Diclofenac-loaded biopolymeric nanosuspensions for ophthalmic application

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Nanomedicine: Nanotechnology, Biology, and Medicine 5 (2009) 90 – 95 www.nanomedjournal.com

Original Article: Pharmacological Nanomedicine

Diclofenac-loaded biopolymeric nanosuspensions for ophthalmic application Sagar M. Agnihotri, PhD,⁎ Pradeep R. Vavia, PhD Pharmaceutical Division, Institute of Chemical Technology, University of Mumbai, Matunga, Mumbai, India

Abstract

Key words:

Polymeric nanoparticle suspensions (NS) were prepared from poly(lactide-co-glycolide) and poly (lactide-co-glycolide-leucine) {poly[Lac(Glc-Leu)]} biodegradable polymers and loaded with diclofenac sodium (DS), with the aim of improving the ocular availability of the drug. NS were prepared by emulsion and solvent evaporation technique and characterized on the basis of physicochemical properties, stability, and drug release features. The nanoparticle system showed an interesting size distribution suitable for ophthalmic application. Stability tests (as long as 6 months' storage at 5°C or at 25°C/60% relative humidity) or freeze-drying were carried out to optimize a suitable pharmaceutical preparation. In vitro release tests showed a extended-release profile of DS from the nanoparticles. To verify the absence of irritation toward the ocular structures, blank NS were applied to rabbit eye and a modified Draize test performed. Polymer nanoparticles seemed to be devoid of any irritant effect on cornea, iris, and conjunctiva for as long as 24 hours after application, thus apparently a suitable inert carrier for ophthalmic drug delivery. © 2009 Elsevier Inc. All rights reserved. Diclofenac sodium; Biodegradable polymer; Nanosuspension; Ophthalmic delivery; Stability

The eyes are among the most readily accessible organs in terms of their location in the body, and yet drug delivery to the tissues of the eye is particularly problematic. Conventional eyedrops deliver drugs in a pulsatile fashion, with a very brief period of overdosing immediately after instillation, followed by a rapid decline to insignificant levels, usually within minutes. As well, most topically applied drugs are extremely limited in their ability to penetrate the various ocular tissues as a result of various anatomical and physicochemical barriers. There is a clear need for effective topical formulations capable of promoting drug penetration and maintaining therapeutic levels with a reasonable frequency of application. Such enhancement could be affected by altering the

Received 10 March 2008; accepted 21 July 2008. The authors wish to express their thanks to Council of Scientific and Industrial Research (government of India, New Delhi) for financial support. ⁎Corresponding author. E-mail address: [email protected] (S.M. Agnihotri).

solubility characteristic of the drug molecule or by manipulation of the delivery system. Increasing the permeability characteristic of a drug will not on its own eliminate the problem of pulsed entry. In fact, this strategy could result in enhancement of side effects that probably would not be acceptable. The development of an appropriate delivery system could increase the contact time of a drug with the eye surface and might promote or facilitate transfer of drug molecules from the tear phase into the eye tissue. In this role, a controlled or sustained delivery of ophthalmic drugs offers many potential benefits.1 Use of polymeric nanoparticles is one of the most interesting approaches to achieving local controlled drug delivery.2-4 Polymer nanosuspensions (NS) of nonsteroidal antiinflammatory agents (NSAIDs) have often been proposed as controlled drug delivery systems able to solve pharmacokinetic problems and/or the gastric damaging effects typical of most of these drugs.5,6 The polymers that seem to have the best potential for this application are poly(lactide-co-glycolide) (PLGA), poly

1549-9634/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.nano.2008.07.003 Please cite this article as: S.M. Agnihotri, P.R. Vavia, Diclofenac-loaded biopolymeric nanosuspensions for ophthalmic application. Nanomedicine: NBM 2009;5:90-95, doi:10.1016/j.nano.2008.07.003

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Table 1 Formulative variables of DS-loaded polymeric nanosuspensions ⁎ Batch no. †

Drug-to-polymer ratio

Particle size (nm ± SE)

PI (nm ± SE)

Drug loading (% ± SE) ‡

ζ-Potential (mV ± SE)

A1

1:1

125 ± 4.2

0.995 ± 0.08

28.12 ± 1.2

–25.2 ± 1.3

A2

1:2

174 ± 3.5

0.801 ± 0.06

36.44 ± 1.4

–25.4 ± 1.2

A3

1:10

130 ± 5.0

0.613 ± 0.08

95.44 ± 1.1

–25.8 ± 1.4

B1

1:1

190 ± 4.5

0.535 ± 0.08

17.18 ± 1.3

–25.4 ± 1.6

B2

1:2

167 ± 5.2

0.416 ± 0.09

32.83 ± 1.6

–25.6 ± 1.7

B3

1:10

160 ± 4.4

0.281 ± 0.06

73.60 ± 1.7

–25.3 ± 1.2

PI, polydispersity index. ⁎Data are the mean of three determinations ± SE. † Nanosuspensions formulated from poly[Lac(Glc-Leu)] polymer (A batches) and PLGA polymer (B batches). ‡ Drug loading % = Total amount of drug taken – drug measured in supernatant/Total amount of drug taken × 100.

(alkylcyanoacrylate) (PACA), and poly(ɛ-caprolactone) (PCL). 7 These polymers have been studied for their applications or interactions with the eye and are found to have good biocompatibility.8 Diclofenac sodium (DS) is one of the few NSAIDs used to treat ocular inflammatory conditions (e.g., to prevent the myosis induced by surgical trauma, as during cataract extraction).9 In this study we evaluated new colloidal systems for DS ophthalmic delivery. Drug-loaded poly[Lac(Glc-Leu)] (PLDL) and poly(lactide-co-glycolide) (PLGA)—DSPLDL and DS-PLGA NS—were prepared by means of a emulsion and solvent evaporation method, to achieve particles with dimensional and stability characteristics compatible with the ocular application.

Methods

a low temperature in an icewater bath. During injection the mixture was vigorously mixed at an agitation speed of 4000 rpm. The resulting emulsion obtained was sonicated in a probe-type sonicator, and further stirring was continued for 60 minutes. Solvent residues were left to evaporate off under a slow magnetic stirring of the NS at room temperature (20°23°C) for 8–12 hours (Table 1). To the formed NS, an aqueous solution containing 5% (w/v) of mannitol solution (5 mL) was added under stirring to give a concentration of 0.1% (w/v) of DS. The final formulation was sterilized by filtration through 0.22-μm membrane filter and filled in presterilized glass vials. For drug content analysis the NS formulations were centrifuged at 20,000 rpm, 5°C for 20 minutes, and the amount of drug in the supernatant was measured by ultraviolet (UV) spectrophotometer at 277 nm. Blank polymeric NS were formulated by similar procedure without drug to be used in the eye irritancy evaluation. Particle size and zeta potential

Materials DS was a gift from Amoli Organics Ltd. (Mumbai, India); PLGA (75:25), MW = 11,000 Da, was obtained from Boehringer Ingelheim Pharma KG, Ingelheim, Germany), and PLDL, MW = 7474 Da, was used as synthesized in our laboratory.10 Tween 80 and benzalkonium chloride (50%, w/v) were obtained from Sigma-Aldrich Chemie GmbH (Steinheim, Germany). All other chemicals were of analytical grade. Preparation of nanosuspensions Drug-containing NS were prepared according to the o/w emulsion method11 using probe ultrasonicator (60 Hz, 20 cycles/3 sec; Branson, Cleveland, Ohio). Briefly, a solution of copolymer (PLDL or PLGA) dissolved in methylene chloride (5 mL) was mixed with a solution of DS dissolved in methanol (2 mL) under stirring This solution was then slowly injected (0.5 mL/min), with a syringe connected to a thin Teflon tube, into 45 mL water containing Tween 80 (0.02%, w/v) and benzalkonium chloride (0.1%, w/v) kept at

The mean particle sizes and polydispersity index (PI) of the formulations were determined by particle size analyzer (Beckman Coulter N+ Plus, Wipro, India) equipped with software N4 Plus. Every sample was appropriately diluted with HPLC-grade water filtered with a 0.22-μm membrane, and the reading was carried out at a 90-degree angle with respect to the incident beam. The zeta potential (ζ) was calculated from electrophoretic mobility using the Henry equation: lE ¼

eff ðKaÞ 6pg

where μE is the electrophoretic mobility, ɛ is the dielectric constant of the medium, ζ is the viscosity of the medium, K is the Debye-Hückel parameter, and f(Ka) is a correction factor that takes into account the thickness of the double layer and particle diameter (a). The K unit is a reciprocal length. 1/K is frequently described as the thickness of the electrical double layer.

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Table 2 Effects of storage time and conditions on the mean size and drug loading values of DS-loaded polymeric formulations ⁎ Batch No.

Mean size (nm) ± SD Initial values

After 6 months at 5°C (±2°C)

After 6 months at 25°C/60% RH

Initial values

After 6 months at 5°C (±2°C)

After 6 months at 25°C/60% RH

A3 †

130 ± 5.0

215 ± 7.3

218 ± 9.8

95.44 ± 1.1

93.33 ± 3.2

93.56 ± 3.8

†

Drug loading (%) ± SD

160 ± 4.4

193 ± 5.2

223 ± 6.0

73.60 ± 1.7

72.36 ± 1.5

71.86 ± 1.2

A3 ‡

134 ± 6.3

220 ± 9.8

225 ± 12.4

95.20 ± 1.0

92.01 ± 1.2

92.39 ± 1.6

B3 ‡

167 ± 4.6

198 ± 6.3

230 ± 7.2

74.86 ± 1.3

72.45 ± 1.4

71.95 ± 1.5

B3

⁎Data are the mean of three determinations ± SE. † Original nanosuspensions. ‡ Freeze-dried nanoparticles.

Solid-state characterization of DS-polymeric systems Infrared spectrophotometry IR spectra of freeze-dried nanoparticles were obtained with a Perkin-Elmer 1600 spectrophotometer (GMI Inc., Ramsey, Minnesota), using the potassium bromide (KBr) disk technique (about 10 mg sample for 100 mg dry KBr). X-ray diffractometry In x-ray studies an automatic x-ray diffractometer (Rigaku Dmax 2500, Rigaku, Tokyo, Japan) equipped with an x-ray generator was used. Nickel filtered Cu kα1 radiation having a wavelength of 1.5106 Å, operating at 35 Kw and 20 ma in the range (2θ) of 5 to 70 degrees was used. X-ray diffractograms were obtained at a scanning rate of 1 degree (2θ) per minute. Differential scanning calorimetry (DSC) Polymeric nanoparticle samples were separately sealed in aluminum cells and set in a Perkin-Elmer DSC6 apparatus (Uberlingen, Germany) between 30°C and 300°C. Thermal analysis was performed at a heating rate maintained at 10°C per minute in a nitrogen atmosphere. Alumina was used as the reference substance. In vitro drug release The in vitro drug release studies were performed in triplicate with some modification as described earlier.12,13 Briefly, drug-loaded NS were suspended in pH 7.4 phosphate buffer in glass vial. The glass vials were placed in a mechanical shaking bath (100 cycles/min), with temperature adjusted to 37°C. At selected time intervals sample was removed and replaced with fresh buffer. The sample was then centrifuged at 20,000 rpm, and supernatant was analyzed using a UV spectrophotometer. Stability studies of the formulations The physical stability of the NS was evaluated after storage for 6 months under different temperature conditions. Exact volumes of each NS were stored in closed glass bottles and placed at 5° ± 2°C (refrigerator) or at

25°C and 60% relative humidity (RH) away from direct light. Aliquots of 2 mL were withdrawn at 1-, 3-, and 6-month time intervals to measure particle size and drug loading, as described above. Freeze-drying and redispersibility of nanosuspensions NS stored at 5° ± 2°C and at 25°C and 60% RH were freeze-dried to verify the physical stability and the following redispersibility. Ten-milliliter aliquots of samples were frozen in liquid nitrogen and lyophilized by a Labconco Freeze-Dryer (FreezZone 1 liter benchtop model) (Labonco Corp., Kansas City, Missouri) for 24 hours at –40°C, and at a pressure of 0.05 mm Hg. The freeze-dried samples were rehydrated with the original volume of distilled water to restore the drug, and polymer concentrations and particle size changes were assessed as described above. For drug content analysis of solid nanoparticle (NP) formulation, the NPs (10 mg) were accurately weighed and dissolved in 2 mL dichloromethane. The polymer was precipitated using methanol (volume up to 10 mL). After centrifugation at 5000 rpm for 5 minutes the clear supernatant obtained was analyzed by UV spectrophotometer for the drug content. Eye irritancy evaluation Animals Male New Zealand albino rabbits weighing 1.8–2.2 kg and free of any signs of ocular inflammation or gross abnormality were used in the study. The study was performed in accordance with the Institutional Animal Ethics Committee (IAEC) constituted as per directions of the Committee for the Purpose of Control and Supervision of Experiments on Animals (87/1999/CPCSEA), under the Ministry of animal welfare division, Government of India, New Delhi. Two groups of ten rabbits each were used. Ocular tolerability The potential ocular irritancy and/or damaging effects of blank formulations were evaluated according to a modified Draize test14 using a slit-lamp. Observation of the ocular tissue condition was performed after 10 minutes, 6 hours, and 24 hours after the end of the experiments. The

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Figure 2. DSC of I: (A) diclofenac sodium, (B) PLDL, (C) physical mixture, (D) DS-PLDL NP system; II: (A) diclofenac sodium, (B) PLGA, (C) physical mixture, (D) DS-PLGA NP system. Figure 1. X-ray diffractograms of I: (A) diclofenac sodium, (B) PLDL, (C) physical mixture, (D) DS-PLDL NP system; II: (A) diclofenac sodium, (B) PLGA, (C) physical mixture, (D) DS-PLGA NP system.

congestion, swelling, and discharge of the conjunctiva were graded on a scale from 0 to 3, 0 to 4, and 0 to 3, respectively. In this hyperemia and corneal opacity were graded on a scale from 0 to 4.15 Blank formulations (50 μL) were topically administered in the right eye every 30 minutes for 6 hours (12 treatments); the left eyes were used as controls. At the end of the treatment, three observations at 10 minutes, 6 hours, and 24 hours were carried out to evaluate the ocular tissues. Corneal integrity was evaluated by methylene blue staining (1%, w/v in saline).16

Results The different formulative variables tested in this study showed a direct effect upon the amount of drug associated with the polymer matrices; drug loading was found to be maximum with a drug-to-polymer ratio of 1:10. Acceptable PI values were obtained for all preparations (Table 1). The average particle size values were measured immediately after the preparation of the NS or after as long as 6 months of storage at 5° ± 2°C, and at 25°C and 60% RH (Table 2). All batches showed mean sizes below 250 nm, therefore suitable for an ocular application.17 Upon storage the NS formed a sediment, which could be easily redispersed by manual agitation. Average size increased a little with respect to the initial values, probably because of particle aggregation, both in the refrigerator and at 25°C, 60% RH. Zeta potential results (Table 1) showed that drug-loaded formulations carried a negative charge around –25 mV, which promotes particle stability because the repulsive forces prevent aggregation with aging.18 It has been

reported elsewhere that the negative charge on PLGA nanospheres is due to the ionization of carboxylic end groups of surface polymer.19 The results presented here are in accordance with those obtained for similar systems.20 As shown elsewhere,6 using benzalkonium chloride in these formulations ensures good microbiological stability over time. The pH value of the prepared NS was always close to that of pure water (5.8–6.5), therefore compatible with ocular administration. No significant changes in drug content were registered up to 6 months after preparation at both of the selected storage conditions. The described polymeric NS could be freeze-dried, forming a solid residue that can be easily redispersed by hand agitation, without evident size and drug content (measured in solid particles) changes. The results of the analysis are shown in Table 2. A preliminary evaluation in the solid state, IR spectrophotometry, powder x-ray diffractometry, and DSC analysis of the prepared NP systems showed that the drug is dispersed in the polymeric matrices in a microcrystalline form, without polymorph change or transition into an amorphous form. The Fourier transform IR spectrum of DS showed N-H stretch at 3369 cm–1, aromatic stretching – CH stretching at 2971 cm–1, and substituted phenyl group stretch at 748 cm–1. These values remained the same in both NP systems, indicating no existence of different association forms of DS with polymers. In powder x-ray diffractometry spectra of freeze-dried suspensions by the increased polymeric weight fraction, the intensities of typical drug peaks were lowered as a result o the dilution effect exerted by the polymer network, but without a qualitative variation of drug diffractogram,21 our results for the analyzed 1:10 drug-to-polymer systems are in agreement with previous studies (Figure 1). This observation was further confirmed by DSC studies (Figure 2). DS

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Discussion

Figure 3. Release profiles of optimized DS polymeric formulation of batches A3 and B3 at pH 7.4. Data are the mean of three determinations ± SE.

shows a degradation endotherm near 280°C, which was identical with DS-loaded NPs of PLDL (batch A3); however, in the case of DS-PLGA NPs (batch B3), because of the dilution effect of the polymer network no drug peak was observed. Moreover, in case of DS-PLDL NPs there was increase in the glass transition temperature from 36.27°C to 83.77°C; these results suggest that dense morphology and higher degree of crystallinity of blended nanoparticles can thereby be considered an improvement of the storage stability of the system.22 The in vitro release profiles of optimized formulations, DS-PLDL (batch A3) and DS-PLGA (batch B3) NS is shown in Figure 3. In both polymeric formulations the release of DS was complete in all cases, and formulations tested showed a biphasic release pattern: one initial fast release followed by a second slow-release phase (extended release). In vitro release tests were repeated in both the polymer systems after six months of storage at 5° ± 2°C and at 25°C, 60% RH, no relevant differences were observed in the amount of DS released. These findings indicate that both the DS-polymer systems have a homogeneous structure that did not change much during storage, although dissolution and particle reaggregation phenomena would have occurred in the external aqueous phase. For a polymeric drug delivery to be proposed as an ophthalmic drug carrier, it is important not only to assay the biopharmaceutical properties but also the ocular tolerability. Therefore, in vivo ocular irritancy toward the PLDL and PLGA NS was determined following a modified Draize test protocol. The in vivo results showed no sign of irritation or damaging effects to ocular tissues in rabbit eyes. The scores for conjunctival swelling and discharge were always zero. Iris hyperemia and corneal opacity scores were zero at all observations. Therefore, the potential clinical interest of the PLDL and PLGA NS is supported because of the absence of irritant effects in vivo.

For in vitro release profiles the burst effect could be attributed to the escape of drug from the surface of the polymeric system; then the DS present in the polymeric matrix diffuses to the release medium through the pores and channels of the polymeric NPs. The type of the polymer also affected drug release; PLDL copolymer is more hydrophilic as compared with PLGA copolymer, but the release rate from the DS-PLGA system was slightly faster as compared with the DS-PLDL system. This phenomenon could be due to the amorphous nature of the PLGA copolymer. Another reason for the rapid release from such systems could be that the drug diffuses easily out of the polymeric systems when colloidal systems are placed in release medium at pH 7.4, a pH value higher than the pKa of the drug.23 pH change promoted DS ionization and distribution of the ionized molecules in the release medium to a faster extent. Taking into account the degradation of the PLDL copolymer10 and PLGA copolymer24 in the release of DS from the polymeric systems, no significant degradation of PLDL and PLGA occurred in buffer solution as measured by molecular weight loss at 37°C after 16 days. For this reason the drug release cannot be explained by the erosion of the polymeric matrix. Furthermore, the homogeneous and finer dispersion of drug molecules in the polymer matrix at lower drug concentration enhances dissolution, allowing a better penetration of the dissolution medium through NPs. Such behavior would indicate that the drug solubility in the type of the release medium is more important than its diffusion through the polymer network in delineating the final dissolution pattern. The dispersion of DS in the polymer matrices led to a gradual dissolution and release of the drug, which became complete within 14 hours. In conclusion, the co-dispersion of DS in selected polymers resulted in NS that showed good mean sizes for ophthalmic applications. The suspensions allowed for improved corneal adhesion and stability upon storage, particularly at low temperatures. For the possibility of modulating the preparation conditions and the stability shown upon storage as the original suspensions, as well as for the very good tolerability, the described formulations may be useful in clinical practice for a valid therapeutic approach for the topical treatment of inflammatory conditions of the eye. References 1. Zimmer A, Serbe H, Kreuter J. Evaluation of pilocarpine-loaded albumin particles as drug delivery systems for controlled delivery in the eye I. In vitro and in vivo characterization. J Control Release 1994;32: 57-70. 2. Li VH, Wood RW, Kreuter J, Harmia T, Robinson JR. Ocular drug delivery of progesterone using nanoparticles. J Microencapsul 1986;3: 13-218. 3. Marchal-Heussler L, Sirbat D, Hoffman M, Maincent P. Poly (ɛ-caprolactone) nanocapsules in carteolol ophthalmic delivery. Pharm Res 1993;10:386-90.

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