Formulation and Characterization of Eudragit RS 100 Nanosuspension for Ocular Delivery of Indomethacin.


Vijay Shinde*, P. Amsa, S. Tamizharasi, D. Karthikeyan, T. Sivakumar and Aniket Kale

Department of Pharmaceutics, Nandha College of Pharmacy and Research Institute, Erode-638052 (India)

*Corresponding Author E-mail:



The aim of this study is to formulate a novel ophthalmic nanosuspension (ONS), an alternative carrier system to traditional colloidal carriers for controlled release (CR) of Indomethacin (IM). In the present study, ONS is employed to avoid some of major disadvantages of colloidal carriers systems such as instability in cul de sac and short half life by increasing efficiency of drug encapsulation as well as by CR. A quassi-emulsion solvent evaporation method was used to prepare IM loaded Eudragit RS 100 ONS with the aim of improved ocular bioavailability and distribution. Five different formulations were prepared and evaluated for pH of ONS, particle size, entrapment efficiency, differential scanning calorimetry (DSC), in vitro release profile, in vivo release studies and stability studies. An average size range of 50 to 500 nm in diameter was obtained and encapsulation efficiency up to 96.0% was observed for all the formulations. Cumulative percent drug released for all formulations after 14 h was between 86.24 to 96.18% indicating effective CR property of ONS. The release profile revealed from best formulation followed Non-Fickian diffusion mechanism. In vivo studies showed that IM concentration in aqueous humor up to 6 h was 73.80, 45.19 and 41.71”g/ml. No appreciable difference was observed in the extent of degradation of product during 90 days in the nanosuspension, which were stored at various temperatures Overall, the study also revealed that ONS was capable of releasing the drug for a prolonged period of time and increased bioavailability.


KEYWORDS: Nanosuspension, Indomethacin, in vivo absorption study, in vitro drug release.



Local application of drugs in the forms of drops, colloidal carrier system, gel, etc., to eye is the most popular and well-accepted route of administration for the treatment of various eye disorders. Bioavailability of ophthalmic drug is however very poor due to efficient protective mechanisms of the eye. Blinking, baseline and reflex lachrymation and drainage remove rapidly foreign substances including drugs from the surface of the eye. Moreover, the anatomy, physiology and barrier function of the cornea compromise the rapid absorption of drugs. Frequent instillations of eye drops are necessary to maintain a therapeutic drug level in the tear film or at the site of action. But the frequent use of highly concentrated solutions may induce toxic side effects and cellular damage at the ocular surface.


Ophthalmic nanosuspension (ONS) can be defined as colloidal dispersions on nano-sized drug particles that are produced by a suitable method and stabilized by a suitable stabilizer; these can prove to be a boon for drugs that exhibit poor solubility in lachrymal fluids.


Eye diseases can cause therapeutic discomfort and anxiety in patients, with the ultimate fear of loss of vision or even facial disfigurement. Many regions of the eye are relatively inaccessible to systematically administered drugs and as a result, topical delivery remains the preferred route of delivery in most cases.


Indomethacin (IM) a model potent anti-inflammatory drug was used in the present study. Although it has been shown to reduce post-operative inflammation and to decrease the intraocular irritation after cataract extraction and in cystoid macular oedema, the clinical use of its marketed most commonly used eye drops is limited due to its poor bioavailability and topical side-effects, including burning sensation, irritation, and epithelial keratitis. Based on these considerations and keeping in mind that enhancement of the precorneal processes such as the slow removal of IM from the absorption site and the improvement of both paracellular and intracellular pathways of epithelial cells would be of great benefit to reduce the dose, its frequent instillation and consequently reduce the local side-effects inherent to this drug.


Considering the drawbacks associated with the current formulation, the present investigation has been dedicated to prolonging the retention time of medication on the eye surface and to the improvement of transcorneal penetration as well as solubility of poorly soluble drug IM by formulating nanosuspension.




Indomethacin drug supplied as a gift sample by Micro Labs Limited, Hosur (India), Eudragit RS 100 polymer purchased from Central Institute of Fisheries Technology, Cochin. Tween 80 was purchased from Himedia Laboratories Pvt. Ltd., Mumbai. Benzalkonium Chloride was purchased from Ranbaxy Fine-Chem. Pvt., Ltd., Mumbai.



Preparation of Eudragit RS 100 nanosuspension:4 -6.

Nanosuspensions were prepared by the quassi- emulsion solvent diffusion technique. Nanosuspension was prepared by using different drug to polymer ratio. Quantity of drug in all formulation was kept constant i.e. 100 mg. The different ratio of drug and polymer is as given in table no.1. The drug and polymer were co- dissolved at room temperature in ethanol (5 ml) and sonicated for 10 minutes. The solution was slowly injected with syringe into 50 ml water containing Tween 80 (0.02 %w/v) and benzalkonium chloride (0.1 % w/v) and kept at low temperature in an ice water bath.


During injection the mixture was mixed by mechanical stirring (propeller 4000 rpm) for one hour. The solution immediately turned into a pseudo-emulsion of the drug and polymer-ethanol solution in the external aqueous phase. The counter diffusion of ethanol and water out of and into the micro droplets. After completion of stirring the solution dispersion was subjected to ultra sonication for a period of 10 minutes. The gradual evaporation of the organic solvent determined the in situ preparation of the polymer and the drug with the formation of matrix type nanoparticles. Ethanol residues were left to evaporate off under slow magnetic stirring of the nanosuspensions at the room temperature for 8-12 hours. Using this above method 5 formulations of nanosuspension FN-1, FN-2, FN-3, FN-4 and FN-5 were prepared by varying polymer concentration.


Table no.01:  Formulation of different batches of Indomethacin Nanosuspension








(% w/w), mg






Eudragit RS 100 (%w/w ), mg






Tween 80

(% w/v)






Benzalkonium chloride (% w/v)






Ethanol (ml)






Water qs to (ml)







Compatibility study:

Compatibility of the IM with Eudragit RS 100 used to formulate nanosuspension was established by Fourier Transform Infra Red spectral analysis, Schimadzu, Japan. FT-IR spectral analysis of IM, Eudragit RS 100 and combination of the IM with Eudragit RS 100 was carried out to investigate any changes in chemical composition of the drug after combining it with the excipients.


Evaluation of pH:5

pH is one of the important factors involved in the formulation process. Two areas of critical importance are the effects of pH on solubility and stability. The pH of ophthalmic formulation should be such that the formulation will be stable at that pH and same time there would be no irritation to the patient upon administration of the formulation. The pH of the prepared formulations was checked by using pH meter.


Particle Size and surface morphology:5, 6

Particle size analysis was done by Scanning Electron Microscopy (SEM).SEM is the most commonly used method for characterizing drug delivery systems, due to simplicity in sample preparation and ease of operation. The three dimensional information about macro – (0.1- 10 nm) meso (1- 100 nm) and nanostructure (10-1,000 nm), is often found within the same micrograph.


SEM has been used to determine particle size distribution, surface topography, texture and to examine the morphology of fractured or sectioned surface. Particle size analysis was done by SEM using JEOL JSM- T330A scanning microscope. Cleaned brass specimen studs were used for mounting the samples. Wet solvent paint was applied on these studs and while the paint was wet, the pellets were placed on each studs and allowed to dry. Then the sample was observed in scanning electron microscope and photographs were taken


Determination of Drug Entrapment Efficiency:6, 7

The percentage of incorporated indomethacin (entrapment efficiency) was determined spectrophotometrically at 320 nm. After centrifugation (5000 r.p.m. for 5 minute) of the aqueous suspension, amount of the free drug was detected in the supernatant and the amount of incorporated drug was determined as the result of the initial drug minus the free drug.

The percentage efficiency (EE %) could be achieved by the following equation

Entrapment efficiency (%) =  

W initial drug – W free drug        W initial drug    X 100


Zeta potential:8, 9

The particle charge is one of the factors determining the physical stability of emulsions and suspensions. The higher particles are equally charged, the higher is the electrostatic repulsion between the particles and the higher is the physical stability.

Typically the particle charge is quantified as called zeta potential, which is measured e.g. via the electrophoretic mobility of the particles in an electrical field. Alternatively the particle charge can be quantified in surface charge per surface unit, determined by colloid titration. Zeta potential is an abbreviation for electrokinetic potential in colloidal systems. In the colloidal chemistry literature, it is usually denoted using the Greek letter zeta, hence ζ-potential. From a theoretical viewpoint, zeta potential is electric potential in the interfacial double layer (DL) at the location of the slipping plane versus a point in the bulk fluid away from the interface.


Differential scanning calorimetry: 10, 11

DSC is an important evaluation technique to find any possible interaction between drug and polymer. Any such interaction leads to reduce entrapment efficiency of polymer and also efficacy of drug. Differential scanning calorimetric analysis was performed using Shimadzu DSC- 60 system. Polymeric sample of nanosuspension was sealed in aluminum cells and set in a Shimadzu DSC-60 apparatus between 30șC - 300șC. Thermal analysis was performed at a heating rate maintained at a 10șC per minute in a nitrogen atmosphere. Alumina was used as the reference substance. Enthalpy changes (ΔH) were calculated from peak areas of samples and to study the polymeric changes in formulations.


In vitro Drug Release Studies: 4, 6

The in vitro release of indomethacin from a formulation was studied through Dialysis membrane-100 (cut of: 350 Da) using modified apparatus. The dissolution medium used was freshly prepared 0.14 M phosphate buffer solution (pH 7.4).Dialysis membrane -100, previously soaked overnight in the dissolution medium was tied to one end of a specifically designed glass cylinder ( open at both end). 5 ml of formulation was accurately placed into this assembly. The cylinder was attached to a stand and suspended in 50 ml of dissolution medium maintained at 37± 1ș C so that the membrane just touched the receptor medium surface. The dissolution medium was stirred at low speed using magnetic stirrer. Aliquots each of 1 ml volume were withdrawn at hourly intervals and replaced by an equal volume of the receptor medium. The aliquots were suitably diluted with the receptor medium and analyzed by UV- VIS spectrophotometery at 320 nm To check the eventual limiting effects of the dialysis membrane on drug dissolution, separate experiments were run in duplicate with a solution of saline, freshly prepared 0.14 M phosphate buffer solution (pH 7.4) in pure indomethacin of the drug concentration in the nanosuspension.


Rabbit eye irritation: 6, 12

Ocular irritation studies were performed on five male albino rabbits each weighing 1.5-2.2 kg. The potential ocular irritation and/ or damaging effects of the nanosuspension under test were evaluated by observing them for any redness, inflammation (or) increased tear production.

Formulation was tested on five rabbits by dispensing nanosuspension in the cul-de-sac of the left eye. Both eyes of the rabbits under test were examined for any signs of irritation before treatment and observed up to 24 hours.


Kinetic modeling:13, 14 ``

a) Zero order kinetics:

Drug dissolution from pharmaceutical dosage forms that do not disaggregate and release the drug slowly, assuming that the area does not change and no equilibrium conditions are obtained can be represented by the following equation –

                              Q t = Q o + K o t

Where Q t = amount of drug dissolved in time t, Q o = initial amount of drug in the solution and K o = zero order release constant.


b) Higuchi model:

Higuchi developed several theoretical models to study the release of water-soluble and low soluble drugs incorporated in semisolids and or solid matrices. Mathematical expressions were obtained for drug particles dispersed in a uniform matrix behaving as the diffusion media and the equation is

                              Q = KH. t 1/2

Where Q t = Amount of drug released in time t, K H = Higuchi dissolution constant.


c) Krosmeyer and Peppas release model:

To study this model the release rate data are fitted to the following equation

                              Mt / M = K.t n

Where Mt / M is the fraction of drug release, K is the release constant, t is the release time and n is the Diffusional exponent for the drug release that is dependent on the shape of the matrix dosage form.

If the exponent n = 0.5 or near, then the drug release mechanism is Fickian diffusion, and if n have value near 1.0 then it is non-Fickian diffusion.


Short term stability:15

Information on the stability of drug substance is an integral part of the systemic approach to stability evaluation. The purpose of stability testing is to provide evidence on how the quantity of a drug substance or drug product varies with time under influence of variety of environmental factors such as temperature, humidity, and light and to establish a re-test period for drug substance or a shelf life for the drug product and recommended storage conditions.


Stability is defined as the extent to which a product remains within specified limit throughout its period of storage and use. A drug formulation is said to be stable if it fulfills the following requirements:

·        It should contain at least 90% of the stated active ingredient.

·        It should contain effective concentration of the added preservatives, if any

·        It should not exhibit discoloration or precipitation nor develops foul odor.

From the 5 batches of IM loaded nanosuspension, formulation FM-3 was tested for stability studies. Formulation was divided into 3 sample sets and stored at:

4°C in refrigerator 27°C±2°C/65% RH±5% RH in humidity control oven. 40°C±2°C/65% RH±5% RH in humidity control oven.

After 90 days drug content of all the samples were determined by the method discussed previously in entrapment efficiency section. In vitro release study of formulation FM-3 was also carried out after 90days storage.


In Vivo Studies:4, 6, 15

Indomethacin is well known anti-inflammatory, reducing prostaglandin synthesis, which found to be most effective against ocular infection. Hence here also an attempt made to determine its anti-inflammatory activity with the help of best formulation (FN3) in comparison standard preparation. The study protocol was approved by institutional Animal Ethical Committee for the use of animal in research (proposal no.688/2/C/CPCSEA). In vivo were performed on groups of six male New Zealand albino rabbits weighing 1.8-2.2 kg, and with no signs of ocular inflammation or gross abnormalities. Animals were divided into two groups one for standard indomethacin as controlled preparation and another for formulation FN-3.


The 50 ”l of preparations were instilled into the conjunctival sac (left eye control preparation and right eye nanosuspension.) at 60,120,240 and 360 minute durations. To perform the paracentesis (The removal of fluid from a body cavity using a needle/puncture of the wall of a fluid filled cavity by means of a hallow needle to draw off the contents), animals were lightly anaesthetized with an  intramuscular injections of ketamine hydrochloride 50 mg/kg, Xylazine 10mg/kg was instilled into the conjunctival sac. Aqueous humor samples from each animal were collected with a 26 gauge needle attached to tuburculine syringe. The needle was introduced into the anterior chamber through the cornea, taking care not damage the iris, the lens and the anterior uvea.


Eye conditions were examined using slit lamp every hour after paracentesis and Cyclopentolate hydrochloride (Cyclogik) eye drops were put to avoid inflammation.50 ”l of aqueous humor were collected and analyzed by HPLC for drug concentration. The aqueous humor samples were frozen immediately and stored at -18 °C. For analysis the sample were mixed with an equal volume of methanol containing 6% v/v perchloric acid. After centrifugation (3 min at 12000 r.p.m), 20 ”l of the supernatant was analyzed by HPLC at detection wavelength 254 nm.


Mobile phase = acetonitrile –water –acetic acid (65:35: 1 v/v)

Flow rate = 1.0 ml /min

Detection = 254 nm

Column = Phenomenex C18 column (Luna 5 μm, 250 Ś 4.6 mm).

The statistical significance of the differences between means of Indomethacin concentration values in aqueous humor was evaluated using an ANOVA test.


Sterility Testing:16

One of the requirements of an ophthalmic preparation is its sterility.  The tests for sterility are intended for detecting the presence of viable forms of microorganisms in ophthalmic preparations. These tests were carried out under conditions designed to avoid accidental contamination of the product during the test.  The tests were based upon the principle that if micro-organisms are placed in a medium which contains nutritive material and water, and kept at a favorable temperature, the organisms will grow and their presence can be indicated by turbidity in the originally clear medium.  In the present study, three media namely, Alternate thioglycolate medium (ATGM), Fluid thioglycolate medium (FTGM) and Soyabean casein digest medium (SCDM) were used to investigate the presence of aerobic, anaerobic organisms and fungi.



Compatibility study:

From the IR spectral analysis, it was found that IR spectrum of pure drug IM and combination of pure drug with polymers like Eudragit RS 100 showed the all characteristic peaks of IM confirming the compatibility of the pure drug and polymer.


pH :

pH values for all the formulations were within acceptable range 5.8-6.4 and hence would not cause any irritation upon administration of the formulation. It was also observed that increase in Eudragit RS 100 polymer causes a slight increase in pH for formulations.


Drug entrapment efficiency:

The drug content in five batches of Indomethacin nanoparticles was studied. The amount of drug bound per 1 ml of nanosuspension was determined in each batch. The maximum entrapment was found in FN-3(96.0%) and lowest entrapment in FN-5 (83.57%). Sonication of the solution after addition of drug in polymer solution also plays important role in drug entrapment efficiency, as sonication leads to uniform distribution of the drug. Uniform distribution of drug will also give consistent entrapment efficiency of the same batch with less deviation


SEM photomicrograph of Indomethacin with Eudragit RS 100


Figure No. 01


Figure No. 02


Differential Scanning Caloritmetry:

The DSC thermograms for drug as well as formulations were represented in Figure 3 and 4. DSC analysis of Indomethacin showed the endothermic peak at its melting point i.e. at 158.6°C (ΔH = 14.94 J/g). DSC curves of the binary systems observed at 147.6șC (ΔH = 84.01J/g). They showed complete disappearance of the melting endotherm of Indomethacin, which could indicate the complete amorphization of the drug as well as loss of drug crystallinity, which indicates the change in melting point, release kinetics and bioavailability.


Figure No. 03: DSC of Indomethacin drug


Figure No. 04: DSC of formulation no.3


Stability Study

Stability study was carried out for the formulation FN3 by exposing it to various temperature 5-8oC, at room temperature and 40 + 2oC for 3 months. The sample was analyzed for drug content at regular interval of three months and it was evident that there was no remarkable change in the drug content of nanosuspension. Results show that formulation FN3 was stable at mentioned temperatures.


Table no.2: Stability study data for FN3

Sr. No.


% R.D.C.


% R.D.C.


% R.D.C.

40 + 2OC













100.00 + 0.00

99.97 + 0.038

99.84 + 0.012

99.63 + 0.011

99.24 + 0.023

98.54+ 0.041

100.00 + 0.00

99.95 + 0.038

99.81 + 0.010

99.54 + 0.031

98.93 + 0.021

98.47+ 0.021

100.00 + 0.00

99.94 + 0.038

99.73 + 0.025

98.44 + 0.026

98.80 + 0.031

98.33+ 0.013

R.D.C. = Remaining Drug Content in 7.4 pH buffer


Graph no.01


In vitro Release Study:

The release study was conducted for all the five formulations. Most of the formulations were found to have a linear release and the formulations were found to provide approximately 84.59% release within a period of 14 hours. Cumulative percent drug released for FN-2, FN-3 after 14 h was 92.39%, 96.18% and for FN-1, FN-4 and FN-5 after 14 h was 89.14%, 88.24% and 86.24%, respectively. The in vitro release of all the five batches of nanosuspensions showed an interesting bi-phasic release with an initial burst effect. In the first hour, drug released was 14.97%, 14.06%, 15.24%, 14.91% and 13.87 % for FN-1, FN-2, FN-3, FN-4 and FN-5, respectively. Afterwards the drug release followed a steady pattern approximating zero order release. The burst release in the first hour can be attributed to the drug loaded on the surface of nanoparticles.


Kinetic modeling:

The various kinetic models were applied to in vitro release data for prediction of the drug release kinetic mechanism. The release constants were calculated from the slope of appropriate plots, and the regression coefficient (r2) was determined.


Table 03:    Release kinetic models of different formulation


Zero order



Slope (K0 )

Correlation ( r2 )

Slope  ( KH )

Correlation ( r2 )

Slope ( n )

Correlation (r2 )






































Table no.4:   In vivo Release of Indomethacin after topical administration of Indomethacin loaded Nanosuspension

Time in hrs

Controlled  sample

Formulation FN3


Conc. ”g/ml


Conc. ”g/ml






















It was found that the in vitro drug release of nanosuspension was best explained by zero order kinetics as the plots shows highest linearity. The correlation coefficient (r2) was in the range of 0.9854 to 0.9978 for various formulations as shown in Table no.4. For formulation FN3 correlation coefficient (r2) is found to be 0.9919, indicating that the drug release was nearly independent of concentration, followed by Higuchi’s (r2 = 0.9916 with KH = 24.5834).


Graph no.2: In vitro drug release for all formulation


In the current study, drug release kinetic according to korsmeyer-peppa’s model is also followed. The values of release rate exponent (n), calculated as per the equation proposed by peppa’s, and all the slope values ranges from 0.5061 to 0.5204 revealed the fact that the drug release follows a Non Fickian Diffusion.


Sterility Testing:

Ultra-Violet radiation was used to sterilize the formulation and sterility testing was carried out under aseptic conditions. It was found visually that the Alternate thioglycolate, Soybean casein digest media; Fluid thioglygolate media containing sterilized formulation was free from turbidity. This confirmed the absence of aerobic organism, anaerobic organism and fungi. From this it confirms the sterility of formulation; therefore, the sterilized formulation was considered suitable for in vivo studies.

Rabbit eye irritation:

The prepared nanosuspension of Indomethacin showed satisfactory Ocular tolerance. No ocular damage or abnormal clinical sings were visible. Only a few sings of increased lacrimation were noted.


In vivo Study:

Formulation FN3 optimal particle size and satisfactory in vitro release was selected for in vivo drug studies. Drug concentration was determined by HPLC method. The drug concentration was determined in each formulation by calculating the peak areas of formulation FN3 and controlled sample from HPLC graphs.


The in vivo study was carried out using best formulation and standard sample. The maximum concentration was found to be 73.80”g/ml where as standard sample showing 13.96”g/ml, which indicate sustained action of formulation as compare to standard sample.


FN3: Formulation No.3; C-1: Controlled Sample



From the above findings it is evident that, polymeric system of IM loaded nanosuspension have achieved the objectives of increased contact time, prolonged release and decreased frequency of administration. Drug release studies indicate that the no significant increase drug release with increase in drug to polymer ratio but also depends on the pH, particle size. Zeta potential was found +45 mv which indicates better stability of IM nanosuspension. In vivo release profile indicated that polymeric system of IM has achieved the objectives of increased contact time, prolonged release, and decreased frequency of administration, avoidance of eye irritation and redness of the rabbit eye



I am thankful to Nandha College of Pharmacy and Research Institute for their financial support throughout my project work



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Received on 19.02.2010       Modified on 03.03.2010

Accepted on 20.03.2010      © RJPT All right reserved

Research J. Pharm. and Tech.3 (3): July-Sept. 2010; Page 854-860