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Development and Characterization of Flurbiprofen loaded niosomal In-situ gel for Ophthalmic Drug Delivery System

Manoj Bisht


, Rimpal Kanyal


Manoj Bhardwaj


, Alok Kumar


, Dipiksha


, Jyotirmay



1,2,3,4,5,6 Devsthali Vidyapeeth College of Pharmacy, Lalpur, Rudrapur, U.S.Nagar, Uttarakhand

* Manoj bisht E-mail address:[email protected] ABSTRACT

Niosome are non-ionic surfactant based liposomes, obtained by the hydration of artificial non-ionic surfactants, without or with addition of cholesterol or additional lipids. The intention of the current study was to organize and evaluate the in-situ niosomal gel loaded with flurbiprofen for the ophthalmic drug delivery system. Flurbiprofen, a nonsteroidal anti-inflammatory drug (NSAIDs) is used to relieve inflammation. Flurbeprofen loaded Niosomes along with cholesterol was successfully prepared by thin film hydration method, and the finding of different grades of span used (20, 40 and 60) were compared with the different ratio of cholesterol. Identification of Niosome were estimated by several parameters viz. drug entrapment efficiency, drug content, particle size and in-vitro diffusion analysis. Study revealed that Niosomes in the ratio (1:1) with span 60 and cholesterol (F9) showed most significant entrapment efficiency (79.2%).

Keywords: Niosomes, in-situ gel, flurbiprofen, TEM.


One of the most difficult challenges confronting the pharmaceutical industry is drug delivery. The currently available dosage forms of ophthalmic solution, suspension, and ointment are simply inadequate to combat some of the most serious infections. Effective drug delivery on the eye is extremely difficult due to the eye's extensive defence mechanisms, which make it difficult to achieve a successful concentration of the drug within the target area of the eye. The use of traditional ophthalmic dosage forms such as solutions, suspensions, and ointments is still permissible. Because of their limited bioavailability, such dose formulations are insufficient to treat different eye disorders such as glaucoma.[1].

Current ophthalmic formulations such as solution, suspension, and ointment have a number of disadvantages, due to reduced drug bioavailability in the ocular cavity. The main aim of designing a therapeutic system is to achieve an exact measure of drug at the target spot at the right time.The physico-chemical properties of a therapeutic drug alongwith anatomy and physiology of the eye are responsible for ocular disposition and elimination.[2].

Variety of methods have been implemented to enhance the bioavailability, onset of time and time of the therapeutic properties of ocular drugs. Following approaches are widely practiced :

● Use of sustained drug delivery systems, which transmit controlled and constant delivery of ophthalmic drugs.

● maximization of corneal drug absorption and minimization of precorneal drug loss[3,4].

Eye drops and ointments are the most frequent ophthalmic preparations available on the market. However, when these formulations are placed in the eye, they quickly lose their adhesion to the corneal layer due to tear flow and naso-lacrimal drainage. Because only a little amount (10 percent) is available for therapeutic efficacy thus frequent administration is required.


Therefore numerous efforts have been implemented to overcome these problems such as the introduction of novel ophthalmic pharmaceutical formulation were came. Which were aimed to improve drug saturation via the cornea and release the drug in a prolonged and regulated manner.[5].

Merits of Ocular Drug Delivery Systems


1. Simplest handling with better patient compliance is obtained because there is no need for any needles, skilled staff to assist with shipment, and self-medication.

2. Rapid absorption and quick onset of action due to high vascularity and bulky absorption surface.

3. Effective in the management of emergency conditions, rather than other routes of administration.

4. Potential dose reduction compared to oral administration because of bypass First pass metabolism.

Demerits of Ocular Drug Delivery Systems


1. Limited permeability of the cornea which results in the low assimilation of ophthalmic drugs.

2. Untoward systemic adverse effects are seen because a chief part of the administered dose is maintained in the tear duct due to draining.

3. The fast excretion of the drug via the result of intermittent and tear flow in a small phase of time.


A niosome is a vesicle based on non-ionic surfactants. Non-ionic surfactant and cholesterol incorporation as an excipient are used to synthesize niosomes. [8] Other excipients can be utilised as well. Niosomes have a greater penetration potential than previous emulsion formulations. [9] It can entrap both hydrophilic and lipophilic drugs in an aqueous layer or a non-ionic surfactant-based vesicular membrane. Niosomes are vesicles considered as novel drug delivery systems.Depending on the manner of preparation, it might be unilamellar or multilamellar. They are formed by incorporation of excipients such as with cholesterol or lipids and without cholesterol or lipids . The hydrophilic ends of the surfactant bilayer are exposed on the outside and inside of the vesicle, while the hydrophobic chains face each other within the bilayer. They have a bilayer structure similar to liposomes, but the ingredients used to prepare niosomes make them more stable.[10]

The size of niosomes is determined by the method of preparation. Thin film hydration, hand shaking, ether injection, reverse phase evaporation, sonication, microfluidization, and transmembrane pH gradient are some of the methods that can be used to make them[11]. Niosomes have a wide range of varieties that are dependent on the process used to prepare them as well as the bilayer composition. However, the principle is the same (i.e., lipid phase development followed by hydration in an aqueous media, resulting in the formation of Niosomes). Vesicle diameter, entrapment efficiency, in-vitro drug release, zeta potential analyses, and stability tests are used to characterise the Niosomes.[12]

Niosomes are microscopic-lamellar, spherical, unilamellar, and multilamellar structures that develop when non-ionic surfactants (span 20, span 40, span 60) and cholesterol are mixed together and then hydrated in distilled water. The hydrophilic ends of surfactant molecules are exposed on the outside and inside of the vesicle, while the hydrophobic chains face each other within the bilayer.[13]


Fig. 1: Niosomes



● As compared to liposomes, the niosomes are osmotically active, chemically stable and have long storage time.

● Because of the functional groups on their hydrophilic heads, their surface formation and modification is quite simple.

● They have high compatibility and low toxicity due to their non-ionic nature. moreover they are biodegradable and non-immunogenic too.

● They can entrap lipophilic drugs into vesicular bilayer membranes and hydrophilic drugs in aqueous compartments.

● They can improve drug molecules' therapeutic performance by protecting them from the biological environment, resulting in better availability and controlled drug delivery by restricting drug effects to target cells in targeted carriers and delaying clearance from circulation in sustained drug delivery.


In situ gels are drug delivery systems that are in solution form before administration in the body, but once administered, undergo gelation in situ, to form a gel.[15]

The administration of ocular medications is the most exciting challenge, and indeed the main problem facing the pharmacist is the drug's speedy precorneal clearance, which results in poor bioavailability and therapeutic response due to the dynamics and resources of the high tear fluid. This problem can be resolved by gel-forming in situ gel release system, prepared from the phase transition from the gel phase to the polymer phase, due to the change in the exact physical-chemical parameter (pH, temperature, ion-sensitive).[16]


Flurbiprofen was obtained as a gift sample from Arochem chemical laboratories , Delhi. Span 20,Span 40,Span 60, Polaxomer 407 and HPMC K4M were purchased by Yarrow Chem Products ,chloroform and Methanol were purchased from Finar reagent, Ahmedabad (Gujarat). All the chemicals and reagents used for study were of analytical grade.



Preparation of flurbiprofen niosomes by Thin Film hydration method-

After dissolving the drug and cholesterol in various ratios of methanol and chloroform, a certain amount of surfactant is added. A rotary vacuum evaporator was used to evaporate the solvent at a temperature of 60 ° C. In the round bottom flask, a thin layer of cholesterol and surfactant was produced. The phosphate buffer solution pH 7.4 (10 ml) was added to the round bottom flask at 60 ° C and stirred for about 15 minutes. This results in a fine dispersion of the mixture and the preparation of niosome.


Table no. 2.1: Niosomes with varying Cholesterol: Surfactant Molar Ratio

S.No Drug(mg) Cholesterol(mg) Surfactant (mg) Solvent (Chloroform:Methanol) (2:1)

1 50 100 100 15

2 50 95 105 15

3 50 90 110 15

4 50 85 115 15

Preparation of niosomal in situ gel

Different types of batches of gel were prepared by optimized formulation of flurbiprofen loaded niosomes using poloxamer 407. For the preparation of gel, Polaxomer 407 and HPMC K4M were dispersed in Niosomes and continuously stirring 100 rpm for 1hour.

Characterization of Niosomes


1.Calibration curve

Preparation of stock solution were done by, first 100mg of drug dissolve in 100 ml in different solvent of varying range of pH (eg.- phosphate buffer 7.4pH, methanol ext.). Prepare the different dilution of 2,4,6,8,10 µg/ml from the stock solution and check the absorbance of the drug by using a UV-visible spectrophotometer. All procedure were done in a triplicate manner. Calculate the calibration curve equation and R2 value.[19]


Fig no. 2.1: Calibration curve of Flurbiprofen in methanol 2. Partition coefficient:

The partition coefficient provides a specific concentration ratio in two different solvent solvents. When a phase is polar and another non-polar phase, i.e. the log P value calculated at that moment.

The drug that has a value of log p> 1 is classified as lipophilic.

Log p <1 is indicated for the hydrophilic drug.

Po / w = (C oil / Caq.) Balance

When using n-octanol and Polar solvent, a partition coefficient study was performed. The drug was added to 50 ml of polar and non-polar phase in the separation funnel. The mixture was stirred continuously for 2 hours until the equilibrium was allowed and maintained throughout the night. The two phases were separate in themselves. Both phases were analyzed for the respective contents of the drug by measuring the absorbance by a UV-visible spectrophotometer.[20]

3. Vesicle size and zeta potential

In this, the niosomes can be controlled by the dynamic diffusion of light (DLS) and by photonic correlation spectroscopy (PCS). The Zeta potential of the formula can be calculated from the Zeta meter in the nm range.[21]

4. Drug entrapment

Ultra centrifugation method is used for the determination of entrapment efficiency of the niosomes.

Entrapment efficiency of drug loaded niosomes was determined after separation of entrapped drug, which was performed by cooling centrifugation at 10,000 rpm for 30 min at 50C. The supernatant liquid was collected for absorbance . The separated vesicles were washed with phosphate buffer solutions and the washings were mixed with supernatant liquid. The vesicles were suspended in 5 mL phosphate buffer solutions and placed in a dialysis bag.The dialysis bag after tying at both ends was immersed in 200 mL phosphate buffer solutions, maintained at 37∘ C, and


stirred overnight by using magnetic stirrer . The absorbance of the drug was estimate by spectrophotometrically at

�max of 244 nm, against phosphate buffer solutions as blank.[22]

Entrapment efficiency = (theoretical amount-entrapped drug) / theoretical amount × 100

5. Drug content

Drug content of the niosomes can be determined using a UV spectrophotometer. It can also be quantified by a modified high performance liquid chromatographic technique.[23]

6. In-vitro drug release kinetics:

In order to consider the drug release mechanism and drug release data were analyzed


Calibration curve of flurbiprofen in phosphate buffer (7.4) for the identification of λmax uses any dilutions and checks the absorbance between the range of 200-400nm . The obtained λmax was observed at 244 nm .[24]

The concentration estimates of pure Flurbiprofen showed linearity (r2= 0.999) over the concentration range of 2- 10µg/ml passing through origin and it follows Lambert-beer's law. Slope and intercept values of calibration curve obtained are 0.13 and 0.002 respectively. The absorbance shown by standard solution Phosphate Buffer (pH7.4) is table 3.1.

The UV absorption data at 244 nm and concentration estimates of pure Flurbiprofen showed linearity (r2= 0.998) over the concentration range of 2-10µg/ml passing through origin and it follows Lambert-beer's law. Slope and intercept values of calibration curve obtained are 0.082 and 0.020 respectively. The absorbance shown by standard solution methanol is given in table no.2.4 and the standard curve is revealed in figure no. 3.1.

The calibration curve equation Y= 0.096x + 0.009 and the r2 value = 0.999

Table 3.1: Calibration curve of flurbiprofen in phosphate buffer (7.4)

S.No Concentration(µg/ml) Absorbance

1 2 0.201±0.2015

2 4 0.392±0.3951

3 6 0.598±0.5981


4 8 0.776±0.775

5 10 0.973±0.975

Fig.3.1: Calibration curve of flurbiprofen in phosphate buffer (7.4)

Table 3.2: calibration curve of flurbiprofen in methanol

S.No Concentration(µg/ml) Absorbance

1 2 0.198±0.195

2 4 0.347±0.349

3 6 0.549±0.5495

4 8 0.745±0.7452

5 10 0.953±0.955


Fig.3.2: Calibration curve of flurbiprofen in methanol 2. Solubility studies of drug in different solvent

Table 3.3: Solubility studies of drug in different solvent

Solvent Result

Water Sparingly soluble

Alcohol Soluble

Methanol Freely soluble

Acetone Freely soluble

Phosphate buffer pH 7.4 Soluble

Chloroform Soluble

● After Solubility analysis it was established that Flurbiprofen drug was liberally soluble in acetone, methanol and phosphate buffer (pH 7.4).

3. Determination of partition coefficient

Table.3.4: Determination of partition coefficient


Organic solvent/

Aq. Phase

Abs. in oil phase Df= 100 Abs. in aq. Phase P = C1/


Log P

N octanol / distilled water

1st 2nd 3rd 1st 2nd 3rd 12.75 1.10

0.152 0.155 0.154 0.011 0.013 0.012

Average value

0.153 0.012

4. FTIR Spectra

Fig.3.3: FTIR Spectra of drug Fig.3.4: FTIR Spectra of Drug+ lipid


Fig.3.5: FTIR Spectra of EXCIPIENT

4. Drug content & Drug Entrapment

● Table 3.1 shows the drug entrapment efficiency of niosome formulations. The formulation with the highest drug entrapment efficiency was 88.20% (F12), while the formulation with the lowest drug entrapment efficiency was 75.71% (F3). The entrapment efficiency of the niosomal formulation improves as the concentration of span increases and the concentration of cholesterol decreases. The entrapment efficiency of Span 60 is higher.

● Table no. 3.1 reveals the drug content formulations. The maximum drug concentration was found to be 89.34% in the (F11) formulation, while the lowest drug content was found to be 65.02% in the (F3) formulation. As the concentration of non-ionic surfactants increases, so does the drug content of niosomal formulations.

● Weight the noisome equivalent to 100 mg of drug dissolve in methanol and after centrifugation the supernatant layer separated and filtered and checked the absorbance in the uv- visible spectrophotometer at 244 nm wavelength.


Table no.3.5:Determine of drug entrapment and drug content

Formulation Percentage of Drug Entrapment Percentage of Drug content

F1 (span20) 80.00 75.20

F2 (span20) 78.20 69.45

F3 (span20) 75.71 65.02

F4 (span20) 78.05 70.51

F5 (span40) 79.51 79.20

F6 (span40) 75.20 80.74

F7 (span40) 76.52 85.12

F8 (span40) 75.42 80.11

F9 (span60) 80.10 85.21

F10 (span60) 82.52 80.33

F11 (span60) 85.21 89.34

F12 (span60) 88.20 85.21


Fig.3.6: Percentage of Drug Entrapment of Niosomes

Fig.3.7: Percentage of Drug Content

5.Particle size by zeta


Fig.3.8: Zeta potential report

The result of the particle size from the above graph showed the particle range varies from 138 nm to 734 nm and the average particle size was 1004 nm for the f5.

6. In-vitro drug release Pure Drug Dissolution

Fig.3.9: %cdr of Flurbiprofen


Fig.3.10:Percentage cumulative amount of drug using span 20 formulation vs time

Fig.3.11:Percentage cumulative amount of drug using span 40 formulation vs time


Fig.3.12:Percentage cumulative amount of drug using span 60 formulation vs time

Fig.3.13:In-vitro drug release of gel pure drug and optimized formulation F5 (0.5%)


Fig.3.14:zero order release

Fig.3.15:First order release



Higuchi model


Korsmyer peppas model


Table -3.6: Release pattern of Flurbiprofen niosomes

Zero order kinetics First order kinetics Higuchi model Korsmeyer-peppas model

r2 r2 r2 r2 N

0.952 0.968 0.951 0.870 0.935

Table -3.7: Evaluation of optimized Niosomal in-situ gel

Formulation Clarity pH Viscosity (cps) %Drug


%Drug Entrapped

F11 in-situ gel Turbid 7.4 5.2 89.34 85.21


Niosomes are versatile, easy to prepare, biocompatible nanocarriers with size ranging from tens of nanometers to few micrometers. The niosomes are utilized for drug and gene delivery to both anterior and posterior segments of the eye with increased corneal permeation, higher ocular bioavailability of the drug, and prolonged drug release. The formulation was found to be transparent and to have a high capacity for in situ gelling. The improved formulation was sterile and demonstrated long-term drug release over an 8-hour period. The formulation demonstrated sustained drug release and a lengthy drug residence time, enhancing the medication's effectiveness by allowing it to be localised at the site of action. Study finding suggested that niosomes could be a better candidate as a novel ocular drug delivery system.

5. CONFLICT OF INTEREST: Author states that there is no conflict of interest.

6. ACKNOWLEDGEMENT: Author is highly thankful to the Department of Pharmacy of Devsthali Vidyapeeth College of Pharmacy, Rudrapur for providing spectral and analytical facilities.



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