Optimization and Characterization of Lipid Based Nano Emulsion of Prednisolone Acetate for Ophthalmic Drug Delivery
Hardik Joshi1*, Dr. Pragna Shelat2, Dr. Divyang Dave2
1PhD Scholar, Kadi Sarva Vishwavidyalaya, Gandhinagar, Gujarat, India.
2K.B. Institute of Pharmaceutical Education and Research, Kadi Sarva Vishwavidyalaya,
Gadhinagar, Gujarat, India.
*Corresponding Author E-mail: hardikjoshi14@gmail.com
ABSTRACT:
Prednisolone acetate is a topical anti-inflammatory agent used in the treatment of steroid responsive inflammation of palpeberal and bulber conjunctiva, cornea and anterior segments of the globe. The aim of the present work is to optimize and characterize lipid based nano emulsion of a poorly water-soluble drug, Prednisolone acetate. Lipid based nanoemulsion was prepared using micro fluidization process. Solubility studies were carried out to identify suitable oil and surfactant. A three level three factor Box-Behenken design was used to optimize lipid based nano emulsion. Prepared formulation was characterized for drug content, pH, globule size, zeta potential, polydispersity index, osmolality and in vitro drug release study by dialysis method using bottle apparatus. Eye irritation study was carried out by Hen’s egg chorioallantoic Membrane test. Stability study of the prepared formulation was performed as per ICH guidelines. Prepared nanoemulsion is transparent with a blue tinge. Optimized batch of lipid based nanoemulsion showed average globule size of 110nm with a poly dispersity index less than 0.11. The results of in vitro drug release study suggest more than 90% drug release over a period of 12h. Developed formulation was found to be non-irritant and stable when stored at 40°C and can be used for ophthalmic delivery.
KEYWORDS: Prednisolone Acetate, Lipid based nanoemulsion, Box-Behenken design, Microfluidization, Hen’s egg chorioallantoic method.
INTRODUCTION:
Prednisolone acetate is a topical anti-inflammatory agent widely used for the treatment of steroid-responsive inflammation of the palpeberal and bulbar conjunctiva, cornea, and anterior segment of the globe. Prednisolone acetate is practically insoluble in water, hence difficult to formulate as aqueous eye drops.
An emulsion-based formulation approach offers an advantage to improve the solubility and bioavaibility of poorly water-soluble drugs. There are two types of emulsion which are commercially available and used as a vehicle for active pharmaceuticals: oil in water (o/w) and water in oil (w/o) emulsion.
For ophthalmic drug delivery, o/w emulsion is common and widely used because of less irritation and better ocular tolerance over w/o emulsion. Nano emulsion is transparent or translucent system covering a size range of 50nm to 200nm. They are defined as thermodynamically stable dispersions of oil-in-water (o/w) stabilized by an interfacial film of surfactant and co-surfactant molecules.[1] They exhibit low viscosity and transparency which make them very attractive delivery system in pharmaceutical field. Nano emulsions are novel drug delivery system having potential application in improving solubility and bioavaibility of poorly water-soluble drug.[1,2]
Conventional dosage forms, such as eye drop solution account for 90% of marketed ophthalmic formulations. The reason may be attributed to ease of administration and patient compliance. The dosage regimen of marketed suspension product of prednisolone acetate is one to two drops into the conjunctival sac two to four times daily. The adverse reaction associated with use of market suspension product is elevation of intraocular pressure (IOP) with possible development of glaucoma and infrequent optic nerve damage, posterior sub capsular cataract formation and delayed wound healing.[11] The disadvantage of topical application of current ophthalmic preparations is poor bioavailability due to pre-corneal processes such as rapid removal of drug from the absorption site and the existence of a cornea that restricts the passage of the drug molecules.[6] Dose uniformity and subsequently the dosing error are key issues of ophthalmic suspension preparation. Also it is difficult to sterilize the ophthalmic suspension as sterile filtration technique cannot be applied to suspension due to undissloved particles.
Lipid-based nano carriers have properties similar to those of three-layered tear film. Lipid components in the lipid-based nano carriers might interact with the lipid layer of the tear film, enabling the carriers to remain in the conjunctival sac for a long time, where they act as a drug depot.[6] The natural biodegradability, nanometer droplet size range, sterilizability and substantial drug solubilization either at the innermost oil phase or at the o/w interface, and improved ocular bioavailability are thus making the lipid emulsion a promising ocular delivery vehicle.[7-9] A lipid emulsion containing cyclosporine A 0.05% (Restasis™, Allergan, Irvine, USA) is now exploited commercially as a vehicle to improve the ocular bioavailability.[10]
The purpose of the present research work was to develop, optimize and characterize the lipid based nano emulsion containing prednisolone acetate. The impact of process variables during crude emulsion preparation and different homogenization techniques were evaluated for final emulsion preparation. The effect of different process variables on globule size and polydispersity index was assessed using the using Box Behnken statistical design and in-vitro characterization was successfully established. The irritation potential of optimized nano emulsion was also assessed by Hen’s Egg Chorioallantoic Membrane Test.
MATERIALS AND METHODS:
Prednisolone Acetate, a water insoluble drug was purchased from Farmabios (Italy). Vegetable oils like Olive oil, Sesame oil, Castor oil, Cotton seed oil and Soybean oil were obtained as a gift samples from Croda chemicals (USA). Miglyol 812 N was obtained as gift sample from Cremer (Oleo, Germany). Surfactants like Tween 80, Tween 20, Tween 85 and SPAN 80 were obtained as gift samples from Croda chemicals (USA). Sodium chloride, Disodium edetate, Sorbic acid, Sodium Acetate trihydrate, Sodium hydroxide and Hydrochloric acid were purchased from Merck (Germany). Other chemicals used were of analytical grade.
Quantification of Prednisolone Acetate:
An HPLC system (LC-2030C, Shimadzu Prominence, Japan) consisting of an auto sampler, pump, column oven, UV detector and data processing software (Empower®3) was used for analysis of drug content. YMC Triart, C18; 250X4.6mm, 5µ column was used. Analysis of prednisolone acetate was carried out using a mixture of water and acetonitrile (65:35 %v/v) as mobile phase at a flow-rate of 2.0mL/min at 25⁰C. Detection was performed at 254nm. A standard solution of prednisolone acetate was prepared by dissolving 25 mg in 50 mL of methanol. 5mL of this solution was further diluted up to 50mL with methanol. Series of solutions were prepared by taking 1, 2, 3, 4 and 5mL of this solution and diluted up to 50 mL with methanol to formulate the solution of 1, 2, 3, 4 and 5µg/mL respectively. The standard curve of peak area was plotted against concentration of solution and linearity, limit of detection and limit of quantification was calculated.
Solubility study of Prednisolone acetate in oil and surfactant:
Approximately 5g of different vegetable oils was taken in small vials and excess amount (0.2g) of prednisolone acetate was added. The vials were tightly closed with help of stoppers and continuously stirred for 48 hours at 37°C±1°C using orbital shaker. The samples were centrifuged at 9000rpm for 30 min. The undissloved suspended particles were removed by membrane filter (pore size 0.45 micron, Millipore, Merck, Germany). Filtered solution (0.5g) was dissolved in dichloro methane and diluted to 50mL. From above prepared solution, 5mL of solution was added in dichloro methane and diluted to 50mL. The concentration of prednisolone acetate was determined by measuring absorbance at wavelength of 243nm using UV spectrophotometer. Each determination was performed in triplicate. The blank solution for each experiment were prepared by dissolving respective vegetable oils in dichloride methane and diluted to appropriate concentration. Similar procedure was adopted for determination of solubility of Prednisolone acetate in different non-ionic surfactants like Span 80, Tween 85, Tween 80 and Tween 20.
Preparation of Lipid based Nano emulsion:
The oil in water emulsion was prepared using the Prednisolone acetate (0.5mg/mL), castor oil (5% w/v) as an oil phase and Tween 20 (5% w/v) as an emulsifying agent. The Lipid nano emulsion was prepared in two steps: (1) Preparation of crude emulsion (2) Preparation of final emulsion. In first step, the weighed quantity of prednisolone acetate was added and dissolved into mixture of castor oil and Tween 20 previously heated at 70⁰C. The crude emulsion of above mixture was homogenized with 250mL of water using high shear homogenizer (Polytron, Kinematica AG, MT 5100 S, Switzerland) at 10,000 RPM for 30 min by maintain temperature of 50⁰C±3⁰C.
In another beaker, weighed quantity of other excipients like sodium chloride, disodium edetate, sorbic acid and sodium acetate trihydrate were added and dissolved in 200mL of water at 50⁰C±3⁰C. In second step, crude emulsion was further homogenized using high pressure homogenizer (Microfludizer, M-110P, Microfluidics Corporation, USA) at inlet pressure of 19500 psi for 4 cycles by maintaining temperature of emulsion at 50⁰C ±3⁰C. After completion of homogenization, emulsion was mixed with excipient phase under continuous stirring. The above emulsion was cooled and pH of emulsion was adjusted to 5.5 using freshly prepared sodium hydroxide and/or hydrochloric acid solution. The final volume of emulsion was adjusted to 500 mL using water and sterilized using filtration with help of 0.22micron PVDF membrane (Millipore, Merck, Germany).
Optimization of formulation using Box-Behenken Design:
A Box-Behenken statistical design was applied for optimization of process variables to develop the stable Nano emulsion for ophthalmic drug delivery. A three factor three level Box-Behenken response surface method using Design expert (Version 9.0, Stat Ease Inc., USA) was used for optimization purpose. The Homogenization pressure (X1), Number of cycles (X2) and Homogenization temperature (X3) were selected as independent variables whereas the responses Globule size (Y2) and Polydispersity index (Y2) were selected as dependent variables. The method was used to explore the response surfaces and for constructing polynomial models to evaluate the main effects, interaction effects and quadratic effects of process variables on Globule size and Polydispersity Index. The design matrix comprising of 15 experimental runs were constructed for which non-linear software generated quadratic model is defined as; Y = b0 + b1X1 + b2X2 + b3X3 + b12X1X2 + b13X1X3 + b23X2X3 + b11X12 + b22X22 + b33X32 where Y is measured response, b0 is an intercept, b1 to b33 are regression coefficients. The term X1, X2 and Xi2 (i = 1, 2 or 3) represent the interaction and quadratic terms. Table 1 shows the design layout with Independent variables and corresponding response of dependent variables.
Characterization:
Globule size and Polydispersity Index:
Globule size and Polydispersity index of the Nano emulsion was determined by zeta sizer (Nano ZS, Malvern Instruments, UK) that analyzes the fluctuations in light scattering due to Brownian motion of the globules. Light scattering was monitored at 25°C at a 173° angle in triplicate. The formulation (0.1 mL) was diluted up to 10mL of water and used for measurement.
Table 1: Independent variables and corresponding response of dependent variables
Formulation |
Independent variables |
Response |
|
||
X1, Homogeniza-tion pressure(°C) |
X2, Homogenization cycles (nos) |
X3, Homogenization temp (°C) |
Y1, Globule size (nm) |
Y2, PDI |
|
PNE1 |
10000 |
4 |
35 |
140.6 |
0.198 |
PNE2 |
15000 |
2 |
65 |
129.2 |
0.148 |
PNE3 |
20000 |
2 |
50 |
118.0 |
0.142 |
PNE4 |
15000 |
4 |
50 |
128.0 |
0.136 |
PNE5 |
20000 |
6 |
50 |
106.0 |
0.116 |
PNE6 |
15000 |
2 |
35 |
132.2 |
0.178 |
PNE7 |
20000 |
4 |
35 |
126.4 |
0.156 |
PNE8 |
15000 |
6 |
65 |
114.5 |
0.104 |
PNE9 |
10000 |
2 |
50 |
142.6 |
0.210 |
PNE10 |
15000 |
6 |
35 |
125.8 |
0.158 |
PNE11 |
10000 |
6 |
50 |
132.2 |
0.161 |
PNE12 |
20000 |
4 |
65 |
102.0 |
0.112 |
PNE13 |
15000 |
4 |
50 |
125.0 |
0.132 |
PNE14 |
15000 |
4 |
50 |
122.4 |
0.136 |
PNE15 |
10000 |
4 |
65 |
127.4 |
0.145 |
Zeta potential:
Zeta potential of the nano emulsion formulation was measured based on electrophoretic mobility of globules as such without dilution using zeta sizer (Nano ZS, Malvern Instruments, UK) in triplicate.
pH:
The pH of the nano emulsion formulation was measured using pH meter (Thermo scientific, USA) in triplicate at 25°C.
Viscosity:
Viscosity of the nano emulsion formulation was measured using Brookfield viscometer (Model: LVDV-II+P, USA) fitted with an S-0 spindle at 25°C. A sample volume of 16 mL was used.
In-vitro drug release study:
The in-vitro drug release profile of nano emulsion was performed by dialysis method using bottle rotating apparatus. The Float-A-Lyzer G2 Device (MWCO-1000 KD Cellulose Ester membrane, Spectrum chemicals, USA) was used for in-vitro drug release study. The simulated tear fluid of pH 7.4 containing 0.75% SLS was selected as dissolution medium. The emulsion was accurately weighed (0.5g) and transferred into treated device. Similarly, the dissolution medium was accurately weighed (0.5g) and transferred to same device. The sample loaded Float-A-Lyzer G2 Devices were placed into bottle (250mL capacity) containing 225 mL of dissolution media by ensuring no leakage. The in-vitro release test was carried at flow rate of 12 RPM by maintaining the temperature at 37°C±2°C. A 5mL of sample was withdrawn at pre-determined time interval up to 12 h and same volume of fresh medium was added to maintain the constant volume. The concentration of prednisolone acetate was determined using HPLC system. The study was carried out in triplicate and average values were taken.
Hen’s Egg Chorioallantoic Membrane Test:
The irritation potential of lipid based nano emulsion formulation was assessed by Hen’s Egg Chorioallantoic Membrane Test. Commercially available fertilized white chicken eggs without micoplasms were used for the test. Eggs were used on the 10th day of incubation, after having been controlled for embryo viability. The eggs were opened near to the air cell using a pair of surgical scissors. The section of the shell was carefully pared off to reveal the highly vascularised chorioallantoic membrane (CAM). After test sample application of 0.3 mL, the CAM surface was observed over a period of 5 min with interval of 0.5 min, 2 min and 5 min. The time of the appearance of haemorrhage, lysis and coagulation occurring on the vascular system was recorded.
Stability study:
The optimized nano emulsion formulation was filled in low density polyethylene (LDPE) bottles and charged for the accelerated as well as long term storage condition as per ICH guidelines (40±2°C/75±5% RH and 25±2°C/60±5% RH) for a period of 3 months and 6 months in stability chambers (Newtronic, India). The samples were evaluated for the various physicochemical parameters in order to check the effect of storage time and temperature.
RESULTS AND DISCUSSION:
Solubility study:
The solubility of Prednisolone acetate in different oils and surfactants was determined in order to formulate an emulsion. Prednisolone acetate exhibited highest solubility in castor oil (3.47±0.78mg/g) and polysorbate 20 (5.70±0.75mg/g). From the solubility results, the castor oil and polysorbate 20 were selected for preparation of lipid based ophthalmic nano emulsion.
Preparation of Lipid based nano emulsion:
The Lipid based nano emulsion was prepared in two steps: (1) Preparation of crude emulsion (2) Preparation of final emulsion.
To investigate the effect of speed during high shear homogenization, the crude emulsion was prepared using high shear homogenization at different RPM by keeping the temperature at 50°C±3°C constant. All crude emulsions were further processed to final emulsion using Microfludizer at constant homogenization pressure of 20, 000 PSI at temperature of 50°C±3°C. The final emulsions were evaluated for globule size and PDI. The globule size and PDI remained unchanged when crude emulsion was prepared at different RPM which clearly indicate that homogenization pressure and number of cycles during final emulsion preparation have direct impact on globule size reduction. Based on study data (not shown), it can be concluded that there is no significant impact of homogenization speed on globule size and PDI during crude emulsion preparation. Hence, processing parameters during crude emulsion preparation was not optimized further.
Two different homogenization technique i.e. high-pressure homogenization and Microfluidization were evaluated to investigate the impact of homogenization techniques on final emulsion preparation. The final emulsion was prepared at pressure of 20,000 PSI and temperature of 50°C±3°C using two different equipments like high pressure homogenizer (SPX Flow Technology, Model 2000, Denmark) and Microfludizer (Microfluidics Corporation, M-110P, USA).
The nano emulsion produced using high pressure homogenizer took longer time for getting desire globule size as compared with Microfludizer. The high-pressure homogenization took around 7 pass to get the globule size of 120nm with PDI of 0.132. However, Microfluidization process took only 2 pass to reach the globule size of 118.4nm with PDI of 0.138. The number of homogenization cycle required for high pressure homogenizer is higher than Microfludizer which will directly increase the total processing time for preparation of nano emulsion.
These findings are attributed to the different mechanism of globule size reduction for both equipments. In high pressure homogenizer, crude emulsion enters in valve (made up of ceramic) at high pressure and low velocity. The product flows through the adjustable close clearance area between the valve and seat with rapid increase in velocity and corresponding decrease in pressure. This intense energy transition will disrupt the globules. In Microfludizer, the fixed geometry interaction chamber with specific size of orifice (made up of Diamond) by design produces uniform and high shear rate for globule size reduction with reproducible and scalable results. Hence, processing parameters of Microfluidization process for final emulsion preparation was further optimized.
Optimization using Box Behenken Design:
The Microfluidization process for preparation of nano emulsion was adopted for optimization of critical process parameters like homogenization pressure (X1), number of cycles (X2) and homogenization temperature (X3). A three factor three level Box Behenken statistical design was applied in order to optimize process parameters with desired physicochemical characteristics. As per the study design suggested by Design Expert®, a total of 15 runs were conducted. The details regarding input factors and output variables are given in Table 1. The data was fitted into different models and the model regression equations representing relationship between input factors and each response can be found as following,
Globule Size Y1= +124.82 -11.30*X1 -5.44*X2 -6.49*X3
Poly dispersity Index (PDI) Y2= +0.13 -0.024*X1 -0.017*X2 -0.023*X3 +5.750E-003*X1X2 +2.250E-003*X1X3 -6.000E-003*X2X3 +0.014*X12 +8.417E-003*X22 +3.917E-003*X32
Effect of independent variables on Globule Size (Y1):
It has been observed that globule size (Y1) follows linear regression relationship with three factors, homogenization pressure (X1), homogenization cycle (X2) and homogenization temperature (X3). In regression equations, a positive sign indicates a positive correlation between response and that particular factor, and a negative sign indicates an inverse relation between response and that particular factor.
From the model equation for Y1 provided above, it is evident that globule size has linear relationship with the three factors with the range included in the study. All the three factors have negative impact on globule size i.e., increasing these parameters reduces average globule size in a given range of input variables. Although all the three factors have significant effect on globule size, homogenization pressure exerts maximum impact on reduction in particle size (p<0.0001) as compared to other two factors.
It is evident from Figure 1 that as all three factors (i.e., homogenization pressure, number of cycles and homogenization temperature) increase, globule size decreases. This is in line with the negative coefficients in linear equation for globule size (Y1).
Effect of independent variables on Polydispersity Index (Y2)
Polydispersity Index (PDI (Y2)) follows quadratic model while fitting with the three factors X1, X2 and X3. In regression equations, a positive sign indicates a positive correlation between response and that particular factor, and a negative sign indicates an inverse relation between response and that particular factor.
From the model equation for Y2 provided above, it is evident that PDI has quadratic relationship with the three factors with the range included in the study. It is evident from the ANOVA that all the three main effects and X12 have statistically significant effect on PDI outcome. All the main effects like X1, X2 and X3 have negative impact on PDI, i.e., increasing these parameters reduces PDI in a given range of input variables.
Figure 1: 2D contour plot for globule size (Y1) at constant homogenization temperature 35°C indicating linear relationship
Figure 2: 2D contour plot for Polydispersity Index (Y2) at constant homogenization temperature 35°C indicating nonlinear relationship
At the same time, X12 have negative impact on Y2.
Two-dimensional contour plots as given in Figure 2 can be used to understand interaction among factors on the responses. These plots represent effect of two independent variables simultaneously on a response, keeping third independent factor at constant level. It can be seen that effects of independent variables PDI is nonlinear due to effects of interaction terms.
Generation of Design Space:
As per the model suggested above, the process was optimized to get formulation with desired globule size (~ 110 nm) and PDI (~ 0.11). In order to confirm the design space (Figure 3), a confirmatory run was conducted with optimized process parameters. This run was selected based on highest predictability suggested by software. The results of confirmatory run are in line with the predicted optimized process parameters which clearly indicate that the developed model for both responses is accurate and reliable.
Figure 3: Overlay plot, yellow region indicates design space for lipid based nanoemulsion
Characterization:
Globule size and Polydispersity Index:
Globule size and Polydispersity index of the Nano emulsion was determined by zeta sizer (Nano ZS, Malvern Instruments, UK) that analyzes the fluctuations in light scattering due to Brownian motion of the globules. The results are enumerated in Table 2.
Zeta potential:
Zeta potential of the nano emulsion formulation was measured based on electrophoretic mobility of globules as such without dilution using zeta sizer (Nano ZS, Malvern Instruments, UK) in triplicate. The results are depicted in Table 2.
pH:
The pH of the nano emulsion formulation was measured using pH meter (Thermo scientific, USA) in triplicate at 25°C. The results are depicted in Table 2.
Viscosity:
Viscosity of the nano emulsion formulation was measured using Brookfield viscometer (Model: LVDV-II+P, USA) fitted with an S-0 spindle at 25°C. A sample volume of 16 mL was used. The results are depicted in Table 2.
Table 2: Results of characterization studies of optimized lipid based nanoemulsion
Method |
Initial |
Globule size (nm) |
Zavg = 114.5 PDI = 0.109 |
Viscosity (cps) |
1.55 |
Zeta potential (mV) |
-1.82 |
pH of emulsion |
5.46 |
In-vitro drug release study:
Due to unique anatomical and physiological functions, ocular surface presents special challenges for design and development of in-vitro release method for ophthalmic formulation. To account for the short ocular residence time, the method ideally should detect drug release at early time points. However, no validated method is currently available to test drug release from ophthalmic formulations, which necessitates developing a sensitive method. Hence, the in-vitro drug release method was developed using bottle rotating apparatus. A flot-a-Lyzer cell (1mL) of cellulose acetate membrane (1mL, 1000 KD MWCO, Spectrum) was used to hold the emulsion formulation.
The marketed formulation of Prednisolone acetate Ophthalmic Suspension was evaluated along with the optimized nano emulsion formulation. The in-vitro drug release profile of nano emulsion formulation and marketed formulation using bottle rotating apparatus is shown in Figure 4. The market formulation is available in concentration of 10mg/mL as micro fine suspension having particle size around 2µ. The 90% of drug was released from nano emulsion formulation within 12 hr. However, around 68% of drug was released from marketed formulation. The drug release from marketed formulation is slower as compare to developed nano emulsion formulation. This result is mainly attributed due to difference in particle size of both formulations. This clearly indicates that the developed dissolution method is capable enough to discriminate the effect of formulation variables like dosage form and globule size.
Figure 4. In-vitro drug release profile of optimized lipid based nano emulsion and marketed formulation using bottle rotating apparatus.
Hen’s Egg Chorioallantoic Membrane Test:
Hen’s Egg Chorioallantoic Membrane Test was used to check the eye irritation potential of optimized lipid nano emulsion formulation of prednisolone acetate. The experimental result of controls and test formulation is presented in Table 3 and Figure 5. After application of 0.3mL of 0.9% sodium chloride solution as negative control showed no effect of Lysis, haemorrhage or coagulation on chorioallantoic membrane. However, after application of 0.3mL of 1% SDS and 1% sodium hydroxide solution as positive controls showed significant damage to chorioallantoic membrane. On the other hand, application of 0.3 mL of nano emulsion formulation absolutely showed no effect on chorioallantoic membrane after 5 minutes with respect to Lysis, hemorrhage or coagulation. The irritation score was calculated using the following formula,
IS = (301- Haemorrhage) X 5 + (301- Lysis) X +
300 300
7 (301- Coagulation) X 9
300
Where haemorrhage is the time taken to start (in seconds) of hemorrhage reactions on CAM; lysis is the time taken to start (in seconds) of vessel lysis on CAM; coagulation is the time taken to start (in seconds) of coagulation formation on CAM.
The irritation score for controls and test formulation is presented in Table 3. The irritation score of HET-CAM test showed that the developed nano emulsion containing the polysorbate 20 with optimized concentration is essentially non-irritating and possess good ocular tolerability.
Table 3: Irritation score of controls and test formulation.
Formulation |
Irritation Score |
0.9% NaCl solution |
0.1 |
1% SDS solution |
7.4 |
1% NaOH solution |
12.6 |
Nano emulsion formulation |
0.4 |
Category of Irritation: - 0-0.9: Non-irritant, 1-4.9: Weak or slight irritation, 5-8.9 or 5-9.9: Moderate irritation, 9-21 or 10-21: Strong or severe irritation
Stability study:
Stability study of optimized nano emulsion formulation was investigated after sterilization by filtration (0.2µ membrane filter) followed by filling in low density polyethylene (LDPE) ophthalmic bottles. The results of stability study are summarized in Table 4.
There was no change in physical appearance of nano emulsion or phase separation observed in both 40°C and 25°C conditions. Table 4 depicts the results of various physico-chemical parameters of optimized formulation at different temperature condition before and after stability study which were found to be in-line with anticipated limits.
Globule size was increased from 108.2nm to 127.8nm at 40°C for 6M. This could be attributed due to change in interfacial property of droplet at higher temperature and induce the droplet coalescence. However, no change in globule size was observed at 25°C for 6M. No change in pH of emulsion, viscosity, zeta potential, osmolality was observed in both 40°C and 25°C conditions. Drug content was decreased at 98.1% to 96.1% at 40°C for 6M. However, no change in drug content was observed at 25°C for 6M.
The zeta potential of nano emulsion formulation is -1.82 mV which is almost neutral. There was no significant change in zeta potential observed in both 40°C and 25°C conditions at 6M. This is due to non-ionic nature of surfactant i.e. polysorbate 20 used in formulation. Stability data supports that optimized nano emulsion formulation is stable although zeta potential was neutral.
Figure 4 : A) CAM treated with 0.9% Sodium chloride solution as Negative control – IS 0.1 B) CAM treated with 1% SDS solution as Positive control – IS 7.4 C) CAM treated with 0.1 N sodium hydroxide solution as positive control – IS 12.6 D) CAM treated with Nano emulsion formulation - IS 0.4.
Table 4: Stability data of Prednisolone acetate lipid emulsion (Mean± SD, n=3)
|
Parameters |
Time period |
|
||||
|
Initial |
40⁰C 3 month |
40⁰C 6 months |
25⁰C 3 months |
25⁰C 6 months |
||
|
Appearance |
White milky aqueous emulsion |
White milky aqueous emulsion |
White milky aqueous emulsion |
White milky aqueous emulsion |
White milky aqueous emulsion |
|
|
pH of emulsion |
5.46 |
5.41 |
5.39 |
5.48 |
5.45 |
|
|
Viscosity (cps) |
1.55±0.01 |
1.52±0.01 |
1.49±0.03 |
1.56±0.04 |
1.54±0.03 |
|
|
Zeta potential (mV) |
-1.82 |
-2.22 |
-2.12 |
-1.96 |
-1.98 |
|
|
Drug content (%) |
98.1±1.33 |
97.2±1.10 |
96.1±1.18 |
99.0±1.21 |
98.2±1.69 |
|
|
Globule size (nm) |
108.2±4.93 |
119.0±3.44 |
127.8±2.42 |
110.6±2.67 |
112.4±2.72 |
|
|
Osmolality (mOsm/kg) |
296 |
301 |
304 |
299 |
298 |
|
CONCLUSION:
The proposed lipid based nano emulsion formulation containing prednisolone acetate was successfully developed and in-vitro characterization using different analytical techniques were established. Using 20, 000 PSI homogenization pressure and 3 homogenization cycles, the speed of high shear homogenization during crude emulsion preparation has no significant impact on globule size and PDI results of final emulsion. The Box-Behenken statistical design was successfully applied for optimization of process variables for nano emulsion preparation. The proposed lipid based nano emulsion formulation was found to be stable and non-irritant. In nut shell, the proposed lipid based nano emulsion formulation has potential to improve the ocular bioavailability and it can be promising vehicle for ophthalmic drug delivery of water insoluble drugs.
CONFLICT OF INTEREST:
The authors declare no conflict of interest.
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Received on 18.10.2019 Modified on 16.12.2019
Accepted on 28.01.2020 © RJPT All right reserved
Research J. Pharm. and Tech 2020; 13(9):4139-4147.
DOI: 10.5958/0974-360X.2020.00731.3