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Contents lists available at ScienceDirect

International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac

Amphotericin-B entrapped lecithin/chitosan nanoparticles for prolonged ocular application

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Yashpal S. Chhonker a,e,1 , Yarra Durga Prasad a,1 , Hardik Chandasana a,e , Akhilesh Vishvkarma a , Kalyan Mitra b,e , Praveen K. Shukla c,d , Rabi S. Bhatta a,d,e,∗ a

Pharmacokinetics and Metabolism Division, CSIR-Central Drug Research Institute, Lucknow 226001, India Electron Microscopy Division, CSIR-Central Drug Research Institute, Lucknow 226001, India c Medical Mycology Division, CSIR-Central Drug Research Institute, Lucknow 226001, India d Department of Pharmaceutics, National Institute of Pharmaceutical Education and Research (NIPER), Raebareli 229010, India e Academy of Scientific and Innovative Research (AcSIR), Anusandhan Bhawan, Rafi Marg, New Delhi 110001, India

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a r t i c l e

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a b s t r a c t

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Article history: Received 30 July 2014 Received in revised form 1 October 2014 Accepted 6 October 2014 Available online xxx

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Keywords: Fungal keratitis Amphotericin-B Lecithin/chitosan nanoparticles

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1. Introduction

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Fungal keratitis is the major cause of vision loss worldwide. Amphotericin-B is considered as the drug of choice for fungal infections. However, its use in ophthalmic drug delivery is limited by the low precorneal residence at ocular surface as a result of blinking reflex, tear turnover and nasopharyngeal drainage. We report Amphotericin-B loaded lecithin/chitosan nanoparticles for prolonged ocular application. The prepared nanoparticles were in the size range of 161.9–230.5 nm, entrapment efficiency of 70–75%, theoretical drug loading of 5.71% with positive zeta potential of 26.6–38.3 mV. As demonstrated by antifungal susceptibility against Candida albicans and Aspergillus fumigatus, nanoparticles were more effective than marketed formulation. They exhibited pronounced mucoadhesive properties. In-vivo pharmacokinetic studies in New Zealand albino rabbit eyes indicated improved bioavailablity (∼2.04 fold) and precorneal residence time (∼3.36 fold) of nanoparticles prepared from low molecular weight of chitosan as compared with marketed formulation. © 2014 Published by Elsevier B.V.

Fungal Keratitis (FK), inflammation of the eye’s cornea, continues to pose a diagnostic and therapeutic challenge to clinicians. It is one of the most frequent causes of ocular morbidity worldwide and if neglected it may lead to permanent vision loss. The incidence of FK has increased over the past three decades perhaps due to the increased use of contact lenses, eye drops containing steroidal drugs, compromised corneal surface observed in case of diabetes and HIV positive cases [1–4]. According to the United States Centers for Disease Control and Prevention, 154 new FK cases were reported between 2005 and 2006, among them 94% cases were associated with contact lens wear and 34% cases required corneal transplantation. Although FK cases were reported all over the world [5–7], the infection is more prevalent in tropical, sub tropical climates and

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∗ Corresponding author at: Pharmacokinetics and Metabolism Division, CSIRCentral Drug Research Institute, Lucknow 226001, India. Tel.: +91 522 2772974; fax: +91 522 2771942x4853. E-mail addresses: [email protected], rabi [email protected] (R.S. Bhatta). 1 These authors contributed equally.

agriculture based nations like India, China and Ghana. The principal causative organisms include Aspergillus, Candida and Fusarium spp. [8–12]. Amphotericin-B (AmB) is a natural polyene antifungal antibiotic produced by the fungal strain Streptomyces nodosus. It has wide spectrum of activity against various fungi including the filamentous fungi Aspergillus spp. and yeasts such as Candida spp. with low incidence of resistance [13]. Currently AmB is available in lyophilized form as Fungizone, Abelcet and AmBisome for intravenous use. As an ‘off-labeled’ eye drop preparation (0.15–0.3%) it is being used as one of the first line therapies in the management of FK administered at every 30–60 min. Since crystalline AmB is insoluble in water, sodium deoxylcholate, a water soluble surfactant is incorporated in the Fungizone to increase the solubility of the AmB. But the drawbacks associated with Fungizone are painful instillation owing to the incorporation of surfactant deoxycholate and high dosing frequency leading to poor patient compliance. Literature survey revealed that several approaches were reported for the ocular delivery of AmB using collagen shields [14], intracameral injection [15–18], solubilised AmB using cyclodextrins [13], liposomal eye drops [19], nanoparticles mediated delivery [20,21], oral delivery through lipid based formulations

http://dx.doi.org/10.1016/j.ijbiomac.2014.10.014 0141-8130/© 2014 Published by Elsevier B.V.

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Table 1 Physico-chemical properties of L/C NPs with different molecular weight CS, each value represents mean ± S.D. (n = 3). Formulation

Particle size (d, nm)

PDI

ZP (mV)

EE (%)

Blank NPs I NPs I with AmB Blank NPs II NPs II with AmB Blank NPs III NPs III with AmB

230.5 ± 3.1 282.7 ± 5.2 201 ± 4.3 274.9 ± 3.1 128.9 ± 3.5 161.9 ± 1.3***

0.141 ± 0.032 0.238 ± 0.023 0.24 ± 0.019 0.221 ± 0.051 0.286 ± 0.025 0.189 ± 0.004

38.3 ± 1.23 37.8 ± 1.97 29.5 ± 3.4 30.25 ± 1.62 27.6 ± 2.52 26.6 ± 1.13**

– 74.6 ± 0.75 – 72.5 ± 0.52 – 70 ± 1.02

** ***

Significant (P < 0.05) difference as compared with NPs I. Significant (P < 0.05) difference as compared with NPs I.

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[22], lipidic emulsion [23]. Collagen shields are associated with limitations like lack of individual fit for patients, chances of expulsion and reduction in visual acuity. Cataracts were observed in some patients treated with intracameral injections [24]. Intravenous administration is the treatment of choice for invasive fungal infections, but this route may cause poor ocular availability and severe nephrotoxicity [25,26]. With simple eye drop solution it is not possible to maintain therapeutic concentration for prolonged time as it is easily cleared off by tear dilution and blinking reflex. Though, better alternative to Fungizone such as liposomal preparation AmBisome is available in the market cost limits its use in developing countries where health care resources are limited. However, there is a dearth of effective antifungal agents in the management of FK. The only FDA approved drug Natamycin is not available in many regions [27] and since FK is a vision threatening infection requires aggressive therapy, there is a clinical need to develop an ophthalmic formulation which is affordable as well as effective. In the search for novel drug delivery systems, colloidal carriers like biodegradable polymeric NPs have been extensively studied in the last two decades [28,29]. Recently, lecithin/chitosan (L/C) nanoparticles (NPs) received increased attention in the field of drug delivery [30–34]. Lecithin (Phosphatidylcholine) is the most abundant groups of phospholipids found in the cell membranes. As a lipophilic matrix it may render higher lipophilic drug loading and prolonged release of encapsulated drug. Chitosan (CS) is a natural polysaccharide widely used in bioadhesive drug delivery systems [35–37]. It is the only polymer that has cationic character among the biodegradable polymers that have a monograph in a pharmacopoeia [38]. Being cationic in nature, CS helps enhance the precorneal contact time of drug through interaction with negatively charged ocular mucosal surface thereby minimizing the ocular drainage. Both the lecithin and CS are known to be biocompatible and biodegradable polymers. Thus, the objective of the present study is to develop a biodegradable polymeric nanoparticulate delivery system having prolonged release as well as mucoadhesive properties in order to decrease the dosing frequency and improve patient compliance. Besides, the effect of molecular weight of CS on the performance of the delivery system was also reported.

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2. Materials and methods

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2.1. Materials

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AmB and natamycin (internal standard, IS for LC–MS/MS analysis) were kindly supplied as a gift samples by Cipla Pvt. Ltd., Mumbai, India. Lecithin (Phospholipon 50) was gifted by Lipoid, Ludwigshafen, Germany. Chitosan (low, medium and high molecular weight with viscosity values of 20 cPs, 200 cPs and 800 cPs, respectively) and mucin (type II, from porcine stomach) were obtained from Sigma, USA. Fungizone® (Nicholas Piramal India Ltd., India) was purchased from local pharmacy store. The Fungizone® powder was dissolved in sterile water for injection at a concentration of 0.15%, w/v of AmB. Calibrated glass capillaries (microcaps) of

10 ␮L were obtained from Dummond Scientific Co., USA. Oasis HLB solid phase cartridges (1 cc, 30 mg) were procured from Waters Corporation Milford, Massachusetts, USA. Sodium acetate and acetic acid AR were purchased from E Merck Ltd., Mumbai, India. HPLC grade methanol and acetonitrile were purchased from Sigma, USA. Ultrapure water (18.2 M cm) was obtained from Milli-Q PLUS PF water purification system. All other reagents were of analytical grade and obtained from standard commercial suppliers. 2.2. Preparation of NPs L/C NPs were prepared by ionic gelation method as described by Sonvico et al. [30]. In brief, AmB (0.2%, w/v) was dissolved in methanol:DMSO (1:1) and added to the methanolic solution of lecithin (2.5%, w/v). A solution of CS (1%, w/v) was prepared in 1% acetic acid and aliquots of this were added to 46 mL of ultrapure water in order to obtain different L/C ratios (10:1) in the prepared NPs (Table 1). NPs suspension (NPs I – prepared from high mol. wt CS; NPs II – prepared from medium mol. wt CS; NPs III – prepared from low mol. wt CS) was obtained by injection (needle size 0.45 mm × 13 mm/26G × 1/2) of AmB dissolved lecithin solution 4 mL into aqueous phase containing pluronic F-127 under moderate stirring at 1000 rpm with mild heating. Finally, the organic solvent was evaporated under reduced pressure (Buchi, Switzerland) at 58 ◦ C and the final volume of the aqueous suspension was adjusted to 10–20 mL. Blank NPs were prepared following the same procedure devoid of AmB. NPs were lyophilized by means of Maxi Dry Lyo (Heto Holten, Denmark) using glucose (5%, w/v) as a cryoprotectant. 2.3. HPLC method The Waters HPLC system, Milford USA consisted of a binary pump (model 1525), auto sampler (model 717 plus) and Dual  absorbance detector (model 2487). The chromatographic separation was performed by using a Spheri-5, Cyano column (30 mm × 4.6 mm, 5 ␮m). The system was analyzed in gradient flow with a mobile phase consisting of acetonitrile (solvent A): 10 mM sodium acetate (solvent B) buffer, (pH adjusted to 4.0 with acetic acid) at a flow rate of 1.0 mL/min. The mobile phase was prepared daily, filtered through 0.22 ␮m membrane filter (Millipore) and degassed ultrasonically (Powersonic 420) prior to use. The initial composition of 75% B till 5 min followed up with linear gradient as follows: 75% to 40% B in 8 min, 40% to 75% B in 15 min. The absorption wavelength was set to 408 nm for AmB and the injection volume was 20 ␮L. The total run time was 15 min and retention time was 10.8 min. Calibration curves were plotted as concentration of drugs versus peak area response. Data collection and analyses were performed by using Empower pro 2 software. 2.4. Physico-chemical characterization 2.4.1. Particle size and zeta potential NPs particle size distribution and zeta potentials were measured using photon correlation spectroscopy (Malvern Nano-ZS, UK). The

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measurements were performed at a scattering angle of 173◦ and temperature of 25 ◦ C using samples appropriately diluted with ultrapure water to minimize particle–particle interactions. The mean particle size was determined by using disposable polystyrene cuvette where as zeta potential was measured using a fixed-glass cell. All measurements were performed in triplicate. 2.4.2. Entrapment efficiency (EE) and drug loading (DL) Ten milligrams of lyophilized NP were dissolved in 1 mL of DMSO, a common solvent for both drug and polymer. This solution was centrifuged at 13,000 × g for 20 min and the supernatant was diluted with methanol. The amount of unentrapped AMB was calculated by using the HPLC method as described in previous section. All the measurements were performed in triplicate. The EE and DL were calculated using the following equations:

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EE %

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Total amount of drug − Amount of drug in supernatamt Total amount of drug used

× 100

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DL % =

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Total amount of drug − Amount of drug in supernatamt Total amount of formulation components

× 100

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2.4.3. NPs morphology Morphologic evaluation of the NP was performed using transmission electron microscopy (TEM; Philips CM-10, Eindhoven, The Netherlands). Samples of the nanoparticle suspension (5–10 ␮L) were dropped onto Formvar-coated copper grids (Plano GmbH, Wetzlar, Germany). After complete drying, the samples were stained using 2% w/v phosphotungstic acid. Digital Micrograph and Soft Imaging Viewer software (Olympus, Singapore) were used to perform the image capture and analysis. 2.4.4. FT-IR In order to disclose the structure of the L/C NPs, the interactions between the drug and polymers were studied by FT-IR using lyophilized NPs. The spectra were recorded by using the potassium bromide (KBr) pellet technique (PerkinElmer spectrophotometer version 10.03.06, USA). The scanning range was 4000–450 cm−1 . 2.4.5. In-vitro drug release The release of AmB from lecithin NPs and L/C NPs was assessed using a dialysis technique under sink conditions over a 10 h period. One mL of NPs suspension equivalent to 0.15% AmB (w/v) was enclosed in dialysis bag (cellulose membrane, mw cut-off 12400, Sigma) and incubated in 30 mL of phosphate buffered saline (PBS), pH 7.4 containing Tween 80 (1%, v/v) at 37 ◦ C under mild agitation in water bath. One mL of the drug releasing medium was removed at predetermined intervals and replaced with equal volume of fresh buffer to maintain sink conditions. The samples were filtered through a 0.22 ␮m membrane filter, appropriately diluted and analyzed for AmB content by using validated HPLC method (n = 3). 2.4.6. Mucoadhesive capacity Mucoadhesive studies were performed in order to obtain an insight into the mechanism of interaction between L/C NPs and

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mucin. A solution of mucin (0.1%, w/v) was prepared and mixed with appropriate volumes of L/C NPs. The resulting dispersion was incubated in a thermostatic oscillating water bath at 37 ◦ C. Two parameters, namely turbidity and zeta potential of the dispersion were taken into consideration for the analysis of mucoadhesive capacity. Samples were withdrawn at predetermined intervals and evaluated for changes in turbidity and zeta potential of the dispersion. The zeta potential of the NPs was measured at given time intervals by using aforementioned zetasizer. The zeta potential of the NPs aqueous dispersion was used as a reference. Theoretically, eventual changes in their zeta-potential values during incubation with mucin could be considered as an indication for their interaction. Turbidimetric analysis was performed by measuring the absorbance of the dispersion at 650 nm and compared with the turbidity of the native mucin by using Evolution 600 UV–visible spectrophotometer (Thermo Scientific, USA). The accurately weighed NPs (10 mg) were added to 10 mL aqueous mucin dispersion (0.1%, w/v) and stirred at 200 rpm. The turbidity of the dispersions was measured at certain time intervals and compared to the turbidity of the native mucin dispersion. The behavior of NPs was also examined in a mucin free aqueous dispersion under the same conditions. 2.4.7. In-vitro antifungal activity 2.4.7.1. Minimum inhibitory concentration. Antifungal susceptibility tests were performed against C. albicans procured from the Microbial Type Culture Collection and Gene Bank (MTCC 183) and A. fumigatus (Patient isolated) fungal strains. The antifungal activity was expressed as MIC90 indicating minimal inhibitory concentration at which 90% of the growth of fungi was inhibited. The concentration tested for both the standard AmB and NPs ranged from 16 ␮g/mL and below. The test was performed with 96-well flat bottom plates by serial dilution of AmB by adding known concentration of NPs suspension in RPMI growth medium with L-glutamine and MOPS-buffer, pH 7.0 (Sigma Chemical Co., St. Louis, Mo.). Cell suspensions of C. albicans and A. fumigatus were prepared in RPMI 1640 medium and adjusted to give final inoculums concentration of 1.0 × 103 –5.0 × 103 CFU/mL and 0.4 × 104 –5.0 × 104 CFU/mL, respectively. Each well was inoculated with 100 ␮L of fungus suspension to give the desired final drug concentration. The plates were incubated at 35 ◦ C for 24 h (C. albicans) or 48 h (A. fumigatus). The MICs were determined by measuring absorbance of the samples at 530 nm with microplate reader (SoftMax® Pro, USA). 2.4.7.2. Zone of inhibition. A layer of Sabouraud dextrose agar (20 mL) seeded with the test microorganism (0.2 mL) was allowed to solidify in the petri plate. Five ␮g disks were prepared by pipetting appropriate volumes of stock solutions of AmB NPs, Fungizone® and AmB powder in dimethyl sulfoxide (DMSO) onto sterile blank disks (Himedia Labs, India). The disks were dried in oven at 35 ◦ C for 5 min. These disks were placed on the solidified agar layer with the help of sterile forceps and kept at 4 ◦ C for 15 min. The plates were incubated at 35 ◦ C for 24 h (C. albicans) or 48 h (A. fumigatus). The diameter of the zone of inhibition was measured by an antibiotic zone finder. Readings were taken in triplicate. 2.5. Ocular irritation test The potential ocular irritancy and/or damaging effects of AmB NPs were evaluated according to modified Draize test [39]. Test substance (0.01 mL) was instilled directly onto the cornea of the right eye every 30 min for 6 h (12 treatments). Right eye served as control and was treated with distilled water. At the end of the treatment, six observations at 12 h intervals were carried out to

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Fig. 1. TEM micrographs of (A) Lecithin NPs and (B) L/C NPs.

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evaluate the ocular tissues. The congestion, swelling, discharge, and redness of the conjunctiva were graded on a scale from 0 to 3, 0 to 4, 0 to 3, and 0 to 3, respectively. Irritation and corneal opacity were graded on a scale from 0 to 4.

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The performance of AmB loaded L/C NPs (0.15%, w/v) after instillation in rabbits eye was evaluated and compared with marketed Fungizone® (0.15%, w/v). Animal studies (n = 3) were carried out as per the approval and guidelines of the local ethics committee on animal experimentation. New Zealand albino rabbits (male, weight range 2.5–3.0 kg) were used in these studies. Ad libitum access to food and water was given during the study. Briefly, 20 ␮L of each sample was instilled into the lower conjunctival sac of left eye. The eyelids were kept closed for 5 s post instillation to prevent the loss of the instilled solution. Tear samples (10 ␮L) were collected by using a small calibrated glass capillary (Dummond Scientific Co, USA.) placed near the eye. Samples were obtained at 5 min, 15 min, 30 min, 45 min, 60 min, 90 min, 120 min, 150 min, 180 min, 210 min, 240 min, 270 min and 300 min post dose. They were stored in micro-centrifuge tubes at −20 ◦ C until LC–MS/MS analysis. Quantitation was performed on a triple quadrupole mass spectrometer (API 4000, Applied Biosystems, MDS Sciex Toronto, Canada) employing electrospray ionization technique (ESI). The analysis was performed using Phenomenex, Luna 3u CN column (100 mm × 2 mm, 3 ␮m). The mobile phase composed of methanol and 3.5 mM ammonium acetate (90:10, v/v) at a flow rate of 0.3 mL/min. Multiple reaction monitoring (MRM) in negative ion mode was used for quantification of ion transitions at m/z 923.5/183.4 and 664.5/137.2 for AmB and IS, respectively [40].

of bias in residual error and mathematical model selection criteria including akaike’s information criterion. 2.9. Statistical data analysis Statistical data analysis was performed using the student’s t-test with P < 0.05 as the minimal level of significance.

2.7. Stability

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The physical stability of the lyophilized NPs was evaluated at 4 ± 2 ◦ C or 25 ± 2 ◦ C for a period of 90 days. Lyophilized NPs (50 mg) were stored in closed amber-coloured glass vials. At different time intervals, 10 mg of the formulation was withdrawn for particle size, zeta potential and EE measurements.

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Ocular pharmacokinetic parameters of AmB in tear were derived from tear concentration time profile using Phoenix WinNonlin software Ver 6.3 (Pharsight Corporation, Mountain view, USA). For compartmental modeling, several models were evaluated and the model which best fit the experimental data was selected on the basis of visual assessment of the predicted versus actual data, lack

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3. Results and discussion

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AmB loaded L/C NPs were prepared by injection of an alcoholic solution of lecithin into an aqueous CS solution. The size and surface charge of the obtained NPs were dependent on the molecular weight of CS. Table 1 illustrates the variation in the particle size, zeta potential and EE of the obtained NPs. With increasing molecular weight of CS, particle size, zeta potential and EE were increased. The increase in particle size is ascribed to the fact that longer molecular chains entangled together resulting in larger size. However, overall size distribution was not altered significantly (polydispersity index (PDI) less than 0.2) indicating the homogeneity of the prepared NPs. With increase in CS molecular weight surface charge was increased. This is because negatively charged lecithin NPs provide the binding force to the positively charged CS layer which transferred the zeta potential into positive. Fig. 1 represents the morphology and shape of NPs was confirmed by using TEM. The NPs were roughly spherical in shape and separated from each other. No indication of aggregation was observed among NPs possibly due to surface charge dependent steric hindrance. 3.2. FT-IR

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Fig. 2 depicts the FT-IR absorption bands of different formulations. As can be seen in Fig. 2A, the spectrum of AmB exhibits a strong stretching band around 3401 cm−1 characteristic of hydrogen bonded AmB molecules. In addition, absorption band at 1645 cm−1 represents C O stretching with a high frequency shoulder. Different molecular weight CS exhibited same spectra (Fig. 2C). In drug-excipients compatibility study of physical mixture of formulation components (Fig. 2D) and L/C NPs (Fig. 2E), the characteristic peak of AmB i.e., absorption band at 3401 cm−1 due to –OH is stretching still present indicating the compatibility between AmB and excipients used in the study. 3.3. In-vitro drug release The drug release from different formulations in the simulated ocular circumstances (35 ◦ C, pH 7.4) was studied. AmB release from

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Fig. 2. FT-IR spectra of (A) Amphotericin-B, (B) lecithin, (C) chitosan, (D) physical mixture and (E) NPs III.

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L/C (different molecular weight) NPs, each containing 0.15% AmB (w/v), was shown in Fig. 3. The release rate of all three L/C NPs showed a biphasic release pattern: one initial burst release followed by a prolonged release. At 1 h, the cumulative release percentage of AmB was 26.26%, 29.0% and 55% from NPs I, NPs II and NPs III, respectively. The initial burst release was ascribed to rapid desorption and diffusion of AmB molecules located at or close to the surface of the NPs. High percentage of drug release as in case of NPs III can be explained by the fact that smaller the particles size smaller

Fig. 3. In-vitro drug release profile of AmB in PBS (mean ± SD, n = 3).

the surface area thereby facilitating the drug release process. An initial burst release is beneficial in terms of antifungal activity as it helps achieve the therapeutic concentration of drug in minimal time period [34]. After the burst release phase, the rate of release fell as the dominant release mechanism was changed to drug diffusion through the lecithin matrix. The amount of AmB released at 10 h was 83%, 86% and 88% from NPs I, NPs II and NPs III, respectively.

3.4. Mucoadhesive capacity The eventual changes in absorbance and zeta potential values of L/C NPs upon incubation with 0.1% mucin dispersion were depicted in Figs. 4 and 5, respectively. The zeta potential of the NPs dropped out over the time. This could be explained by adsorption of negatively charged mucin on the surface of the positively charged L/C NPs thereby decreasing the surface charge. The results presumed that the interaction was electrostatic in nature. Apart from electrostatic interaction, there might be hydrogen bonding and physical entanglement also involved. On the other hand, turbidity of the mucin NPs dispersion went up as compared with the turbidity of the native mucin as a result of the interaction of NPs with mucin. Among the three formulations, NPs I have high absorbance value indicating high adhesion with mucin followed by NPs II and NPs III. This can be explained by the fact that increase in molecular weight of the chitosan has resulted in higher number of positive charged

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of CS. However, no ocular damage or clinically abnormal signs were observed in the cornea, conjunctiva or iris upon administration of NPs. Only grades 0 and occasionally 1 were recorded according to modified Draize test. No differences between control and treated eyes for each group of rabbits were observed. The eye irritation scores for all groups were less than 1, indicating excellent ocular tolerance of L/C NPs.

3.7. In-vivo precorneal retention

Fig. 4. Mucoadhesive measurements using turbidimetric assay (mean ± SD, n = 3). Table 2 In-vitro antifungal activity of various AmB-entrapped vehicles against Candida albicans and Aspergillus fumigatus. Formulation

AmB Fungizone® NPs I NPs II NPs III

C. albicans

A. fumigatus

MIC (␮g/mL)

Z. I (mm)

MIC (␮g/mL)

Z. I (mm)

0.002 0.004 0.002 0.002 0.004

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0.06 0.25 0.12 0.12 0.25

1.21 0.85 1.01 1.15 0.95

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units in polymer for stronger interaction with negatively charged mucin.

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The results of antifungal activity were summarized in Table 2. AmB was slightly more effective in-vitro when dissolved in DMSO. MIC90 of AmB encapsulated L/C NPs was almost similar with that of MIC90 value of Fungizone® . A probable explanation is that the drug dissolved in DMSO is readily available; however, the drug release from NPs is restricted by L/C matrix. The diameter of the zone of inhibition was shown in Fig. 6. Clear zones of inhibition were obtained. Results revealed that standard AmB dissolved in DMSO has a slightly larger zone of inhibition as compared with others. There was no significant difference between the zone of inhibition of marketed formulation and NPs formulation. These results indicating the potential antifungal efficacy of prepared L/C NPs.

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Upon topical ocular instillation, Fungizone® produced slight irritation, redness and increase in secretion indicating poor tolerance. NPs I showed precipitation due to the high molecular weight

Fungal keratitis is an ocular emergency that requires immediate intervention to prevent ocular morbidity and vision loss. Formulation used for the treatment of fungal keratitis should have a long tear retention time and be able to give a gradual and prolonged release of the medication. Precorneal retention profile can be used to evaluate the mucoadhesiveness of ophthalmic formulation and it may provide useful information for prediction of bioavailability in intraocular section. Fig. 7 and Table 3 list AmB concentrations in precorneal region following topical instillation of AmB loaded L/C NPs. Compartmental PK analysis was performed on mean AmB concentration data in rabbit tears. Based on the selection criteria employed, one-compartment model with bolus input and firstorder elimination from the central compartment provided the best fit to the observed mean data. Compared with Fungizone® , L/C NPs have lower Cmax . This may be due to the immediate availability of AmB from Fungizone® while the burst effect observed in case of NPs was unable to attain such high levels initially. On the contrary, L/C NPs achieved significantly higher t1/2 and longer MRT (mean residence time) than Fungizone® . In this study, the exposure of AmB measured as the area under the curve (AUC) in the tear fluid following a single instillation was 556.20 min ␮g/mL, 646.92 min ␮g/mL, 845.74 min ␮g/mL and 1139.46 min ␮g/mL in case of Fungizone® , NPs I, NPs II and NPs III respectively. The increase in AUC was higher for L/C NPs over the marketed formulation. Most importantly the clearance was low for NPs in comparison with Fungizone® . This suggests that the entrapment of AmB in NPs was essential to prolong the precorneal retention of the drug. Moreover the presence of CS layer was prominent factor affecting the exposure of AmB to cornea. The prolonged precorneal retention time of L/C NPs may provide an intimate contact between drug and ocular surface tissues, thus, facilitate the penetration of drug into cornea and aqueous humor. The surface of cornea and conjunctiva is covered by a thin fluid layer called mucus. The primary component of mucus is mucin, a high molecular mass glycoprotein which is negatively charged at physiological pH. Thus, the positively charged CS can provide binding force to the lecithin NPs to interact with negatively charged mucin components such as sialic acid residues so as to provide prolonged precorneal exposure of entrapped medication. The mucoadhesion

Fig. 5. Estimation of the zeta potential of NPs in (A) water and (B) incubation with 0.1% mucin (mean ± SD, n = 3).

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Fig. 6. Zone of inhibition of (A) AmB, (B) Fungizone and (C) NPs III against A. fumigatus; (D) AmB, (E) NPs III and (F) Fungizone against C. albicans. Bar graphs (G) and (H) represent zone of inhibition of different formulations against A. fumigatus and C. albicans, respectively (Mean ± SD, n = 3).

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of CS is not simply attributed to the positive charge but also its inherent nature which is promoted by the presence of free amine and hydroxyl groups which form hydrogen bonds with ocular surface [41]. Several factors were considered in the finalization of AmB nanoformulation. The foremost factor was the pharmacokinetic profile of NPs upon ocular instillation. An ideal formulation should exhibit higher AUC, MRT and lower clearance for prolonged ocular drug exposure. As evident from ocular tear concentration–time

profile and PK estimates, the formulation NPs-III exhibit significantly low clearance and high AUC and MRT as compared with NPs-I (Table 3). A probable explanation is that increase in molecular weight has resulted in increased particle size which eventually increases ocular clearance due to shear force of tear flow and eye blinking. The ocular shear force of tear and eye blinking is important mechanism of ocular clearance of foreign particles. Shear force increases with increase in particle size. Thus bigger the particle higher will be clearance.

Table 3 Pharmacokinetic parameters of AmB following topical instillation in rabbit eyes. Parameter

Units

Fungizone®

NPs I

NPs II

NPs III

Cmax AUC0–∞ MRT Cl %RB

␮g/mL min ␮g/mL min mL/min

19.81 ± 1.9 556.209 ± 74.51 18.79 ± 0.858 0.05 ± 0.00 100%

13.57 ± 1.8 646.92 ± 123.39 41.16 ± 5.90** 0.04 ± 0.00*** 116.31%

14.63 ± 2 845.74 ± 173.85 49.49 ± 6.20** 0.03 ± 0.00*** 152.05%

15.38 ± 2.27 1139.46 ± 155.70** 63.29 ± 3.99** 0.02 ± 0.00*** 204.86%

MRT and Cl of NPs III was found significantly different from NPs I and NPs II, Relative Bioavailability (%RB) = (AUC(NPs) × Dose(Fungizone)/AUC(Fungizone) × Dose(NPs)) × 100. ** Significant (P < 0.05) difference as compared with Fungizone® . *** Significant (P < 0.05) difference as compared with Fungizone® .

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Acknowledgements The authors are thankful to Director, CSIR-CDRI, India, for providing funding and facilities for the study. Authors H.C. and Y.S.C. are also thankful to CSIR and Indian Council of Medical Research respectively for fellowship. Y.D.P and A. V. are grateful to NIPER-Raebareli, India for providing fellowship. Ms. Garima Pant is acknowledged for technical assistance. CSIR-CDRI communication number is 8807. References

Fig. 7. Ocular pharmacokinetic profile of AmB following topical instillation in rabbit eyes (Mean ± SD, n = 3).

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The optimum formulation should have balance between two opposite forces i.e., mucoadhesion and shear force for prolong drug exposure. NPs-I upon ocular instillation shows mild visible precipitation in tear fluid, which may be due to higher zeta potential causing strong aggregation of oppositely charged NPs-I particles and mucin. Thus higher tubidity was observed in incubation of NPsI with mucin (Fig. 5). Although NPs-I has higher zeta potential and mucoadhesion but also experiences high clearance due to higher shear force as compared with significantly smaller particle NPs-III formulation. Other parameters such as drug release rate and ocular irritation were also compared. As observed from in-vitro drug release profile, lowering the molecular weight of CS results in rapid drug release rate need to maintain ocular therapeutic drug concentration (Fig. 3). Based upon these considerations, NPs-III was selected as optimum formulation for prolonged ocular application of AmB.

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Stability studies indicated that three different types of NPs exhibited preferable physicochemical stability under study conditions (data not shown). No indication of aggregation or precipitation was observed over a period of 90 days. Insignificant changes in the particle size ad zeta potential were observed. The EE was significantly decreased probably due to the leaching of AmB from NPs. 4. Conclusion In conclusion, AmB release was significantly prolonged by using L/C NPs. The CS in the formulation rendered the NPs positive surface charge as well as mucoadhesive property. The precorneal retention was higher for L/C NPs as compared with Fungizone® . Different molecular weights of CS NPs have different mucoadhesive properties. Based on the in-vitro release and pharmacokinetic performance, NPs-III was selected as optimum formulation for prolonged ocular application of AmB. Overall, L/C NPs achieved high AUC and MRT in comparison with marketed formulation. Thus, L/C NPs could be viable alternative for the existing formulation in terms of both prolonged exposure and ocular tolerance. However, further studies are needed to correlate this data to real patients.

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chitosan nanoparticles for prolonged ocular application.

Fungal keratitis is the major cause of vision loss worldwide. Amphotericin-B is considered as the drug of choice for fungal infections. However, its u...
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