Covalent Functionalized Self-Assembled Lipo ... - ACS Publications

Feb 4, 2014 - In the present work,. AmB has been formulated in lipo-polymerosome (L-Psome) .... magnetic stirring (Remi, Mumbai, India) for 24 h at ro...
0 downloads 0 Views 2MB Size
Download PDF
Article pubs.acs.org/molecularpharmaceutics

Covalent Functionalized Self-Assembled Lipo-Polymerosome Bearing Amphotericin B for Better Management of Leishmaniasis and Its Toxicity Evaluation Pramod K. Gupta,† Anil K. Jaiswal,‡ Vivek Kumar,† Ashwni Verma,† Pankaj Dwivedi,† Anuradha Dube,‡ and Prabhat R. Mishra*,† †

Pharmaceutics Division and ‡Parasitology Division, Council of Scientific and Industrial Research-Central Drug Research Institute, B 10/1, Sector 10, Jankipuram Extension, Sitapur Road, Lucknow, India 226031 S Supporting Information *

ABSTRACT: Amphotericin B remains the preferred choice for leishmanial infection, but it has limited clinical applications due to substantial dose limiting toxicities. In the present work, AmB has been formulated in lipo-polymerosome (L-Psome) by spontaneous self-assembly of synthesized glycol chitosanstearic acid copolymer. The optimized L-Psome formulation with vesicle size of 243.5 ± 17.9 nm, PDI of 0.168 ± 0.08 and zeta potential of (+) 27.15 ± 0.46 mV with 25.59 ± 0.87% AmB loading was obtained. The field emission scanning electron microscopy (FESEM) and high resolution transmission electron microscopy (HRTEM) images suggest nearly spherical morphology of L-Psome. An in vitro study showed comparatively sustained AmB release (66.082 ± 1.73% within 24 h) and high plasma stability compared to commercial Ambisome and Fungizone, where glycol chitosan content was found to be efficient in preventing L-Psome destabilization in the presence of plasma protein. In vitro and in vivo toxicity studies revealed less toxicity of AmB-L-Psome compared to commercialized Fungizone and Ambisome favored by monomeric form of AmB within L-Psome, observed by UV−visible spectroscopy. Experimental results of in vitro (macrophage amastigote system) and in vivo (Leishmania donovani infected hamsters) illustrated the efficacy of AmB-L-Psome to augment effective antileishmanial properties supported by upregulation of Th-1 cytokines (TNF-α, IL-12 and IFN-γ) and inducible nitric oxide synthase, and downregulation of Th-2 cytokines (TGF-β, IL-10 and IL-4), measured by quantitative mRNA analysis by real time PCR (RTPCR). Conclusively, developed L-Psome system could be a viable alternative to the current less stable, toxic commercial formulations and developed as a highly efficacious drug delivery system. KEYWORDS: cholesterol, glycol chitosan, lipo-polymerosome, molecular organization, stearic acid

■

INTRODUCTION Amphotericin B (AmB), a polyene macrolide antibiotic, is highly effective against various Leishmania species and is currently recommended as first line treatment for the visceral form.1 Amphoteric nature of AmB contributed by its hydrophobic (the polyene hydrocarbon chain) and hydrophilic (the polyhydroxyl chain) domains is responsible for poor aqueous and organic solubility, and intercalation into cell membranes to elicit drug’s pharmacological effects.2 To sort out insolubility and nonabsorbability problems conventional AmB formulation Fungizone (a complex with bile salt deoxycholate) is available. However, toxic side effects, in particular, hemotoxicity and azotemia, which occurs in around 80% cases, and renal tubular damage at therapeutic doses have often limited its clinical applications.3 The appropriate delivery system would have a unique advantage in overcoming toxic manifestations on normal body functions caused by direct exposure of the drug and to improve its therapeutic efficacy. It © 2014 American Chemical Society

was thought that lipid based formulations could be the best approach where association of the drug within the lipid molecules would protect the vital organs from direct exposure of the drug. In some earlier reports, it was claimed that liposomal carriers were much less toxic than Fungizone, which opened the gate for development of AmB-liposomal formulations.4 This ultimately resulted in three lipid based products (Abelcet, Ambisome and Amphocil) which claimed to give an overall efficacy at the level of Fungizone but were less toxic. Based on the pharmacokinetics and efficacy data, Ambisome is the most efficient formulation in terms of usage spectrum and is least toxic among all three formulations, but it has a stability problem and is exorbitantly highly priced due to Received: Revised: Accepted: Published: 951

October 14, 2013 January 29, 2014 February 4, 2014 February 4, 2014 dx.doi.org/10.1021/mp400603t | Mol. Pharmaceutics 2014, 11, 951−963

Molecular Pharmaceutics

Article

Table 1. Effect of Various Process Parameters Affecting the Size of L-Psomea no.

parameter changed

variables

remarks

1 2 3 4 5 6 7 8 9 10 11

degree of substitution concentration of copolymer aqueous phase volume copolymer to lipid ratio type of lipid concentration of lipid volume of DMAc type of size reducing method time of sonication amplitude of sonication time of homogenization

GC-SA (10%,25%,50%) GC-SA25% (0.1%, 0.2%, 0.3%, 0.4% w/v) 1 mL, 2 mL, 4 mL 2:1, 4:1, 6:1, 8:1 Chol, PC, lecithin, DMPG, DSPG, HSPC, DMPC 0.1%, 0.2%, 0.3%, 0.4% w/v 0.1 mL, 0.2 mL, 0.3 mL, 0.4 mL sonication and homogenizer 30 s, 60 s,120 s, 180 s,240 s 10%, 20%, 30%, 40% 2 min, 5 min, 10 min,15 min

sonication (60 s at 20% strength), copolymer:lipid (4:1) sonication (60 s at 20% strength), copolymer:lipid (4:1) sonication (60 s at 20% strength), copolymer:lipid (4:1) sonication (120 s at 20% strength) sonication (120 s at 20% strength), copolymer:lipid (4:1) sonication (120 s at 20% strength), copolymer:lipid (4:1) sonication (120 s at 20% strength), copolymer:lipid (4:1) conditions for all are given belowb sonication (20% strength), copolymer:lipid (4:1) sonication (120 s), copolymer:lipid (4:1) shear homogenization speed 10000 rpm

a Chol: cholesterol. PC: α-phosphatidylcholine. DMPG: dimyristylphosphoglyceride. DSPG: disteroylphosphoglyceride. HSPC: hydrogenated soya phosphatidylcholine. DMPC: dimyristylphosphatidylcholine. Constant parameters: probe sonicator (Misonix, Berlin, Germany); membrane dialysis bag; stirring speed, 1200 rpm; magnetic bead-20 mm; primary L-Psome: in 5 mL vial. bHomogenization (IKAT25 digital ULTRA-TURRAX) at 10000 rpm for 5 min. Probe Sonicator (Misonix, Berlin, Germany) at 20% capacity for 2 min.

the cost of phospholipids, thus posing limitations. Moreover, low-priced AmB disk formulations containing cationic lipid dioctadecyldimethyl have been developed, but such formulations have been reported to possess low drug-loading capacity.4 Polymer glycol chitosan (GC) is a commercially available, water-soluble derivative of biocompatible and degradable natural polymer chitosan and has been used as a scaffold for drug delivery or diagnostic imaging.5,6 The long acyl chain of stearic acid (SA) provides stronger interaction between copolymer and AmB, responsible for more sustained release for intercalated AmB. In addition, SA prevents self-association of free AmB molecules and shows reduced drug related toxicity.7 The GC-SA copolymer was synthesized in a manner similar to the small molecular weight single-chain amphiphiles that are known to form vesicles.8 This type of polymer system with amide linkage has been used to prepare nanocarriers, able to self-assemble in aqueous solution to form stable nanocarriers,9 and they have been utilized for successful delivery of various types of therapeutic agents such as genes,10 peptides11 and anticancer drugs.12 Furthermore, Chol was incorporated as a hydrophobic filler in between amphiphilic copolymer bilayer which has the ability to provide greater stability and reduced aggregation and enables the formation of vesicles.13 In previous studies, our research group has prepared liposomes14 and nanocapsules15 as an efficient AmB delivery system toward leishmaniasis treatment. In the present study, a stable vesicular drug carrier system lipo-polymerosome (LPsome) made up of amphiphilic copolymer and lipid that can be a good parallel drug carrier for AmB was formulated. The amphiphilic properties of L-Psome due to hydrophilic and hydrophobic part of copolymer, and lipid moiety, can result easily and with high AmB loading and greater stability and be short of toxicity compared to commercial formulations. However, this type of nanoengineered drug delivery system was not explored for this kind of drug, and effects of various formulation parameters on physicochemical properties were not studied in detail, which has a major influence on toxicities and stability. In continuation of this and considering all the facts, AmB loaded novel L-Psome using the amphiphilic copolymer of GC and SA, with various lipids including cholesterol, was designed, developed and evaluated for various pharmaceutical studies, i.e., drug release kinetics, stability, AmB molecular organization and in vitro and in vivo toxicity along

with antileishmanial and immunomodulatory activity, compared with marketed formulations Ambisome and Fungizone.

■

MATERIALS AND METHODS Materials, Parasite, Cell Line and Animals. Glycol chitosan (GC, Mw 90 kDa, 82.7% degree of deacetylation), [1ethyl-3-(dimethylamino)propyl]carbodiimide hydrochloride (EDC), stearic acid (SA), cholesterol, α-phosphatidylcholine (PC), soya lecithin, dimyristylphosphoglyceride (DMPG), disteroylphosphoglyceride (DSPG), hydrogenated soya phosphatidylcholine (HSPC), dimyristylphosphatidylcholine (DMPC), phosphotungstic acid, sodium deoxycholate (SDC), 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), bovine serum albumin (BSA), dialysis membrane (Mw 12 kDa) and Tween 80 were purchased from SigmaAldrich (St. Louis, MO). Dimethylacetamide (DMAc), HPLC grade methanol and acetonitrile were from SD Fine Chem Ltd. (Mumbai, India). Amphotericin B (AmB) was kindly provided as a gift sample from Emcure pharmaceutical Ltd. (Pune, India). All other reagents were of analytical grade. The WHO reference strain of L. donovani (MHOM/IN/80/ Dd8) was used for both in vitro and in vivo experiments. Leishmania parasites and the macrophage cell line J774A were maintained in RPMI-1640 medium (Sigma, USA) supplemented with 10% heat inactivated fetal bovine serum (HIFBS), 100 U/mL penicillin and 100 μg/mL streptomycin at 37 °C in humidified atmosphere of 5% (v/v) CO2/air mixture. Syrian golden male hamsters (Mesocricetus auratus, 45−50 g) were used to study the antileishmanial effects of the AmB formulations, while in vivo toxicity studies were performed in male Swiss mice (18−20 g) with prior approval of the Animal Ethics Committee of CSIR-Central Drug Research Institute and according to regulations of the Council for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Ministry of Social Justice and Empowerment, Government of India. The Indian Animal Ethics Committee (IAEC) approval no. is CDRI/2012/38. Synthesis and Characterization of GC-SA. The GC-SA copolymer was synthesized using the EDC coupling method with slight modifications.16 Briefly, SA (50%, 25% and 10% with respect to per sugar residue of GC) and EDC (1.2 mol/ mol SA) were dissolved in 10 mL of ethanol followed by vortexing to get a clear solution. This solution was added dropwise in 10 mL of aqueous GC solution (0.02 g/mL) under 952

dx.doi.org/10.1021/mp400603t | Mol. Pharmaceutics 2014, 11, 951−963

Molecular Pharmaceutics

Article

Table 2. Drug Loading, Encapsulation Efficiency and Drug Recovery of Different AmB Loaded Formulationsa formulations VF1b (copolymer + Chol): AmB, 6:1 VF2 (copolymer + Chol): AmB, 4:1 VF3 (copolymer + Chol): AmB, 2:1 VF4c (copolymer + DMPC): AmB, 6:1 VF5 (copolymer + DMPC): AmB, 4:1 VF6 (copolymer + DMPC): AmB, 2:1

drug loading (%) 8.7 13.0 25.6 2.4 4.5 8.3

± ± ± ± ± ±

0.5 0.6 0.9 0.1 0.1 0.4

encapsulation efficiency (%) 52.2 85.8 89.6 2.6 4.9 24.8

± ± ± ± ± ±

drug recovery (%)

0.7 0.8 1.3 0.3 0.05 0.6

4.6 3.8 2.8 14.9 8.9 3.8

± ± ± ± ± ±

0.4 0.1 0.1 0.2 0.1 0.3

size (nm) 263 294 341 450 635 758

± ± ± ± ± ±

20 16 12 22 29 26

PDI 0.17 0.19 0.20 0.36 0.40 0.26

± ± ± ± ± ±

0.06 0.03 0.02 0.10 0.12 0.08

zeta potential (mV) (+) (+) (+) (+) (+) (+)

24.8 23.8 19.3 16.1 14.3 12.1

± ± ± ± ± ±

0.4 0.2 0.8 0.3 0.5 0.9

a Each data represents the mean ± standard deviation (n = 3). bVF1−VF3: Copolymer:Chol was taken as 4:1. cVF4−VF6: Copolymer:DMPC was taken as 4:1.

mL) was spread on a carbon tape glued to an aluminum stub and allowed to air dry. The multiple images of various areas of the FESEM stubs were recorded under high vacuum for LPsome size analysis. For the HRTEM measurement, samples (1 mg/20 mL) were prepared as a thin aqueous film supported on a 300-mesh copper grid. Negative staining was performed using a droplet of 2% (w/v) phosphotungstic acid. Direct imaging of dried samples was executed at a 200 kV acceleration voltage using HRTEM. Determination of AmB Loading, Entrapment and Recovery Efficiency. To optimize AmB loading in delivery system, various formulations were prepared by varying copolymer to drug ratio and type of lipids (Table 2). The AmB content was determined by extracting the drug from the AmB-L-Psome using DMAc, diluted with methanol, filtered through a 0.45 μm membrane filter and analyzed using HPLC method as described in our previous publication.17 In brief, The HPLC system was equipped with 10 ATVP binary gradient pumps (Shimadzu, Japan), a Rheodyne model 7125 injector (Rohnert Park, CA, US) with a 20 mL loop and SPD-M10 AVP UV detector (Shimadzu, Japan). Separation was accomplished on a Lichrosphere Lichrocart C18 column (250 × 4 mm, 5 μm; Merck, Darmstadt, Germany) using acetonitrile and potassium dihydrogen phosphate buffer at pH 4 (60:40, v/v) as mobile phase at 1.0 mL/min flow rate, and column effluent was detected with a UV detector at 407 nm. Data were acquired and processed using class-VP software. The percentage of drug loading, encapsulation efficiency (EE) and recovery was calculated using the following formulas:

magnetic stirring (Remi, Mumbai, India) for 24 h at room temperature (RT, 25 °C). Then the GC-SA was separated from unreacted SA and EDC with dichloromethane (3 × 15 mL) followed by lyophilization under freeze−drying. Synthesized GC-SA copolymer was characterized by FTIR (Perkin-Elmer, Buckinghamshire, United Kingdom) and FTNMR (Bruker, Rheinstetten, Germany) spectroscopy at RT. 1H NMR spectra were obtained using D2O, CDCl3 and CF3COOD with D2O as solvent for GC, SA and GC-SA copolymer, respectively and utilized to characterize synthesized copolymer and degree of SA substitution on GC per 100 deacetylated GC. Preparation of AmB Loaded L-Psome. Self-assembled LPsome was prepared through a single-step nanoprecipitation method. In this method, lipid, typically Chol and synthesized copolymer GC-SA were dissolved in 250 μL of ethanol and 100 μL of DMAc, respectively, and both solutions mixed gently. This solution was added dropwise into 1 mL of aqueous phase (distilled water) under 5 min constant stirring followed by vortexing. Thereafter, size reduction of this dispersion was done using a probe sonicator (Misonix, Berlin, Germany) at 20% amplitude for 120 s, followed by dialysis against 1000 mL of water for 2 h using a dialysis membrane bag for complete removal of free molecules and solvent. For loading of AmB in L-Psome, subsequently AmB solution (1 mg/100 μL DMAc) was added dropwise in L-Psome dispersion under stirring, followed by solvent evaporation. For removal of free drug, AmB loaded L-Psome (AmB-L-Psome) was ultracentrifuged at 40000g for 30 min (Thermo scientific, Sorvall, Wx ultra 100, Germany), and obtained pellets were dried under vacuum. Effects of various formulation and process parameters affecting the size of L-Psome studied are listed in Table 1. Characterization of L-Psome. The cumulative mean of LPsome size, polydispersity index (PDI) and zeta potential were determined by dynamic light scattering using Zetasizer (Nano ZS, Malvern Instruments, Worcestershire, U.K.) after suitable dilution. The zeta potential of particles depends on the dispersion medium; therefore, the surface charge has been measured in double distilled water adjusted to 50 μS/cm using 0.9% NaCl solution and in the original dispersion medium. The zetasizer measures the electrophoretic mobility of the particles, which was converted into the zeta potential using the Helmholtz−Smoluchowski equation built into the Malvern Zetasizer software. All of the measurements were performed in triplicate. The surface morphology of optimized L-Psome was ascertained using field emission scanning electron microscopy (FESEM, SUPRA 40VP, Carl Zeiss NTS GmbH, Oberkochen, Germany) and high resolution transmission electron microscopy (HRTEM, Tecnai G2 F20, Eindhoven, The Netherlands). For FESEM microphotographs, formulation sample (1 mg/

loading efficiency =

wt of (total − free) AmB in L‐Psome × 100 wt of L‐Psome

(1)

encapsulation efficiency =

wt of (total − free) AmB in L‐Psome × 100 wt of total AmB

recovery efficiency wt of free AmB in remaining solution × 100 = wt of total AmB

(2)

(3)

Drug Release Study. The release of AmB from AmB-LPsome was assessed using a dialysis membrane under sink conditions. For release study, various release media consisting of phosphate-buffer solution (PBS, pH 7.4)18 with varying strengths of Tween 80 (0.5, 1 and 2%, v/v), sodium lauryl sulfate (SLS) (0.5, 1 and 2%, w/v) or SDC (1 and 2%, w/v) were tried to find out a compatible one. In this study, 2 mg of AmB equivalent formulation (AmB-L-Psome, Fungizone and 953

dx.doi.org/10.1021/mp400603t | Mol. Pharmaceutics 2014, 11, 951−963

Molecular Pharmaceutics

Article

= 6) by intraperitoneal route. The animals were examined for mortality over the next 8 h. In Vitro Anti-Amastigote Assay. The activity of AmB formulations against intracellular amastigotes was evaluated as per protocol described earlier.20 Briefly, J774A macrophages (1 × 105 cells/well) in 24-well plates were infected with promastigotes expressing green fluorescent protein (GFP) at multiplicity of 10 parasites per macrophage. After 12 h incubation 24-well plates were washed thrice with PBS (pH 7.2) to remove nonphagocytosed promastigotes and resupplemented with RPMI-1640 complete medium. The cells were incubated with AmB-L-Psome and commercial Fungizone as well as Ambisome at different drug concentrations, and blank L-Psome with the same amount of formulations in triplicate for 48 h. After 48 h treatment, cells were removed, washed in PBS and quantitated by flow cytometry equipped with a 20 mW argon laser with excitation at 488 nm and emission at 515 nm followed by multiparametric data analysis by Kaluza analysis software (Beckman Coulter). The parasite growth inhibition was determined by comparing the fluorescence levels of drugtreated parasites with that of untreated control parasites, and the 50% inhibitory concentration (IC50) of each compound was calculated by linear regression analysis. The 90% inhibitory concentration (IC90) was calculated by GraphPad Prism6. In Vivo Assay in L. donovani Infected Hamsters. The in vivo AmB-formulation efficacy was studied against L. donovani amastigotes in a golden hamster model.21 Briefly, 5 to 6 day old stationary phase cultures of L. donovani promastigotes were pelleted and washed twice with PBS and finally resuspended in PBS at 1 × 108/mL. For the resuspended parasites, inoculation of 1× 107 promastigotes per 100 μL of PBS was immediately used for infection into the hamsters intracardially. After 30 day postinfection of established Syrian golden hamster model, hamsters (n = 5 in each group) were intraperitonealy dosed 1 mg/kg with AmB-L-Psome, Ambisome, Fungizone and formulation without drug for 5 consecutive days into each hamster. Treated animals were sacrificed one week after treatment and compared with the infected untreated control group (means ± SD) (n = 5). The splenic dab smear of all performed animals was monitored microscopically using Giemsa-stained imprints, in which parasite burdens were measured by counting the number of amastigotes per 100 macrophage nuclei. The percentage of inhibition (PI) was calculated using the formula

Ambisome) was kept in a dialysis bag, sealed and placed in 200 mL of release medium under continuous shaking at 100 rpm at 37 °C. A definite volume (1 mL) of the release medium was withdrawn at regular time intervals (30 min and 1, 2, 3, 4, 6, 8, 12, 24, 48, 72 h) and replenished with an equal volume of fresh medium. The amount of the AmB in the collected samples was analyzed using the HPLC method as described previously.17 AmB Release Kinetics. AmB release kinetics of various formulations were determined using various dissolution models as given in Table S1 in the Supporting Information. Release profile data were processed and plotted according to the equations of different models, and regression analysis was carried out. Apart from the analysis of complete profile, first 24 h release data was also analyzed. The criterion for selecting the most appropriate model was based on best goodness-of-fit (R2 values). Slope of the plots was release rate constants for particular model, and these values were also utilized to describe the mechanism. Stability Study of Optimized AmB-L-Psome. Stability study of AmB formulations (AmB-L-Psome and commercial Ambisome and Fungizone) was performed by measuring size using Zetasizer, encapsulation efficiency and drug release from formulations using the HPLC method at different time points (1, 7, 15, 30, 90 and 180 days) at 4, 25 and 40 °C in PBS. In addition, stability of formulations was also evaluated after incubation with 10 vol % BSA and 10 vol % plasma (separated from Wistar rat blood) at RT under gentle stirring at a concentration of 1 mg/mL. All the measurements were performed in triplicate. Toxicity Assay and AmB Molecular Organization in the Formulations. Molecular aggregation state of AmB was identified by UV−visible spectroscopy (UV-1700 pharmaSpec, Shimadzu) and these states of the drug dictate the toxicity of the formulation.19 The UV−visible spectra of AmB-L-Psome and commercial Ambisome and Fungizone formulations were recorded at 10 μg/mL AmB equivalent concentration in distilled water using UV−visible spectrophotometer. The samples were scanned in the range of 200−500 nm at a rate of 405 nm/min and a scan step of 0.1 nm. We obtained spectra in which the final absorbance band was centered around 407 nm, and the ratio of the first to last peak in the absorbance spectrum served as an indicator of relative aggregation state. The hemolysis and J774A cell cytotoxicity assay was performed in 5 to 20 μg/mL AmB concentrations of AmB-LPsome, Fungizone and Ambisome, and equivalent blank LPsome formulations using methods reported previously.17 Subacute toxicity assay was performed in four mouse groups (n = 3), receiving AmB-L-Psome (5 mg/kg), Ambisome (5 mg/kg), Fungizone (1 mg/kg) and saline (control group) intravenously in a constant volume of 200 μL of sterile water for injection daily for 15 days. Twelve hours after the last treatment, mice were euthanized and blood was collected by cardiac puncture and centrifuged at 2000g followed by biochemical analysis. Transaminase activities, urea and creatinine were determined using an automatic Hitachi 912 apparatus (Roche Diagnostics Corporation, Indianapolis, IN, USA). The kidney tissue samples from control and treated animals were preserved in 10% formalin, embedded in paraffin, sectioned and stained with hematoxylin and eosin for histopathological examination. In order to assess the acute toxicity of AmB-L-Psome (5−20 mg/kg), Fungizone (1−5 mg/kg) and Ambisome (5−20 mg/ kg) were administered in increasing doses to mouse groups (n

PI = (PP − PT/PP) × 100

(4)

where PP is the number of amastigotes per 100 macrophage nuclei before treatment whereas PT is the number of amastigotes per 100 macrophage nuclei after treatment in spleen. In Vivo Immunomodulatory Assay. Quantitative real time PCR (qRT-PCR) was performed in triplicate to assess the expression of mRNAs for various cytokines (TNF-α, IL-12, IFN-γ, IL-4, IL-10 and TGF-β) and inducible NO synthase (iNOS) in splenic cell smear of differently treated L. donovani infected hamsters used in the above-mentioned in vivo assay. mRNA from splenocytes of experimental hamsters was isolated using Tri reagent (Invitrogen, USA) followed by cDNA synthesis using first strand cDNA synthesis kit (Fermentas, USA) as per the manufacturer’s protocol. qRT-PCR was conducted under these conditions: initial denaturation at 95 °C for 2 min followed by 40 cycles, each consisting of denaturation at 95 °C for 30s, annealing at 55 °C for 40 s, and extension at 954

dx.doi.org/10.1021/mp400603t | Mol. Pharmaceutics 2014, 11, 951−963

Molecular Pharmaceutics

Article

Figure 1. Effect of parameter variation on size and PDI of L-Psome (n = 3): (a) degree of SA substitution on GC and (b) concentration of copolymer (constant parameters: sonication time 60 s, amplitude 20% and copolymer:lipid::4:1), (c) copolymer to lipid ratio (constant parameters: 0.2% w/v GC-SA25%, sonication time 120 s and amplitude 20%), (d) type of lipid (constant parameters: 0.2% w/v GC-SA25% concentration, copolymer:lipid::4:1, sonication time 120 s and amplitude 20%), (e) concentration of lipid (constant parameters: 0.2% w/v GC-SA25% concentration, copolymer:lipid::4:1, sonication time 120 s and amplitude 20%), (f) DMAc volume, (g) sonication time (constant parameters: 0.2% w/v GC-SA25% concentration, 0.2% w/v lipid concentration, copolymer:lipid::4:1 and amplitude 20%), (h) amplitude of sonication (constant parameters: 0.2% w/v GC-SA25% concentration, 0.2% w/v lipid concentration, copolymer:lipid::4:1 and sonication time 120 s) and (i) homogenization time (constant parameters: homogenization speed 10,000 rpm, 0.2% w/v GC- SA25%, 0.2% w/v cholesterol, copolymer:lipid::4:1 and volume of DMAc 0.1 mL).

72 °C for 40 s per cycle using the iQ5 multicolor real-time PCR system (Bio-Rad, USA). cDNAs from infected hamsters were used as comparator samples. Quantification of the PCR signals was performed by comparing the cycle threshold (CT) value of the gene of interest with that of hypoxanthine phosphoribosyltransferase (HPRT), the reference genes. Values are expressed as fold change (FC) of mRNA relative to those in unstimulated cells. Statistical Analysis. All results are presented as mean ± standard deviation (SD) of three independent measurements. Statistical significance of differences was analyzed by one-way analysis of variance (ANOVA), and p value of 3-fold) in LPsome compared to DMPC, due to the fact that steroidal hydrophobic head of Chol provides stronger AmB intercalation than DMPC.33 The VF3 formulation with the highest EE (89.6 ± 1.25%) was found to be optimum for delivering therapeutically active dose (Table 2). The reason behind higher drug loading is the AmB intercalation between hydrophobic core and lipid monolayer at the interface of the amphiphilic GC shell and SA.34 Additionally, the higher wall thickness of L-Psome (15.4 nm) is anticipated to provide extra space for AmB residence.

Figure 3. In vitro drug release study of different AmB formulations (AmB-L-Psome, Ambisome and Fungizone) in PBS with 2% w/v sodium deoxycholate (SDC), pH 7.4 at different time intervals.

The AmB release profile was analyzed using various kinetic models to know the release mechanism.35,36 Modeling of release profile was carried out, and results are represented in Table S3 in the Supporting Information, where R2 and K are correlation coefficient and release rate constant, respectively, for particular model. AmB release from Fungizone is believed to occur by diffusion mechanism as it has a matrix type system (micellar formulation). As per expectation, Fungizone had maximum R2 in the case of the Korsmeyer−Peppas model as well as in the Higuchi model. Ambisome shows release in zero order fashion, and n values near to 1 in the Korsmeyer−Peppas model also signify the same. Weibull is the best model to understand release pattern from AmB-L-Psome, which seems to be affected by more than one parameter for complete as well as 24 h release. No release profile was found to fit in the Hopfenberg, Baker−Lonsdale and Hixson−Crowell models as the former describes release from eroding system and the latter is for a porous matrix system and none of the systems are believed to have either characteristic.37 In Vitro Stability Study. Time and temperature dependent stability of AmB formulations was monitored on the basis of variation in vesicle size and encapsulation efficiency of AmB in formulations (Figure 4a,b) and drug release from formulations (Figure S4 in the Supporting Information). AmB-L-Psome has shown negligible increase in size and drug release and insignificant changes in encapsulation efficiency of AmB at 25 957

dx.doi.org/10.1021/mp400603t | Mol. Pharmaceutics 2014, 11, 951−963

Molecular Pharmaceutics

Article

Figure 4. Stability study of AmB-L-Psome, Ambisome and Fungizone after incubation (a) for different times at RT in PBS, (b) for 180 days at various temperatures (4 °C, 25 °C, 40 °C) in PBS, (c) for different times with 10% BSA at RT and (d) for different times with 10% rat plasma at RT, showing change in size and encapsulation efficiency of formulation (n = 3).

°C after six months, while maximum change was observed in Ambisome, whereas Fungizone was not much affected during six months storage period. The polymeric shell made up of GC provides tremendous stability to AmB-L-Psome formulation compared to Ambisome. The same results were observed for stability study during formulation storage at various temperatures for 180 days. Since in vivo drug carriers’ stability in biological fluid is an important criterion for their use in animal models, change in AmB-L-Psome size and drug content in the presence of 10% BSA solution and 10% rat plasma solution was monitored over time and compared to that of Ambisome and Fungizone. As shown in Figure 4c,d, the AmB-L-Psome was stable while a slight increase in size and insignificant increase in drug content were observed in the case of Fungizone. In contrast, Ambisome had a dramatic increase in size from 110 to 290−320 nm within 20 min of incubation in BSA and rat plasma while drug content of Ambisome was changed radically. Consistent with the previous reports, we observed that the presence of the GC shell prevented protein adsorption on the surface of L-Psome and resulted in reduction of its biofouling as similar to hydrophilic PEG nanoparticles.38 Furthermore, stearate hydrophobic domain of copolymer also imparts the stability to L-Psome necessary for a vehicle.7 Molecular Organization of AmB in Formulations and Assessment of Its Toxicities. AmB is known to assemble in various forms at the molecular level, viz., monomeric, dimeric

and multimeric, or these forms can coexist. It is reported that in such aggregated state (dimeric or multimeric), porelike structures, the polar mycosamine head groups are exposed at two opposite sides. This can be accountable for membrane binding and trans-membrane localization of AmB aggregated structures with regard to the cholesterol-containing lipid bilayer membranes. Since only aggregates can form ion channels in membranes, primarily responsible for toxicity toward mammalian cells,3,39 and the AmB aggregate generation depends on the processing step during the development. The molecular states of AmB in tested formulation are shown in Figure 5i. The spectral modifications induced by aggregation may be represented by the ratio of the first (346 nm) to the fourth (408 nm) peaks, I/IV (A346/A408), indicative of the aggregated/ monomeric ratio which enables analysis of formation of molecular forms. Therefore, the degree of aggregation of AmB may be easily monitored by using this ratio.40 It can be observed in Figure 5i.a that Fungizone shows the peaks typically at 346, 362, 384 and 408 nm and the aggregated/ monomeric ratio value was >2, which indicates the presence of aggregated forms of AmB, particularly dimeric, consistent to previous reports.41 On the other hand, the A346/A408 ratio for Ambisome and formulated AmB-L-Psome was less than 2 (about 0.25) (Figure 5i.b,c), indicating the presence of monomeric AmB. The SA domain prevents self-association of monomeric AmB molecules due to strong interaction among 958

dx.doi.org/10.1021/mp400603t | Mol. Pharmaceutics 2014, 11, 951−963

Molecular Pharmaceutics

Article

Figure 5. (i) Molecular organization of AmB in different formulations, (a) Fungizone, (b) Ambisome and (c) AmB-L-Psome (10 μg/mL), from UV−visible spectroscopy. (ii) The effect of Fungizone, Ambisome, AmB-L-psome and L-Psome equivalent to amphotericin B concentration of 5, 10 and 20 μg/mL on (a) RBCs collected from Wistar rat’s blood and (b) J774A macrophage cell line (n = 3). Serum biochemical analysis of (c) aspartate aminotransferase (ASAT), alanine aminotransferase (ALAT), serum creatinine and blood urea of mice that received saline (control group), Fungizone (1 mg/kg), Ambisome (5 mg/kg), AmB-L-Psome (5 mg/kg) and L-Psome intravenously in a constant volume of 200 μL of sterile water for injection daily for 15 days (n = 3). (d) Percent mortality rate of nine mouse group (n = 6) treated with Fungizone (1, 2, 5 mg/kg), Ambisome (5, 10, 20 mg/kg) and AmB-L-Psome (5, 10, 20 mg/kg) through intraperitoneal route. (iii) Histopathology of kidney from treated mice after iv dose of (a) control group (normal saline), (b) Ambisome group (5 mg of AmB/kg), (c) Fungizone group (1 mg of AmB/kg) and (d) AmB-L-Psome group (5 mg of AmB/kg).

GC-SA copolymer and AmB, thus reducing the aggregated forms.7 Moreover, it can also be observed that absorbance of AmB-L-Psome is much less than the other two formulations, which indicates that AmB is well intercalated in the L-Psome wall, indicating that the formulation under study is anticipated to show less toxicity. The rank order of hemolysis (Figure 5ii.a) and cytotoxicity (Figure 5ii.b) of the AmB-formulations was Fungizone > Ambisome > AmB-L-Psome. As shown in Figure 5ii.a the hemolysis induced by Fungizone was higher even at a minimum concentration used in the experiment as compared to others. Other two formulations of AmB, i.e., AmB-L-Psome (P < 0.05,

compared to Fungizone) and Ambisome, have shown negligible hemolysis, possibly due to the presence of low concentrations of dimeric and multimeric aggregated forms of AmB, which are mainly responsible for hemolysis.42 Furthermore, Figure 5ii.b demonstrated that AmB-L-Psome formulation is less cytotoxic (higher % cell viability) compared to Fungizone (p < 0.05), while slightly less toxic compared to Ambisome owing to the existence of monomeric form of AmB. This is in agreement with previous results showing that the aggregated form of AmB, as in Fungizone, is more toxic against human erythrocytes and other cultured mammalian cells than the monomeric form of the drug. 42 These findings suggest that the L-Psome 959

dx.doi.org/10.1021/mp400603t | Mol. Pharmaceutics 2014, 11, 951−963

Molecular Pharmaceutics

Article

formulation under study is hemocompatible and may show minimum hemolysis in vivo and be safe against J774A cells. Biochemical analysis revealed that Fungizone and Ambisome showed elevated levels of serum aspartate aminotransferase (ASAT) and alanine aminotransferase (ALAT) (Figure 5ii.c), responsible for hepatotoxicity.4 This data complies with previous reports which showed that the composition and proportion of the lipid associated with AmB (Ambisome)4 affects toxicity. In contrast, administration of AmB-L-Psome in mice did not show any significant increase in the serum ASAT and ALAT levels. The Fungizone treated mice had significant increase (>3-fold) (p < 0.05) in the level of serum creatinine and blood urea over control, while Ambisome and AmB-LPsome had comparable values in renal function parameters as shown in Figure 5ii.c. In accordance with serum creatinine results, the histopathological analysis of kidney tissues revealed a normal pattern in all treated groups (Figure 5iii.a,b,d) except the Fungizone group, which showed patchy tubular epithelial necrosis that varied in extent from focal to extensive (Figure 5iii.c). Further, mice showed relatively more tolerance to the AmB at high tolerated dose (20 mg/kg) when injected with AmB-L-Psome than that of Ambisome, while 100% mortality was observed in mice treated with Fungizone only at 5 mg/kg dose (Figure 5ii.d), preventing further escalation of the dose. It is evident that low toxicity of AmB-L-Psome formulation observed toward erythrocytes, macrophage cells and kidney tissues and reduction of mortality rate in mice was due to slow and sustained release of AmB from core shell type of selfassembly of L-Psome.43 The low toxicity of AmB-L-Psome in biochemical analysis was further supported by stability of the AmB-L-Psome, as core shell self-assembly cannot rupture and dissociate AmB freely in biological fluids. The AmB-L-Psome was safe with diminished nephrotoxicity even at higher doses because of the presence of the monomeric form of AmB, concordant with previous lipid-complexed AmB formulation studies.44 Assessment of Antileishmanial and Immunomodulatory Efficacy. Results of in vitro amastigote inhibitory activity are presented in the form of dose response curve (Figure 6a) and in terms of inhibitory concentration (Figure 6b) as well. The rank order of antileishmanial efficacy of the four formulations was AmB-L-Psome > Ambisome > Fungizone > L-Psome. These results supported the output from in vivo studies in golden hamsters in which the inhibition of L. donovani infection was significantly greater with AmB-L-Psome (66.46 ± 2.08% inhibition) (p < 0.05) compared to Fungizone (56.54 ± 3.91% inhibition), whereas 62.37 ± 3.13% parasite inhibition was observed with Ambisome, while the blank LPsome had 4.35 ± 0.64% parasite inhibition (Figure 6c). The marked improvement of antileishmanial efficacy of AmB-LPsome over Ambisome and Fungizone was supported by a surge in iNOS, IFN-γ, TNF-α and IL-12 mRNA levels (Figure 7b,c) and downregulation of the Th-2 cytokine (IL-4, IL-10 and TGF-β mRNA) level (Figure 7a) in the treated hamsters, as protection against Leishmania is associated with Th2-to-Th1 switching for complete parasite clearance. Chitosan and its derivatives have been shown to induce production of proinflammatory cytokines and inducible nitric oxide synthase (iNOS) from macrophages.45,46 The results in this study demonstrated that hydrophobically modified glycol chitosan (GC-SA) component of L-Psome formulation induced significant elevated release of the TNF-α, IFN-γ and IL-12 cytokine from splenic macrophages, ensuring Th1-mediated

Figure 6. (a) In vitro dose−response curve of AmB-L-Psome, LPsome, Fungizone and Ambisome with different concentrations of formulations against L. donovani amastigote infected macrophages observed after 48 h of incubation (n = 3), (b) In vitro antileishmanial activity (IC50 and IC90) of AmB-L-Psome, L-Psome, Fungizone and Ambisome in L. donovani amastigote infected macrophages observed after 48 h of incubation (n = 3). (c) In vivo antileishmanial activity of AmB-L-Psome, L-Psome, Fungizone and Ambisome (p < 0.05) in the established Syrian golden hamster model infected with L. donovani amastigotes at an intraperitoneal dose of 1 mg of AmB/kg body weight of hamster (n = 5). The mean parasite burden in the spleen of untreated, infected control animals was 454 ± 38 amastigotes per 100 cell nuclei of macrophages (n = 5) (* p < 0.05, ** p < 0.01).

protection. These findings were supported by previous reports that show elevated release of TNF-α by hydrophobically modified glycol chitosan (HGC) based nanoparticles.46 Conversely, results showed GC-SA25% based L-Psome mediated favorable decrease in Th-2 cytokine gene expression, which are potent inhibitors of macrophage activation and killing of Leishmania parasites.47 Additionally, the commercial formulation Ambisome contains specific phospholipids which are not cost-effective and makes treatment of leishmaniasis very costly,48 whereas AmBL-Psome is simply composed of polymer and cholesterol that are relatively more economical, and the process thereof is simple and reproducible. Since AmB-L-Psome has shown greater stability, low toxicity and improved efficacy with low cost as compared to commercial formulation, it opens a new era of polymer−lipid based drug delivery systems in Leishmania chemotherapy.

■

CONCLUSIONS A novel type of L-Psome has been fabricated with great scalability. This carrier system is suitable for delivery of a wide range of drugs or combination (both hydrophilic and 960

dx.doi.org/10.1021/mp400603t | Mol. Pharmaceutics 2014, 11, 951−963

Molecular Pharmaceutics

Article

Figure 7. In vivo immunomodulatory responses regarding splenic iNOS and cytokine (TNF-α, IL-12, IFN-γ, IL-4, IL-10 and TGF-β) mRNA expression in infected control and treated (AmB-L-Psome, L-Psome, Fungizone and Ambisome) Syrian golden hamsters by quantitative RT-PCR. Analysis showing the relative fold changes (FC) of iNOS and cytokine expression level ± SD (n = 5 hamsters/expression of mRNA) in comparison to reference gene (HPRT).

Notes

hydrophobic) at a time in inner hydrophobic polymeric (SA) core and outer hydrophilic polymeric (GC) shell of L-Psome formulation. The L-Psome vesicles were spherical in morphology with a unimodal size distribution and able to provide high drug payload with sustained release of drug. The AmB-L-Psome proved to be a biologically safe and stable delivery system with improved efficacy compared to commercial formulations. LPsome bearing AmB formulations can be an alternative low cost treatment for better management of leishmaniasis with immunomodulatory components advantageous for eradication of Leishmania parasites.

■

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS P.K.G. thankfully acknowledges CSIR, New Delhi, India, for awarding fellowship. Authors are thankful to Electron Microscope Facility at Department of Anatomy, All India Institute of Medical Sciences (AIIMS, New Delhi, India), for providing electron microscope analysis. The authors are also thankful to CSIR for funding under network project BIOCERAM (ESC 0103). This is CSIR-CDRI communication no. 8612.

■

ASSOCIATED CONTENT

S Supporting Information *

The synthetic scheme for glycol chitosan-stearic acid (GC-SA) copolymer preparation and its characterization by IR and NMR spectroscopy. The drug release of AmB-L-Psome, Ambisome and Fungizone at different temperatures after 180 days storage in PBS is presented in figure. The degree of substitution of amino group of GC by SA per mole calculated from NMR spectroscopy given in table at different molar ratios of GC and SA. The different release models for study of release kinetics of various formulations and their equations also provided. This material is available free of charge via the Internet at http:// pubs.acs.org.

■

REFERENCES

(1) Dea-Ayuela, M. A.; Rama-Iniguez, S.; Sanchez-Brunete, J. A.; Torrado, J. J.; Alunda, J. M.; Bolas-Fernandez, F. Anti-leishmanial activity of a new formulation of amphotericin B. Trop. Med. Int. Health 2004, 9, 981−990. (2) Milhaud, J.; Ponsinet, V.; Takashi, M.; Michels, B. Interactions of the drug amphotericin B with phospholipid membranes containing or not ergosterol: new insight into the role of ergosterol. Biochim. Biophys. Acta 2002, 1558, 95−108. (3) Wasko, P.; Luchowski, R.; Tutaj, K.; Grudzinski, W.; Adamkiewicz, P.; Gruszecki, W. I. Toward understanding of toxic side effects of a polyene antibiotic amphotericin B: fluorescence spectroscopy reveals widespread formation of the specific supramolecular structures of the drug. Mol. Pharmaceutics 2012, 9, 1511− 1520. (4) Larabi, M.; Pages, N.; Pons, F.; Appel, M.; Gulik, A.; Schlatter, J.; Bouvet, S.; Barratt, G. Study of the toxicity of a new lipid complex formulation of amphotericin B. J. Antimicrob. Chemother. 2004, 53, 81−88. (5) Uchegbu, I. F.; Sadiq, L.; Arastoo, M.; Gray, A. I.; Wang, W.; Waigh, R. D.; Schatzlein, A. G. Quaternary ammonium palmitoyl

AUTHOR INFORMATION

Corresponding Author

*Tel: +91 522 2612411-18. Fax: +91 522 2623405. E-mail: [email protected]; [email protected]. 961

dx.doi.org/10.1021/mp400603t | Mol. Pharmaceutics 2014, 11, 951−963

Molecular Pharmaceutics

Article

glycol chitosan–a new polysoap for drug delivery. Int. J. Pharm. 2001, 224, 185−199. (6) Cho, Y. W.; Park, S. A.; Han, T. H.; Son, D. H.; Park, J. S.; Oh, S. J.; Moon, D. H.; Cho, K. J.; Ahn, C. H.; Byun, Y.; Kim, I. S.; Kwon, I. C.; Kim, S. Y. In vivo tumor targeting and radionuclide imaging with self-assembled nanoparticles: mechanisms, key factors, and their implications. Biomaterials 2007, 28, 1236−1247. (7) Adams, M. L.; Kwon, G. S. Relative aggregation state and hemolytic activity of amphotericin B encapsulated by poly(ethylene oxide)-block-poly(N-hexyl-L-aspartamide)-acyl conjugate micelles: effects of acyl chain length. J. Controlled Release 2003, 87, 23−32. (8) Yoshioka, T.; Skalko, N.; Gursel, M.; Gregoriadis, G.; Florence, A. T. A non-ionic surfactant vesicle-in-water-in-oil (v/w/o) system: potential uses in drug and vaccine delivery. J. Drug Targeting 1995, 2, 533−539. (9) Nam, H. Y.; Kwon, S. M.; Chung, H.; Lee, S. Y.; Kwon, S. H.; Jeon, H.; Kim, Y.; Park, J. H.; Kim, J.; Her, S.; Oh, Y. K.; Kwon, I. C.; Kim, K.; Jeong, S. Y. Cellular uptake mechanism and intracellular fate of hydrophobically modified glycol chitosan nanoparticles. J. Controlled Release 2009, 135, 259−267. (10) Yoo, H. S.; Lee, J. E.; Chung, H.; Kwon, I. C.; Jeong, S. Y. Selfassembled nanoparticles containing hydrophobically modified glycol chitosan for gene delivery. J. Controlled Release 2005, 103, 235−243. (11) Park, J. H.; Kwon, S.; Nam, J. O.; Park, R. W.; Chung, H.; Seo, S. B.; Kim, I. S.; Kwon, I. C.; Jeong, S. Y. Self-assembled nanoparticles based on glycol chitosan bearing 5beta-cholanic acid for RGD peptide delivery. J. Controlled Release 2004, 95, 579−588. (12) Kim, J. H.; Kim, Y. S.; Kim, S.; Park, J. H.; Kim, K.; Choi, K.; Chung, H.; Jeong, S. Y.; Park, R. W.; Kim, I. S.; Kwon, I. C. Hydrophobically modified glycol chitosan nanoparticles as carriers for paclitaxel. J. Controlled Release 2006, 111, 228−234. (13) Liang, X. F.; Wang, H. J.; Luo, H.; Tian, H.; Zhang, B. B.; Hao, L. J.; Teng, J. I.; Chang, J. Characterization of novel multifunctional cationic polymeric liposomes formed from octadecyl quaternized carboxymethyl chitosan/cholesterol and drug encapsulation. Langmuir 2008, 24, 7147−7153. (14) Singodia, D.; Verma, A.; Verma, R. K.; Mishra, P. R. Investigations into an alternate approach to target mannose receptors on macrophages using 4-sulfated N-acetyl galactosamine more efficiently in comparison with mannose-decorated liposomes: an application in drug delivery. Nanomedicine 2012, 8, 468−477. (15) Kansal, S.; Tandon, R.; Dwivedi, P.; Misra, P.; Verma, P. R.; Dube, A.; Mishra, P. R. Development of nanocapsules bearing doxorubicin for macrophage targeting through the phosphatidylserine ligand: a system for intervention in visceral leishmaniasis. J. Antimicrob. Chemother. 2012, 67, 2650−2660. (16) Nakajima, N.; Ikada, Y. Mechanism of amide formation by carbodiimide for bioconjugation in aqueous media. Bioconjugate Chem. 1995, 6, 123−130. (17) Asthana, S.; Jaiswal, A. K.; Gupta, P. K.; Pawar, V. K.; Dube, A.; Chourasia, M. K. Immunoadjuvant chemotherapy of visceral leishmaniasis in hamsters using amphotericin B-encapsulated nanoemulsion template-based chitosan nanocapsules. Antimicrob. Agents Chemother. 2013, 57, 1714−1722. (18) Choi, K. C.; Bang, J. Y.; Kim, P. I.; Kim, C.; Song, C. E. Amphotericin B-incorporated polymeric micelles composed of poly(d,l-lactide-co-glycolide)/dextran graft copolymer. Int. J. Pharm. 2008, 355, 224−230. (19) Espada, R.; Valdespina, S.; Alfonso, C.; Rivas, G.; Ballesteros, M. P.; Torrado, J. J. Effect of aggregation state on the toxicity of different amphotericin B preparations. Int. J. Pharm. 2008, 361, 64−69. (20) Mookerjee Basu, J.; Mookerjee, A.; Banerjee, R.; Saha, M.; Singh, S.; Naskar, K.; Tripathy, G.; Sinha, P. K.; Pandey, K.; Sundar, S.; Bimal, S.; Das, P. K.; Choudhuri, S. K.; Roy, S. Inhibition of ABC transporters abolishes antimony resistance in Leishmania Infection. Antimicrob. Agents Chemother. 2008, 52, 1080−1093. (21) Gupta, S.; Dube, A.; Vyas, S. P. Antileishmanial efficacy of amphotericin B bearing emulsomes against experimental visceral leishmaniasis. J. Drug Targeting 2007, 15, 437−444.

(22) Park, K.; Kim, J. H.; Nam, Y. S.; Lee, S.; Nam, H. Y.; Kim, K.; Park, J. H.; Kim, I. S.; Choi, K.; Kim, S. Y.; Kwon, I. C. Effect of polymer molecular weight on the tumor targeting characteristics of self-assembled glycol chitosan nanoparticles. J. Controlled Release 2007, 122, 305−314. (23) Felix, L.; Hernandez, J.; Arguelles-Monal, W. M.; Goycoolea, F. M. Kinetics of gelation and thermal sensitivity of N-isobutyryl chitosan hydrogels. Biomacromolecules 2005, 6, 2408−2415. (24) Yuan, H.; Lu, L. J.; Du, Y. Z.; Hu, F. Q. Stearic acid-g-chitosan polymeric micelle for oral drug delivery: in vitro transport and in vivo absorption. Mol. Pharmaceutics 2011, 8, 225−238. (25) Park, K.; Kim, K.; Kwon, I. C.; Kim, S. K.; Lee, S.; Lee, D. Y.; Byun, Y. Preparation and characterization of self-assembled nanoparticles of heparin-deoxycholic acid conjugates. Langmuir 2004, 20, 11726−11731. (26) Jiang, G. B.; Quan, D.; Liao, K.; Wang, H. Novel polymer micelles prepared from chitosan grafted hydrophobic palmitoyl groups for drug delivery. Mol. Pharmaceutics 2006, 3, 152−160. (27) Liu, C. G.; Desai, K. G.; Chen, X. G.; Park, H. J. Linolenic acidmodified chitosan for formation of self-assembled nanoparticles. J. Agric. Food Chem. 2005, 53, 437−441. (28) Gaumet, M.; Vargas, A.; Gurny, R.; Delie, F. Nanoparticles for drug delivery: the need for precision in reporting particle size parameters. Eur. J. Pharm. Biopharm. 2008, 69, 1−9. (29) Ferdous, A. J.; Stembridge, N. Y.; Singh, M. Role of monensin PLGA polymer nanoparticles and liposomes as potentiator of ricin A immunotoxins in vitro. J. Controlled Release 1998, 50, 71−78. (30) Jain, J. P.; Kumar, N. Self assembly of amphiphilic (PEG)(3)PLA copolymer as polymersomes: preparation, characterization, and their evaluation as drug carrier. Biomacromolecules 2010, 11, 1027− 1035. (31) Kucerka, N.; Kiselev, M. A.; Balgavy, P. Determination of bilayer thickness and lipid surface area in unilamellar dimyristoylphosphatidylcholine vesicles from small-angle neutron scattering curves: a comparison of evaluation methods. Eur. Biophys. J. 2004, 33, 328−334. (32) Kilicarslan, M.; Baykara, T. The effect of the drug/polymer ratio on the properties of the verapamil HCl loaded microspheres. Int. J. Pharm. 2003, 252, 99−109. (33) Kucerka, N.; Marquardt, D.; Harroun, T. A.; Nieh, M. P.; Wassall, S. R.; de Jong, D. H.; Schafer, L. V.; Marrink, S. J.; Katsaras, J. Cholesterol in bilayers with PUFA chains: doping with DMPC or POPC results in sterol reorientation and membrane-domain formation. Biochemistry 2010, 49, 7485−7493. (34) Adams, M. L.; Andes, D. R.; Kwon, G. S. Amphotericin B encapsulated in micelles based on poly(ethylene oxide)-block-poly(Lamino acid) derivatives exerts reduced in vitro hemolysis but maintains potent in vivo antifungal activity. Biomacromolecules 2003, 4, 750−757. (35) Berry, M. R.; Likar, M. D. Statistical assessment of dissolution and drug release profile similarity using a model-dependent approach. J. Pharm. Biomed. Anal. 2007, 45, 194−200. (36) Costa, P.; Sousa Lobo, J. M. Modeling and comparison of dissolution profiles. Eur. J. Pharm. Sci. 2001, 13, 123−133. (37) Dash, S.; Murthy, P. N.; Nath, L.; Chowdhury, P. Kinetic modeling on drug release from controlled drug delivery systems. Acta Pol. Pharm. 2010, 67, 217−223. (38) Thevenot, J.; Troutier, A. L.; David, L.; Delair, T.; Ladaviere, C. Steric stabilization of lipid/polymer particle assemblies by poly(ethylene glycol)-lipids. Biomacromolecules 2007, 8, 3651−3660. (39) Huang, W.; Zhang, Z.; Han, X.; Tang, J.; Wang, J.; Dong, S.; Wang, E. Ion channel behavior of amphotericin B in sterol-free and cholesterol- or ergosterol-containing supported phosphatidylcholine bilayer model membranes investigated by electrochemistry and spectroscopy. Biophys. J. 2002, 83, 3245−3255. (40) Barwicz, J.; Christian, S.; Gruda, I. Effects of the aggregation state of amphotericin B on its toxicity to mice. Antimicrob. Agents Chemother. 1992, 36, 2310−2315. (41) Charvalos, E.; Tzatzarakis, M. N.; Van Bambeke, F.; Tulkens, P. M.; Tsatsakis, A. M.; Tzanakakis, G. N.; Mingeot-Leclercq, M. P. Water-soluble amphotericin B-polyvinylpyrrolidone complexes with 962

dx.doi.org/10.1021/mp400603t | Mol. Pharmaceutics 2014, 11, 951−963

Molecular Pharmaceutics

Article

maintained antifungal activity against Candida spp. and Aspergillus spp. and reduced haemolytic and cytotoxic effects. J. Antimicrob. Chemother. 2006, 57, 236−244. (42) Legrand, P.; Romero, E. A.; Cohen, B. E.; Bolard, J. Effects of aggregation and solvent on the toxicity of amphotericin B to human erythrocytes. Antimicrob. Agents Chemother. 1992, 36, 2518−2522. (43) Adams, M. L.; Kwon, G. S. The effects of acyl chain length on the micelle properties of poly(ethylene oxide)-block-poly(N-hexyl-Laspartamide)-acyl conjugates. J. Biomater. Sci., Polym. Ed. 2002, 13, 991−1006. (44) Yardley, V.; Croft, S. L. A comparison of the activities of three amphotericin B lipid formulations against experimental visceral and cutaneous leishmaniasis. Int. J. Antimicrob. Agents 2000, 13, 243−248. (45) Canali, M. M.; Porporatto, C.; Aoki, M. P.; Bianco, I. D.; Correa, S. G. Signals elicited at the intestinal epithelium upon chitosan feeding contribute to immunomodulatory activity and biocompatibility of the polysaccharide. Vaccine 2010, 28, 5718−5724. (46) Choi, M.; Cho, M.; Han, B. S.; Hong, J.; Jeong, J.; Park, S.; Cho, M. H.; Kim, K.; Cho, W. S. Chitosan nanoparticles show rapid extrapulmonary tissue distribution and excretion with mild pulmonary inflammation to mice. Toxicol. Lett. 2010, 199, 144−152. (47) Melby, P. C.; Tryon, V. V.; Chandrasekar, B.; Freeman, G. L. Cloning of Syrian hamster (Mesocricetus auratus) cytokine cDNAs and analysis of cytokine mRNA expression in experimental visceral leishmaniasis. Infect. Immun. 1998, 66, 2135−2142. (48) Sundar, S.; Chakravarty, J.; Agarwal, D.; Rai, M.; Murray, H. W. Single-dose liposomal amphotericin B for visceral leishmaniasis in India. N. Engl. J. Med. 2010, 362, 504−512.

963

dx.doi.org/10.1021/mp400603t | Mol. Pharmaceutics 2014, 11, 951−963