) efect of the factors A and C on entrapment efciency, (c) efect of the factors A and B on particle size, (d) efect of the factors A and C on particle size, (e) efect of the factors A and B on drug release rate constant, and (f ) efect of the factors A and C on drug release rate constant.( * Te data that were displayed were the average of three observations, with a statistical signifcance threshold of p < 0.05 ).

Figure 5 :Figure 6 :
Figure 5: Desirability plot showing the maximum desirability for the set desirability criteria.
Study.Tis study was conducted on the optimized LMV-ACNs that were prepared with PLGA RG503H.Te reason behind the use of the HeLa cell lines is that these cells are resistant to cell death by natural apoptosis, and they are destroyed only due to the Plasma drug concentration (µg/mL)

Figure 7 :
Figure 7: (a) Plot of time versus plasma drug concentration of LMV; (b) plot of time versus liver drug concentration of LMV. ( * Te data presented were the mean of three observations, and the comparison was made at a signifcance limit of p < 0.05 ).

Table 1 :
Description of the three formulation factors and their levels taken in this work.

Table 2 :
Combinations of the formulation factors according to the selected factorial design and the observed results * of characterization studies of LAM-ACNs.
* Te results were expressed as average ± standard deviation for n � 3.

Table 3 :
Results of ANOVA for the quadratic model for three selected response variables.
Note. a Sum of squares; b degrees of freedom; c mean sum of squares; d p value less than 0.05 indicates model terms are signifcant; e polymer concentration (% w/w); f surfactant concentration in secondary emulsion (%w/v); g concentration of glycerol in external phase (%v/v); h entrapment efciency; i release rate constant.

Table 4 :
Results showing various pharmacokinetic parameters after noncompartmental analysis of plasma and liver data obtained from in vivo biodistribution studies of LMV-ACNs.
t 1/2 : elimination half-life; AUC: area under the time-plasma drug concentration curve; MRT: mean residence time; V ss : steady state volume of distribution; Cl T : total body clearance.( *