http://informahealthcare.com/drt ISSN: 1061-186X (print), 1029-2330 (electronic) J Drug Target, Early Online: 1–10 ! 2014 Informa UK Ltd. DOI: 10.3109/1061186X.2014.939983

ORIGINAL ARTICLE

Methazolamide-loaded solid lipid nanoparticles modified with low-molecular weight chitosan for the treatment of glaucoma: vitro and vivo study Fengzhen Wang1,2, Li Chen1,3, Dongsheng Zhang4, Sunmin Jiang5, Kun Shi6, Yuan Huang1, Rui Li1, and Qunwei Xu1 Journal of Drug Targeting Downloaded from informahealthcare.com by RMIT University on 09/09/14 For personal use only.

1

School of Pharmacy, Nanjing Medical University, Nanjing, Jiangsu, China, 2Department of Pharmaceutics, China Pharmaceutical University, Nanjing, Jiangsu, China, 3Zhenjiang First People’s Hospital, Affiliated Hospital of Jiangsu University, Zhenjiang, Jiangsu, China, 4The First School of Clinical Medicine, Nanjing Medical University, Nanjing, Jiangsu, China, 5Wuxi Hospital for Maternal and Child Health Care, Affiliated Hospital of Nanjing Medical University, Wuxi, Jiangsu, China, and 6Department of Orthopedics, Xuzhou Central Hospital, Xuzhou, Jiangsu, China Abstract

Keywords

The aims of this study were to design and characterize methazolamide (MTZ)-loaded solid lipid nanoparticles (SLN) with and without modification of low molecular weight chitosan (CS) and compare their potentials for ocular drug delivery. Low molecular weight CS was obtained via a modified chemical oxidative degradation method. SLN with CS (CS-SLN-MTZ) and without CS (SLN-MTZ) were prepared according to a modified emulsion-solvent evaporation method. SLN-MTZ and CS-SLN-MTZ were 199.4 ± 2.8 nm and 252.8 ± 4.0 nm in particle size, 21.3 ± 1.9 mV and +31.3 ± 1.7 mV in zeta potential, respectively. Physical stability studies demonstrated that CS-SLN-MTZ remained stable for at least 4 months at 4  C, while SLN-MTZ no more than 2 months. A prolonged in vitro release profile of MTZ from CS-SLN-MTZ was obtained compared with SLN-MTZ. Furthermore, CS-SLN-MTZ presented a better permeation property in excised rabbit cornea. In vivo studies indicated that the intraocular pressure lowering effect of CS-SLN-MTZ (245.75 ± 18.31 mmHg  h) was significantly better than both SLN-MTZ (126.74 ± 17.73 mmHg  h) and commercial product Brinzolamide Eye Drops AZOPTÕ (171.17 ± 16.45 mmHg  h). The maximum percentage decrease in IOP of CS-SLN-MTZ (42.78 ± 7.71%) was higher than SLN-MTZ (27.82 ± 4.15%) and was comparable to AZOPT (38.06 ± 1.25%). CS-SLN-MTZ showed no sign of ocular irritancy according to the Draize method and the histological examination.

Intraocular pressure-lowing effect, low molecular weight chitosan, methazolamide, ocular irritancy, solid lipid nanoparticle

Introduction Methazolamide (MTZ) is a neutral, lipophilic carbonic anhydrase inhibitor (CAI) and has been used in clinic for anti-glaucoma treatment for over half a century [1,2]. However, systemically administered MTZ has a limited use due to its poor bioavailability and the systemic side effects such as central nervous system depression, diuresis, and vomiting [3,4]. To overcome these shortcomings, topical administration directly to the eye was investigated to enhance its bioavailability and minimize or eliminate the systemic side effects [5]. Because of factors such as lachrymation, nasolachrymal drainage and metabolic degradation, the residence time of common commercial eye drops for topical

Address for correspondence: Rui Li or Qunwei Xu, School of Pharmacy, Nanjing Medical University, Lane 818, East Tianyuan Road, Nanjing, 211166, Jiangsu, China. Tel: +86 25 86868478. E-mail: [email protected] (R. Li); [email protected] (Q. Xu)

History Received 11 March 2014 Revised 20 May 2014 Accepted 26 June 2014 Published online 21 July 2014

application is no more than 5 min. Moreover, the relative impermeability of cornea leads to only 1–5% of the applied drug penetrating into intraocular area [6]. As for MTZ solution, it also showed a fast elimination in the precorneal region and an impermeability of the corneal epithelial membrane. To enhance the residence time of MTZ on the cornea and prolong its pharmacological activity, new colloidal systems for ocular application were developed, such as liposome, calcium phosphate nanoparticle, in situ gel, and solid lipid nanoparticle (SLN) [7–9]. SLN, initially developed at the beginning of the 1990s, is prepared from oil-in-water (o/w) emulsion with lipids that are solid at ambient temperature. As a widely-investigated colloidal particle system, it was developed with the potentiality for the delivery of both hydrophilic and lipophilic drugs [10]. The mean diameter of SLN is in the range of approximately 50 and 1000 nm [11]. In addition to its desirable properties including high monodisperisity, long temporal stability, good biodegradation, low toxicity, enhanced biocompatibility, and improved efficacy, SLN combines all the advantages of fat emulsion, polymeric nanoparticle and liposome while overcoming their shortcomings such as drug leakage, hydrolysis and un-stability drug

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storage [12–14]. Besides, myriads of studies have reported that SLN possessed the ability to significantly prolong the residence time, sustain the release profile and increase the bioavailability for topical administration [15–18], especially for the ocular application [11,17]. With the optimization of multiple preparation methods (e.g. high pressure homogenization, solvent evaporation, solvent emulsification diffusion method, etc.), it had no problems with respect to large-scale production for SLN. Our previous studies demonstrated that SLN incorporating MTZ was a suitable ocular drug delivery system and showed a favorable decreasing effect on the intraocular pressure (IOP) of rabbit eyes upon topical application [8]. However, the major challenge we encountered was the poor corneal permeability. Negatively charged nanoparticles were reported to be difficult to interact with the negatively charged cornea surface. In order to address the problem, the particle surface charge turnover from negative to positive was recommended [19–21]. One of the most frequently-used approaches recently developed was to modify the surface of the nanoparticles with cationic polymers. Chitosan (CS), composed of 2-amino-2-deoxy-h-d-glucan combined with glycosidic linkages, is a natural polysaccharide with positive charge, which is recognized as a good candidate for modification of SLN [20]. Over the last decade, CS has attracted extensive attention for its biocompatibility, biodegradability, non-toxicity, mucoadhesiveness and the convenience to be obtained [22,23] Due to the favorable characteristics of CS, it could be used as absorbefacient for ophthalmic purpose in which the positive charged groups of CS were able to interact with cornea, reversibly unfold tight junction to enhance transportation of drug [24,25]. In this case, drug bioavailability could reach an improvement through prolongation of residence time in absorption region and enhancement of penetration across mucosal barriers [26,27]. However, cytotoxic extent of CS was dependent upon their molecular weight and increased with increasing molecular weight [28]. The high molecular weight, high viscosity, and low water solubility of CS restrict its uses resulting in the depolymerization to produce low molecular weight CS with decreased toxicity, and good solubility in physiological pH environment. Currently, we prepared a series of water-soluble, low molecular weight CS being used for SLN surface-modifying by hydrogen peroxide in order to combine the advantages of this desirable cationic mucoadhesive property of CS with the features of SLN. SLN with and without CS modification were respectively prepared by modified emulsification-solvent evaporation and low temperature-solidification method. We assessed their physical characteristics, in vitro release and ex vivo transcornea penetration profiles as well as ocular irritation. Furthermore, we evaluated the ability of CS-SLN-MTZ in the anti-glaucoma effect compared with unmodified SLN-MTZ, making a more comprehensive research on the effect of CS on SLN-mediated ophthalmic transfer of MTZ. Altogether, the aims of this study were to compare the properties of SLN with CS and without CS modification and evaluate their efficiency for anti-glaucoma treatment, offering a promising avenue to fulfill the need for an ophthalmic drug delivery system.

J Drug Target, Early Online: 1–10

Materials and methods Chemicals and animals MTZ, of 99% purity, was purchased from AOYIPOLLEN (Hangzhou, China). CS (Mw: 50–120 kDa, D.D95%, viscosity: 30–100 Mpa S) was purchased from Haidebei Marline Bioengineering Co., Ltd. (Jinan, China). Phospholipids (Lipoid S100) were provided by Lipoid (Ludwigshafen, Germany). Tween 80 was purchased from Jiujiu Bio Tech. Co., Ltd. (Jiangsu, China). Polyethylene glycol 400 (PEG 400) was supplied by the Dow Chemical Company (Shanghai, China). HPLC grade methanol was obtained from Jiangsu Hanbon Sci. & Tech. Co., Ltd (Nanjing, China). Deionized water was purified by Hitech-K flow Water Purification System (Hitech Instruments Co., Ltd. Shanghai, China). All other chemicals and solvents were of analytical grade or higher. New Zealand albino rabbits, weighing 2.5–3.0 kg with normotensive eyes, were provided by Animal Experimental Center of Nanjing Medical University. The rabbits kept in individual cages were fed ad libitum and maintained in a temperature-controlled room on a 12-h light/dark cycle (light period from 6:00 AM to 6:00 PM). All procedures were conducted in accordance with the Principles of Laboratory Animal Care and approved by the local ethics committees for animal experimentation. Preparation of low molecular weight of CS, SLN and CS-SLN Preparation of low molecular weight of chitosan Water-soluble CS of low molecular weight was produced with a modified chemical oxidative degradation method [29,30]. A weighed amount of CS was dissolved in 0.35 mol/l acetic acid solution at 60  C. Then 30% hydrogen peroxide solution (2.5 ml H2O2/g CS) was added for degradation. Excessive hydrogen peroxide was scavenged with a certain amount of sodium bisulfite (NaHSO3) following test with starch KI test paper (San’aisi Chemical Co., Ltd, ShangHai, China). The mixture was then dialyzed (MWCO ¼ 1.0 K) against deionized water at 4  C to remove irrelevant small molecular weight substances. After this process, the degraded CS was lyophilized. A series of low-molecular weights of CS were obtained under different degradation times (1.5 h, 2.5 h, 3.5 h and 4.5 h, respectively) and their mean-viscosity molecular weights were determined with Ubbelohde viscometer. Preparation of SLN and CS-SLN SLN and CS-SLN loaded with MTZ were produced according to a modified emulsion-solvent evaporation and low temperature-solidification method. In brief, MTZ (35 mg), glyceryl monostearate (100 mg) and lecithin (10 mg) were dissolved in 5 ml ethanol in the test tube which was kept in 70  C water bath and then the solution was added dropwise to 15 ml aqueous surfactant solution (1% PEG400 and 1% Tween80, w/v) under vigorous stirring (RET Control-visc C, IKA, Staufen, Germany) at the same temperature. After evaporating to 5 ml, the mixture was then quickly poured into 25 ml secondary phase under stirring over ice bath for 30 min. The secondary phase was based on mannitol (50 mg/ml) dissolved in pH 4.5 acetic acid solution for SLN-MTZ

Methazolamide-loaded solid lipid nanoparticles

DOI: 10.3109/1061186X.2014.939983

preparation. To coat SLN, the prepared CS (2.5 g/ml) were added into secondary phase for CS-SLN-MTZ preparation. The dispersion was finally filtered through a millipore filter (0.45 mm) and the samples were stored at 4  C. Blank SLN and blank CS-SLN were prepared in a similar way without the addition of MTZ. MTZ solution was prepared with 35 mg MTZ completely dissolved into 30 ml deionized water through sonication. Characterization

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Average particle size and zeta potential The average particle size, size distribution (polydispersity index, PI) of SLN-MTZ and CS-SLN-MTZ dispersions were determined by photon correlation spectroscopy and the zeta potential was analyzed by laser Doppler anemometry. Both measurements were made with a ZetaPlus Zeta Potential Analyzer (Brookhaven Instruments Corporation, Holtsville, NY). All samples were diluted appropriately with deionized water to get optimum concentration for measurements. In each case, the experiment was repeated in triplicate (n ¼ 3).

microscopy (TEM, JEM-200 CX, JEOL, Tokyo, Japan). A drop of the nanoparticle dispersion was mounted on a carboncoated copper grid, air-dried and then examined. Osmotic pressure and pH The osmotic pressure and pH for SLN-MTZ and CS-SLNMTZ were determined with FM-9X freezing-point osmometer (Instrumental Factory of Shanghai Medical University, Tianjin, China) and PHS-3C precise pH instrument (Shanghai precision & Scientific Instrument Co. Ltd., Shanghai, China) separately to confirm the biocompatibility for ophthalmic conditions. All analyses were repeated in triplicate (n ¼ 3). Physical stability SLN-MTZ and CS-SLN-MTZ dispersions were transferred into ampoules (10 ml), respectively, which were sealed for storage at 4  C in refrigerator for 6 months. The average size, zeta potential, entrapment efficiency and drug loading were determined with an interval time of 1 month.

Drug encapsulation efficiency and drug loading

In vitro studies

The drug encapsulation efficiency (EE) and drug loading (DL) were measured by ultrafiltration method. Briefly, 1 ml of SLN-MTZ/CS-SLN-MTZ colloidal solution was placed in the upper chamber of a centrifuge tube matched with an ultrafilter (Amicon Ultra 15, MWCO 100KD, Millipore, Cork, Ireland) and centrifuged for 10 min at 14 000 rpm at 4  C using a Sigma-3k30 High Speed Refrigerated Centrifuge (Sigma-Aldrich, Steinheim, Germany). After this process, liquid phase containing free drug moved into the sample recovery chamber through filter membrane, which was detected to determine the mass of free drug. The total drug in SLN/CS-SLN was obtained as follows: 1 ml of SLN-MTZ/ CS-SLN-MTZ, diluted in methanol to reach a volume of 25 ml, was sonicated for 15 min to dissolve the lipid ingredient and the obtained suspension was then filtrated through 0.45-mm membrane. The amounts of MTZ were detected by HPLC method which was reported by Li et al. [8]. The entrapment efficiency (EE %) and drug loading (DL %) were calculated separately by Equations (1) and (2) as follows:

In vitro drug release study

EE% ¼

DL% ¼

Wtotal drug  Wfree drug  100% Wtotal drug

ð1Þ

Wtotal drug  Wfree drug  100% Wtotal drug  Wfree drug þ Wemulsifiers þ Wlipid ð2Þ

where Wtotal drug is the mass of total MTZ in SLN/CS-SLN, Wfree drug is the mass of free MTZ detected in the sample recovery chamber after centrifugation of the aqueous dispersion, Wemulsifiers and Wlipid are the mass of emulsifiers and lipid initially used. Morphological examination The morphologies of SLN-MTZ and CS-SLN-MTZ nanoparticles were observed using a transmission electron

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The release of MTZ from nanoparticle dispersions was examined by dialysis method in simulated tear fluid (STF, composition: NaCl 0.68 g, NaHCO3 0.22 g, CaCl22H2O 0.008 g, KCl 0.14 g, and distilled deionized water to 100 ml) [31]. Two milliliters of MTZ solution, SLN-MTZ and CSSLN-MTZ suspensions were respectively poured in the dialysis membrane (12 000–14 000 MWCO, Sigma) with the openings tied and then suspended in 100 ml STF at 35 ± 0.5  C with agitation speed of 50 rpm. Aliquots (1 ml) were withdrawn at a predetermined time interval, meanwhile, 1 ml isothermal STF was added. All samples were filtered through a millipore filter (0.45 mm) and analyzed by HPLC. Each releasing experiment was performed in triplicate. The cumulative percentage of released drug Fn was plotted as a function of time (t, min), and calculated based on the following: Qn ¼ Cn  V0 þ

n1 X

Ci  Vi

ð3Þ

i¼1

Fn % ¼

Qn  100% ðC0  V0 Þ

ð4Þ

where Qn (mg/ml) stands for the cumulative amount of MTZ, Cn for the drug concentration of the dissolution medium at each sampling time, Ci for the drug concentration of the ith sample, V0 and Vi are the volumes of the dissolution medium and the sample, respectively. Ex vivo transcornea penetration study The ex vivo transcornea penetration property of SLN-MTZ and CS-SLN-MTZ was investigated on isolated rabbit corneas of New Zealand albino rabbits. Rabbit corneas were excised immediately after sacrifice and were placed between the epithelial donor and the endothelial acceptor chambers of modified Franz diffusion cells with an effective diffusion area

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J Drug Target, Early Online: 1–10

of 0.68 cm2 which was equilibrated with Glutathione-Sodium Bicarbonate Ringer’s solution buffer (GBR). GBR buffer is commonly applied to preserve freshly excised corneas and widely used in the corneal permeability studies [32–34]. It was prepared by mixing equal volume (1000 ml) of one buffer solution containing 12.4 g NaCl, 0.716 g KCl, 0.206 g NaH2PO4H2O, 4.908 g NaHCO3 with a second buffer supplemented with 0.23 g CaCl22H2O, 0.318 g MgCl25H2O, 1.8 g glucose and 0.184 g glutathione [35]. One milliliter of each sample (MTZ solution, SLN-MTZ and CS-SLN-MTZ) and 7.8 ml GBR were added into donor side and receptor side of the cornea, respectively. GBR was maintained at 35 ± 1  C with a constant magnetic stirring of 600 rpm. Every time 1 ml was withdrawn from the receptor at a predetermined time interval and an equal volume of GBR at the same temperature was added. The amount of drug solubilized in the acceptor phase was assayed by HPLC. All the experiments were conducted in three repeats. The cumulative penetration quantity at different intervals (Qn, mg/cm2), steady-state flux (Jss, mg/cm2/h) and apparent permeability coefficient (Papp, cm/h) were calculated by Equations (3), (4) and (5) as follows: ! n¼1 X 1 Vr C n þ Vs Ci Qn ¼ ð5Þ A i¼1 Jss ¼

DQ Dt

ð6Þ

Jss C0

ð7Þ

Papp ¼

where Vr (mL) is the volume of receiving compartment; A (cm2) is the area of the penetrating region; Cn (mg/mL) is the drug concentration in the donative compartment at different intervals; Vs (mL) is the sampling volume; DQ/Dt (mg/h) is the linear portion of the slope; C0 (mg/mL) is the initial drug concentration in the donative compartment [20]. Corneal hydration level Corneal hydration level was assessed with the gravimetric method [36]. At the end of penetration experiment, the cornea was removed from the apparatus. After the remaining sclera for fixation being carefully removed, the cornea was gently blotted dry and weighed. The corneal sample was then desiccated at 70  C for 12 h and weighed again. The percent corneal hydration level (HL%) was calculated by the following equation:   1  Wb HL% ¼  100% ð8Þ Wa where Wa and Wb are the wet corneal weight and dry corneal weight, respectively. In vivo studies Pharmacodynamics study The pharmacodynamics study was conducted in a crossover, randomized and double-blinded design. The IOP was measured as a function of time using an indentation tonometer

(YJI, Suzhou Min Green Medical Apparatus and Instruments Co. Ltd., Suzhou, China). Since the contact of the tonometer can produce some discomfort, rabbit eyes were anesthetized topically with 1 drop of 0.2% lidocaine hydrochloride (Shun Jian Pharmaceutical Co., Ltd., Guizhou, China) before the measurement. In the first stage, a drop of treatment solution was placed on the left eyes of the rabbits while the right eyes were instilled with the same volume of physiological saline served as control. The eyes were checked regularly and no inflammation was observed during the experiment. One week time was given for drug clearance before the second stage began. In this case, the left eyes were treated with physiological saline and the right eyes were treated with treatment solution. Three readings of IOP were taken on each eye and the mean IOP was calculated (IOPcontroleye and IOPtreatment eye). In this study, the treatment solutions include blank CSSLN, SLN-MTZ, CS-SLN-MTZ and commercial Brinzolamide Eye Drops (AZOPTÕ ). All IOP measurements were made at the same time of day with the same operator and DIOP % could be calculated by the following equation: DIOP% ¼

IOPcontrol eye  IOPtreatment eye  100% IOPcontrol eye

ð9Þ

The pharmacodynamic parameters [37] taken into consideration were maximum percentage decrease in IOP (DIOP %), area under percentage decrease in IOP versus time curve (AUC0–8 h), and mean residence time (MRT). Dates were analyzed by Bioavailability Program Package (BAPP 2.2) software (Nanjing, China). Ocular irritation Ophthalmic irritation is a common drawback in ocular drug development and often restricts their clinical use. In this study, 18 New Zealand albino rabbits were used to study the acute ocular tolerance to SLN-MTZ, CS-SLN-MTZ and Brinzolamide Eye Drops (AZOPT) according to a modified Draize test [38]. Before experiment, the animal models were examined to ensure that their ocular surface structures were normal and no clinical signs existed. They were randomly divided into three groups of six animals, and each received 50 ml of one of the three sterilized formulations in the lower conjunctival sac of the left eye every 30 min for 8 h. The contralateral eye was used as control and received the same amount of physiological saline. To prevent loss of test formulations, the upper and lower eyelids were gently held together for approximately 30 s. Then, animal discomforts and symptoms in the conjunctiva, cornea, and lids were evaluated with a slit lamp microscope. Histological examination Twelve albino New Zealand rabbits of either sex with no ophthalmic problems were randomly divided into three groups. Each group received a sample of SLN-MTZ, CSSLN-MTZ or Brinzolamide Eye Drops individually three times a day for 14 consecutive days. The right eyes were administered with samples while the left eyes receiving the same amount of saline served as control. The animals were then euthanized by air embolism after being deeply anesthetized with intravenous injection of anesthetic.

DOI: 10.3109/1061186X.2014.939983

Eyeball and lid tissues were removed, fixed in 10% formaldehyde solution, and embedded in paraffin for a pathology study. Histological sections (5 mm) were made, stained with hematoxylin and eosine (H and E), and microscopically observed for any pathological modifications using a microscope (BX51W1, Olympus Optical Co., Ltd, Tokyo, Japan).

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Statistical analysis of the results was performed using Student’s t-test and one-way analysis of variance (ANOVA). The statistical analysis was computed with the SPSSÕ software. Differences were considered significant when p50.05.

demonstrated that in the crystal regions of CS, the reaction occurred at the outer layer which was peeled off when it was sufficiently depolymerized to become soluble in the reaction medium, while in the amorphous regions, it occurred by the penetration of the medium [30,40]. The molecular weights of CS at degradation times 1.5 h, 2.5 h, 3.5 h and 4.5 h were 10220 Da, 6569 Da, 4468 Da and 2251 Da, respectively, according to the Mark-Houwink equation [Z] ¼ 8.443  103M0.847 with Ubbelohde viscometer [30]. With the increase in the degradation time, the smaller molecular weight of CS was obtained. In this study, we selected 6569 Da CS for the modification of SLN by optimization (data not shown).

Results and discussion

Characterization

Preparation and molecular weight determination of chitosan

Zeta potential and average diameter

Statistics

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Methazolamide-loaded solid lipid nanoparticles

Degradation of CS with hydrogen peroxide is based on the formation of reactive hydroxyl radicals to break the 1,4-b-Dglucoside bonds of CS [39]. A peeling-off depolymerization pattern was proposed initially by Schweiger. And myriads of subsequent researches have validated this hypothesis and Figure 1. Zeta potential, particle size and transmission electron micrograph of SLNMTZ (a, c, e) and CS-SLN-MTZ (b, d, f).

The mean zeta potential and diameter of the nanoparticles are exhibited in Figure 1. The addition of CS in the preparation of SLN-MTZ resulted in statistically larger particle size (p50.05). Such increase is attributable to the CS coating around the surface of SLN-MTZ. CS has the ability to swell in with the presence of water [41]. The PI values of both

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Table 1. Physical–chemical properties of MTZ-loaded solid lipid nanoparticles (SLNs) prepared without and with the modification of chitosan (mean ± SD, n ¼ 3).

Table 2. Effects of storage time (at 4  C) on the stability of CS-SLN-MTZ and SLN-MTZ. SLNs

SLNs

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SLN-MTZ CS-SLN-MTZ

pH

Osmotic pressure (mOsm/l)

EE%

DL%

4.9 ± 0.2 5.2 ± 0.2

286.7 ± 4.5 307.7 ± 3.5

54.0 ± 4.3 58.3 ± 3.6

10.3 ± 0.8 9.9 ± 0.6

SLN-MTZ and CS-SLN-MTZ were within 0.1–0.2, indicating a narrow particle size distribution. SLN-MTZ showed a zeta potential of 21.3 ± 1.9 mV. After the coating with CS, a surface charge conversion of SLN was accomplished. The coating mechanism involved hydrogen bonding and electrostatic interaction between the polysaccharide and the phospholipid. The result of zeta potential (+31.3 ± 1.7 mV) of CS-SLN-MTZ also supported the conclusion that CS adsorption occurred. Drug encapsulation efficiency and drug loading The EE and DL of both SLN and CS-SLN at different MTZ feeding were investigated (data not shown). With increase in MTZ feeding, the EE decreased slightly, meanwhile, the DL first increased markedly and then kept almost constant (about 10%). In order to load MTZ as much as possible, a high amount of MTZ (35 mg) was used in the preparation. The DL and EE of the optimized nanoparticles for the next experiments were shown in Table 1. From the data, it is not difficult to find that there was no difference in the EE of SLN and CSSLN. As a hydrophobic drug, MTZ was located within the hydrophobic core of SLNs. CS was only expected to interact with the shell of SLN. In this case, CS coating has no significant influence on MTZ loading. Morphological examination TEM of SLN and CS-SLN with MTZ are presented in Figure 1. Both microscopic images showed particles of analogous spherical morphologies and compact structures with smooth surface. There was obvious halo at the surface of CS-SLN-MTZ while little at the surface of SLN-MTZ, which may be caused by the adhesion of CS. The diameters of the SLN-MTZ and CS-SLN-MTZ nanoparticles based on the TEM were about 150 nm and 200 nm both smaller than the diameters determined by photon correlation spectroscopy. For the TEM experiments, nanoparticles were in dry state. While for the photon correlation spectroscopy experiments, they were dispersed in an aqueous phase, in this case, hydrodynamic layers might form around the particles which may lead to overestimated sizes [42]. Osmotic pressure and pH In order to accomplish comfort and ease for ophthalmic application, the proper pH (3.5–8.5) and osmotic pressure (100–640 mOsm/l) are suggested as the tear is able to adjust the pH and osmolality to physiologic levels after administration [43]. In this study, formulations of CS-SLN-MTZ and SLN-MTZ presented mild acidic pH values and their osmotic pressures were about 300 mOsm/l (Table 1). Both pH and

CS-SLN-MTZ 0d 1 month 2 months 3 months 4 months 5 months 6 months SLN-MTZ 0d 1 month 2 months 3 months 4 months

EE%

Particle size (nm)

Zeta (mV)

62.82 62.32 57.12 52.97 57.32 56.84 57.68

244.4 242.9 249.5 256.0 254.2 321.2 882.8

+31.30 +29.33 +32.28 +33.64 +28.16 +20.11 +10.63

54.81 55.47 59.50 61.81 55.55

193.4 201.3 277.5 818.7 892.1

20.94 21.77 20.94 14.31 11.27

osmolality ere within the acceptable range and were suitable for in vivo study. Physical stability The results of the physical stability experiments for SLNMTZ and CS-SLN-MTZ are shown in Table 2. SLN-MTZ exhibited an extreme increase in particle size and the zeta potential dropped to 14.31 mV during the third month. CSSN-MTZ maintained a relative stable state with particle sizes within 350 nm and zeta potentials above +20 mV during the first 5 months after preparation. But for the sixth month, CSSLN exhibited a gargantuan average diameter of 882.8 nm due to coagulation. And the zeta potential dropped to +10.63 mV abruptly. For both SLN-MTZ and CS-SN-MTZ, the encapsulation efficiencies did not seem to be affected distinctly as MTZ was expected to be incorporated within the core of SLNs. In appearance, SLN-MTZ got mildewed after 4 months’ storage making it unable to be further analyzed while CSSLN-MTZ showed no significant changes in physical state and was equally opalescent except some aggregating deposits after 6 months’ storage. The different phenomenon could properly be attributed to CS’s anti-bacterial property [44]. Compared with SLN-MTZ, CS-SLN-MTZ showed a better stability. In vitro studies In vitro drug release study The results of the drug release experiments (dialysis bag method) from SLN-MTZ and CS-SLN-MTZ suspensions are shown in Figure 2 with MTZ solution as control. An accumulated quantity of 99.92% was released within 60 min for MTZ solution compared to 45.22% and 38.83% for SLNMTZ and CS-SLN-MTZ. SLN-MTZ and CS-SLN-MTZ both represented a sustained drug release behavior over a period of 8 h. By comparison, CS-SLN-MTZ showed a longer release profile of MTZ than SLN-MTZ. It is probably due to the adhesive property of CS, which can form hydrophilic compact matrix layer around nanoparticle leading to the retardant effect of the lipophilic drug of MTZ [45].

Methazolamide-loaded solid lipid nanoparticles

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DOI: 10.3109/1061186X.2014.939983

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Figure 2. In vitro release profiles of MTZ solution, SLN-MTZ and CS-SLN-MTZ (mean ± SD, n ¼ 3). Figure 4. Percentage decrease in IOP after administration of CS-SLN without loading MTZ (CS-SLN), SLN-MTZ, CS-SLN-MTZ and commercial product (mean ± SD, n ¼ 6).

Furthermore, the positive charge of CS-SLN-MTZ facilitates its binding to negatively charged cornea, which may be beneficial for cellular uptake and internalization by transcellular pathway [47]. The results from this work indicated that the coating of CS affected markedly the interaction of CS-SLN-MTZ with the ocular mucosa and CS-SLN-MTZ had a positive effect on its in vivo success as a drug carrier. Evaluation of corneal hydration levels

Figure 3. In vitro transcorneal permeation of SLN-MTZ and CS-SLN-MTZ (mean ± SD, n ¼ 3).

Table 3. Steady-state flux (Jss), apparent permeability coefficient (Papp) and corneal hydration levels (HL) of CS-SLN-MTZ and SLN-MTZ (mean ± SD, n ¼ 3).

Formulations

Jss  102 (Jss, mg/cm2/h)

Papp  105 (cm/h)

HL%

CS-SLN-MTZ SLN-MTZ

4.02 ± 0.36 1.71 ± 0.50

3.45 ± 0.31 1.47 ± 0.43

76.40 ± 1.27 76.05 ± 1.69

Ex vivo transcornea penetration study The transcornea penetration profile of CS-SLN-MTZ was significantly different from that of SLN-MTZ (Figure 3, p50.05). Although the release rate of MTZ from SLN-MTZ was faster than that of CS-SLN-MTZ, the results of cornea penetration rate were reversed. As listed in Table 3, the apparent permeability coefficient (Papp) of CS-SLN-MTZ was about 2–3 times higher than that of SLN-MTZ, reflecting CS-SLN-MTZ performed better drug permeability than SLNMTZ. The mechanism for enhancing corneal permeation of CS-SLN-MTZ may involve the intrinsic abilities of CS [46]. As a penetration enhancer, CS can promote CS-SLN-MTZ to cross the mucosal barriers through paracellular pathway by reversibly disrupt the epithelial tight junctions [6,47].

The hydration level (HL %) of the corneal tissue has been considered as a sensitive indictor of tissue integrity [31]. It is reported that a limited speed of fluid uptake may be expected for the excised eye cornea from the surrounding environment, which is determined by the low permeability of the epithelium and endothelium suggesting the integrity of cornea, while damage of the epithelium causes the hydration to increase [48] The normal water weight contained in the rabbit cornea ranges between 75% and 78%. A corneal damage being indicated by hydration levels increased to 83% or more [33,49]. After the experiment of ex vivo transcornea penetration study, each cornea was carefully examined of its integrity in appearance. The HL% values for SLN-MTZ and CS-SLN-MTZ were listed in Table 3. All values were less than 78%, indicating that no damage had ever occurred on the corneas during the studies [50]. In vivo studies Pharmacodynamics study Different formulations (blank CS-SLN, SLN-MTZ, CS-SLN-MTZ and the commercial Brinzolamide Eye Drops (AZOPTÕ )) were administered ocularly to evaluate their therapeutic efficacies. The decrease in IOP at serial time points generated the data shown in Figure 4. The effect of CSSLN-MTZ on IOP reduction could sustain for more than 8 h, while SLN-MTZ about 6 h. The relevant pharmacokinetic parameters are presented in Table 4. The mean residence time (MRT) of CS-SLN-MTZ was longer than that of MTZ-SLN.

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Table 4. Pharmacodynamic parameters after administration of AZOPT, SLN-MTZ and CS-SLN-MTZ (mean ± SD, n ¼ 6). Pharmacodynamic parameters Formula AZOPT SLN-MTZ CS-SLN-MTZ

DIOP%max

AUC0–8 h (mmHg  h)

MRT (h)

38.06 ± 1.25 27.82 ± 4.15 42.78 ± 7.71

171.17 ± 16.45 126.74 ± 17.73 245.75 ± 18.31a

3.91 ± 1.83 3.92 ± 1.97 5.13 ± 2.03

AUC0–8 h (area under the percentage decrease in IOPtime curve); MRT (mean residence time). a Indicates significant difference from both the group of SLN-MTZ and AZOPT (p50.05).

Table 5. Results of irritant ocular test of different preparations.

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Degree of irritation Group

AZOPT

MTZ-SLNs

MTZ-CS-SLNs

1 Right eye Left eye

1 0

1 0

0 0

Right eye Left eye

0 0

0 0

2 0

Right eye Left eye

2 0

0 0

0 0

Right eye Left eye

1 0

1 0

0 1

Right eye Left eye

0 0

0 0

0 0

1 0 5 1 0 0

1 0 3 0.5 0 0 Non-irritant

1 0 4 0.7 0 0

2 3 4 5 6 Right eye Left eye Total scores of right eyes Average scores of right eyes Total scores of left eyes Average scores of left eyes Results

The difference may be ascribed to the mucoadhesive property of CS, which was reported as a potential component for enabling increased precorneal drug residence time [51]. The enhanced MRT can lead to significant increases in bioavailability and reduced drug wastage. In this study, the area under the percentage decrease in IOP (AUC0–8 h) of CS-SLN-MTZ was 2 times as high as that of SLN-MTZ and even higher than that of AZOPTÕ (about 0.5 times higher in AUC0–8 h). Besides, the maximum percentage decrease in IOP of CSSLN-MTZ was higher than SLN-MTZ and was similarly equal with AZOPTÕ. Typically, the poor bioavailability of ocular formulation arises from fast elimination due to preocular loss factors (lachrymation, tear turnover, nasolachrymal drainage, metabolic degradation and non-productive desorption/absorption) and the relative impermeability of the cornea [52]. CSSLN-MTZ with hydrophilic and positively charged surface showed considerable advantages compared with SLN-MTZ. At physiological pH, the corneal epithelium is negatively charged [53] which favored the transcellular permeability of positively charged particles. The ability to reversibly open up the epithelial tight junctions promotes the paracellular transportation of drug [6,47]. Besides electronic interaction, the highly adhesive nature of CS would ensure the adherence of CS-SLN-MTZ to the surrounding membranes preventing drainage with tears and providing sustained release of drug. In short, all the results above indicated the enhancing effect of CS on the transcorneal permeation as we expected. CS is a potentially suitable candidate for the modification of SLNs to enhance their bioavailabilities. Ocular irritation The ocular irritation of multiple administrations among albino rabbits is summarized in Table 5 according to the Draize method [53]. No significant differences were discovered in any test formulation-exposed eyes compared to control eyes (physiological saline) except some of them showing a slight reflex lacrimation. MTZ-SLN, MTZ-CS-SLN and

Figure 5. Pathological photos of iris and retina of rabbit eyes under the investigation of irritation. a, b, c and d represent the iris of rabbit eyes given saline, CS-SLN-MTZ, SLN-MTZ and AZOPTÕ respectively. a0 , b0 , c0 and d0 represent the retina of rabbit eyes given saline, CS-SLN-MTZ, SLN-MTZ and AZOPT, respectively. Iris and retina from both control and treated eyes displayed normal cell layers and morphology. No signs of tissue edema were observed in any structure studied after exposure to CS-SLN-MTZ, SLN-MTZ or AZOPT compared with controls.

DOI: 10.3109/1061186X.2014.939983

Brinzolamide Eye Drops (AZOPT) were well tolerated in rabbit eyes, and no ocular damage or clinically abnormal signs in various ocular structures (including cornea, conjunctiva or iris) were observed, indicating excellent ocular tolerance.

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Histological examination Histological sections of rabbit eyeballs are presented in Figure 5 to discover the pathological changes in the experimental rabbit eyes treated with different formulations. For all groups, each rabbit was convinced with no angiogenesis, pigment off in iris and no cellular necrosis was found in retina. Cornea, lens, ciliary body and optic nerve were also examined. Cornea kept its epithelial integrity and showed no signs of stromal keratitis, stromal hyperplasia and texture thickness. There was no turbidity, epithelial hyperplasia in lens. Ciliary body and optic nerve showed no edema or necrosis. All these observations conclude that SLN and CSSLN are non-toxic to the ocular tissues.

Conclusion The results obtained in this work suggest that SLN with the modification of CS have a great potential for topical delivery due to its positive zeta potential, high physical stability, enhancement of transcorneal permeation and absence of cytotoxicity in vivo. For ophthalmic applications, CS-SLN not only provides ease of application just like eye drops but also circumvents the problems associated with the conventional systems, with the added advantages of an extending period of time and being patient-friendly. The coating of CS endowed SLN with favorable biological properties and CS-SLN is potentially useful as a drug carrier for the treatment of local ophthalmic diseases.

Declaration of interest This study was financially supported by the National Natural Science Foundation of China (No. 81100977). The authors report no conflict of interest.

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