Formulation and Optimization of Ethosomes loaded with Ropinirole Hydrochloride: Application of quality by Design Approach

 

Sneh Priya, Divya Jyothi*, Jainey P James, Amala Maxwell

Nitte Gulabi Shetty Memorial Institute of Pharmaceutical Sciences, Nitte Deemed to be University, Deralakatte, Karnataka, India.

 

ABSTRACT:

The objective of the present study was to formulate and understand ethosomal formulations of Ropinirole (ROP) using quality by design (QbD) approach. To assess the effect of independent variable such as soya phosphatidyl choline and ethanol concentration on dependable variable such as vesicle size and percentage entrapment efficiency, a 3² factorial design were employed. Ethosomes were prepared by cold method and evaluated for the following parameter; vesicle size, zeta potential, polydispersity index and percentage entrapment efficiency. In vitro drug release of optimized formulation was performed and compare with pure drug solution. Based on QbD approach, design space (DS) was optimized with a combination of selected variables with entrapment efficiency > 50% w/w and a particle size < 400 nm. The results of multivariate regression analysis illustrated that vesicle size and % entrapment efficiency of ethosomes were affected by ethanol concentration and phosphatidyl choline amount. The optimized formulation had ethanol concentration of 49.9% and Soya phosphatidyl choline amount 380 mg with vesicle size of 320.45 nm and entrapment efficiency of 65.69 ± 3.5 %. In vitro drug release after 24 h from optimized formulation was found to be 64.8% when compared to pure drug which released 99.2% within 2 h. The conclusion of the study substantiates that by using a QbD approach, the effect of formulation variation in responses can be easily found based on the knowledge gained by regression analysis, the concept of ‘‘desirability function”.

 

 

INTRODUCTION:

The QbD approach accentuates the comprehension of different components of the formulation for improved command over wanted yield. Design of experiments (DoE) and multivariate statistical data analysis are essential elements of QbD, recognized by the recent International Conference of Harmonization Q8 guideline ^1,2. These tools facilitate the simultaneous variation of all the formulation variables, allowing quantification and prioritization of the consequences made by these variables in the defined design space, together with any possible interaction between them. Using QbD approach, the effect of formulation variables on vesicle size and the percentage entrapment efficiency of ethosomal formulations was studied. Based on the data gained by multivariate analysis, the idea of ‘‘desirability function’’ was applied to attain optimum formulation conditions within the defined design space³.

 

Parkinson’s disease (PD) is a neurological that affects extrapyrimidal system of brain. It occurs due to the death of vital nerve cells in brain called neurons, which are present in substantial nigra. These dying neurons produce dopamine. As the amount of dopamine produced in the brain decreases simultaneously symptoms of Parkinson’s disease will be intensified⁴.

 

Ropinirole hydrochloride (ROP) is a new nonergoline dopamine agonist recently introduced drug to treat Parkinson’s disease. Because of its extensive first pass metabolism, it has a lower oral bioavailability (50-55 percent), short elimination half-life of 4-6 h⁵.

 

The conventional multi dose antiparkinson’s therapy leads to re-emergence of Parkinson’s symptoms, due to the fluctuations in serum levels of drug, which occurs due to rapid hepatic first pass metabolism⁶. The rational strategy to overcome this drawback is to minimize the fluctuations by fabricating the drug in sustained release formulations such as niosomes⁷, liposomes⁸, ethosomes⁹, transferosomes¹⁰ etc. Hence aim of present was conducted to optimize the ethosomal formulation of ROP by QbD approach.

 

MATERIAL AND METHODS:

Materials

Ropinirole Hydrochloride (ROP) was received as a generous gift from Orchid Pharmaceuticals Pvt. Ltd., (Chennai, India). Soy phosphatidylcholine was purchased from Hi Media laboratories, (Mumbai). Cholesterol was procured from MercSpecialities Pvt. Ltd, (Mumbai, India). Chloroform, ethanols were procured from Sigma Aldrich Chemicals Pvt. Ltd., (USA). All other chemicals used were of analytical grade and procured from LobaChemie Pvt. Ltd. (Mumbai, India).

 

Methods:

Experimental design:

Based on literature survey ethosomal vesicles were formulated with Soy Phosphatidyl Choline (SPC) with concentration in the range of 3-4% and ethanol in the range of 45-50%. Concentration of lipid and ethanol plays most important role in formation of ethosomes, which affect vesicles size and also in the drug entrapment. Thus, these two factors were considered for design of experiment^3,11. A 3² factorial design was adopted to optimize their concentrations, with the vesicle size and percentage of entrapment efficiency being the dependent variables. Multiple regression assessment of the responses obtained of the nine formulation was carried out using ''Design Expert'' software (Stat-Ease, Inc., Minneapolis, MN) (version 11)¹².

 

Table 1: Factors and their level

Independent factor	Level
	Low (-1)	Medium (0)	High (1)
Concentration of ethanol (%V/V) (X1)	45	47.5	50
Amount of lipid in mg (X2)	300	350	400
Dependable factor	Vesicle Size in nm (Y1)
	% Entrapment efficiency (Y2)

 

Different factor combinations were obtained and experimentally run to measure the responses. The composition and quantities of different formulations are given in Table 2.

 

Formulation and characterization of ethosomes loaded with Ropinirole:

Preparation of ethosomes:

Cold method was used to prepare ethosomes loaded with Ropinirole Hydrochloride¹³. Soy Phosphatidyl Choline and cholesterol were dissolved in ethanol by keeping on a magnetic stirrer. This ethanolic mixture was heated at 30°C in water bath during which propylene glycol was added. Then the water-soluble drug Ropinirole Hydrochloride was dissolved in 6 ml water and added at 700 rpm to the ethanol mixture. The solution was continuously stirred for 15 min to allow the formation of ethosomal vesicles.

 

Table 2: Formulation composition batches of ethosomes as per 3² full factorial design for 10 ml formulations

Formulation Code	Ethanol 3ml of (%v/v)	Soy Phosphatidyl Choline (mg)	Cholesterol (mg)	Drug (mg)	Propylene Glycol (ml)
F1	45	300	25	180	1
F2	47.5	300	25	180	1
F3	50	300	25	180	1
F4	45	350	25	180	1
F5	47.5	350	25	180	1
F6	50	350	25	180	1
F7	45	400	25	180	1
F8	47.5	400	25	180	1
F9	50	400	25	180	1

 

Characterization of ROP loaded ethosomes:

The ROP loaded ethosomal formulations obtained from 3² full-factor model were characterized to know the impact of formulation factors on the responses.

 

Vesicle size, size distribution and zeta potential:

Malvern zetasizer (Nano ZS, Malvern Instruments, UK), was used to determine the vesicle size, size distribution and zeta potential of ethosomes.Zeta potential values help’s to assess the formulation's stability.

 

Percentage entrapment efficiency:

Take 8 ml of the ethosomal suspension in Tarsus centrifuge tube of 15 ml capacity and it is centrifuged by cold centrifugation at 12000 rpm for 1 h at 4°C. After centrifugation the supernatant and the sediment are separated. The concentration of Ropinirole Hydrochloride present in the supernatant were analysed by UV spectroscopic method at 249nm. The percentage entrapment efficiency was calculated using the following formula.

 

---- (2)

 

Formulation of Optimized Batch

In order to optimize the formulation parameters, the impact of method variables on the responses was evaluated statically using one-way ANOVA, by the commercially available software package Design-Expert version 11. To achieve the optimal ethosomal formulation, minimum vesicle size and maximum %EE were designated with desirability more than 0.8. On the basis of this approach, optimized ethosomal formulation was prepared with ethanol concentration of 48.9 percent v / v and SPC 380 mg with desirability of 0.811, which was formulated in the same way as factorial batches

 

Characterization of optimized ethosomes batch:

Various parameters were evaluated for optimized batch which include Vesicle size, size distribution and zeta potential, % Entrapment Efficiency, Optical High resolution microscopy and Scanning Electron Microscopy.

 

Vesicle size, size distribution and zeta potential, and % Entrapment Efficiency of optimized formulation was performed as similar manner as the factorial batches.

 

Scanning electron microscopy

Scanning Electron microscopy was used to conduct the surface morphological analysis of optimized ethosomes. In this technique, a drop of ethosomal preparation was installed on a transparent glass stub, air dried and covered with sputter coater Polaren E 5100 and visualized under scanning electron microscope

 

In vitro diffusion study:

The in vitro drug diffusion studies were carried out using two sides opening boiling tube with cellophane membrane (MW cut-off range 12,000-14,000) on one end. 2ml of ethosomal suspension containing Ropinirole Hydrochloride is placed on the cellophane membrane. The receptor compartment consists of 30ml of phosphate buffer pH 7.4 and the medium was mixed with magnetic stirrer at constant speed and the temperature was adjusted to 37±0.5°C. At predetermined time intervals, 5ml sample was withdrawn from reservoir compartment and absorbance was measured spectrophotometrically at 249 nm. The reservoir compartment was replenished with the same quantity of phosphate buffer pH 7.4 and the study was conducted for 24 h¹⁴.

 

Drug release kinetics and mechanism of drug release:

Release kinetic of drug was obtained by subjecting release data to first order (log cumulative percentage of drug v’s time), and zero order kinetics (cumulative amount of drug released v’s time). The drug release mechanism was determined by fitting release data to Korsmeyer-Peppas model (log cumulative percentage of drug released v’s log time) and Higuchi’s matrix model (cumulative percentage of drug released v’s square root of time).

 

RESULTS AND DISCUSSION:

Statistical analysis of experiment design:

ROP loaded ethosomes were effectively prepared by cold method through utilizing 3² full factorial to comprehend the impacts of the ethosomal constituents (SPC and ethanol) on its traits. The independent elements, for example concentration of SPC and ethanol at three levels were assessed for their impact on vesicle size, and % EE. In Table 3 responses were mentioned. Every one of the responses examined was to a great extent influenced by the independent variable picked as reflected from the consequences of regression analysis.

 

Fig.1: Formulated ROP loaded ethosomes

 

Vesicle size:

For the infusion of ethosomes through skin, vesicle size shows a vital role as per literature detailed. The impact of ethanol and SPC on the vesicle size of preparation got from the 3² full factorial design (Table 3) demonstrate that rise in ethanol concentration from 45% to 50v/v, considerably diminished the vesicle size at all SPC quantity. Additionally, at particular ethanol concentration, increment in SPC quantity, the vesicle size of ethosomal preparation expanded considerably.

 

Further to understand the impact of formulation variable concurrently on the vesicle size, regression analysis was applied.

 

Quadratic model implied significant with model f-value of 191.22. The Predicted R² of 0.9629 is in sensible agreement with the Adjusted R² of 0.9917; i.e. the difference is less than 0.2.The results are condensed in Table 4. The following quadratic equation was obtained from the outcomes of the analysis:

 

Vesicle Size= +341.89 -29.0 (A)* +21.83 (B)* -7.50(AB)* - 3.33 (A²) + 9.17 (B²)*            (3)

 

Where A and B are the concentration of ethanol and soya phosphatidyl choline respectively, the standardized beta coefficient in this equation is represented by numbers and the asterisk symbol shows significance of the variables. The achieved regression model was found to be statistically substantial (p<0.05) with a great adjusted R² value of 0.9917. The implication of quadratic terms B² on the vesicle size designates that the model has a curvature at advanced levels of formulation variables. The effect of direct and interaction terms on the vesicle size is also signified concurrently in response surface in Fig 2a and 2b.

 

Entrapment efficiency:

The effects of ethanol concentration and SPC concentration on the EE of formulations obtained from the full-factorial design are shown in Table 3. It was observed that at respective SPC concentration, EE increased with the increase in ethanol concentration from 40% to 47.5% v/v, which could be due to the cosolvent effect of ethanol,facilitating the accommodation of drug in the aqueous core of the vesicle. However, at 50% v/v ethanol concentration, EE of formulations decreased. This could be attributed to partial fluidization of lipid bilayers by ethanol, resulting in leakage of entrapped drug from these formulations. At respective ethanol concentrations, EE of formulations increased with increase in SPC concentration from 300 to 350. This could be explained based on the fact that at higher SPC concentration, more volume of aqueous phase is entrapped around lipid bilayer to hydrate SPC. Because ROP is in the aqueous phase of the vesicles, increasing the volume of entrapped aqueous phase will result in increased EE.

 

To understand the effect of formulation variables on EE simultaneously, regression analysis was performed. Quadratic model implied significant with model f-value of 51.22. The Predicted R² of 0.8595 is in reasonable agreement with the Adjusted R² of 0.9691; i.e. the difference is less than 0.2. The results of regression analysis are summarized in Table 4 and the following equation was obtained for EE:

 

Entrapment efficiency= +63.49 + 4.05 (A)* +9.08 (B)* -0.525(AB) - 8.18 (A²)^* – 0.783 (B²) (4)

where the terms A, B and the coefficients have been defined earlier in Equation (3). The regression model was found to be statistically significant (p <0.05) with high adjusted R² value of 0.9691. It can be observed from Table 6 and Equation (4) that EE is significantly affected by both linear (A and B) and quadratic (A² and B²) effect of ethanol concentration and PC:CH ratio. The significance of quadratic terms A² on the entrapment efficiency indicates that the model has a curvature at higher levels of formulation variables. The effect of linear and interaction terms on the vesicle size is also represented simultaneously in response surface in Fig. 3a and 3b.

 

Table 3: Results of responses of ethosomes as per 3² full factorial

Form. Code	X1	X2	Y1	Y2	PDI	Zeta Potential
	Ethanol (%)	B:SPC (mg)	Vesicle size (nm)	EE (%)
F1	45	300	349	40.7	0.422	-11.9
F2	47.5	300	327	54.9	0.476	-10.2
F3	50	300	304	48.9	0.461	- 9.65
F4	45	350	367	50.2	0.518	-7.44
F5	47.5	350	341	63.7	0.704	-12.0
F6	50	350	311	60.2	0.421	-10.7
F7	45	400	405	61.3	0.734	-9.11
F8	47.5	400	376	70.3	0.331	-10.3
F9	50	400	331	67.4	0.754	-7.9

 

Table 4: Summary of regression analysis and ANOVA.

Sl No	Factor	Vesicle size (Adjusted R2 = 0.9917)		%Entrapment efficiency (Adjusted R2=0.9691)
		Estimated beta coefficient	p value	Estimated beta coefficient	p value
1.	Intercept	341.89	0.0006*	63.49	0.0042*
2.	A-Ethanol	-29.00	0.0002*	4.05	0.0098*
3.	B-Soya phosphatidyl choline	21.83	0.0004*	9.08	0.0009*
4.	AB	-7.50	0.0147*	-0.5250	0.5780
5.	A²	-3.33	0.2084	-8.18	0.0064*
6.	B²	9.17	0.0218*	-0.7833	0.5585

*Statistical significance of independent variables.

 

Figure 2 a) 2D and b) 3D Response surface curve depicting the effect of SPC and ethanol on the vesicle size of ethosomes.

 

Figure 3 a) 2D and b) 3D Response surface curve depicting the effect of SPC and ethanol on the Entrapment efficiency of ethosomes.

 

Formulation and characterization of Optimized Batch

Optimized batch was formulated as per solution given by software with concentration of 380 mg of soya lecithin and 48.9 ml ethanol respectively in the similar manner as the factorial batches. Observed value of vesicle size and entrapment efficiency was forund to be 320.46 nm and 65.69 ± 3.5 % respectively, which was within 95 % CI of Predicted value as shown in Table 7. The solution given software was acceptable.

 

Table 5: Point Prediction of otimized formulation with 95% confidence intervale

Response	Predicted Mean	Observed	95% CI low for Mean	95% CI high for Mean
Vesicle Size	323.351	320.46 ± 5.34	316.977	329.725
Entrapment efficiency	64.7934	65.69 ± 3.5	61.0723	68.5144

 

Scanning electron microscopy:

The vesicle shape and surface morphology of the formulations was determined by the Scanning electron microscopy (SEM). The particles observed were spherical and uniform and the surface of the vesicles was found to be smooth. SEM analysis also supported that the vesicle size was less than 500nm.

 

Fig.4 SEM of optimized ethosomal vesicle

 

In vitro drug release profile of ethosomes:

A comparative release profile of ethosomes and drug solutions are illustrated in Fig. 5. Cellophane membrane was used that only allowsthe diffusion of drug in solution form, while it withholds ethosomal vesicles. Therefore, to diffuse the entrapped drug through cellophane membrane, first it should release from the vesicles to surrounding medium.In present study result showed alomost 100 % drug relased from drug solution within 2 h while significantly less amount of drug was released from ethosomal vesicle i.e., 64.8%±2.01% at the end of 24 h. This indicating that diffusion of drug from vesicles to the surrounding medium is the rate- determining step in the release of drug through the cellophanemembrane. From the above results, it can conclude that theethosomal formulations provides a controlled and sustained delivery of drug through cellophane membrane compared to drug solutions, where, the lipid vesicles acts as a reservoir of drug and provides a continuous delivery of drug for prolonged time period

 

Drug release kinetics:

The release kinetics of the optimized formulation and pure drug was studied by various kinetic models. The interpretation of data was based on the value of resulting regression coefficients. The optimized formulation followed first order release kinetics with high regression coefficient (R²) of 0.893 when compared to zero order kinetic model which has a regression coefficient (R²) of 0.27. The mechanism of drug release from ethosomes was studied by fitting the data in korsemeyer peppas exponential model and Higuchi model. Good linearity was observed with regression coefficient (R²) of 0.949 for optimized formulation as per the release plotted for korsemeyer peppas equation. The release exponent (n) was found to be 0.77 which is above 0.45 showed that release can the characterized by Non – Fickian (anomalous) diffusion which may indicate that the drug release rate is controlled by more than one mechanism, i.e. diffusion coupled with erosion mechanism.

 

Fig 5. Comperetive in vitro drug release study of optimized ethosomal formulation with drug solution

 

Table 6: Comparison of in vitro drug release kinetics

Formulation code	Kinetic models
	Zero order		First order		Higuchi		Korsmeyer-peppas
	R2	K	R2	K	R2	k	R2	K	N
Pure drug	0.104	6.557	0.635	-0.049	0.721	33.087	0.802	1.81	0.345
optimized Ethosomal suspension	0.027	0.471	0.893	-0.018	0.920	15.317	0.949	0.869	0.77

 

CONCLUSION:

The study distinctively demonstrated the impact of the SPC and ethanol on vesicle size and entrapment efficiency of ethosomes. The effect of formulation variation in responses can be easily understood by using QbD approach. The in vitrorelease study showed,slow and sustained release of drug from vesicle may be due to more compact wall around the drug by biodegradable lipid matrix. The slow release of the ROP revealed that drug remains will be in in systemic circulation for longer time to achieve the better therapeutics efficacy.

 

ACKNOWLEDGMENTS:

We acknowledge Nitte (deemed to be university),Mangaluru, for providing financial support (Under NU Faculty Short-term Research Grant: NUFR2/2017/06/18)  to conduct research work.. Authors are thankful to Orchid Pharmaceuticals Pvt. Ltd., Chennai, India, for providing us with the gift sample of Ropinirole hydrochloride.

 

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Accepted on 16.03.2020           © RJPT All right reserved

Research J. Pharm. and Tech 2020; 13(9):4339-4345.