Sparfloxacin-loaded PLGA nanoparticles for sustained ocular drug delivery

POTENTIAL CLINICAL RELEVANCE

Nanomedicine: Nanotechnology, Biology, and Medicine 6 (2010) 324–333

Original Article

Sparfloxacin-loaded PLGA nanoparticles for sustained

Himanshu Gupta, B.Pharm, M.Pharma,b,1, Mohammed Aqil, M.Pharm, PhDa,⁎,Roop K. Khar, M.Pharm, PhDa, Asgar Ali, M.Pharm, PhDa,Aseem Bhatnagar, MBBS, MDb, Gaurav Mittal, MSc, PhDb

aDepartment of Pharmaceutics, Faculty of Pharmacy, Jamia Hamdard, New Delhi, IndiabDepartment of Nuclear Medicine, Institute of Nuclear Medicine and Allied Sciences, Ministry of Defence, New Delhi, India

Received 14 June 2009; accepted 8 October 2009

www.nanomedjournal.com

Abstract

Poor ocular bioavailability of drugs (b1%) from conventional eye drops (ie, solution, suspension, and ointments) is mainly due to thephysiologic barriers of the eye. In general, ocular efficacy is closely related to ocular drug bioavailability, which may be enhanced byincreasing corneal drug penetration and prolonging precorneal drug residence time. In our current work, we develop and evaluate a newcolloidal system, that is, poly(dl-lactide-co-glycolide) (PLGA) nanoparticles for sparfloxacin ophthalmic delivery, to improve precornealresidence time and ocular penetration. Nanoparticles were prepared by nanoprecipitation technique and characterized for variousproperties such as particle size, zeta potential, in vitro drug release, statistical model fitting, stability, and so forth. Microbiological assaywas carried out against Pseudomonas aeruginosa using the cup-plate method. Precorneal residence time was studied in albino rabbits bygamma scintigraphy after radiolabeling of sparfloxacin by Tc-99m. Ocular tolerance of the developed nanosuspension was also studiedby the Hen Egg Test-Chorioallantoic Membrane (HET-CAM) method. The developed nanosuspension showed a mean particle size inthe range of 180 to 190 nm, suitable for ophthalmic application with zeta potential of –22 mV. In vitro release from the developednanosuspension showed an extended release profile of sparfloxacin according to the Peppas model. Acquired gamma camera imagesshowed good retention over the entire precorneal area for the developed nanosuspension compared with that of a marketed formulation.The marketed drug formulation cleared very rapidly from the corneal region and reached the systemic circulation through thenasolacrimal drainage system, as significant radioactivity was recorded in kidney and bladder after 6 hours of ocular administration,whereas the developed nanosuspension cleared at a very slow rate (P b .05) and remained at the corneal surface for longer duration, asno radioactivity was observed in the systemic circulation. HET-CAM assay with 0 score in 8 hours indicates the nonirritant property ofthe developed nanosuspension. The developed lyophilized nanosuspension was found to be stable for a longer duration of time than theconventional marketed formulation with a good shelf life.

From the Clinical Editor: Poor ocular bioavailability of drugs (b1%) from conventional eye drops is mainly due to the eye physiologicalbarriers. In this study, a new colloidal system, PLGA nanoparticle for sparfloxacin ophthalmic delivery was demonstrated to improveprecorneal residence time and ocular penetration. The developed lyophilized nanosuspension was found to be stable for longer duration oftime than conventional marketed formulations.© 2010 Elsevier Inc. All rights reserved.

Key words: Gamma scintigraphy; Nanoparticle; Ocular; PLGA; Sparfloxacin

No conflict of interest was reported by the authors of this article.⁎Corresponding author: Department of Pharmaceutics, Faculty of

Pharmacy, Jamia Hamdard, New Delhi 110062, India.E-mail address: [email protected] (M. Aqil).1 H. Gupta was the recipient of a Senior Research Fellowship from the

Council of Scientific and Industrial Research (Government of India), NewDelhi, India.

1549-9634/$ – see front matter © 2010 Elsevier Inc. All rights reserved.doi:10.1016/j.nano.2009.10.004

Please cite this article as: H. Gupta, M. Aqil, R.K. Khar, A. Ali, A. Bhatnagardrug delivery. Nanomedicine: NBM 2010;6:324-333, doi:10.1016/j.nano.2009.

Poor ocular bioavailability of drugs (b1%) from conven-tional eye drops (ie, solution, suspension, and ointments) ismainly due to the precorneal loss factors that include rapid tearturnover, nonproductive absorption, transient residence time inthe cul-de-sac, and the relative impermeability of the drugs tothe corneal epithelial membrane.1,2 This poor ocular bioavail-ability imparts the need for frequent instillation to achieve thetherapeutic effect, which may sometimes lead to undesirable

, G. Mittal, Sparfloxacin-loaded PLGA nanoparticles for sustained ocular10.004

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side effects caused by systemic drug absorption. In general,ocular efficacy is closely related to ocular drug bioavailability,which may be enhanced by increasing corneal drug penetrationand prolonging precorneal drug residence time.3 A variety ofocular drug delivery systems such as inserts4 and collagenshields5 and colloidal systems such as liposomes,6,7 nano-particles,3,8 and nanocapsules9,10 have been designed andinvestigated for improved ocular bioavailability. The use ofnanotechnology-based drug delivery systems such as micro-emulsions, nanosuspensions, nanoparticles, solid lipid nanopar-ticles, niosomes, dendrimers, and liposomes has led to thesolution of various solubility-related problems of poorly solubledrugs, such as dexamethasone, budesonide, ganciclovir, and soon.11 Polymeric nanoparticle formulation is one of the strategiescurrently used to improve drug absorption across biologicalmembranes.12 Based on literature data, the three mostcommonly used polymers in ophthalmic drug formulations arepoly(alkyl cyanoacrylates), polycaprolactone, and poly(lacticacid)/poly(lactic-co-glycolic acid). Other polymers with oculardrug delivery application include chitosan, Eudragit RL/Eudragit RS, polystyrene, and poly(acrylic acid). Much of thepublished data suggests that in the case of ophthalmic drugdelivery, an appropriate particle size and a narrow size range,ensuring low irritation, adequate bioavailability, and compati-bility with ocular tissues, should be sought for every suspendeddrug.13,14 Hence, the best known class of biodegradablepolymers for sustained drug delivery is poly(dl-lactide-co-glycolide) (PLGA). PLGA is a biodegradable and biocompat-ible polymer that is hydrolytically degraded into nontoxicoligomer and monomer, lactic acid and glycolic acid.15 This isthe reason that it has been used extensively in nanoparticulatedrug delivery systems. Recently, Agnihotri and Vavia devel-oped and evaluated diclofenac sodium–loaded PLGA nanopar-ticles for ocular use and found good biocompatibility with theeye.16 Flurbiprofen-loaded PLGA nanoparticles were alsostudied extensively in various aspects for their application inocular inflammation17,18 and proved to have good stability andocular tolerance.

Sparfloxacin is a newer-generation hydrophobic fluoroqui-nolone used in bacterial conjunctivitis. Poorly water solubledrugs are difficult to develop as a conventional ocular drugdelivery system.14 Sparfloxacin is reported to be more active invitro than ciprofloxacin against mycobacteria and gram-positivebacteria, including Streptococcus pneumoniae and other strep-tococci and staphylococci.19 Nanotechnology can be used toformulate such poorly water soluble drugs as a nanosuspensionand offers the opportunity to address many of the deficienciesassociated with such class of drug.20 Many published reportshave shown the importance of particle size in ocularbioavailability.21,22 Attempts have been made to improve theocular bioavailability and the therapeutic effectiveness of suchpoorly water soluble drugs for ophthalmic use, but no literaturewas found on preparation and evaluation of sparfloxacin-PLGAnanoparticles for improving ocular bioavailability.

Hence, in our current work we develop and evaluate a newcolloidal system, that is, PLGA nanoparticles for sparfloxacinophthalmic delivery, to improve precorneal residence time andocular bioavailability.

Methods

Materials

Sparfloxacin was received as a kind gift from Micro LabsLtd. (Chandigarh, India). PLGA (50:50), IV = 0.2 dl/g, wasobtained from Purac Biomaterial (Singapore). Polyvinyl alcohol(PVA; MW 95,000) was obtained from Sigma-Aldrich ChemieGmbH (Steinheim, Germany). All other chemicals were ofanalytical grade.

Preparation of nanoparticles

Nanoprecipitation technique was applied to prepare spar-floxacin nanoparticles with slight modification of a previouslyreported process.23 Typically, different ratios of drug andpolymer (keeping drug constant at 10 mg and varying polymerconcentration; Table 1) were dissolved in acetone (5 mL) atroom temperature. The resulting solution was slowly droppedwith a constant speed (0.5 mL/min) into water (20 mL)containing PVA (1.5% wt/vol) with continuous magnetic stirringat 1800 rpm. Acetone and some water were evaporated, and thefinal volume of the aqueous suspension was collected. Thenanosuspension was then centrifuged at 18,000 rpm, 20°C, for 1hour (Remi, Mumbai, India). Nanoparticles were collected andwashed (three times) with distilled water using a previouslydescribed centrifugation approach and then lyophilized bymeans of Christ Alpha 1–4 lyophilizator (Christ, Osterode,Germany) using 1% wt/vol mannitol as lyoprotectant.

Nanoparticle recovery (%)

Freeze-dried nanoparticle was weighed accurately, andnanoparticle recovery (%) was calculated using Eq. (1):

Nanoparticle recovery kð Þ=

Mass of nanoparticles recovered × 100Mass of polymer; drug; and other excipients used

ð1Þ

The individual values for three replicates were determined,and their mean values are reported.

Determination of drug incorporation efficiency

Freeze-dried nanoparticles were dissolved in acetonitrile (10mL) (a common solvent for PLGA and drug). Quantity ofsparfloxacin in the solution was measured by ultravioletspectroscopy (UV 1601; Shimadzu, Kyoto, Japan) at 290 nm.Drug incorporation efficiency was expressed as drug entrapment(%); represented by Eq. (2):

Drug entrapment kð Þ = Mass of drug in nanoparticles × 100Mass of drug used in formulation

ð2Þ

The individual values for three replicates were determined,and their mean values are reported.

Table 1Various formulation evaluated for drug-loaded nanoparticles⁎

Batch no. Drug-to-polymerratio

Particle size(nm ± SE)

Polydispersity index(nm ± SE)

Drug entrapment(%± SE)

Zeta potential(mV ± SE)

SN1 1:1 210 ± 3.8 0.489 ± 0.07 25.18 ± 1.2 –22.8 ± 1.3SN2 1:2 198 ± 4.4 0.373 ± 0.08 44.83 ± 1.7 –22.2 ± 1.1SN3 1:5 190 ± 2.5 0.327 ± 0.01 51.83 ± 1.2 –22.6 ± 0.8SN4 1:10 181 ± 3.2 0.238 ± 0.06 86.60 ± 1.8 –22.5 ± 1.8SN5 1:20 232 ± 2.2 0.527 ± 0.02 60.60 ± 0.9 –22.7 ± 1.4

⁎ Data are the mean of six determinations ± SE.

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Physicochemical characterization

Particle size and zeta potentialNanoparticle size distribution and zeta potential were

determined using photon correlation spectroscopy (Zetasizer,HAS 3000; Malvern Instruments, Malvern, UK). The sizedistribution analysis was performed at a scattering angle of 90degrees and at a temperature of 25°C using samples appropri-ately diluted with filtered water (0.2-μm filter; Minisart,Gottirgen, Germany), whereas zeta potential was measuredusing a disposable zeta cuvette. For each sample, the meandiameter/zeta potential ± standard deviation of six determina-tions was calculated applying multimodal analysis.

Particle morphologyMorphologic evaluation of the nanoparticles was performed

using transmission electron microscopy (TEM; Philips CM-10,Eindhoven, The Netherlands). Samples of the nanoparticlesuspension (5 to 10 μL) were dropped onto Formvar-coatedcopper grids (Plano GmbH, Wetzlar, Germany). After completedrying, the samples were stained using 2% wt/vol phospho-tungstic acid. Digital Micrograph and Soft Imaging Viewersoftware (Olympus, Singapore) were used to perform the imagecapture and analysis.

Solid-state characterization

Infrared spectrophotometryInfrared (IR) spectra of freeze-dried nanoparticles were

obtained with a Perkin-Elmer 1600 spectrophotometer (GMIInc., Ramsey, Minnesota) using the potassium bromide (KBr)disk technique (about 10 mg sample for 100 mg dry KBr). IRspectra of sparfloxacin, PLGA, and their physical mixture weretaken for comparison.

X-ray diffractometry (XRD)In x-ray studies, an automatic x-ray diffractometer (Philips

PW 3710; Eindhoven, The Netherlands) equipped with a PWR30 x-ray generator was used. Nickel-filtered Cu kα1 radiationhaving a wavelength of 1.5106 Å, operating at 35 kW and 20 mAin the range (2θ) of 5 to 70 degrees, was used. X-raydiffractograms 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 350°C. Thermalanalysis was performed at a heating rate maintained at 10°C per

minute in a nitrogen atmosphere. Alumina was used as thereference substance.

In vitro drug release

The in vitro drug release study was performed in triplicatewith some modification as described earlier.16 Briefly, drug-loaded nanosuspension was suspended in simulated tear fluid(STF), pH 7.4, in a glass vial. The glass vials were placed in amechanical shaking bath (100 cycles/min), with temperatureadjusted to 37°C. A 1-mL sample was removed at predeter-mined time intervals and replaced with an equal quantity offresh STF. The sample was then appropriately diluted andquantitatively analyzed using a UV spectrophotometer at 290nm. A marketed formulation was also tested and compared withthe developed nanosuspension.

Statistical analysis

The results of in vitro data were analyzed by statisticalsoftware PCP Disso (version 3.0) Maharashtra, India, to obtainthe best fit kinetic model for in vitro drug release.

In vitro transcorneal permeation study

Goat corneas were used to study the transcorneal permeabilityof sparfloxacin from the developed formulation.24 Fresh wholeeyeballs of goats were obtained from a local butcher's shop andtransported to the laboratory in cold condition in normal saline.Corneas were then carefully removed along with 5 to 6 mm ofsurrounding scleral tissue and stored in freshly prepared artificialtear solution, pH 7.4. The study was carried out in a modifiedFranz diffusion chamber. The upper chamber served as a donorcompartment in which 100 μL of drug solution/formulationunder study was placed. The excised goat cornea was fixedbetween clamped donor and receptor compartments of the Franzdiffusion cell in such a way that its epithelial surface faced thedonor compartment. The lower chamber served as a receivercompartment that was infused with freshly prepared simulatedtear fluid. The whole system was maintained at 37 ± 0.5°C. Theperfusate was collected at periodic time intervals for up to 4hours in preweighed microcentrifuge tubes and subjected to thequantification of sparfloxacin by UV at 290 nm.

Microbiological studies

The microbiological studies ascertained the biological activityof the optimized formulation and of the marketed eye dropsagainst a microorganism (Pseudomonas aeruginosa). A layer of

Table 2Scoring chart for HET-CAM test

Effect Scores Inference

No visible hemorrhage 0 NonirritantJust visible membrane discoloration 1 Mild irritantStructures are covered partially due to

membrane discoloration or hemorrhage2 Moderately irritant

Structures are covered totally due tomembrane discoloration or hemorrhage

3 Severe irritant

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nutrient agar (20 mL) seeded with the test microorganism (0.2mL) was allowed to solidify in the Petri plate. Cups were made onthe solidified agar layer with the help of a sterile borer at 4 mmdiameter. Then, a volume of the formulations (optimizedformulation and marketed eye drops) containing equivalentamount of drug was separately poured into two cups. Afterkeeping Petri plates at room temperature for 4 hours, the plateswere incubated at 37°C for 24 hours. The zones of inhibition wereobtained. The diameter of the zone of inhibition was measured byan antibiotic zone finder. Readings were taken in triplicate.

Ocular tolerance test (HET-CAM test)

For analyzing the ocular tolerability of the developedformulation, a modified HET-CAM test as reported earlier byour group was carried out.24 The HET-CAM test has been shownto be a qualitative method of assessing the potential irritancy ofchemicals. The potential irritancy of compounds may be detectedby observing adverse changes that occur in the chorioallantoicmembrane of the egg after exposure to test chemicals.25 Briefly,fertilized hen's eggs were obtained from a poultry farm. Threeeggs for each formulation weighing between 50 and 60 g wereselected and candled to discard the defective ones. These eggswere incubated in a humidified incubator at a temperature of 37 ±0.5°C for 3 days. The trays containing eggs were rotatedmanually in a gentle manner after every 12 hours. On the thirdday, 3 mL of egg albumin was removed by using steriletechniques from the pointed end of the egg. The hole wasimmediately sealed by 70% alcohol-sterilized Parafilm (Amer-ican Can Company, Neenah, Wisconsin) with the help of aheated spatula. The eggs were kept in the equatorial position forthe development of chorioallantoic membrane (CAM) awayfrom the shell. The eggs were candled on the fifth day ofincubation, and every day thereafter nonviable embryos wereremoved. On the tenth day, a window (2 × 2 cm) was made onthe equator of the eggs through which formulations (0.5 mL)were instilled directly onto the CAM surface and left in contactfor 5 minutes. The membrane was examined for vasculardamage, and the time taken for injury to occur was recorded. An0.9% NaCl solution was used as a control as it is reported to bepractically nonirritant. The scores were recorded according to thescoring schemes as shown in Table 2.

In vivo ocular retention study: Gamma scintigraphy

In vivo precorneal drainage of the developed formulation wasassessed by gamma scintigraphy. Male New Zealand albinorabbits of either sex weighing 1.8 to 2.5 kg and free of any signsof ocular inflammation or gross abnormality were used in thestudy. Animals were procured from the animal house of theInstitute of Nuclear Medicine & Allied Sciences (INMAS),Delhi, India, and had free access to food and water. The studywas carried out under the guidelines of CPCSEA (Committee forthe Purpose of Control and Supervision of Experiments onAnimals, Ministry of Culture, Government of India), and all thestudy protocols were approved by the local institutional animalethics committee. Utmost care was taken to ensure that animalswere treated in the most humane and ethically acceptablemanner. Sparfloxacin was dissolved in 0.1 mL of 1 M NaOH

diluted with normal saline in an amber-colored bottle andradiolabeled with Tc-99m by direct labeling method usingstannous chloride as reducing agent. This radiolabeled drugsolution was used to prepare PLGA nanosuspension as describedearlier. A gamma camera (Millenium VG, Milwaukee, Wiscon-sin), autotuned to detect the 140 KeV radiation of Tc-99m, wasused for scintigraphy study. Rabbits were anesthetized usingketamine HCl injection given intramuscularly in a dose of 15 mg/kg body weight. The rabbits were positioned 5 cm in front of theprobe, and 50 μL of the radiolabeled formulation was instilledonto the left corneal surface of each rabbit. Recording was started5 seconds after instillation and continued for 30 minutes using128 × 128 pixel matrix. Sixty individual frames (60 × 30seconds) were captured by dynamic imaging process. Region ofinterest (ROI) was selected on one frame of the image, and time-activity curve was plotted to calculate the rate of drainage fromthe eye. A single whole-body static image was also taken after 6hours of instillation of drug/formulation. Each formulation wastested on three rabbits.

Stability studies of the formulations

The physical stability of the lyophilized nanoparticle wasevaluated after storage for 6 months. Fifty milligrams oflyophilized nanoparticles was stored in closed amber-coloredglass vials at 5 ± 2°C (refrigerator) away from direct light. Tenmilligrams of the formulation was withdrawn at 1-, 3-, and 6-month time intervals to measure particle size and drug content, asdescribed earlier. For quantitative analysis, ultra performanceliquid chromatography (UPLC) was performed using a WatersAcquity system equipped with a binary solvent delivery pump,an autosampler, and a tunable UV detector (Waters, Milford,Massachusetts). The chromatographic separation was performedusing a Waters Acquity HSS T-3 (100 × 2.1) mm; 1.8 μm, C18column. The mobile phase containing a mixture of 0.1% aqueousortho-phosphoric acid on a timed gradient program T (min)/%B(1/10, 2/10, 3/25, 4/10, 5/10) with a flow rate of 0.5 mL/min wasused. The detection was obtained at a wavelength of 290 nm. Theinjection volume was 1 μL; acetonitrile solution was used as adiluent while the column was maintained at 50°C.26

Results

The amount of drug (ie, 10 mg) was kept constant, andconcentration of PLGA was varied accordingly (ie, 10, 20, 50,100 mg, etc.). Formulation SN4 with the drug:polymer ratio of1:10 was found to be appropriate with particle size of 181 nm,

Figure 1. (A) Graph showing particle size distribution of optimizednanosuspension. (B) Graph showing the zeta potential of optimized nanosus-pension. (C) Transmission electron micrograph of optimized nanosuspension.

328 H. Gupta et al / Nanomedicine: Nanotechnology, Biology, and Medicine 6 (2010) 324–333

nanoparticle recovery of 70%, encapsulation efficiency of86.6%, drug loading of 12.3%, polydispersity index of 0.238,and zeta potential of –22.5. (Table 1; Figure 1) Optimizedformulation SN4 was prepared in six replicates for furthercharacterization. The results obtained here were found to be inaccordance with a previously published study.27-29 The mor-phology of the prepared PLGA nanoparticles was found to bespherical as seen in TEM image (Figure 1, C).

In the compatibility study of sparfloxacin and PLGA alone,physical mixture, and nanoparticles, characteristic peaks ofsparfloxacin in FTIR spectra were obtained, that is, C = Ostretching around 1715 cm-1, C = C at 1620 cm-1, and –CH(1440 to 1500 cm-1) along with the characteristic peak of PLGAat 1746 cm-1. This infers two facts: First, the FTIR curve

Figure 2. (I) XRD diffractograms: A, sparfloxacin; B, PLGA; C, physicalmixture; D, sparfloxacin-PLGA nanoparticle system. (II) DSC thermo-grams: A, sparfloxacin; B, PLGA; C, physical mixture; D, sparfloxacin-PLGA nanoparticle system.

Figure 3. (A) In vitro drug release profile of optimized sparfloxacinnanosuspension and marketed formulation. (B) PCP Disso (version 3) bestfitted model for in vitro drug release of sparfloxacin nanosuspension.

Figure 4. Transcorneal permeation study for the marketed eye drops and theoptimized nanosuspension.

Figure 5. Diameter of zone of inhibition for the marketed eye drops and theoptimized nanosuspension (n = 3).

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obtained is similar to that of pure drug and excipients confirmingthat there is no interaction between PLGA and sparfloxacin.Second, it shows the crystalline nature of the drug. The drugpeak shown in FTIR spectra of the nanoparticle can be attributedto the fact that during preparation of the KBr disk ofnanoparticles under high pressure and mixing, the nanoparticleswere crushed and drug came out. The drug entrapment is furthercharacterized by XRD and DSC. It reveals the state of theencapsulated drug whether it is dispersed in a microcrystallineform, without polymorph change or transition change inamorphous form. In XRD analysis, XRD diffractogram ofpure PLGA and sparfloxacin was compared with that of physicalmixture and of freeze-dried nanoparticles. Drug peak innanoparticles was diminished or suppressed and not seen; thiscan be attributed to the dilution factor due to high concentrationof polymer without any qualitative fraction. This is inaccordance with previous studies16,29 that the drug is in matrixof PLGA polymer (Figure 2). Similar results were observed byDSC studies (Figure 2). Degradation endotherm of sparfloxacinwas observed at 262°C, in DSC of pure drug and physicalmixture of drug with PLGA, whereas no drug peak wasobserved in PLGA nanoparticle system because of dilutionfactor and entrapment of drug in matrix system. This also showsthe storage stability of the system.30

In vitro release

The in vitro release profiles of optimized formulation, SN4,and marketed formulation is shown in Figure 3. The formulationSN4 shows a two-step release pattern: one initial burst release

followed by a second slow-release phase (extended release). Aninitial burst release is beneficial in terms of antibacterial activityas it helps achieve the therapeutic concentration of drug inminimal time followed by constant release to maintain sustainedand controlled release of the drug. Developed PLGA nanopar-ticles released 37.8% sparfloxacin in 2 hours, 57.13% in 6 hours,and 85.8% in 24 hours compared with 75.33% and 96.7% in 2hours and 6 hours, respectively, of the marketed formulation.Curve fitting of in vitro release data of the optimized formulationwas compared with a different release model to select the bestfitting model using PCP Disso version 3.0 software. The best fitkinetic model was the Peppas model [R = 0.9841, t-test =14.655 (passes) (Figure 3) with a critical value of n ∼0.5],suggesting non-Fickian diffusion process. In vitro transcornealpermeation studies conducted on the developed formulationshow a higher permeation across goat cornea after 4 hours(47.93%) compared with that of the marketed formulation(36.24%) (Figure 4).

Microbiological studies

The optimized SN4 nanosuspension formulation was testedmicrobiologically by cup-plate technique. Clear zones ofinhibition were obtained. The diameter of the zone of inhibitionis shown in Figure 5. Diameter of zone of inhibition by themarketed formulation was 11.43 ± 0.25mm and 12.63 ± 0.15mm

Table 3Scores obtained in HET-CAM test

Formulations Time (min)

0 5 15 30 60 120 240 480 1440

Scores

Normal saline as control Egg1 0 0 0 0 0 0 0 0 0Egg2 0 0 0 0 0 0 0 0 0Egg3 0 0 0 0 0 0 0 0 0Mean 0 0 0 0 0 0 0 0 0

Developed formulation Egg1 0 0 0 0 0 0 0 0 0Egg2 0 0 0 0 0 0 0 0 0Egg3 0 0 0 0 0 0 0 0 1Mean 0 0 0 0 0 0 0 0 0.33

Figure 6. Gamma scintigraphic static whole-body images after 6 hours of administration: (A) marketed formulation; (B) sparfloxacin-PLGA nanosuspension.

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at 12 hours and 24 hours compared with 14.23 ± 0.15 mm and15.56 ± 0.11mm for themarketed and test formulation at 12 hoursand 24 hours. Results revealed prolonged microbial efficacy ofdeveloped nanosuspension compared with marketed eye drops.

Ocular tolerability test (HET-CAM test)

A mean score of 0 was obtained for normal saline.25

Nanosuspension was nonirritant up to 8 hours (mean score 0)while the mean score was found to be 0.33 up to 24 hours(Table 3). The study shows that the formulation is nonirritantand is well tolerated.

Gamma scintigraphyFor scintigraphic studies, sparfloxacin was radiolabeled with

radionuclide Tc-99m. The latter was chosen as it emits low-

energy gamma rays that do not lead to serious health hazards.Sparfloxacin was instantaneously labeled with Tc-99m. Labelingefficiency was checked by instant thin layer chromatography(ITLC) using 100% acetone as mobile phase. The Rf value offree Tc is∼0.9, so it reaches to the top of the ITLC strip, whereasthe complexed Tc (drug-Tc complex) cannot travel much due todifference in molecular weight and is retained at the base of theITLC strip. Thus, from the difference in the top and bottomcounts, labeling efficiency can be calculated. After prelabelingefficiency studies, which include labeling parameters such asstannous chloride concentration and pH, were optimized, a 50 μgstannous chloride concentration at pH 7.0 was found to give themaximum labeling efficiency (95.2%). In these conditions,minimum colloids (1%) were produced. After administration ofthe radiolabeled formulation, a good spreading was observedover the entire precorneal area. The marketed formulation

Figure 7. Time-activity curves showing precorneal drainage.

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cleared very rapidly from the corneal region and reached into thesystemic circulation via the nasolacrimal drainage system assignificant activity was recorded in kidney and bladder after 6hours of ocular administration (Figure 6, A), whereas PLGAnanosuspension formulation was retained at the corneal surfacefor longer duration as no significant radioactivity was observedin the systemic circulation (kidney and bladder) even after 6hours (Figure 6, B). The curve of the remaining activity on thecorneal surface as a function of time (time-activity curve) wasplotted and is shown in Figure 7. The marketed formulationshows a quick fall in radioactivity counts with respect to time oncorneal surface compared with the nanosuspension showing thatthe nanosuspension was retained for longer duration givingextended release compared with the marketed eye drops.

Stability studies

There were negligible alterations in the initial values ofparticle size, encapsulation efficiency, and drug loading of theformulation over storage for 180 days. The samples wereanalyzed for drug content by UPLC. Again the drug degraded toa negligible extent, and the degradation rate constant for theoptimized formulation was low (0.9 × 10−4). Because the overalldegradation is b5%, a tentative shelf life of 2 years may beassigned to the optimized formulation.31

Discussion

In our current work, we have prepared PLGA nanoparticles ofsparfloxacin using a modified nanoprecipitation technique. Wehave tried different drug-to-polymer ratios to obtain low particlesize with maximum encapsulation efficiency. Both particle sizedistribution and polydispersity can influence the nanoparticulatedrug delivery. A particle size below 250 nm32 with apolydispersity index near 0.2533 was considered optimum forocular administration. Zeta potential is an important physico-chemical parameter that influences stability of the nanosuspen-sion. Extremely positive or negative zeta potential values causelarger repulsive forces, whereas repulsion between particles withsimilar electric charge prevents aggregation of the particles andthus ensures easy redispersion.34,35 In case of a combinedelectrostatic and steric stabilization, a minimum zeta potential

of ± 20 mV is desirable.33 All formulations showed nearlysimilar zeta potential of –22 mV.

Sparfloxacin can be adsorbed and/or dispersed into thepolymeric matrix system of PLGA nanoparticles. This can beexplained on the basis of the hydrophobic nature of sparfloxacin.It readily precipitated in aqueous medium and gets encapsulatedby the PLGA matrix preventing its diffusion in external phase.36

Generally, DSC and XRD studies are carried out to determinewhether the drug was incorporated in the nanoparticulate systemas crystalline, amorphous, or bound form. The analysis showedthat the drug is encapsulated in the polymer matrix rather thanphysically adsorbed on the nanoparticle surface, and the drugalso maintains its crystalline nature inside the matrix.

The drug from the biodegradable particles generally releasesthrough several mechanisms such as desorption of the surface-bound/adsorbed drug; disintegration; diffusion through theparticle matrix; diffusion through the polymer wall, in casedrug is encapsulated in the core; surface and bulk degradation;and a combined degradation/diffusion process.37 PLGA isknown to undergo bulk degradation. Release of the entrappedtherapeutic agent from the PLGA matrix can occur throughdiffusion with degradation mediated process. It has been shownthat during the early phases, release occurs mainly throughdiffusion in the polymer matrix, whereas during the laterphases, release is mediated through both diffusion of thetherapeutic agent and degradation of the polymer matrixitself.38,39 PVA is a swellable, hydrophilic macromolecule,and it is possible that PVA present on the surface could form ahydrogel barrier to the diffusional release of drug resulting inslower release as in the case of PVA-stabilized nanoparticles.40

The viscosity of the solution may increase dramatically inpresence of the polymer, but the transport conditions for smalldrug molecules are apparently the same as they are in water.

It is important to evaluate ocular tolerability of the developednanosuspension. Ocular irritation of the developed formulationwas checked by hen's egg chorioallantoic membrane test, whichis a rapid, sensitive, and inexpensive test. Testing with incubatedeggs is a borderline case between in vivo and in vitro systemsand does not conflict with the ethical and legal obligations. Thechorioallantoic membrane of the chick embryo is a completetissue including veins, arteries, and capillaries and is technicallyvery easy to study. It responds to injury with a completeinflammatory process, a process similar to that induced in theconjunctival tissue of rabbit eyes.25 The developed formulationwas tested by using this method, and the result was comparedwith those obtained using normal saline, which was used ascontrol that is supposed to be practically nonirritant. The resultsshow a mean score of 0.33 up to 24 hours, which indicates thenonirritant property of the formulation.

PLGA is an anionic polymer, and hence it is non-mucoadhesive in nature. But due to the small size of thePLGA nanoparticles, the nanoparticles are able to be retained onthe eye.32,41,42 Moreover, PVA-stabilized PLGA here may alsohelp in retention of nanoparticles on the eye. A good spreadingand retention of the formulation was observed in gammascintigraphy studies compared with the marketed formulation.In the time-activity curve, there is a minimal fall in counts/second of the formulation compared with the rapid fall of the

332 H. Gupta et al / Nanomedicine: Nanotechnology, Biology, and Medicine 6 (2010) 324–333

marketed formulation, further showing that the nanoparticlesstay longer on the eye.

Many factors affect the stability of a pharmaceutical product,including the stability of the active ingredient(s); the potentialinteraction between active and inactive ingredients; the manufac-turing process; the dosage form; the container-liner-closuresystem; handling; and length of time between manufacture andusage. To calculate shelf life of the formulation, extensive stabilitydata were collected according to International Conference onHarmonization (ICH) guidelines on lyophilized nanoparticles.Degradation rate constant for the optimized formulation was low(0.9 × 10−4) and hence shows that the formulation is stable inlyophilized form for longer time (∼2 years).

In conclusion, the developed PLGA-sparfloxacin nanosuspen-sion gives appropriate particle size, extended release with bettertolerability, and prolonged retention at the corneal site. Hence thedeveloped sparfloxacin-PLGA nanosuspension is suitable forsustained ocular drug delivery in treatment of conjunctivitisand can proceed to clinical evaluation and application.

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