RESEARCH ARTICLE – Pharmaceutics, Drug Delivery and Pharmaceutical Technology

Formulation and Characterization of Atropine Sulfate in Albumin–Chitosan Microparticles for In Vivo Ocular Drug Delivery EVELYN ADDO,1 RODNEY C. SIWALE,2 ALADIN SIDDIG,3 ALPHIA JONES,4 RUHI V. UBALE,5,6 JANET AKANDE,7 HENRY NETTEY,8 NEIL J. PATEL,6 MARTIN J. D’SOUZA,4 RICHARD T. ADDO6 1

Union University College of Education and Human Studies, Jackson, Tennessee 38305, USA Western New England University College of Pharmacy, Springfield, Massachusetts 01119, USA 3 University of Charleston School of Pharmacy, Charleston, West Virginia 20304, USA 4 Mercer University College of Pharmacy and Health Sciences, Atlanta, Georgia 30341, USA 5 Lake Erie College of Osteopathic Medicine, School of Pharmacy, Bradenton, Florida 34211, USA 6 Union University School of Pharmacy, Jackson, Tennessee 38305, USA 7 Lonza Inc., Alpharetta, Georgia 30004, USA 8 University of Ghana School of Pharmacy, Legon-Accra, Ghana 2

Received 7 August 2014; revised 24 December 2014; accepted 8 January 2015 Published online in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/jps.24380 ABSTRACT: The overall study goal was to produce a microparticle formulation containing atropine sulfate for ocular administration with improved efficacy and lower side effects, compared with that of the standard marketed atropine solution. The objective was to prepare an atropine sulfate-loaded bovine serum albumin–chitosan microparticle that would have longer contact time on the eyes as well as better mydriatic and cycloplegic effect using a rabbit model. The microparticle formulation was prepared by method of spray-drying technique. The percent drug loading and encapsulation efficiency were assessed using a USP (I) dissolution apparatus. The particle sizes and zeta potential were determined using laser scattering technique and the surface morphology of the microparticles was determined using a scanning electron microscope. The product yield was calculated from relative amount of material used. In vitro cytotoxicity and uptake by human corneal epithelial cells were examined using AlamarBlue and confocal microscopy. The effects of the microparticle formulation on mydriasis in comparison with the marketed atropine sulfate solution were evaluated in rabbit eyes. The prepared microparticle formulation had ideal physicochemical characteristics for delivery into the eyes. The in vivo studies showed that the microparticles had superior effects C 2015 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci on mydriasis in rabbits than the marketed solutions  Keywords: microparticles; micropheres; nanoparticles; ocular delivery; albumin; chitosan; atropine; mydriasis

INTRODUCTION The protective physiological mechanism existing on the precornea area and the anatomical structure of the cornea surface puts severe limitations on topical application of drugs into the eye. These limitations that lead to considerable drug loss are serious constraints on the effectiveness of drug delivery systems for topical and transcorneal treatment. These conditions make it difficult to provide adequate concentration of drug on the eye for the proper duration of time in order to achieve the required bioavailability of the drug needed for its therapeutic effectiveness. As a result, targeted administration of drugs to the anterior and posterior segments of the eye remains a significant challenge in ocular drug delivery. Within the last decade, drug retention in the cornea has received great attention with the intent to maximize the residence time of the drug delivery vehicles on the eyes, thus solving a specific absorption window issue as well as for localized and targeted drug delivery.1 One approach involves the creation of bioadhesive systems that strongly adhere to the mucus or cell surfaces. This has involved the adherence of drug-loaded Correspondence to: Richard T. Addo, Associate Professor of Pharmacy (Telephone: +404-358-4518; Fax: +731-661-5980; E-mail: [email protected]) Journal of Pharmaceutical Sciences  C 2015 Wiley Periodicals, Inc. and the American Pharmacists Association

microparticles coated with mucoadhesive polymers or specific ligands to the epithelial membrane.2 Since then, a number of reports have shown that mucoadhesive polymers can increase the residence time of drugs on the ocular surface.3,4 One of these polymers is chitosan (CSN), which has been found to possess favorable biological characteristics such as biodegradability, biocompatibility, and nontoxicity. This makes it suitable for use in biomedical and pharmaceutical preparations.5–13 Many researchers have developed different CSN-based carriers to enhance the transport of drugs across intestinal mucosal barriers,14 as artificial kidney membranes,15 as vehicles for compressed tablets,16–18 for gene therapy,19–21 as suture and wound healing materials,22 for vascular grafts,23 and cartilage regeneration.24 In addition, it has also been used for its antacid and antiulcer activities that prevent or weaken drug irritation of mucosal surfaces,25 among many other applications such as hydrogels preparation26,27 and as topical ocular drug delivery systems.28 Chitosan is a natural polymer with one primary amino and two free hydroxyl groups for each carbon 6 unit. It has been found that easy availability of these amino groups gives it a positive charge and thus enables it to easily react with many negatively charged surfaces.29 This imparts strong bioadhesive and particularly mucoadhesive characteristics.30,31 It has been reported that addition of bioadhesive polymers such as Addo et al., JOURNAL OF PHARMACEUTICAL SCIENCES

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RESEARCH ARTICLE – Pharmaceutics, Drug Delivery and Pharmaceutical Technology

CSN as a coating for particulate delivery systems, prolongs the contact time by significantly increasing the half time of its clearance.32,33 This makes it a promising candidate for ocularretentive drug delivery system.34 Furthermore, CSN possesses film-forming capacity and has been used for immobilizing enzymes, living cells, and in ophthalmology.34 As such, it has been identified as the biopolymer of choice for the development of both soft and hard contact lenses.35 In line with that, it has also become an important ingredient in ocular bandage lenses used as a protective device for acutely or chronically traumatized eyes.35,36 Moreover, cationic materials, such as CSN, have been shown to offer several benefits, including the ability to facilitate cellular uptake of drug compounds through contact with the negatively charged cellular membrane.37 One possibility for mucoadhesive systems is the employment of small particles of micron or submicron range called microparticles. Microparticles made from mucoadhesive polymers exhibit a prolonged residence time at the site of application or absorption, facilitate an intimate contact with the underlying absorption surface, and thus contribute to improved therapeutic performance of drugs. De Campos et al.38 reported an intense interaction of CSN with ocular surfaces that results in uptake and transport across the cell layers when the polymer is in microparticulate/nanoparticulate form.35 Microparticles are particularly relevant for ocular drug delivery because for patients’ comfort, it is reported that solid particles intended for ophthalmic use should not exceed 5–10 :.39 It has been reported that previous work by a number of researchers showed that poly (alkylcyanoacrylate) nanoparticles40,41 and poly-g-caprolactone nanocapsules42–44 were able to increase the intraocular penetration of drugs, while reducing their systemic absorption.38 A more positive result was reported when the microparticles were made of CSN. Genta et al.45 reported an increased and prolonged corneal penetration of acyclovir encapsulated in CSN microspheres. Furthermore, Felt et al.34 reported that the presence of CSN significantly prolonged cornea contact time of tobramycin following topical instillation to rabbits.38 This improved penetration was attributed to the microparticle/nanoparticle sizes of the delivery systems.46 In this project, CSN was combined with bovine serum albumin (BSA) to form a copolymer matrix for the atropine sulfate model drug. Albumin microparticles have been extensively investigated in controlled-release systems as vehicles for the delivery of therapeutic agents.47,48 Its exploitable features include its reported biodegradation into natural products, its lack of toxicity and its nonantigenicity49 The model drug for this project was atropine sulfate that is currently used for various ophthalmic therapies as a solution formulation. Atropine is a muscarinic antagonist that is used topically as a cycloplegic agent to temporarily paralyze the eye’s accommodation reflex and as a mydriatic to dilate the pupil.50 It acts by blocking the contraction of the circular sphincter muscle, stimulated by acetylcholine, thereby allowing the radial papillary dilator muscle to contract to dilate the pupil.51 Of all the drug compounds, atropine is reported to provide the greatest amount of cycloplegia and is considered as the gold standard for treatment.50 Cycloplegic refraction is reported by ophthalmologists as invaluable in the evaluation of patients with decreased vision and ocular deviation.51 Furthermore, the eyes of today’s children have no time to relax because of reading, writing, watching television, using the computer, surfing the internet, or playing video games. Not surprisingly, about Addo et al., JOURNAL OF PHARMACEUTICAL SCIENCES

half of school children are reported to be shortsighted, whereas indications for cycloplegic refraction are limited in adults.51 It has recently been reported that regular usage of atropine eye drops can retard the progression or completely stop myopia in children.52 However, conventional eye drops typically act transiently and enter the eye by either diffusion across the cornea or the sclera.53–56 As a result, atropine is well absorbed into the systemic circulation and has been reported to exert severe systemic side effects after ocular administration of solution formulations.51 Serious side effects of the drug include fever, tachycardia, convulsion, and even death, and it is contraindicated in patients with Down syndrome and albinism.57–59 Therefore, a sustained-release drug delivery system such as microencapsulation that protects and controls the release of the drug but having similar or superior pharmacodynamics effect than the standard formulations on the market will be ideal in minimizing both the dose and the spread of the drug with its accompanying ectopic toxicity after administration. The goal of this project, therefore, was to formulate a microparticle drug delivery system that has improved efficacy, but with lower side effects than the standard marketed atropine solution for delivery into the eyes. The objectives were to prepare microparticle with BSA and CSN copolymer matrix for the drug that would have longer contact time, be taken up by human corneal epithelial cells, and have better mydriatic and cycloplegic effect on the eye using rabbit models. The cycloplegic and mydriatic effects of atropine sulfate were measured by the novel use of pupil to corneal ratio in rabbit model. The production of the microparticles was by the spray-drying method. Spray drying is robust with high-product yield.48,60–65 Furthermore, the technique avoids the toxic chemicals used in solvent evaporation method that would be of great limitation in ophthalmic application.66

MATERIALS Chemicals Bovine serum albumin (Fraction V, DNAase, RNAase, and Protease-free), glutaraldehyde (25% in water), atropine sulfate powder, formaldehyde, Triton X-100, and sodium bisulfite were obtained from Fisher Scientific (Norcross, Georgia). Trypsin, sterile deionized water, methanol, and phosphatebuffered saline (1× PBS) pH 7.4 were obtained from Sigma– Aldrich chemicals (St. Louis, Missouri). CSN glutamate was obtained from Pronova (Drammen, Norway). The standard 1% atropine sulfate ophthalmic solution and natural tear fluid were obtained from Alcon Pharmaceuticals (Fort Worth, Texas). Human Corneal Epithelial cells were provided by the Ciba Vision (Duluth, Georgia). Ultra culture growth media were obtained from ATCC (Manassas, Virginia). Infra-Red Dye 800 CW was obtained from Li-cor Biosciences (Lincoln, Nebraska). Equipment ¨ The Buchi 191 Mini Spray Dryer (used in the spray-drying ¨ process) was obtained from Buchi Corporation (Newcastle, Delaware). The Horiba LA920 laser scattering particle size distribution analyzer (used to determine microsphere particle size) was obtained from Horiba Instruments Incorporated (Irvine, California). The Malvern Zetasizer Nano ZS (for zetapotential analysis) was obtained from Malvern Instruments DOI 10.1002/jps.24380

RESEARCH ARTICLE – Pharmaceutics, Drug Delivery and Pharmaceutical Technology

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(Worcestershire, UK). The BioTeK ELx808 Absorbance Microplate Reader (used for atropine assay analysis) was from BioTek instruments Inc. (Highland Park, Winooski, Vermont). The JEOL JSM-5800L scanning electron microscope (for surface morphology analysis) was obtained from JEOL USA (Peabody, Massachusetts). The Distek dissolution system model 2100C, USP Dissolution apparatus I, with a rotating basket (used for the microsphere formulations release studies) was obtained from Distek Inc. (North Brunswick, New Jersey). Lambda 4B UV/Vis spectrophotometer (PerkinElmer, Waltham, Massachusetts) was used for reading the release R multi-well plate reader sestudy samples. The Cytofluor ries 4000 (used for the AlamarBlue cytotoxicity assay) was obtained from PerSeptive Biosystems (Framingham, Massachusetts). Zeiss Confocal microscope LSM410 equipped with argon-krypton laser (Carl Zeiss Micro-Imaging, Thornwood, New York) was used for the uptake studies. The Odyssey infrared imaging system was from Li-cor Biosciences.

METHODS Preparation of Atropine Sulfate in Albumin–CSN Microparticles Bovine serum albumin–CSN solution (1%, w/v) was prepared and cross-linked with 0.75% glutaraldehyde for 24 h. The excess glutaraldehyde was neutralized with sodium bisulphite. Atropine sulfate was added to the cross-linked BSA–CSN matrix, to achieve 10% (w/w) drug loading with respect to the concentration of the polymer matrix. For the blank microparticles, no atropine sulfate was added to the cross-linked BSA–CSN polymer matrix. The cross-linked solutions were spray dried using a Buchi 191 Mini Spray Dryer. The settings of the various parameters for the spray dryer were: inlet temperature, 110°C; outlet temperature, 80°C; aspirator, 55%; compression flow rate, 800 psi; pump rate, 2.5% for optimum drug encapsulation and yield. The product yield for the microparticles was calculated using the following formula:

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Zeta Potential The zeta potential of the particles was measured using a Malvern Zetasizer Nano ZS (Malvern Instruments). The measurements were performed on six sample (n = 6) in 0.9% saline solution (normal saline) at a final microparticle concentration of 2 mg/mL (pH 6.8). Surface Morphology A scanning electron microscope (JEOL JSM 5800LV) was used to evaluate surface morphology of the microparticles. Microparticles were coated for 1 min under 80 mTorr of vacuum with gold/palladium at 15 mA. The micrographs were obtained using a 3-kV accelerating voltage at a 10,000 magnification. Release Profile and Mechanism of Drug Release In vitro release studies of the atropine sulfate-loaded microparticles were carried out at 37°C using natural tear fluid pH 7.4 (100 mL) in the modified USP type I dissolution apparatus. Atropine sulfate (25 mg) microparticles were suspended in 3 mL of natural tear fluid inside a dialysis bag with a molecular weight cut off of 12–14 kDa. The dialysis bag was sealed at both ends, enclosed into the dissolution baskets, and immersed into the natural tear fluid in the beakers. The dissolution apparatus was set at 100 rpm, and samples were taken at predetermined time intervals from each of the six baskets (n = 6). An equivalent volume of fresh tear fluid was replaced at each sampling time. The samples were analyzed for atropine sulfate by a Lambda 4B UV/Vis spectrophotometer (PerkinElmer) at 220 nm. In order to find out the mechanism of drug release from the microparticles, data obtained from in vitro release studies were fitted to various kinetic models. The following kinetic models were used: zero-order equation, first-order equation, and Higuchi model. Plots of Qt versus t (zero-order model), log (Q0 –Qt ) versus t (first-order model), and Qt versus t (Higuchi model) were made, where Qt is the drug release at time t and Q0 is the initial amount of drug present in the microparticles.

Product yield (%) =

Weight of microparticles obtained from spray dryer Weight of the total amount of solid in the feed ×100

Preparation of Standard Atropine Sulfate Solutions for Dose–Response Evaluation Atropine sulfate solutions (1.0%, 0.66%, and 0.33%) were used for this study. The 0.33% and 0.65% solutions were prepared by diluting the 1% solution with normal saline. Physicochemical Characterization of Microspheres Particle Size Distribution The particle size distribution of BSA–CSN microparticles was measured using the Horiba LA920 laser scattering particle size distribution analyzer. Briefly, the microparticles were suspended in Milli-Q pure water (2 mg/mL) containing 0.1% Tween 20. The particle sizes were determined after sonication. The results were calculated from six (n = 6) samples reading. DOI 10.1002/jps.24380

Encapsulation Efficiency To determine the encapsulation efficiency of the atropine sulfate in albumin–CSN microparticles, 5 mg of the dry microparticles powder containing the drugs were suspended in 200 :L of a buffer containing 50 mM Tris–HCl, pH 8.0, and 10 M ethylenediaminetetraacetic acid. The suspension was vortexed repeatedly to generate a homogeneous suspension and incubated overnight in a water bath at 37°C. To the above suspension, an equal volume of a buffer containing 200 mM NaOH, 1% SDS (w/v), and 100 :g/mL of proteinase K (DNase-free, RNase-free; Roche, Branchburg, New Jersey) was added and vortexed repeatedly. This reaction mixture was further incubated at 37°C for an additional period of 5 h. The concentration of atropine sulfate was determined by absorbance at 220 nm in a Lambda 4B UV/Vis spectrophotometer. Encapsulation efficiency was calculated as per the following formula:

Encapsulation efficiency (%) =

Actual drug loading ×100 Theoretical drug loading

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RESEARCH ARTICLE – Pharmaceutics, Drug Delivery and Pharmaceutical Technology

In Vitro Cytotoxicity, Internalization, and Localization Studies of Albumin–CSN Microparticles by Human Corneal Epithelial Cells

Confirmatory Studies of the In Vitro Internalization and Localization by Z-Stacking Method

Cell Culture

To ascertain whether the microparticles were actually taken into the cells and not just on their surfaces, a confirmatory study using the Z-stacking method was carried out. In this study, human corneal epithelial cells were exposed for 60 min to 0.5 mg/mL BSA–CSN microparticles containing FITC dye. Confocal serial images along the z-axis of human corneal epithelial cells were taken at 1.5-:m intervals from top to the bottom of the cell monolayer.67

Human corneal epithelial cells were seeded in six-well culture plates at approximately 3.0 × 105 cells/mL in ultraculture growth media containing 5% glutamine with no antibiotics. The human corneal epithelial cell cultures were incubated at 37°C and 5% CO2 . The cells were grown until they were 80%–100% confluent.

Determination of Cytotoxicity of Albumin–CSN Microparticles on Human Corneal Epithelial Cells To evaluate the cytotoxicity of the formulated albumin–CSN microparticles in human corneal epithelial cells, the cells were exposed to increasing concentrations of microparticles varying from 0.01 to 2 mg/mL for 24 h as previously described by Addo and coworkers.67 The negative control was human corneal epithelial cells in growth media and the positive control was human corneal epithelial cells in media containing 0.005% benzalkonium chloride (BAC). BAC is a quaternary ammonium cationic surface acting agent used as a bactericide/microbicide because of its cellular membrane lipid bilayer disruptive properties. After the 24-h incubation, the media were removed, and the cells were then washed several times with (1× strength solution) Dulbecco’s phosphate buffer saline (DPBS; pH 7.4), and incubated with a 1:10 dilution of AlamarBlue in 1× DPBS (pH 7.4) for 2 h. AlamarBlue contains a specific (fluorometric/colorimetric) REDOX indicator that both fluoresces and undergoes colorimetric change in response to cellular metabolic reduction. The extent of redox is, therefore, a measure of cellular metabolic reductive activity. The plates were then read R multiwell plate reader at 530 nm excitation using a Cytofluor and 580 nm emission. The fluorescence was proportional to the conversion of AlamarBlue by viable human corneal epithelial cells.

Uptake Study of Microparticles into Epithelial Cells Using Confocal Microscopy Ultra culture growth medium supplemented with 5% glutamine was used to suspend FITC-labeled albumin–CSN microparticles at 50 :g/mL. In order to exclude the possibility of autofluorescence by formaldehyde and nonspecific staining of cells by free FITC, the growth media supernatant of FITClabeled BSA–CSN microparticles was vigorously shaken repeatedly for 2 h and centrifuged. The microparticles were then incubated with the cells to check for any leakage of dye from the microparticles, which might stain cells before they were used. Human corneal epithelial cells at approximately 1 × 105 cells/plate were seeded and incubated overnight to allow for cell adhesion. The washed FITC-labeled microparticles were incubated with the cell at a concentration of 10 microparticles per cell for 24 h followed by 5× washing with PBS. Cells were then fixed onto glass slide with formaldehyde. Images were captured by using a 488-nm fluorescein, and 568-nm rhodamine filters. Differential interference contrast using a Zeiss Confocal microscope LSM410 equipped with argon-krypton laser was overlaid to obtain images to determine localization of microparticles inside the cell compartments. Addo et al., JOURNAL OF PHARMACEUTICAL SCIENCES

Quantitative Evaluation of the Uptake of Microparticles by the Human Corneal Epithelial Cells In these studies, about 1010 BSA–CSN–FITC-labeled microparticles were placed on filters of the transwell plates containing previously seeded and confluent human corneal epithelial cells. Total of 3 :m pore size of filter inserts were used. The transport epithelial electrical resistance across the cell monolayer was monitored using an ohmmeter (Millicell ETS) as mark of monolayer formation and as an indication of the formation and integrity of tight junctions between the cells. The FITC-labeled microparticles were applied to the apical side of the inserts and microparticles transported through the cell layer were collected in the basolateral compartment by sampling at 0.25, 0.5, 1, 2, 4, 8, 12, and 24 h time intervals. The amount of microparticles taken up and retained in the cells were also determined at the same time intervals by washing off the unattached microparticles form the cells with PBS and lysing the cells with Triton X (1%, w/v). Samples were analyzed using the fluorescent microplate reader and a cytofluorometer. Evaluation of the In Vitro Mechanism of Microparticle Transport Across Human Corneal Epithelial Cells This study was to determine whether the uptake of the microparticles into the corneal cells was temperature and energy dependent. Confluent corneal cell cultures on transwell plates were exposed to 150 :L of FITC-labeled microparticles (0.5 mg/mL) for predetermined time intervals from 15 min to 2 h, at 4°C and 37°C in the presence or absence of 100 mM sodium azide, (a known metabolic inhibitor). The amount of microparticles taken up and retained in the cells was also determined at the same time intervals as indicated in section Quantitative Evaluation of the Uptake of Microparticles by the Human Corneal Epithelial Cells above. In Vivo Evaluation of the Atropine Sulfate Formulation Standardization of the Novel Procedure for Measuring the Degree of Mydriasis Using Rabbit Model The objective of this study was to develop a standardized procedure to measure mydriasis using pupil to corneal ratio of the eye of rabbits. The pupil length and the cornea length of the eyes were measured without the addition of any external drug in the presence or absence of light to determine whether any constant parameter could be obtained that would serve as a baseline for comparison. Briefly, the eyes were videotaped from a fixed distance using Panasonic 30× digital camera with an inbuilt flashlight, with or without light. The video recording was downloaded into a window moviemaker. Grids with standard dimensions were also photographed from the same distance as the eyes. The pictures were then fixed and imported onto DOI 10.1002/jps.24380

RESEARCH ARTICLE – Pharmaceutics, Drug Delivery and Pharmaceutical Technology

Microsoft power point, where the pupil and cornea measurements were carried out by superimposing the grids on the pictures of the eyes. The ratios of diameter of the pupil to that of the corneal were calculated. A graph of pupil to corneal ratio on the y-axis and time on the x-axis was plotted. Dose–Response Study of Atropine Sulfate Solution Formulations on Mydriasis in Rabbits’ Eye Atropine sulfate drops (0.33%, 0.66%, and 1%) prepared in tear solutions (from the marketed 1% solution) were applied to different groups of rabbit eyes (n = 12). One eye of each animal received the test solution, whereas the other eye served as a control and received blank tear solution. Two drops of each solution were added to each eye with the aid of a dropper. Each rabbit’s eye was videotaped with the Panasonic 30× digital camera with and without light. The designated time for video tapping was 0.25, 0.5, 0.75, 1.0, 2.0, 3.0, 6.0, 8.0, and 24 h. The video recording was downloaded into window moviemaker. The pictures were then fixed and imported onto Microsoft power point, where the pupil and cornea measurements were carried out and analyzed as in section Standardization of the Novel Procedure for Measuring the Degree of Mydriasis Using Rabbit Model above. Effect of the Atropine Sulfate Microparticle Formulations on Mydriasis in Rabbits Eye The atropine sulfate microparticles were used to determine their effect on mydriasis for comparison with the marketed solution formulation. Briefly, the microparticles were suspended in a tear fluid to produce 0.33% and 0.66% atropine sulfate concentration, respectively. Two drops of each were administered in the eyes of different groups of Harlan rabbits (n = 12), with the aid of a dropper. One eye served as a test and the other as the control (which received blank tear solution) in each animal. Each rabbit’s eye was videotaped using Panasonic 30× digital camera with an in-built flashlight, with and without light. The designated times for video tapping analysis were as indicated in the above sections Standardization of the Novel Procedure for Measuring the Degree of Mydriasis Using Rabbit Model and Dose–Response Study of Atropine Sulfate Solution Formulations on Mydriasis in Rabbits’ Eye. The video recording was downloaded into window moviemaker. The pictures were then fixed and imported onto Microsoft power point, where the pupil and corneal measurements were carried out with the aid of a standardized grid. A graph of pupil to cornea ratio on the y-axis and time on the x-axis plotted. Subjects were observed for any obvious signs of ocular irritations such as redness and lachrymation. In our initial in vivo rabbit studies, death was observed at a higher atropine sulfate concentration via ocular administration because of the possibility of tachycardia. We also realized that

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the microparticulate formulation resulted into a greater permeability and therefore a greater bioavailability. One of the main aims of this study was to developed an alternative formulation that could elicit a response similar to the already market formulation but at a lower concentration. In other words, the authors also wanted to see whether a lower concentration (strength) of our microparticle encapsulated formulation of atropine sulfate could produce better pharmacologic effect than the standard marketed solution formulations. This we believe will lead to decrease in atropine sulfate toxicity. We therefore found it prudent not to use the 1% atropine microparticle formulation in the animal study as described in this section. Statistical Analysis Statistical analyses were carried out for both in vitro and in vivo work. All comparisons were made to the control group (marketed 1% atropine sulfate ophthalmic solution), and to each other. Summary of onset of action, duration of action, area under the curve (AUC), and maximum effect (pupil–corneal ratio) were calculated. Statistical significance was determined with the use of ANOVA between groups, and a p value of less than 0.05 was considered statistically significant.

RESULTS Physicochemical Characterization of Microparticles Particle Size The atropine–BSA–CSN microparticles with a mean particle size of approximately 2 :m (Table 1) and a polydispersity index of 0.268 were obtained from spray drying (Fig. 1). Spray drying has been traditionally used to produce particles with a narrow size distribution. This is evident from the results. Interestingly, the addition of CSN to the matrix of the microparticles, compared with the atropine–BSA microparticles without the CSN, reduced the particle sizes from 2.40 to 1.99 :m. This potentially may not have occurred if the CSN had just been used to coat preformulated and packaged BSA microparticles. Therefore, incorporation of the CSN as part of the polymeric matrix has a positive effect on particle size reduction. The reduced size brings with it an extra advantage of large surface to volume ratio that is good for controlled-release delivery of insoluble drugs. Zeta Potential The average zeta potential for the BSA–CSN atropine microparticles was 43.1 mV. A clear inversion of the zeta potential was found between BSA microparticles without the CSN and the BSA–CSN microparticles from a negative value of −38.4 to positive value of 43.1, respectively.

Table 1. Effect of Polymer Type on Particle Size Distribution (:m), Zeta Potential (mV), and Encapsulation Efficiency of Microparticles Formulation Atropine BSA–CSN MS Atropine BSA MS with no CSN Atropine alone

Mean size (:m)

Zeta Potential (mV) Drug-Loaded MS

Zeta Potential (mV) Blank MS

Encapsulation Efficiency (%)

Product Yield (%)

1.99 ± 0.13 2.40 ± 0.57 NA

43.1 ± 2.1 −38.4 ± 2.0 −2.7 ± 1.6

48.3 ± 1.2 −48.4 ± 2.8 NA

98 ± 0.62 96 ± 0.97 NA

86 ± 1.9 85 ± 1.3 NA

The following parameters were kept constant: solvent, stirring speed, and pH. DOI 10.1002/jps.24380

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Figure 1. Particle size distribution (mean 1.99 :m) of the BSA–CSN microparticles containing atropine sulfate.

Figure 2. Scanning electron micrograph of atropine sulfate albumin– CSN microparticles. The microparticle samples were coated for 1 min under 80 mTorr of vacuum with gold/palladium at 15 mA. The monographs were obtained using a scanning electron microscope (JEOL JSM 5800LV) with a 3-kV accelerating voltage at 10,000× magnification.

Surface Morphology Figure 2 shows a scanning electron micrograph of the atropine sulfate BSA–CSN microparticles. The microparticles appeared dimpled or highly porous with a collapsed center and “raisinlike” appearance. They are either round or flat with external scaffold. This differentiates them from traditional microspheres that have dense structure and are spherical in shape. Release Profile The percent cumulative release of the atropine sulfate from the BSA–CSN microparticles is shown in Figure 3a. Approximately 30% and 72% of the atropine sulfate drug was released in the first 5 and 25 h, respectively. However, the release of the drug was extended throughout the study period showing a pattern of extended release. This is an indication of a biphasic pattern Addo et al., JOURNAL OF PHARMACEUTICAL SCIENCES

Figure 3. (a) In vitro release of atropine sulfate from BSA–CSN microparticles in tear fluid. (b) Higuchi plot of in vitro release of atropine sulfate-loaded BSA–CSN microparticles in tear fluid.

of release—an initial burst period (first 5 h) and a subsequent more controlled-released period. Evaluation of the Mechanism of Drug Release from the Formulation by Higuchi Plot Analysis The drug release data were fitted to different kinetic models as mentioned in section Release Profile and Mechanism of Drug Release, and it fitted best in the Higuchi plot answering our concern of knowing the release pattern. Higuchi in 196168 developed an equation for the release of solid drugs dispersed in homogeneous matrix dosage systems. The equation indicates that for a release based on a diffusion mechanism, the amount of drug released, Q, is proportional to DOI 10.1002/jps.24380

RESEARCH ARTICLE – Pharmaceutics, Drug Delivery and Pharmaceutical Technology

A, the square root of the total amount of drug in unit volume of matrix, D, the diffusion coefficient of the drug, Cs , the solubility of drug in the polymeric matrix, and t, the time of release (Q = 2ADCs t)1/2 . Because for a given formulation, A, D, and Cs are constant, for a dissolution and release based on diffusion, a plot of Q, the amount or cumulative percentage of drug released by the square root of them t, should follow a straight line.48 Figure 3b shows the Higuchi plot analysis of the pattern of release of the atropine sulfate from the microparticles for the first 25 h. The graph shows that the pattern followed a straight line with an R2 value of 0.9982, thus indicating a primarily diffusion controlled release for that time period. Upon incubation of microparticles that follow diffusion controlled release in an aqueous medium, drug molecules located at or near the particle surface are dissolved by the penetrating waterfront and diffuse out into the surrounding medium within a short period of time (burst release). In the case of this study, this happened within the first 5 h where 30% of the atropine sulfate was released.

In Vitro Cytotoxicity, Internalization, and Localization Studies of Albumin–CSN Microparticles by Human Corneal Epithelial Cells Cytotoxicity of Albumin–CSN Microparticles in Human Corneal Epithelial Cells In this study, a 2-mg/mL, 10% atropine sulfate-loaded albumin– CSN microparticle suspension in growth media showed no toxicity to human corneal epithelial cells. Atropine sulfate microparticle cytotoxicity in human corneal epithelial cells at 48 h was determined using an AlamarBlue cytotoxicity assay. Cell viability was measured as a percentage of the fluorescence emitted in the negative control comprising human corneal epithelial cells in growth media without microparticles (n = 6). Figure 4 shows the cell viability in increasing concentrations of microparticles. There was no significant difference in cell viability as the microparticle concentration increased from 0.01 to 2 mg/mL. This is an indication that no inherent toxicity can be attributed to the microparticles at concentrations as high as 2 mg/mL.

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Figure 5. Image of the uptake of FITC-labeled BSA–CSN microparticles (50 :g/mL) by human corneal epithelial cells. The cells were exposed to the microparticles for 24 h. Cells were washed 5× with PBS and fixed onto glass slide with formaldehyde. Image was captured by the use of a confocal microscope.

Uptake Study of Microparticles into Epithelial Cells Using Confocal Microscopy Figure 5 shows the uptake of the fluorescence-labeled BSA– CSN microparticles by the human corneal epithelial cells after 24 hours exposure. The possibility of nonspecific staining of cells by BSA–CSN FITC through leakage from the microparticles had been excluded in the method validation. The figure demonstrates the uptake of the microparticle by the human corneal epithelial cells. Confirmatory Study of the Microparticle Internalization and Localization by the Z-Stacking Method Using Confocal Microscopy To confirm the internalization of the microparticles, human corneal epithelial cells were exposed for 60 min to 0.5 mg/mL

Figure 4. Cytotoxic effect of varying concentrations (from 0.01 to 2 mg/mL) of atropine sulfate-loaded BSA–CSN microparticles on human corneal epithelial cells. The study was carried out for 48 h. DOI 10.1002/jps.24380

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Figure 6. Confocal serial images along the z-axis of human corneal epithelial cells. Cells were exposed for 60 min to 0.5 mg/mL FITC-labeled BSA–CSN microparticles. A representative gallery of eleven serial micrographs showing the green fluorescence of the labeled microparticles at 1.5 :m intervals along the z-axis, from top to the bottom of the cell monolayer is shown.

of FITC-labeled BSA–CSN microparticles. Figure 6 is a representative gallery of eleven serial micrographs showing the green fluorescence at 1.5-:m intervals along the z-axis, from top to bottom of the cell monolayer. The results show that the FITC-labeled microparticle was located in the cells and not on the outside of the cells. This is evident from the fact that there was a gradual increase in the amount of florescence from one side of the cell surface at 0 :m to around the middle of the cell at 7.5–9.0 :m. There onwards, the florescence intensity gradually decreased to the other side of the cell at 15.0 :m. Quantitative Evaluation of the Uptake of Microparticles by Human Corneal Epithelial Cells Figure 7a shows the general trend of the percent particles transported into and across the human corneal epithelial cells with time. The figure shows that the number of particles taken into and across the cell layer increased with time. This is expected because the particles had enough contact time with the cells. A closer quantitative evaluation of the percent uptake at equal time intervals, as shown in Figure 7b, shows timedependent changes in the rate of particle uptake and transport. There is a burst uptake of the microparticles within the first 4 h of incubation. After this time point, the pattern plateaus out. This is an indication of a transport system that gets saturated. Evaluation of the In Vitro Mechanism of Microparticles Transport Across Human Corneal Epithelial Cells As shown in Figure 8a, the human corneal epithelial cells demonstrated a higher uptake and transport of particles at Addo et al., JOURNAL OF PHARMACEUTICAL SCIENCES

37°C than at 4°C (p < 0.05). Transport of particles at 37°C was more than double that at 4°C after 60 min. Figure 8b shows the effect of sodium azide, a metabolic inhibitor, on the uptake of the microparticles at 37°C. The controls were without the sodium azide compound. The figure shows that the presence of the compound significantly inhibited the microparticle uptake from the first half hour onwards. It can, therefore, be inferred from the study that the transport mechanism seems to be primarily energy-dependent active transcytosis. However, both graphs show a basic level of uptake of the microparticles at both 4°C and in the presence of the sodium azide. Therefore, it can be deduced that there is a basic underlining passive diffusion mechanism taking place. The passive diffusion is boosted by the increase in energy and metabolism. In Vivo Evaluation of the Atropine Formulation Standardization of the Procedure for Measuring the Degree of Mydriasis Using Rabbit Model The objective of this study was to develop a standardized procedure to measure mydriasis using pupil to corneal ratio of the eye of rabbits. The pupil length and the cornea length of the eye were measured without the addition of drugs and in the presence or absence of light to determine whether a constant parameter could be obtained that will serve as a baseline for comparison. The result of the study is shown in Figure 9. It was observed that the ratio of the pupil length to the corneal length was constant in the rabbits. The average ratio came to be 0.527. In addition, it was determined that the pupil to cornea ratio was more reproducible in the presence of light than in its absence. Plot of the effect of light on the ratio of the pupil to DOI 10.1002/jps.24380

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Figure 7. (a) Evaluation of the quantitative uptake of the atropine sulfate BSA–CSN microparticles across human corneal epithelial cells in vitro using transwell plates. (b) Evaluation of the quantitative uptake of atropine sulfate BSA–CSN microparticles by human corneal epithelial cells at equal time intervals.

corneal lengths of rabbit eyes showed that the ratio of these two parameters was fairly constant. Hence, for all subsequent studies, the pupil to cornea ratio was used in the presence of light (after exposure to light). This is the first time the ratio of the pupil to corneal lengths in rabbit eyes has been reported to be constant.

Mydriatic Effect of the Atropine Sulfate Solutions in Rabbit Model Figure 10 is the plot of the effect of 1% atropine sulfate standard solution on the ratio of the pupil to corneal lengths of rabbit eyes. The effect of the drug on ratio was seen after 0.25 h and the time of maximum effect (Tmax ) was seen at 0.5 h with a maximum ratio (Cmax ) of 0.596. The effect started to wane from then onwards. Figure 11 is a plot of the effect of the atropine sulfate solutions with drug concentrations of 0.33% and 0.66% as compared with the 1% standard solution. As expected, there is a welldefined dose–response relationship between concentration and effect on the ratio of pupil length to cornea length. The Tmax for all three concentrations was 0.5 h. DOI 10.1002/jps.24380

Comparative Effect of the Atropine Sulfate Microparticle Formulations on Mydriasis in Rabbit’s Eye Figure 12 shows the comparative effect of the microparticle formulations with drug loading of 0.33% and 0.66% with that of the standard 1% solution. First, there was a clear dose– response effect between the microparticle formulations with a higher response from the 0.66% than the 0.33% drug loading. The results also show that although the recorded onset of action was at 0.25 h for the formulations and the standard solution, the Cmax of 0.63 ratio for the microparticles with 0.66% drug loading was at 0.25 h. This was 0.25 h earlier than the standard solution and 0.33% drug-loaded microparticles. Table 2 shows the duration of action, AUC, and the Cmax of the three formulations. All the parameters show that there was a greater effect from the microparticles with 0.66% drug loading than the 0.33% drug-loaded microparticles and the 1% standard solution (p < 0.05). The 0.66% microparticles had the highest Cmax of 0.63 as compared with the 0.596 of the standard solution and 0.51 of the 0.33% microparticles. The 0.66% microparticles had an AUC of 10.67, whereas the 1% standard solution and 0.33% microparticles had an AUC of 10.02 and 8.46, Addo et al., JOURNAL OF PHARMACEUTICAL SCIENCES

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Furthermore, the ratio–time curves for the two microparticle formulations showed plateau sections not observed in the solution from 0.25 to 3 h. This is a reflection of the controlledrelease properties of the microparticle formulations.

DISCUSSION

Figure 8. (a) Effect of temperature on the uptake of atropine sulfateloaded BSA–CSN microparticles into corneal epithelial cells. Cellassociated fluorescence after BSA–CSN microparticles exposure at 4°C or 37°C, expressed as percentage of microparticles (MS) available for uptake was significantly reduced at 4°C at 15, 30, 60, and 120 min incubation time. p  0.05 calculated by ANOVA comparing the two groups. (b) Effect of metabolic inhibition with 100 mM sodium azide on the atropine sulfate-loaded BSA–CSN microparticles uptake into human corneal epithelial cells. The microparticles uptake was quantified as the fluorescence of cell lysates after microparticles exposure at 37°C in the absence or presence of sodium azide. Presence of sodium azide significantly inhibited the atropine sulfate-loaded BSA–CSN microparticles uptake at 30, 60, and 120 min incubation times. p  0.05 calculated by ANOVA comparing the two groups.

respectively. The duration of action for the 0.66% microparticles was 8 h. The effect was 2 h greater than that of 6 h for the standard solution and 0.33% microparticles.

Figure 9. Plot of the effect of light on the ratio of the pupil to corneal lengths of rabbit eyes. The ratio of these two parameters was seen to be constant for the first time ever at normal conditions. p  0.05 calculated by ANOVA comparing the two groups. Addo et al., JOURNAL OF PHARMACEUTICAL SCIENCES

The results of this study showed that the BSA–CSN microparticles were an effective delivery system for atropine sulfate administration into the cornea cells of the eye. The formulation had ideal physicochemical characteristics for a controlledrelease delivery into the eyes. Spray drying has been traditionally used to produce particles with a narrow size distribution. This is evident from particle size of approximately 2 :m obtained in this study. Results also showed that the incorporation of the CSN as part of the polymeric matrix reduced the particles size substantially. It is reported that for effective delivery and patients’ comfort, solid particles intended for ophthalmic use should not exceed 5–10 : in diameter, with a narrow size range (low polydispersity index).39,69 The particle sizes of 2.0 :m and polydispersity index of 0.268 obtained in this project are, therefore, ideal for ocular delivery. The small microparticles have an extra advantage of large surface to volume ratios that is good for controlled-release delivery of insoluble drugs. The surface charges of microparticles as represented by the zeta potential are very important for the stability of the formulation in suspensions and in their interaction with the mucosal surfaces. CSN has one primary amino and two free hydroxyl groups for each C6 building unit. The easy availability of the free amino groups confers a positive charge on the surfaces of CSN and makes it a cationic polysaccharide. The molecular attractive forces formed by electrostatic interaction between the positively charged CSN and negatively charged sialic acid residue on mucosal surfaces confer mucoadhesive properties on CSN-coated microparticles.29 CSN has been reported to also show good bioadhesive characteristics and reduce the rate of clearance of drugs from mucosal surfaces such as the nasal cavity, thereby increasing the bioavailability of drugs incorporated in it.70 Therefore, the positively charged microparticle surface is advantageous for the maximum contact time on the eye surface. Moreover, adequate stability of microparticles dispersed in aqueous solutions can be achieved by either electrostatic stabilization or steric stabilization or by a combination of both.71 Adequately high zeta potential values beyond ±30 mV, as obtained in this study, are reported to indicate a stable colloidal dispersion.72 Loss of stability may lead to cloudiness, which may cause impairment of vision upon application to the eyes. The raisin-like and porous appearance of the microparticles obtained in this study is different from the traditional microspheres that have dense structure and are spherical in shape. Compared with traditional microspheres, porous microparticles show many unique properties such as large specific surface area for efficient drug absorption or release and low density.73 These characteristics may be ideal for ocular delivery as it will lead to large contact surface on the eyes and reduction in patients discomfort because of the low density. Drug release from the microparticles showed a biphasic profile with an initial burst followed by a more controlled period. The second slower phase of release depends on microparticle porosity and hydrophilicity as well as molecular interaction forces between the polymer matrix and drug molecules.48,74 In DOI 10.1002/jps.24380

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Figure 10. Plot of the mydriatic effect of 1% atropine sulfate standard solution before and after exposure to light in rabbit eyes. p  0.05 calculated by ANOVA comparing the two groups.

Figure 11. Dose–response studies of the atropine sulfate standard marketed solution showing the extent of mydriasis at three different concentrations (0.33%, 0.66%, and 1%) of atropine sulfate solution in rabbit’s eyes. p  0.05 calculated by ANOVA comparing the groups with each other.

porous and hydrophilic microparticles (such as that made of BSA and CSN) or if there is affinity between the drug and polymer matrix, water penetration into the particles and drug dissolution/diffusion out of the matrix are facilitated. As a result, a second phase of continuous release may follow the burst, leading to a final drug release before the particle erosion sets in or reached an advanced stage—biphasic release pattern.75 Results from this study indicate that the pattern of release of atropine sulfate from the BSA–CSN microparticles follow the biphasic release profile with longer phase of extended release. If the bioadhesive characteristic of the CSN is effective in reducing the rate of clearance of the microparticles from the eye and thereby increasing the contact time, then there is a guarantee of continuous supply of drug into the eye for the duration of contact. The lack of inherent toxicity of the microparticles confirms a similar finding reported by De Campos et al.38 The nontoxicity DOI 10.1002/jps.24380

of the formulation to human corneal epithelial cells coupled with the antiulcer and nonirritant nature of CSN25,76 makes the microparticles ideal for topical and transcorneal ophthalmic drug delivery. The uptake of the microparticles into the human corneal epithelial cells indicated an intracellular transport system that gets saturated. This is very important in ocular delivery systems where contact time on the eyes is limited. Prego et al.77 reported that CSN nanoparticles interaction with Caco-2 cells was saturable in approximately 30 min. However, it was also shown by the same group that the presence of mucus in MTXE12 cell monolayers strongly increased the association with the CSN nanoparticles.77 De Campos et al.38 reported an intense interaction of CSN with ocular surfaces (cornea and conjunctiva) when the polymer is in microparticulate/nanoparticulate form. This is very relevant in the finding obtained in this study because the fact that more BSA–CSN microparticles were Addo et al., JOURNAL OF PHARMACEUTICAL SCIENCES

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Figure 12. Comparison of the mydriatic effect of BSA–CSN microparticles with 0.33% and 0.66% drug loading with the standard 1% atropine sulfate solution after exposure to light in rabbit’s eyes. p  0.05 calculated by ANOVA comparing the groups with the control 1% atropine sulfate solution group.

Table 2. Summary of Comparison of the Onset of Action, Duration of Action, Area Under the Curve (AUC), and Maximum Effect (Pupil–Corneal Ratio) of Microparticles with 0.33% and 0.66% Drug Loading with the Standard 1% Atropine Sulfate Ophthalmic Solution Formulation Microparticles 0.33% 0.66% Standard 1% solution

Onset of Action (h)

Duration of Action (h)

AUC

Maximum Effect (Pupil–Corneal Ratio)

0.25 0.25 0.25

6.00 8.00 6.00

8.46 10.67 10.02

0.511 0.630 0.596

transported into the cell up to 4 h shows the important role the potential mucoadhesive properties of the CSN and microsizes of the particles may play in ocular delivery. The study also showed that the cellular uptake is energy-dependent but with a basic underlining passive diffusion mechanism. This passive diffusion was boosted by the increase in energy and metabolism. The in vivo studies showed that the microparticles had superior effects on mydriasis in rabbits than the marketed solutions. The intensity of action as demonstrated by Cmax was higher and about 0.25 h earlier for the microparticles than the standard marketed solution. This is important for ocular delivery because of limited contact time. Moreover, one of the reasons adduced against the use of atropine sulfate as a cycloplegic agent is the delay in the onset of cycloplegia. It is reported that atropine sulfate in standard marketed formulations needs at least 3 h to reach peak effect and, therefore, must be used for 3 days to produce complete cycloplegia. As a result, it takes 8–14 days for its effect to wash out from the pupil and ciliary body.51 The earlier onset of effect demonstrated in this study may, therefore, be more effective in the ocular delivery of the drug. The result obtained in this study showed that all the basic pharmacokinetic parameters such as duration of action, AUC, and the Cmax were better in the 0.66% drugloaded microparticles than the standard marketed 1% solution formulation. The results, therefore, demonstrate that our microparticle formulations, though lower in drug concentration than that of the standard marketed solution, had superior effect on mydriasis in rabbit eyes. The 0.66% microparticle formulation may, Addo et al., JOURNAL OF PHARMACEUTICAL SCIENCES

therefore, be more effective in delivering the required effect at a shorter onset time, with sustained-release and less drug characteristics. This may be effective in ensuring better compliance from users.

CONCLUSIONS The overall results showed that BSA–CSN microparticles are an effective delivery system for atropine sulfate administration into the cornea cells of the eyes. The addition of CSN increased the contact time and a significantly higher amount of drug was delivered in a sustained-release manner into the eye. As compared with the standard 1% atropine sulfate solution on the market, the microparticle formulation containing 0.66% atropine sulfate showed superior effect on mydriasis in rabbit eyes. Bovine serum albumin and CSN are both considered as generally safe materials as they are polymers that possess biocompatible and biodegradable properties. The combination of the polymers in the preparations of microparticles targeting effective drug delivery into the eyes as shown in this study will go a long way in addressing the inefficient drug delivery and safety issues associated with current drug administration into the eyes. The in vivo results demonstrated that the atropine microparticle formulation, though lower in strength of atropine sulfate than that of the standard marketed solution, was superior to that of the standard marketed solution and well tolerated in human corneal epithelial cells in vitro. DOI 10.1002/jps.24380

RESEARCH ARTICLE – Pharmaceutics, Drug Delivery and Pharmaceutical Technology

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DOI 10.1002/jps.24380