Nanoparticle Laden in situ gel for sustained ocular drug delivery

7/30/2019 Nanoparticle Laden in situ gel for sustained ocular drug delivery

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162 Journal of Pharmacy and Bioallied Sciences April-June 2013 Vol 5 Issue 2

Department ofPharmaceutics, Facultyof Pharmacy, JamiaHamdard, 1Departmentof Nuclear Medicine,Institute of NuclearMedicine and AlliedSciences, Ministry ofDefence, New Delhi, India

Address for correspondence:Dr. Mohammed Aqil,E-mail: [email protected];[email protected]

Hyperacute bacterial conjunctivitis is a severe, sightthreatening ocular infection that warrants immediate

ophthalmic work-up and management. Bacterial conjunctivitisrequires treatment with antibiotics for 5-7 days that may resultin poor patient compliance with conventional dosage forms dueto greater frequency of drug administration, that is, 2-3 dropsevery 2-3 h.[1] This is because in ocular delivery, the physiologicalconstraints imposed by the protective mechanisms of the eyelead to low absorption of drugs, resulting in short duration of thetherapeutic effect. When a drug solution is dropped into the eye,the effective tear drainage and blinking action of the eye result ina 10-fold reduction in the drug concentration within 4-20 min.[1]The availability of medicament at corneal surface would besignificantly improved if the precorneal residence time of drugs

could be increased. Several new preparations such as inserts, [2]collagen shields,[3] and colloidal systems, such as liposomes,[4,5]

nanoparticles,[6,7] and nanocapsules[8,9] have been developed forophthalmic use, not only to prolong the ocular contact time ofthe vehicle but also to slow down drug elimination.[10] The use ofnanotechnology-based drug delivery systems like microemulsions,nanosuspensions, nanoparticles, solid lipid nanoparticles,niosomes, dendrimers, and liposomes has led to the solution ofvarious solubility-related problems of poorly soluble drugs, likedexamethasone, budesonide, ganciclovir, and so on.[11] However,these novel preparations have some disadvantages, such as poorcompliance. Out of these novel formulations; in situ gel andnanoparticles have been established as the promising one. In ourprevious work, we found that these formulations have their owndisadvantages. For example, in situ gel remains for 12-15 h,[12]

whereas poly lactic co glycolic acid (PLGA) nanoparticles arenonmucoadhesive, so are drained out of eyes quickly.[13] In ourpresent work, we try to combine both these formulation strategiesas a nanoparticles suspended in liquid dosage form suitable to beadministered by instillation into the eye which, upon exposureto physiological conditions, changes to the gel phase, thusincreasing the precorneal residence time of the nanoparticles andenhancing ocular bioavailability. Our group has coined the termnanoparticle laden in situ gel for this formulation.[14]

Nanoparticles laden in situgel for sustained

ocular drug delivery

Himanshu Gupta, Mohammed Aqil, Roop K. Khar, Asgar Ali , Aseem Bhatnagar1,

Gaurav Mittal1

ABSTRACT

Proper availability of drug on to corneal surface is a challenging task. However, due to ocular physiological

barriers, conventional eye drops display poor ocular bioavailability of drugs (1%). To improve precornealresidence time and ocular penetration, earlier our group developed and evaluated in situgel and nanoparticles

for ocular delivery. In interest to evaluate the combined effect of in situgel and nanoparticles on ocular

retention, we combined them. We are the first to term this combination as nanoparticle laden in situgel,

that is, poly lactic co glycolic acid nanoparticle incorporated in chitosan in situgel for sparfloxacin ophthalmic

delivery. The formulation was tested for various physicochemical properties. It showed gelation pH near pH 7.2.

The observation of acquired gamma camera images showed good retention over the entire precorneal areafor sparfloxacin nanoparticle laden in situgel (SNG) as compared to marketed formulation. SNG formulation

cleared at a very slow rate and remained at corneal surface for longer duration as no radioactivity was

observed in systemic circulation. The developed formulation was found to be better in combination and can

go up to the clinical evaluation and application.

KEY WORDS: Gamma scintigraphy, in situgel, nanoparticle, nanoparticle laden in situgel, ocular, PLGA,

sparfloxacin

Received : 18-03-13

Review completed : 22-03-13

Accepted : 27-03-13

How to cite this article: Gupta H, Aqil M, Khar RK, Ali A, Bhatnagar A, Mittal G. Nanoparticles laden in situ gel for sustained ocular drug delivery. J Pharm

Bioall Sci 2013;5:162-5.

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DOI:

10.4103/0975-7406.111824

Short Communication


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Gupta, et al.: Nanoparticle laden in situgel

Journal of Pharmacy and Bioallied Sciences April-June 2013 Vol 5 Issue 2 163

In our study, we used third generation flouroquinolonesparfloxacin for formulation evaluation. Hence, in the presentwork we develop and evaluate new system, that is, sparfloxacinPLGA nanoparticle laden in situ gel for ophthalmic deliveryto improve precorneal residence time and thus ocularbioavailability.

Materials and Methods

Materials

Sparfloxacin was received as kind gift from MicroLabs Ltd. (Chandigarh , India) ; PLGA (50:50) ,intravenous0.2 dL/g, was obtained from Purac Biomaterial,Singapore), polyvinyl alcohol (PVA, MW 95,000) was obtainedfrom Sigma-Aldrich Chemie GmbH (Steinheim, Germany).Chitosan (practical grade, 75%-85% deacetylated, and molecularweight 150 kDa) was obtained as kind gift from M/s India SeaFoods, India. All other chemicals were of analytical grade.

Preparation of nanoparticle laden in situgel

Nanoparticles were prepared as per our previous publishedwork.[13] Briefly, nanoprecipitation technique was applied toprepare sparfloxacin nanoparticles. Drug and polymer in ratioof 1:10 (keeping drug 10 mg and PLGA 100 mg) were dissolvedin acetone (5 mL) at room temperature. The resultedsolution was slowly dropped with speed manually (approx.0.5 mL/min) into water (20 mL) containing PVA (1.5% w/v)with continuous magnetic stirring at 1800 rpm. Acetoneand some water were evaporated, and the final volume ofthe aqueous suspension was collected. The nanosuspensionwas then centrifuged at 18,000 rpm, 20C for 1 h (Remi,India). Nanoparticles were collected and washed (3 times)

with distilled water using previously described centrifugationapproach and then lyophilized by means of Christ Alpha1-4 lyophilizator (Christ, Germany) using 1% w/v mannitolas lyoprotectant. Whereas, in situ gel base is prepared bydissolving 0.5% w/v chitosan in 1% v/v acetic acid, pH adjustedto 5.5-6.0 with buffer saline.[12]

Weighed quantity of drug-loaded freeze-dried nanoparticles(equivalent to the prescribed dose of sparfloxacin 0.3% w/v) weretaken and dispersed in the respective 1 ml of placebo in situ gelbase (0.5%w/v chitosan solution) to form nanoparticle ladenin situ gel.Freeze-dried nanoparticles were weighed accuratelyon the basis of its drug loading and release pattern [Table 1].[13]

Physicochemical characterization

Physicochemical characterization of the developed nanoparticlesladen in situ gel were carried out and compiled in Table 2.

Clarity

The clarity of the formulations after and before gelling wasdetermined by visual examination of the formulations underlight alternatively against white and black backgrounds.

Gelation pH

Gelation pH is the pH at which the solution form of theformulation was changed to gel. Formulation was taken in abeaker and 1M NaOH was added dropwise with continuousstirring. pH was checked using pH meter. The pH at whichsudden change in viscosity was observed and noted is recordedas gelation pH.

Viscosity

Viscosity of formulations were determined by using Brookfieldsviscometer (model DV II, spindle no. 02, at 20 rpm), onformulation pH (6.0) and gelation pH near 7.0.

In vivo

ocular retention study-Gamma scintigraphy

Gamma scintigraphy is used to assess in vivo precorneal drainageof the developed formulation. Male New Zealand albino rabbitsof either sex weighing 1.8-2.5 kg and free of any signs of ocularinflammation or gross abnormality were used in the study.

Animals were procured from the animal house of Institute ofNuclear Medicine and Allied Sciences Delhi, India and all studyprotocols were approved by local institutional animal ethicscommittee. Sparfloxacin was radiolabeled with Tc-99m by directlabeling method using stannous chloride as reducing agent asper previous reported method.[13] Gamma camera (MilleniumVG, USA), autotuned to detect the 140 KeV radiation of Tc

99m

was used for scintigraphy study. Rabbits were anesthetized

using ketamine HCl injection given intramuscularly in a doseof 15 mg/kg body weight. The rabbits were positioned 5 cm infront of the probe and 50 PL of the radiolabeled formulation wasinstilled onto the left corneal surface of the rabbits. Recordingwas started 5 s after instillation and continued for 30 min using128u128 pixel matrix. Individual 60 frames (60u30 s) werecaptured by dynamic imaging process. Region of interest wasselected on one frame of the image and time-activity curve wasplotted to calculate the rate of drainage from eye. A single wholebody static image was also taken after 6 h of instillation of drug/formulation. Each formulation was tested on three rabbits.

Table 1: Final composition of sparfloxacin nanoparticles

ladenin situgelIngredients Concentration (w/v) (%)

Sparfloxacin nanoparticles 0.4

Chitosan 0.5

Sodium chloride (NaCl) 0.45

Methylparaben 0.1

Water/base (q.s.) 100

Table 2: Physicochemical characterization of nanoparticles

ladenin situgelParameter Nanoparticle ladenin situgelClarity Very slight turbid solution

pH 6.08r0.07Gelation pH 7.1r0.109Viscosity (at pH 6.0) 42.83r2.041 cpsViscosity (at pH 7.2) 248.33r4.84 cps


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Results and Discussion

In situ gel and nanoparticles have their own merits and demerits.In situ gel stays only for 12 h, whereas PLGA and conjunctiva isanionic in nature and hence PLGA nanoparticles are not ableto be retained for the longer time. To increase the duration,we entrapped PLGA nanoparticles in cationic chitosan in situ

gel system. Chitosan is a cationic polymer and hence will makelink with both PLGA nanoparticles and conjunctiva and thusincreases retention. As chitosan act both as pH sensitive andpermeation enhancer, the viscosity of the chitosan increaseswhen it reaches to eye pH at 7.4. This combination will allownanoparticles to stay for longer duration and provide optimumdrug release. To achieve most out of these two delivery systems,we have combined both the system to develop nanoparticlesladen in situ gel. The nanoparticle size was near 180 nm inconcordant with our previously reported results.[13] The quantityof nanoparticles was calculated on the basis of drug loading andrelease, we have taken 40 mg of SN to prepare 1 ml of respectivenanoparticles laden in situ gel (SNG). Final composition of SNG

is given in Table 1. The developed nanoparticles ladenin situ

gelswere then evaluated for various physicochemical characteristicsas given in Table 2. The preparation is well-dispersed with slight

turbidity. After mixing, no difference is found in gelation pHand viscosity of the formulation.

Gamma scintigraphy

Pharmacoscintigraphy is a tool to analyze the retention time ofthe formulation in cul-de-sac. For gamma scintigraphic studies,

sparfloxacin was labeled with radionuclide Tc-99m using stannouschloride as reducing agent. Tc-99m was chosen for the purposebecause of its moderate half life (6 h). Further it emits gammarays, which have relatively low energy as compared toD andE rays,so leads to no serious health hazards to the workers. Sparfloxacinwas instantaneously labelled with Tc-99m. Labelling efficiencywas checked by instant thin layer chromatography (ITLC)using 100% acetone as mobile phase. The Rf value of free Tcis ~0.9, so it reaches to the top of the ITLC strip while thecomplexed Tc (drug-Tc complex) cannot travel much due todifference in molecular weight and is retained at the base ofITLC strip. Thus, from the difference in the top and bottomcounts, labelling efficiency was calculated. After prelabelingefficiency studies which include labelling parameters likestannous chloride concentration and pH were optimized and a 50Pg stannous chloride

concentration at pH 7.0 was found to give

the maximum labelling efficiency (95.2%). In these conditions,minimum colloids (1%) were produced. The observation of theacquired gamma camera images showed a good spreading overthe entire precorneal area for developed ocular formulations ofsparfloxacin [in situ gel (SG), nanosuspension (SN), nanoparticleladen in situ gel (SNG)], immediately after administration ascompared to marketed formulation. Marketed formulationcleared very rapidly from the corneal region and reached into systemic circulation via nasolacrimal drainage system assignificant activity was recorded in kidney and bladder after 6 hof ocular administration [Figure 1a], whereas formulations SG,

SN, and SNG were retained for longer duration at corneal surface.No significant radioactivity was observed in kidney and bladderafter 6 h of administration of these formulations [Figure 1b-d].To differentiate between these formulations, the curves of the% radioactivity remained on the corneal surface as a function oftime (30 min dynamic imaging) was also plotted [Figure 2]. Theretention on cornea follows the following sequence:

Figure 2: Gamma scintigraphy dynamic study

Figure1:5VCVKEKOCIGUQHTCDDKVCHVGTJQHCFOKPKUVGTKPIURCTQZCEKP

formulations (a) marketed, (b) in situgel, (c) nanosuspension, and

(d) nanoparticles laden in situgel

dc

ba


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Journal of Pharmacy and Bioallied Sciences April-June 2013 Vol 5 Issue 2 165

Marketed formulation nanosuspension in situ

gelnanoparticle laden in situgel

It shows that the nanoparticles laden in situ gel retainedfor the longer duration in the eye giving extended releasethan nanosuspension and in situ gel alone, whereas themarketed formulation drained out of the eye with in

30 min. Due to mucoadhesive property of chitosan,in situ

gel-based formulation cleared at slowest rate as compare tonanosuspension and retained at corneal surface for longesttime duration. Further, the viscosity of chitosan is raised dueto change in physiological conditions of pH (!7) and ionicconcentration of the formulation upon instillation in to eye asa result of buffering action of the tear fluid. Chitosan also actsas penetration enhancer that increases the transport of drugacross cornea.

Conclusion

Our previous study shows that the PLGA nanoparticles, due tosmaller size, are able to provide prolong retention on the eyethan the marketed formulation. PLGA is an anionic polymer andhence it is nonmucoadhesive in nature. To enhance the efficacyof the nanoparticulate formulation, we incorporate them incationic chitosan in situ gel to make nanoparticle laden in situgel, which helps in retention of nanoparticles on the eye. A goodspreading and retention of formulation was observed in gammascintigraphy studies as compared to marketed formulation.In time activity curve, there is a minimal falls in counts/s offormulation as compared to rapid fall of marketed formulationfurther shows that the nanoparticles laden in situ gel stay forlonger time on the eye.

Acknowledgment

Authors are thankful to Council of Scientif ic and Indus tria lResearch (CSIR), New Delhi to provide Senior Research Fellowshipto Himanshu Gupta for conducting this work.

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Source of Support: Dr. Himanshu Gupta was recipient of Senior Research

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Nanoparticle Laden in situ gel for sustained ocular drug delivery