Gelatin Coated Hybrid Lipid Nanoparticles for Oral Delivery of

Article pubs.acs.org/molecularpharmaceutics

Gelatin Coated Hybrid Lipid Nanoparticles for Oral Delivery of Amphotericin B Sanyog Jain,* Pankaj U. Valvi, Nitin K. Swarnakar, and Kaushik Thanki Centre for Pharmaceutical Nanotechnology, Department of Pharmaceutics, National Institute of Pharmaceutical Education and Research (NIPER), Sector 67, SAS Nagar (Mohali), Punjab-160062, India S Supporting Information *

ABSTRACT: Amphotericin B (AmB) loaded polymer lipid hybrid nanoparticles (AmB-PLNs) comprised of lecithin (anionic lipid) and gelatin (Type A, cationic below its isoelectric point 7.0−9.0) were prepared by a two-step desolvation method to improve the oral bioavailability of AmB. The optimized AmB-PLNs were found to have particle size 253 ± 8 nm, polydispersity index (PDI) 0.274 ± 0.008, and entrapment efficiency 50.61 ± 2.20% at 6% w/w of initial theoretical drug loading. Scanning electron microscopy (SEM) revealed spherical shaped nanoparticles whereas confocal laser scanning electron microscopy (CLSM) and fluorescent resonance energy transfer (FRET) analysis confirmed the orientation of the lecithin (located in the core) and gelatin (exterior coat) within the system. The developed formulation exhibited a sustained drug release profile with a release pattern best fitted to Higuchi kinetics. Experiments on Caco-2 cell lines revealed a 5.89-fold increase in the intestinal permeability of AmB-PLNs whereas in vivo pharmacokinetic studies exhibited a 4.69-fold increase in the oral bioavailability upon incorporation of AmB into PLNs as compared to that of free drug. The developed formulation showed significantly lesser hemolytic toxicity as compared to the free drug, Fungizone (micellar solution of AmB) and Fungisome (liposomal formulation of AmB). Furthermore, blood urea nitrogen (BUN) and plasma creatinine levels, indicative of nephrotoxicity, were also found to be significantly lesser for developed PLN formulation as compared to free drug and Fungizone while comparable to that of Fungisome. The histopathology of the kidney tissues further confirmed the absence of any changes in the morphology of the renal tubules. KEYWORDS: amphotericin B, gelatin, lecithin, polymer lipid hybrid nanoparticles (PLNs), oral delivery, nephrotoxicity rats.6 AmB was incorporated in peceol (mixture of mono- and diglycerides of oleic acid), which predominantly leads to increased absorption of drug by the lymphatic transport mechanism. Additionally, the existence of AmB in its native monomeric form in the lipidic environment in contrast to the aggregated form in a conventional micellar solution has been attributed to the significantly lesser toxicity of lipid based formulations.5,7 An exhaustive review on the lipid based approach for oral delivery of AmB has recently been reported.2,8 Apart from lipid based drug delivery systems, various other drug delivery systems, such as polymeric nanoparticles and nanosuspension approaches, have also been utilized for oral delivery of AmB.9−11 A cochelate complex of AmB (Bioral), prepared by precipitation of drug with phosphatidylserine and calcium cations, is presently under clinical trials for its oral delivery.12

1. INTRODUCTION Amphotericin B (AmB) is considered as one of the gold standards for the treatment of systemic fungal infections and leishmaniasis. Although the oral delivery of AmB has been under development for the last five decades, unfortunately, no such product has been placed on the market to date. This is because of the peculiar properties of AmB, that include both poor solubility and intestinal permeability and its acid labile nature in the biological milieu.1,2 Therefore, only intravenous administration of drug has been available; however, this route is also not satisfactory owing to the fact that AmB causes severe hemolytic toxicity and nephrotoxicity, which in extreme cases is exaggerated and requires termination of the AmB therapy.3 In the majority of cases, the self-aggregation capability of AmB has been identified as the linker of these toxicities, and it is recommended to have AmB in monomeric form in the formulation.4,5 Hence, AmB is considered as one of the most challenging difficult-to-deliver drugs. Several attempts have been made to enhance the oral bioavailability of AmB. Recently, a lipid based self-emulsifying drug delivery systems had been developed which showed significant antifungal activity in Aspergillus f umigates infected © 2012 American Chemical Society

Received: March 24, 2012 Accepted: July 30, 2012 Published: July 30, 2012 2542

dx.doi.org/10.1021/mp300320d | Mol. Pharmaceutics 2012, 9, 2542−2553

Molecular Pharmaceutics

Article

Table 1. Optimization of AmB-PLNsa ser. no.

a

optimization param

1

lecithin/gelatin ratio (w/w)

2

stirring speed (rpm)

3

pH of gelatin solution

4

% theoretical drug loading (w/w)

variables 2:1 1:1 1:2 1:3 600 1200 1800 2 3 4 6% 8% 10%

particle size (nm) 852 242 253 283 629 253 266 237 253 275 253 286 304

±9 ±7 ±8 ±5 ± 17 ±8 ±9 ±6 ±8 ±4 ±8 ±7 ± 16

PDI

% entrapmt eff

0.363 ± 0.010 0.234 ± 0.009 0.274 ± 0.008 0.295 ± 0.009 0.353 ± 0.03 0.274 ± 0.008 0.255 ± 0.008 0.239 ± 0.006 0.274 ± 0.008 0.285 ± 0.007 0.274 ± 0.008 0.330 ± 0.02 0.420 ± 0.07

61.27 ± 6.14 46.27 ± 1.56 50.61 ± 2.20 49.07 ± 2.97 46.13 ± 1.39 50.61 ± 2.20 45.00 ± 1.40 52.97 ± 2.63 50.61 ± 2.20 45.31 ± 2.91 50.61 ± 2.20 38.22 ± 2.30 17.53 ± 5.90

remarks stirring speed = 1200 rpm pH of gelatin solution = 3 6% w/w theoretical drug loading lecithin/gelatin ratio = 1:2 w/w pH of gelatin solution = 3 6% w/w theoretical drug loading lecithin/gelatin ratio = 1:2 w/w stirring speed = 1200 rpm 6% w/w theoretical drug loading lecithin/gelatin ratio = 1:2 w/w stirring speed = 1200 rpm pH of gelatin solution = 3

Values represents mean ± SD (n = 6).

were purchased from Rankem Fine Chemicals (New Delhi, India). Ultrapure water (LaboStar, Siemens water technologies, USA) was used for all the experiments. All other excipients used were of analytical grade and were utilized as obtained without any further modification. 2.2. Preparation of AmB Loaded PLNs (AmB-PLNs). The AmB-PLNs were prepared by the two-step desolvation method previously reported by Coester et al., with slight modifications as per laboratory conditions.20 Briefly, 200 mg of gelatin (type A, 300 bloom) was dissolved in distilled water (10 mL) at 40 °C. High molecular weight (HMW) gelatin was precipitated by addition of acetone (10 mL), as desolvating agent. The precipitated HMW gelatin was redissolved in distilled water (10 mL) at 40 °C. The pH of the gelatin solution at the desolvation step was adjusted between 2 and 4 with 0.1 N HCl. AmB (12 mg) was dissolved in 500 μL of DMSO and added to the 100 mg of lecithin previously dissolved in 5 mL of methanol. The resulting drug/lecithin solution was added to the HMW gelatin solution dropwise under constant stirring. Methanol was allowed to evaporate for about 12 h, and subsequently, acetone (10 mL) was added under constant stirring to precipitate gelatin. This resulted in formation of AmB loaded PLNs. An aqueous solution of glutaraldehyde (2% v/v) in the volume of 50 μL was implemented as a cross-linking agent to harden the PLNs. The resulting dispersion was then stirred for 12 h to evaporate acetone at room temperature. The unentrapped AmB was separated by centrifuging the AmB-PLN dispersion at low speed (5000 rpm for 5 min). This process was validated in-house to ensure that AmB-PLNs did not settle upon this treatment. The obtained formulation was washed repeatedly with distilled water to remove the traces of DMSO. The cross-linking was monitored by assessing the surface free amino groups estimated by the 2,4,6-trinitrobenzenesulfonic acid (TNBS) method.21 2.3. Optimization of AmB-PLNs. Various process variables, including lecithin/gelatin ratio (2:1, 1:1, 1:2, and 1:3 w/w), stirring speed (600, 1200, and 1800 rpm), pH of gelatin solution (2, 3, and 4), and % theoretical drug loading (6−10% w/w), were optimized, as shown in Table 1, in order to obtain a final formulation with particle size ≤ 250 nm, polydispersity index (PDI) ≤ 0.3, and entrapment efficiency > 50%. All process variables were optimized at room temperature (25 °C). Additionally, AmB also tends to aggregate at various pH conditions; hence, the monomeric form was confirmed by

However, a novel approach of pharmaceutical composition can be applied for oral delivery of AmB, which is essentially a composition of lipid and polymer commonly referred to as polymer lipid hybrid nanoparticles (PLNs). This particular delivery system is a main focus presently, owing to the dual advantage of its vesicular and particulate nature. The drug is efficiently encapsulated within lipid, which is further stabilized by a polymer coat, subsequently increasing the system’s payload capability and providing additional protection in the gastrointestinal fluids. Additionally, the attraction is further enhanced owing to high biocompatibility of lipids along with the contribution of polymer for structural integrity. Recently, it was shown that the antibiotic loaded PLNs posed superior drug loading capacity compared to plain polymeric nanoparticles formulated without nonlipidic surfactants.13 Various methods have been reported for preparation of PLNs of drugs, which broadly include combined self-assembly techniques or nanoprecipitation14−17 and emulsification18,19 techniques. However, the choice of method depends upon the drug, polymer, and lipid used. The present study aimed at development of PLNs for oral delivery of AmB. It inculcates lecithin as lipid, owing to its proven capability of increasing the deliverability of AmB with gelatin as a polymer due to its wide applications in nanopharmaceuticals along with the advantage of easy availability due to inexpensive cost. Apart from these advantages, the present formulation design has a high level of industrial scalability because of simple manufacturing process and excipients.

2. MATERIALS AND METHODS 2.1. Materials. Gelatin (type A, from porcine skin, 300 bloom) was purchased from Sigma, USA. Lecithin and AmB were generously availed as a gift sample from Cargill, Germany, and Ambalal Sarabhai Enterprises, Vadodara, India, respectively. Rhodamine b-isothiocyanate (RBITC), coumarin-6, mannitol, dialysis bag (MWCS 12000 Da), heparin, pancreatin, pepsin, trypsin-ethylenediaminetetraacetic acid (EDTA), and Triton X100 were obtained from Sigma, USA. Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), penicillin, streptomycin, and Hank’s balanced salt solution (HBSS) were purchased from PAA, Austria. Tissue culture plates and 96 well plates were procured from Tarsons and Costars, Corning Incorporated, respectively. Acetonitrile (HPLC grade), methanol (HPLC grade), dimethyl sulfoxide (DMSO), and acetone 2543



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its in vitro hemolytic toxicity as described in the subsequent section. 2.4. Characterization of AmB-PLNs. 2.4.1. Particle Size and ζ Potential Measurement. The mean particle size, PDI, and ζ potential of the AmB-PLNs were evaluated using Zeta Sizer (Nano ZS, Malvern Instruments, U.K.). 2.4.2. Entrapment Efficiency. The PLN dispersion was pelletized by centrifuging the dispersion at 21000 rpm for 10 min at 15 °C. The resulting pellet was dissolved in a methanol/ DMSO mixture (50:50 v/v) and sonicated for 5 min. The solution was centrifuged at 10000 rpm for 5 min, and the supernatant was analyzed by a validated HPLC method.22 In a further part of the investigation, accountability of the drug was calculated by mass balance in the pellet and supernatant; and it was found to be more than 95% in all cases. % entrapment eff =

characterized for the appearance of the cake, reconstitution time, Sf/Si ratio (ratio of particle size obtained after lyophilization to particle size before lyophilization), PDI, and entrapment efficiency. The selected cryoprotectant was then further optimized for its concentration. 2.6. Stability Studies. 2.6.1. Stability in Simulated Gastrointestinal Fluids. The AmB-PLNs were evaluated for their stability in simulated gastric fluid (SGF, pH 1.2) and simulated intestinal fluid (SIF, pH 6.8), prepared as per USP.29 Each milliliter of the formulation was added to 9 mL of simulated gastrointestinal fluids and incubated for 2 h and 6 h, respectively, in SGF and SIF.30 In a parallel study, the formulation was incubated with SGF for 2 h as described above, followed by transfer of the entire contents to SIF and reincubation for an additional 6 h to simulate the effect of actual in vivo conditions. The samples were then evaluated for changes in particle size, PDI, ζ potential, and entrapment efficiency. 2.6.2. Storage Stability. Freeze-dried AmB-PLNs were also assessed for storage stability over a period of 3 months as per ICH guidelines. Briefly, sealed glass vials containing freezedried AmB-PLNs were stored at 4 °C and room temperature (25 °C with relative humidity ≤55%). Formulations were monitored for changes in particle size, PDI, and entrapment efficiency in addition to physical appearance and ease of reconstitution. 2.7. In Vitro Drug Release Study. The AmB-PLNs equivalent to 1 mg of the entrapped drug were added to an activated dialysis bag (MWCS 12000 Da, Sigma). The dialysis assembly was then suspended in 10 mL of PBS pH 7.4, containing 0.25% w/v of sodium lauryl sulfate (SLS) at 37 °C in a shaking water bath at 100 rpm. Aliquots of 200 μL of sample were withdrawn at 0, 1, 2, 4, 6, 8, 10, 12, 24, 36, 48, 60, and 72 h and estimated by an HPLC method for % cumulative drug release. The cumulative % release of AmB was then plotted against time. In order to assess the effect of the gelatin coating, the release profile of AmB from plain lecithin particles (liposomes) was also evaluated, and hence, a marketed liposomal formulation of AmB, Fungisome, was employed for the same and subsequently compared with that of AmB-PLNs. 2.8. Caco-2 Cell Permeation Studies. Caco-2 cell culture experiments were implemented to assess the uptake and transport of the AmB-PLNs. Briefly, Caco-2 cells (American Type Culture Collection) were grown in 25 cm2 tissue culture flasks and maintained in 5% CO2 atmosphere at 37 °C. The cell medium was supplemented with Dulbecco’s Modified Eagle’s culture medium (DMEM), 20% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 μg/mL streptomycin (PAA, Austria). The media was changed at every 2 day interval, and cells were harvested in 0.25% trypsin-EDTA solution (Sigma) once 90% confluence in the cell culture medium was attained. The cells were then cultured in a 96 well plate (Costars, Corning Incorporated) at a density of 50000 cells/well for subsequent studies. The culture medium was then removed and cells were washed with Hank’s Buffered Salt (HBS) Solution (PAA, Austria) three times. After removing the HBS solution from the plates, 10 μM of free AmB and AmB-PLNs (equivalent to 10 μM of free AmB) was added to each plate and subjected to predefined incubation periods for the apical uptake studies. HBS solution was employed for all dilutions, and blank HBS solution was used as the negative control. After the incubation period, the supernatant was removed and cells were washed three times with HBS solution. Subsequently, 300 μL of ice

amt of AmB in nanoparticles × 100 amt of AmB used in formulation

practical loading (%) amt of AmB in nanoparticles = × 100 wt of PLNs

2.4.3. Shape and Morphology of AmB-PLNs. The shape and surface morphology of the AmB-PLNs were determined by scanning electron microscopy (SEM) (S-3400N, Hitachi, Japan). A drop of colloidal dispersion of AmB-PLNs was deposited on a glass coverslip previously adhered to a metallic stub by a biadhesive carbon tape. The drop was air-dried and coated with gold so as to obtain a conducting surface. Finally, this sample was analyzed by scanning electron microscopy in vacuum. The formation of AmB-PLNs was confirmed by confocal laser scanning microscopy (CLSM) study using fluorescent dyes. The hydrophilic red dye rhodamine Bisothiocyanate (RBITC) was incorporated in the aqueous phase of gelatin while a hydrophobic green dye, coumarin-6, was incorporated in lecithin solution. Preliminary confirmation for formation of core and shell was visualized through 3D optical sectioning while the exact location of lipid and polymer was ascertained by fluorescent resonance energy transfer (FRET) analysis. In the latter case, microparticles were intentionally prepared to clearly visualize the transition of dyes between the surfaces. 2.4.4. UV−Visible Spectral Analysis. The UV visible spectral analysis was performed to evaluate the aggregation state of AmB, which is the principal reason for its toxicity.23−26 Briefly, 10 μg/mL concentrations of Fungizone, AmB-PLNs, and free AmB (dissolved in DMSO) were scanned in the range 250− 500 nm. The ratio of the absorbance of the first peak (I) to the last peak (IV) was used to monitor the aggregation state of AmB.27 2.5. Lyophilization of AmB Loaded PLNs. AmB loaded PLNs were freeze-dried (Vir Tis, Wizard 2.0, New York, USA) following an optimized universal stepwise freeze-drying process, developed and patented by our group.28 Briefly, 5 mL of AmBPLN dispersion was added to glass vials containing 5% w/v of cryoprotectants such as sucrose, dextrose, trehalose, and mannitol. The samples were then subjected to freezing until −60 °C in 8 h through an 8 step process, followed by primary drying until 20 °C in 36 h through 8 steps. The secondary drying was carried out at 25 °C for 6 h. The condenser temperature was −60 °C, and the pressure applied in each step was 200 Torr. The freeze-dried AmB-PLNs were then 2544



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groups, each containing six animals. Different formulations, viz. control (vehicle treated), AmB dissolved in DMSO, Fungizone, Fungisome, and AmB-PLNs, were administered intravenously at doses equivalent to 3 mg/kg on alternate days (day 1, day 3, and day 5). The weight of each animal was recorded before each dosing. On day 6, all animals were sacrificed humanely and blood was collected in heparinized microcentrifuge tubes and centrifuged at 3000 rcf for 5 min to collect plasma. The plasma samples were then evaluated for BUN and plasma creatinine levels using commercially available kits. 2.10.3. Renal Histology. Kidney tissue specimens were also collected and fixed in 10% neutral buffered formalin and subjected to tissue processing followed by embedding in paraffin. The sections were further sliced into layers with 5 μm thickness (Leica, Wetzlar, Germany) and stained with hematoxylin and eosin (H&E) for microscopic examination (Olympus, Tokyo, Japan). 2.11. Statistical Analysis. All the data are expressed as mean ± standard deviation (SD) for all in vitro results and as mean ± standard error of mean (SEM) for all in vivo results. Statistical analysis was performed using SigmaStat (version 3.5) utilizing one-way ANOVA followed by a Tukey−Kramer multiple comparison test. P < 0.05 was considered as a statistically significant difference.

cold methanol was added to the plate and cells were incubated for 2 h at 4 °C. After the incubation period, cell extract was collected and homogenized with tissue homogenizer followed by centrifugation at 21000 rpm for 10 min at 4 °C. The supernatant was further treated with 250 μL of methanol and vortexed for 10 min. The resulting mixture was then centrifuged at 21000 rpm for 10 min and the supernatant was subjected to HPLC analysis for quantification of AmB.31 2.9. In Vivo Pharmacokinetics. The pharmacokinetics of AmB-PLNs was performed in male Sprague−Dawley rats (200−250 g), fasted overnight. All the protocols for animal studies were duly approved by the Institutional Animal Ethics Committee (IAEC), National Institute of Pharmaceutical Education & Research (NIPER), India. Free AmB and AmBPLNs (equivalent to an AmB dose of 10 mg/kg) were dispersed in 1 mL of distilled water and administered orally. Blood samples (500 μL) were collected, from retro-orbital plexus in the heparinized microcentrifuge tubes at periodic intervals of 1, 2, 4, 8, 12, 24, 36, 48, and 72 h, under mild anesthesia. Collected blood samples were centrifuged at 3000 rcf for 5 min to separate the plasma as supernatant. The plasma samples were analyzed for drug content using the validated HPLC method reported previously.22 Briefly, 125 μL of plasma of each time point was taken in a microcentrifuge tube containing 25 μL of 1-amino-4-nitronaphthalene as internal standard in methanol (2.5 μg/mL). The mixtures were vortexed for 10 min, and 500 μL of methanol was added to precipitate the proteins. The supernatant collected after centrifugation was vacuum-dried (40 °C). The obtained residues were then reconstituted in 60 μL of methanol, vortexed, and sonicated for 5 min and then centrifuged again at 10000 rpm for 10 min. The 50 μL of supernatants was then injected into an HPLC instrument for determination of plasma AmB concentrations. 2.10. Toxicity Studies. 2.10.1. In Vitro Hemolytic Toxicity. Whole human blood was collected in heparinized microcentrifuge tubes and subjected to centrifugation at 3000 rcf for 5 min at 4 °C to separate red blood cells (RBCs). The supernatant along with buffy clot was discarded and RBCs were washed thrice with isotonic PBS, pH 7.4. The stock of RBCs was prepared by mixing three volumes of RBCs with 11 parts of the PBS. A 100 μL of this stock was mixed with 1 mL of Fungisome, Fungizone, and AmB-PLNs equivalent to 5 and 10 μg/mL of AmB. RBCs mixed with distilled water and PBS were employed as positive and negative control, respectively. The samples were incubated at 37 °C for 3 h in a shaker bath and then centrifuged at 3000 rcf for 5 min to separate supernatant, which was allowed to stand at room temperature for 30 min to oxidize hemoglobin (Hb). The absorbance of oxygenated hemoglobin (Oxy-Hb) was measured spectrophotometrically at 540 nm, and percentage hemolysis was calculated by using following equation: %hemolysis =

3. RESULTS 3.1. Preparation and Optimization of AmB-PLNs. During the preparation of AmB-PLNs, the critical parameters were carefully optimized in the specified range as summarized in Table 1 and the influence on particle size, PDI, and entrapment efficiency was studied. Although the highest entrapment efficiency of 61.27 ± 6.14% was obtained at higher concentration of lecithin, particle size and PDI were not in the acceptable range. With the increase in the gelatin concentration, the particle size and PDI were significantly reduced with a complementary decrease in the entrapment efficiency. A comparable particle size and PDI were obtained in the lecithin/gelatin ratio at 1:1, 1:2, and 1:3 w/w; however, a slightly higher entrapment efficiency was observed in the case of the 1:2 w/w ratio. Henceforth, further optimization studies were undertaken with the lecithin/gelatin ratio 1:2 w/w. Further, it was found that stirring at 1200 rpm yielded satisfactory parameters for PLNs. At lower stirring speed (600 rpm), formulations of higher particle size (629 ± 17 nm) with high PDI (0.353 ± 0.03) were obtained, while a slight increase in the particle size and reduction in the entrapment efficiency was observed at high stirring speed (1800 rpm). Hence, 1200 rpm was optimized and used for further studies. The particle size and PDI were found to be decreasing with decrease in pH of the gelatin solution. Although AmB-PLNs prepared at pH 2 showed good particle size (237 ± 6 nm), PDI (0.239 ± 0.006), and entrapment efficiency (52.97 ± 2.63%), owing to the high level of aggregation of AmB molecules at pH ≤ 2, as evident by the relatively higher hemolytic toxicity of PLNs formulated at pH 2 as compared to pH 3 and 4 (data not shown), pH 3 was selected for development of the formulation. The increase in the particle size at pH 4 might be attributed to the weak aggregation of the AmB molecules. AmB-PLNs prepared at 6− 10% w/w theoretical drug loading exhibited comparable particle sizes in all cases, but a significant increase in PDI (p < 0.05) and a concomitant decrease in entrapment efficiency were obtained with a proportionate increase in drug loading. Therefore, on the basis of optimization studies, 6% w/w

ABs × 100 AB100

where ABs is the absorbance of the sample and AB100 is the absorbance of the control.9 2.10.2. In Vivo Nephrotoxicity. The nephrotoxicity of AmBPLNs was assessed in Swiss albino mice (25−30 g). Blood urea nitrogen (BUN) and plasma creatinine levels were used as biochemical markers to evaluate the nephrotoxicity of the formulations. The animals were randomly divided into different 2545



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value of the peak I to peak IV ratio, i.e. 0.68 as compared to that of 1.006 of AmB-PLNs and 1.38 of Fungizone (Figure 4). 3.3. Lyophilization of AmB-PLNs. Figure 5 shows the cake formation with different cryoprotectants, of which mannitol was able to conserve the quality attributes of the original formulation and could form a fluffy cake with easy reconstitution capability. Hence, different concentrations of mannitol were screened for identification of the optimum concentration of cryoprotectant required for AmB-PLNs. Table 2 represents the comparative results of the different concentrations of mannitol and its influence on the particle size and PDI after freeze-drying. The higher Sf/Si ratio and significant increase in the PDI (p < 0.05) were observed at concentrations of mannitol higher than 2.5% w/v while aggregation was recorded at 1% w/v upon reconstitution with water. Thus, batches lyophilized with 2.5% w/v mannitol were used for further studies. 3.4. Stability Studies. 3.4.1. Stability in Simulated Gastrointestinal Fluids. Table 3 summarizes the results of the stability of AmB-PLNs in gastrointestinal fluids, which reveals a generalized decrease in the particle size and entrapment efficiency in all cases; however, the decrease was insignificant (p > 0.05) in the case of SIF and the combination of SGF + SIF in contrast to SGF alone. 3.4.2. Storage stability. Table 4 represents the results of storage stability, and insignificant changes (p > 0.05) in the quality attributes of the formulations were observed after 3 months, being indicative of stability of the formulation. 3.5. In Vitro Drug Release. In vitro drug release from the prepared AmB-PLN formulation was carried out in PBS (pH 7.4) containing 0.25% SLS and was compared with that of Fungisome (Figure 6). A biphasic release was observed in both cases with rapid release up to 12 h followed by sustained release until 72 h. However, the retardation was very prominent in the case of AmB-PLNs as compared to that from Fungisome, suggesting the significance of gelatin coating over the lecithin core (liposomes). Only 13.9% drug release was achieved in the case of AmB-PLNs in contrast to 19.4% from Fungisome after 12 h. Table 5 reflects the release kinetics of AmB from different formulations upon fitting the release data to various release models. Higuchi kinetics of the release pattern was found to have the highest value of the correlation coefficient in the case of AmB-PLNs, in contrast to Hixson−Crowell for Fungisome. 3.6. Uptake of AmB-PLNs by Caco-2 Cells. The cell viability was found to be greater than 90% in all groups when measured by sulfo-rhodamine B colorimetric assay until 3 h. Figure 7 reveals the apical uptake of free AmB and AmB-PLNs by Caco-2 cell monolayers. It was found that the uptake of AmB-PLNs was significantly higher (p < 0.05) as compared to that of free drug at all the time points. Further, a plateau was reached after 2 h, showing insignificant increment in percentage uptake. 3.7. In Vivo Pharmacokinetics. Figure 8 depicts the pharmacokinetic profiles of free AmB and AmB-PLNs. The assessment of the pharmacokinetic parameters revealed a significant increase in the Cmax from 35.92 ± 4.30 ng/mL for free AmB to 94.38 ± 7.25 ng/mL from AmB-PLNs (∼2.62-fold increase). A rapid decline in the plasma concentration of free AmB was observed within 36 h and exhibited only 600 ± 91.62 ng·h/mL of AUCtot whereas AmB-PLNs showed AUCtot of 2813 ± 291.43 ng·h/mL (∼4.69-fold increase). Drug was detectable in plasma even at 72 h in the case of PLNs.

theoretical drug loading was selected for further studies. However, the practical drug loading of 3% was achieved in this case. The optimized formulation of AmB-PLNs has a ζ potential of +15.3 ± 2.4 mV. The % free amine group on the surface of AmB-PLNs was kept in the range of 60−70%, measured by the TNBS method to have uniform cross-linking. The residual glutaraldehyde was practically absent in the formulation when estimated by Brady’s test. 3.2. Characterization of AmB-PLNs. 3.2.1. Shape and Morphology. SEM studies showed the spherical shape and smooth surface of the prepared PLNs (Figure 1). A good

Figure 1. SEM image of AmB-PLNs.

correlation was observed between the particle size obtained by SEM and that measured with a Zeta Sizer. Further, the formation of the PLNs was confirmed by 3D optical sectioning of CLSM (Figure 2) and FRET analysis (Figure 3). The

Figure 2. 3D optical sectioning of PLNs showing the lipidic core and polymeric shell.

colored dots in Figure 3A represent the intensity of interaction between two dyes whereas Figure 3B depicts the distance (in nanometers) between two dyes. Further, the scatter plots (Figure 3C) suggest linear interaction between two dyes with high correlation coefficient (96%). These data suggest the formation of PLNs with a lecithin core coated with a gelatin polymer. 3.2.3. UV−Visible Spectroscopy. AmB molecules tend to be in monomeric form in organic solvents such as DMSO, and methanol and exhibits four peaks in the spectrum, having maxima at 350, 368, 388, and 412 nm.4 The ratio of peaks determines the aggregation state of AmB and can be used as a quantitative tool to assess aggregation. The spectrum of AmB dissolved in methanol (monomeric form) showed the lowest 2546



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Figure 3. FRET analysis of AmB-PLNs: (A) colored dots represent the intensity of the interaction between the both dyes (FRET efficiency), and the inset scale dictates the intensity of interaction with respect to color; (B) colored dots represent the distance between both dyes, and the inset scale represents the actual distance between two dye molecules in nanometers; (C) scatter plot for interaction profile between dyes.

AmB formulations expressed as % hemolysis, as compared to that of control. The RBCs were completely lysed in positive control (distilled water) because of osmotic difference and was considered as 100% hemolysis while, in the case of negative control (PBS, pH 7.4), no hemolysis was observed because of equal osmotic pressure. Incubation of RBCs with Fungizone (5 μg/mL and 10 μg/mL) led to significant hemolysis as compared to negative control and AmB-PLNs (p < 0.001). Furthermore, Fungisome (10 μg/mL) also showed significant (p < 0.05) hemolysis as compared to AmB-PLNs (10 μg/mL) whereas insignificant differences were observed between Fungisome and AmB-PLNs at 5 μg/mL. Figure 10 reveals the SEM micrographs of the RBCs upon treatment with control, AmB-PLNs (10 μg/mL), Fungisome (10 μg/mL), and Fungizone (10 μg/mL). It can be clearly visualized that the AmB-PLN formulation was comparable to that of control even at the highest dose strength of 10 μg/mL. 3.8.2. In Vivo Nephrotoxicity. Figure 11 reveals that plasma creatinine and BUN levels in the case of free AmB were significantly higher than those for control and Fungizone (p < 0.001). Further, the levels observed with Fungizone were significantly higher than that with AmB-PLNs and Fungisome, indicative of the relatively higher nephrotoxicity of Fungizone and free AmB. However, there was no significant difference (p > 0.05) among control, Fungisome, and AmB-PLNs in the levels of plasma creatinine and BUN, revealing negligible nephrotoxicity of the developed formulation, being comparable to that of the marketed liposomal formulation. 3.8.3. Renal Histology. The histopathology studies of the kidney specimens revealed a normal pattern of morphology in the case of control, AmB-PLNs, and Fungisome. However, in the case of free AmB and Fungizone, necrotic tubules and their

Figure 4. UV spectrum of methanolic-AmB, Fungizone, and AmBPLNs at the concentration 0 μg/mL.

Figure 5. Freeze-dried AmB-PLNs with different cryoprotectants.

3.8. Toxicity Studies. 3.8.1. In Vitro Hemolytic Toxicity. Figure 9 represents the hemolytic toxicity profile of various

Table 2. Freeze Drying of AmB-PLNs Using Mannitol at Different Concentrationsa before freeze-drying

a

after freeze-drying

conc of mannitol (%)

particle size (nm)

PDI

particle size (nm)

0 1.0 2.5 5.0 10.0 15.0

253 ± 8

0.274 ± 0.008

aggregation aggregation 338 ± 19 367 ± 21 404.5 ± 9 410.5 ± 17

PDI

ratio Sf/Si

± ± ± ±

1.33 1.45 1.59 1.62

0.314 0.556 0.417 0.293

0.024 0.016 0.009 0.028

Values represents mean ± SD (n = 6). 2547



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Table 3. In Vitro Gastric Stability Study of AmB-PLNsa before

a

after

GIT fluid

time

particle size (nm)

PDI

% entrapment efficiency

particle size (nm)

PDI

% entrapment efficiency

SGF (pH 1.2) SIF (pH 6.8) SGF + SIF

2h 6h 2+6h

253 ± 8

0.274 ± 0.008

50.61 ± 2.20

185 ± 12 232 ± 10 217 ± 9

0.295 ± 0.010 0.248 ± 0.007 0.276 ± 0.011

38.20 ± 2.00 48.67 ± 0.76 42.61 ± 2.06

Values represent mean ± SD (n = 6).

Table 4. Storage Stability Data of Freeze Dried AmB-PLNs at 4 and 25 °Ca 4 °C particle size (nm)

time 0 1 2 3 a

month month months months

338 358 383 390

± ± ± ±

19 9 12 15

25 °C particle size (nm)

PDI 0.314 0.329 0.346 0.357

± ± ± ±

0.024 0.014 0.015 0.020

338 353 395 406

± ± ± ±

19 9 6 13

PDI 0.314 0.318 0.357 0.365

± ± ± ±

0.024 0.007 0.012 0.016

Values represent mean ± SD (n = 6).

Figure 7. Uptake of AmB-PLNs by caco-2 cells (n = 6).

physicochemical properties of nanocarriers prepared with two forms of gelatin type A and B have been observed, a slightly narrower PDI was obtained in the case of type A gelatin.32 Hence, the same was implemented as polymer in the present study. Further, lecithin, owing to its superior encapsulation efficiency, proven efficacy in increasing the deliverability of AmB, inexpensiveness, and established compatibility with AmB, was utilized as lipid for formation of PLNs. The AmB-PLNs were prepared by a two-step desolvation method using gelatin (type A, 300 bloom) as polymer (cationic polymer) and lecithin as lipid (anionic lipid), in which the lecithin core was coated with a biodegradable polymer, gelatin. Gelatin suffers from a disadvantage of a very large molecular weight distribution, which could be attributed to the heterogeneity in polypeptide chains, which alters the reproducibility of the manufacturing process, especially in the case of nanopharmaceuticals.33,34 Hence, uniform molecular weight gelatin was prepared prior to its use. This technique is commonly referred to as the two step desolvation method.20 In this method, low- and variable-molecular-weight gelatin chains were discarded in the first desolvation step, and subsequently obtained high- and uniform-molecular-weight gelatin was used for the formulation of AmB-PLNs, which ensured nanoparticles with more uniform size distribution. The second desolvation step was implemented to coat these gelatin polymer chains on the lecithin core. In addition, gelatin acquires positive charge below its isoelectric point, pH 7−9 (+19.4 mV at pH 3.0), which was exploited to initiate electrostatic interaction between gelatin and lecithin (ζ potential about −43.3 mV at pH 3.0). This interaction was very much essential in formation of stable PLNs. However, no undesirable interaction was observed between gelatin and lecithin, as evidenced from DSC thermograms, provided in the Supporting Information. Further, the pH of the gelatin solution was adjusted before addition of AmB and lecithin, to avoid the self-aggregation of AmB. The aggregation behavior of AmB could be attributed to acquisition of specific charge at specific pH due to the presence of two

Figure 6. In vitro release profile of AmB-PLNs and Fungisome (values represent mean ± SD, n = 6).

Table 5. Correlation Coefficient for Different Release Models of Drug Release from Various AmB Loaded Formulations

a

release model

AmB-PLNs

Fungisome

zero order first order Hixson−Crowell Higuchi Korsemeyer Peppas

0.8062 0.8293 0.9384 0.9653 0.9251a

0.9015 0.9347 0.9795 0.9261 0.9572

Slope was found to be 0.548.

subsequent debris were observed (Figure 12), which further corroborated the results of nephrotoxicity studies.

4. DISCUSSION Lipid based systems have shown promising results in improving the deliverability of various difficult-to-deliver drugs, such as AmB. However, some lacunae have been observed with its oral delivery, which includes stability of these systems in gastrointestinal fluids. Therefore, the present paper focuses on the formulation design and development of the polymer lipid hybrid nanoparticles, which can efficiently stabilize these systems in biological milieu. Gelatin is preferred in the nanopharmaceuticals, owing to its biocompatible and biodegradable nature, low immunogenicity, and ease of availability at inexpensive cost. Although no significant difference in the 2548



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Figure 8. Pharmacokinetic profile of AmB-PLNs and free drug (n = 6).

(Table 1). However, higher entrapment efficiency could be achieved by increasing the lipid component, leading to increased solubilization of AmB in the lipid matrix, but the particle size and PDI was compromised owing to nonuniform coating of gelatin over lecithin. Further, the stirring speed primarily controlled the agitation intensity during the formation of AmB-PLNs. The higher particle size and low entrapment efficiency obtained at lower stirring speed could be attributed to the insufficient interaction between gelatin and lecithin leading to aggregation of the particles rather than effective coating. In contrast, the increased stirring speed may lead to the disruption of the lecithin core and interference in the electrostatic interaction (Table 1). Further, a proportionate decrease in the particle size and PDI and an increase in the entrapment efficiency was observed with a decrease in pH, which could be attributed to the increased electrostatic interaction between gelatin and lecithin. The hemolytic toxicity became very prominent at pH ≤ 2 (data given as Supporting Information). The probable reason could be the fact that at low pH values (pH ≤ 2) possible rotation of the positively charged mycosamine shell considerably affects electric charge distribution along the conjugated double bond system of the polyene chain of AmB molecule. This induction of the dipole moment on the polyene leads to increased aggregation tendency among the molecules via the chromophore−chromophore interactions.25 Therefore, pH 3.0 was selected for further studies. A maximum of 6% w/w theoretical drug loading was optimized after extensive optimization studies. At higher drug loading, the leaching of the drug occurred, resulting in a steep fall in entrapment efficiency. CLSM studies (Figure 2) reveal the exact location of the gelatin (containing rhodamine, exhibiting red fluorescence) and lecithin (containing coumarin-6, exhibiting green fluorescence) in the PLNs. A slight yellowish color developed, signifying the overlapping of the red and green fluorescence, which is indicative of the interaction between gelatin and lecithin at the periphery. Furthermore, to confirm the coating of the lipidic

Figure 9. Hemolytic toxicity of various formulations (***p < 0.001) (n = 6).

ionizable groups, viz. amino and carboxylic acid groups. Therefore, AmB acquires either net negative charge above pH 10 or net positive charge below pH 3 and exists in monomeric form.35 Hence, the pH of the gelatin solution was precisely monitored. The interaction between lipid and polymer was presumed to be due to ionic and hydrophobic interactions, later being among the hydrophobic chains of lipid and that of hydrophobic residues of polymer.36−38 The weaker interactions were further strengthened by cross-linking the gelatin polymer using glutaraldehyde. The formulation prepared without crosslinking was found to be unstable and tended to aggregate upon aging (data not shown), which was in accordance with the results obtained with previous studies.32 This might be attributed to the swelling of the hydrophilic polymer upon evaporation of the organic phase during the manufacturing process. The optimization studies revealed that a specific ratio of lecithin to gelatin was essential to achieve maximum entrapment efficiency with satisfactory particle size and PDI 2549



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Figure 10. SEM micrographs of RBCs treated with (A) control, (B) AmB-PLNs (10 μg/mL), (C) Fungisome (10 μg/mL), and (D) Fungizone (10 μg/mL).

Figure 11. Creatinine and BUN levels, in vivo nephrotoxicity markers, in mice treated with various AmB formulations (3 mg/kg) via an intravenous route of administration (n = 6).

each other, which means that the fluorophores must be brought together via very close interactions.39 As evident from Figure 3, dyes were interacting only at the borders of particles, and hence, it was concluded that the particles had core−shell

core by polymer, FRET analysis was performed, which involves distance-dependent excited state interaction in which emission of one fluorohore is coupled to the excitation of another and occurs only when the two fluorophores are within 20−100 Å of 2550



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Figure 12. Micrographs of kidney specimens of animals treated with (A) control, (B) free AmB, (C) Fungizone, (D) AmB-PLNs, and (E) Fungisome.

structure with gelatin coating over lecithin cores. The findings were further confirmed by the ζ potential of the formed AmBPLNs, which was about +15.3 ± 2.4 mV, indicative of the presence of cationic polymer, gelatin, at the surface. Lyophilization of the AmB-PLNs was implemented using a novel stepwise freeze-drying process developed and patented by our group.28 Mannitol was selected for the stabilization of the AmB-PLNs because of its capability to render the product with no aggregation upon freeze-drying, to higher elegance in the formed cake (Figure 5), and to maintain the original quality attributes upon reconstitution (Table 2). Stability in the various simulated gastrointestinal fluids revealed reduction in the particle size upon incubation with different sets of conditions, which could be due to the synergistic effect of the harsh environment conditions of the gastrointestinal tract and to some extent the drug release from the system (Table 3). The SGF (pH 1.2) may affect the gelatin shell via two different mechanisms; viz., solubility may increase at lower pH (type A, isoelectric point 7−9), and second, pepsin may also have detrimental effects on the gelatin shell, leading to direct exposure of the lecithin core to the acidic environment. Although a significant decrease in the particle size was observed, the majority of the drug remained protected, as only a marginal change in the entrapment efficiency was recorded. This further gives an indication that drug was preferentially encapsulated in the lecithin core. In the case of SIF (pH 6.8), an insignificant (p > 0.05) decrease in the particle size and entrapment efficiency was observed, which could be attributed to the fact that the gelatin shell offers protection from the digestive action of pancreatic lipase present in the small intestine. The results were in accordance with the fact that, upon incubation of AmB-PLNs in SGF and subsequent transfer in SIF, insignificant changes in particle size and entrapment efficiency were observed as compared to those in SGF alone. However, a slight increase in the PDI could be attributed to partial solubilization of the gelatin shell in the acidic media. The freeze-dried product also has the tendency to crystallize out and collapse into a denser amorphous mass which upon aging forms a crystalline mass.40 Henceforth, the formulations were subjected to accelerated stability studies and found to be stable for a stipulated period of time (Table 4). The in vitro drug release revealed significant retardation in the drug release from the PLN formulation as compared to the Fungisome (Figure 6). This clearly signifies the importance of a gelatin coating on the lecithin core, i.e. liposomes. Assessment of the release kinetics revealed that AmB-PLNs showed Higuchi kinetics of drug release, reflecting release from the matrix system whereas Hixson Crowell kinetics were observed in the case of Fungisome, reflecting surface erosion as the mechanism of drug release (Table 5). The slope of the Korsemeyer peppas model was found to be 0.548, indicative of anomalous (non-Fickian) diffusion from the developed PLNs. The matrix type of drug release pattern could be attributed to the presence of gelatin coating over lecithin. Mechanistically, it

can be postulated that the gelatin partially cross-linked with glutaraldehyde acts as a barrier for diffusion of drug in contrast to plain lecithin particles (liposomes) where it has to diffuse only from the lipid vesicles. The greater retardation in drug release could be fruitfully exploited to decrease the toxicity profile of AmB formulation, owing to the reduced exposure of drug to the physiological constituents.41 The cell culture experiments using Caco-2 monolayers demonstrated an ∼5.89-fold increase in the overall uptake of AmB-PLNs as compared to free drug (Figure 7). In vivo pharmacokinetic studies further revealed a 2.62-fold increase in the Cmax and a 4.69-fold increase in the AUCtot in the case of AmB-PLNs as compared to free drug (Figure 8). The results are in good correlation with the increased permeation of AmBPLNs across the Caco-2 cell monolayers. These results are in line with our previous findings, which revealed that the nanoparticles are preferentially absorbed via membranous epithelial cells (M-cells) of the Peyer’s patches in the gutassociated lymphoid tissue (GALT).42,43 These findings are further supported by the stability of AmB-PLNs in biological milieu until 8 h, by which time Cmax was achieved. The toxicity profile of the AmB in the PLN formulation was predicted with the help of the aggregated state assessed by UV−visible spectral studies.44 The peak I to peak IV ratio was considered as the marker of the aggregated form of AmB in a particular system. This ratio was about 1.006 for AmB-PLN, in comparison to 0.68 for methanolic solution (ideal situation) and 1.38 for Fungizone (highest aggregated state in the form of a colloidal micellar system). Furthermore, AmB-PLNs showed a broad and shorter peak at 350 nm similar to methanolic AmB, which represented the existence of the monomeric form, in contrast to the case of Fungizone, which exhibited a very prominent and sharp peak, suggesting the aggregated micellar system (Figure 4). The results clearly indicated that drug was entrapped in the lipidic core of AmB-PLNs and was in the monomeric form, thereby predicting the better safety profile of AmB-PLNs as compared to that of Fungizone. However, the slight shifting of the peaks may be due to the difference in aggregation state of AmB in formulations and the polarity of solvents. In vitro hemolytic toxicity studies revealed that AmBPLNs posed significantly (p < 0.001) lesser hemolysis than that of Fungizone, consisting of sodium deoxycholate, which itself causes hemolysis in addition to that of AmB (Figure 9).9 The hemolytic toxicity of PLNs was comparable to that of the liposomal formulation (Fungisome), which could be attributed to lesser exposure of AmB to RBCs due to slower drug release and encapsulation into the carrier system. The results were further supported by SEM studies showing significant hemolysis in Fungizone (Figure 10). AmB has only slightly higher selectivity for ergosterol (in comparison to cholesterol), a fungal membrane component which is the principal target in the antifungal action of AmB. This selectivity is observed only for the monomeric form while in the aggregated form it loses its capability to distinguish ergosterol from cholesterol; hence, 2551



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Notes

it shows toxicity to mammalian tissues, among which kidneys are the principal organs.5,45 Therefore, in vivo nephrotoxicity studies were performed, and an intravenous route of administration was implemented for studies to normalize the comparison with the marketed products. BUN and plasma creatinine levels in the case of the developed formulation were found to be significantly lower as compared to the free drug and Fungizone (Figure 11). This could be attributed to the slower drug release from the system and the existence of the monomeric form in the PLN formulation.41,45,46 These data were further corroborated by histological studies of the kidney specimens, which revealed significant necrotic tubules and debris in the case of free AmB and Fungizone (Figure 12). The results are in line with the previous reports on the toxicity of Fungizone at similar doses.47 The findings revealed significantly lesser (p < 0.05) toxicity of AmB-PLNs in comparison to the Fungizone, which could also be attributed to the monomeric form of AmB in the PLN formulation and slower release of drug in contrast to the aggregated state of AmB in Fungizone. The results clearly suggest the potential of AmB-PLNs in increasing the oral bioavailability of AmB without compromising the safety profile even in the extreme cases of direct administration to the central compartment.

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS The authors are thankful to the Director, NIPER for providing necessary infrastructure facilities and to the Department of Science & Technology (DST), Government of India, New Delhi, India, for financial support. The authors are also thankful for the technical support rendered by Mr. Dinesh Singh and Mr. Rahul Mahajan.

■

5. CONCLUSION A novel approach combining the lipid and polymer into a single system was successfully employed to improve the oral bioavailability of AmB. The limitations of two carrier systems can be successfully overcome by this unique composition. The biocompatibility of lipid and the structural integrity of the polymer were utilized to formulate the AmB-PLNs to increase the oral bioavailability of AmB to a significant level without compromising the safety profile. However, it has been anticipated that the present system is slightly sensitive to the harsh gastrointestinal environment; therefore, further attempts can be made for alternate strategies, such as enteric coating of the formulation to bypass the acidic degradation in the stomach. The significant increase in the oral bioavailability with this novel formulation inspires one to evaluate the efficacy of the same in the various in vivo and in vitro models for antifungal and antileishmaniasis activity. In addition, the multiple dose kinetics and its effect on the therapeutic efficacy can also be explored. Furthermore, the passive targeting capability can also be exhibited by the system upon intravenous administration for leishmaniasis to subside the toxicity profile to a greater extent. In a nutshell, the present formulation strategy can be set to a prototype for all difficult-to-deliver drugs.

■

ASSOCIATED CONTENT

S Supporting Information *

Aggregation behavior of AmB at different pH conditions, hemolytic effect of AmB-PLNs at different pH conditions, hemolysis study of AmB-PLNs prepared at different pH conditions, and DSC thermogram of developed formulations. This material is available free of charge via the Internet at http://pubs.acs.org.

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REFERENCES

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AUTHOR INFORMATION

Corresponding Author

*E-mail addresses: [email protected], sanyogjain@ rediffmail.com. Telephone: +91 172 2292055. Fax: +91 172 2214692. 2552



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Gelatin Coated Hybrid Lipid Nanoparticles for Oral Delivery of

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