Fabrication and in-vitro evaluation of liposomal quercetin and its optimization

 

Suman Saha¹*, Amit Roy¹, Sanjib Bahadur¹, Ananta Choudhury²

¹Department of Pharmaceutics, Columbia Institute of Pharmacy, Raipur, Chhattisgarh

²Department of Pharmaceutics, Assam Down Town University, Assam

 

ABSTRACT:

This study intended to explore the influence of formulation factors on the physico-chemical properties of quercetin-loaded liposomes and optimize the fabrication environment. Thin film hydration technique was employed to prepare liposome and optimization was done by 32 factorial designs combined with desirability function. Nine preparations were prepared by using altered drug: lipid and soyphosphatidylcholine: cholesterol (SPC: cholesterol) ratios and assessed for entrapment efficacy and vesicle size. The findings were the mean diameter and drug encapsulation efficiency. Results exhibited that SPC concentration and SPC: cholesterol molar ratio had a solid impact on liposome size. Increasing the lipid ratio produced a reduction in size. The degree of quercetin charging depended on the factors evaluated. Increasing SPC concentration and lipid ratio pointed lyboosted quercetin entrapment. However, higher quercetin concentrations had a negative effect on drug entrapment. Based on this, an optimized design was determined, prepared and investigated. The entrapment efficiency and vesicle size were found to be very adjacent with the predicted values. The formulation was found to be globular shape and also shows sustained release pattern. These results backing the fact that 32 full factorial designs with desirability function might be efficiently used in optimization of quercetin loaded liposome.  The overall results showed that SPC concentration and lipid ratio were the key features influencing particle size, while entrapment efficiency was affected primarily by quercetin concentration.

 

 

INTRODUCTION:

Plant-derived composites are supposed to exert a favourable influence on human wellbeing and used as medicines. Quercetin is a flavonol, a type of flavonoid, which is frequently present in numerous foods including onions, fruits, and vegetables. Quercetin, at nontoxic concentrations, is known to have a multitude of known biologic effects in which many of its mechanisms remain unidentified. There is significant proof that quercetin is a molecule with numerous biologically valuable properties. These properties backing quercetin’s role, as a treatment; for oxidative damage, cancer, inflammation, bacterial and viral infections, cardiovascular diseases and diabetes.

 

The indication for each of these categories differs in the level that it has been investigated. A decisive result of quercetin’s benefits, to our knowledge, has not completed all the rigidities of pharmaceutical clinical trials. Although there are, many quercetin analogues and dietary flavonoids with varying signal to support their entitlements^1-4.

 

Liposomes are globular vesicles composed of lepidic ampiphiles, typically phospholipids, which establish themselves in water to customise an aqueous core bounded by lipid bilayers. This exclusive structure permits the liposomes to encapsulate mutually hydrophilic and lipophilic ingredients. In doing so, this carrier structure protects the trapped molecules from degradation and weakening down in the systemic flow. Because of their properties, liposomes when made, their physiochemical properties like size, lamellarity, membrane rigidity, etc. are capable to influence and boost the performance of products by increasing component solubility, improving ingredient bioavailability, increase intracellular uptake, alter pharmacokinetics and bio distribution and in-vitro and in-vivo stability^{5, 6}.

 

MATERIAL AND METHOD:

Quercetin and cholesterol were purchased from Alfa-aser (UK). SPC was purchased from Hi media (Mumbai). All other chemicals used were of analytical grade and the solvents were of analytical grade.

 

Preparation of quercetin-loaded liposomes:

Thin film hydration method was used to prepare quercetin loaded liposome^{7, 8}. In this method, lipid, cholesterol and quercetin were firstly dissolved in chloroform, methanol mixture in different molar ratio (Table 1).

 

Table1: 32 Factorial designs with measured responses.

Batch	Independent Variables		Dependent Variables
	X1	X2	Y1 (nm)	Y2 (%)
B1	1	1	374	80.31
B2	-1	-1	328	51.8
B3	0	1	342	72.31
B4	-1	0	345	58.04
B5	1	0	504	71.42
B6	1	-1	546	70.67
B7	0	-1	482	61.08
B8	0	0	351	66.15
B9	-1	1	322	61.42

X1 = Drug: Lipid (Molar ratio), X2 = SPC: Cholesterol (% of total lipid)

Y1 = Vesicle size (nm), Y2 = Entrapment efficiency (%)

 

The solvent was vaporized at 60°C for 1 h under vacuum at 150 rpm by rotary evaporator to produce a thin lipid film. The dried thin lipid layer was hydrated by addition of phosphate buffer solution (PBS) pH 6.8 at 45°C in rotary vacuum evaporator rotated at 100 rpm till the dispersion of entire lipids in the aqueous part. To reduce the vesicle size, dispersion was exposed to bath sonication for 20-30 min. The milky suspension containing multilamellar vesicles was further size reduced in a probe sonicator. Afterward, the mixture was kept for 1 h at room temperature to form vesicles followed by 4°C for 24h in an inert atmosphere. Then preparation was centrifuged for 1h at 15000 rpm in a cooling centrifuge (Remi Instruments, Mumbai, India). Then, the supernatant comprising the vesicles was separated and taken for further studies in a suspended form⁹.

 

3² factorial designs:

The preparations were optimized by 3² factorial designs containing of drug: lipid molar ratio (X1) and SPC: cholesterol (X2) as an independent variables while vesicle size (Y1) and entrapment efficiency (Y2) as response (Table 1). Nine formulations were fabricated and evaluated for response. The found data were treated with Design Expert software 9.0.4.

 

Purification and size reduction of quercetin-loaded liposomes:

For purification, the quercetin-loaded liposomes were centrifuged at 15000rpm for 15 minutes. The supernatant comprising the non-entrapped drug was separated. Remaining sediment denoted as the quercetin-loaded liposomes. A volume of buffer solution was added to yield 2 mL of liposome dispersion, and then the mixture was vortexed for homogenization^{10, 11}.

 

Morphology of liposome:

Shape and lamellarity of liposome was detected by retaining the suspension under microscope (Olympus BX 41, USA). Photographs were taken by a camera attached to the optical microscope in 10x100 magnifications^{9, 10}.

 

Vesicle size and Zeta potential:

The optimized formulation, successively diluted 100-fold with Double distilled water, was used to determine mean vesicle size and polydispersity index (PDI) using Zetasizer (Malvern, UK).Zeta potentials of the optimized formulation were measured by the same instrument at 25°C.

 

Entrapment efficiency:

Liposome suspension was centrifuge at 15000 rpm to separate unentrapped drug. Free drug present in supernatant was determined using UV spectrophotometer. EE (%) was calculated by following equation:

 

EE (%) = [(C_{total –free})/C_total] x100

Where, C_total = total drug added, C_free= unentrapped drug

 

In-vitro drug release from Quercetin liposomes:

Release studies were carried out according to the dialysis method. The release studies were carried out in a 250 mL beaker containing 200 mL of the medium. The medium was stirred using a magnetic needle. Dialysis membrane (Hi-Media, India) was used as a barrier to isolate the donor and the receptor phase. Liposomal dispersion was placed in a dialysis bag (Hi-Media) of cellulose acetate, which was immersed in the medium and magnetically stirred. The stirring was carried out at 100 rpm at 37°C ± 0.5 °C. The simulation of GI transit condition was achieved by altering the pH of the dissolution medium at different time intervals. For the initial 2 h, the pH of the dissolution medium was adjusted to 1.2 using 0.1 N HCl. Subsequently, the pH of the dissolution medium was adjusted to 7.4 with 0.1 N NaOH and buffer salts, and maintained up to 20 h. Samples taken from the receiver solution at predetermined times were replaced with equal volumes of fresh buffer and spectrophotometrically assayed for  drug content^9-11.

 

Stability Study:

Quercetin loaded liposomes were kept in glass vialsat 4-8°C, 25±2°C and 37±2°C for one month. The trials were taken after completion of one month and entrapment efficiency was measured as described earlier¹¹.

 

RESULTS AND DISCUSSION:

Experimental design:

The three level two factor design is an effective approach for investigating variables at different levels with a limited number of experimental runs (Table 2). The vesicle size and EE of total 9 batches showed a wide variation from 322 to 546 nm and 51.8 to80.31%, respectively.

 

Table 2 Variables in 3² Factorial designs for liposome

Variable	Levels
	Low (-1)	Medium (0)	High (+1)
Independent variables
1 =Drug: Lipid (Molar ratio)	-1 (1:5)	0 (1:10)	+1 (1:15)
X2 = Lipid: Cholesterol (% of total lipid)	-1 (70:30)	0 (60:40)	+1 (50:50)

 

Optimization of formulation:

The search for the optimized formulation structure was carried out  by using desirability function approach with Design expert software The optimization process was performed by setting the Y1 at minimum and Y2 at maximum while X1 and X2 within the range obtained. The optimized formulation was achieved at X1=1:9.5, X2=50:50 with the corresponding desirability (D) value of 0.812. This factor level combination predicted the responses Y1=463 nm, Y2=73.43%¹².

 

Checkpoint Analysis:

The assessment of predicted and experimental results illustrates very close agreement, representing the accomplishment of the design combined with a desirability function for the evaluation and optimization of liposome formulations (Table 3).

 

Characterization of Optimized Formulation:

Vesicle size and shape:

Vesicle dimension determination is important parameters for application of liposome. Numerous methods exist for preparation of liposome with diverse size, composed of one or additional lipid bilayer. Usually, lipid film hydration is employed for preparation of multilamellar vesicles.

 

Sonication was done to yield small unilamellar vesicle. The optimized liposome (B10) was globular in nature and found to be unilamellar to multilamellar. The average vesicle size was found to be 475 nm with 0.453 polydispersity index

 

Zeta potential:

Zeta potential of liposome confirms stability and entrapment efficiency and used to envisage in-vivo behaviour. Entrapment efficiency was amplified due to electrostatic pull between charged molecule and liposomes. Any successive alterations of the liposomal surface, such as cholesterol amalgamation, also affect zeta potential. The higher values of zeta potential boost the stability of liposome and by preventing clump formation. Liposome prepared with diverse lipids acquires diverse surface charge. On the contrary, in this study liposome prepared by SPC have a little negative charge (-2.65 mV). It might be due to the outcome of cholesterol on surface charge.

 

Entrapment efficiency:

Drug can be fused into liposome by numerous ways depending on several properties like polarity as well as solubility. It can be adsorbed on superficially on the membrane, trapped in lipid bilayer, captured in inner aqueous core, attached among polar head, or secured by a hydrophobic tail. Technique of preparation and structure of lipid can as well influence the entrapment efficiency. The present work shows 76.14%entrapment efficiency signifying decent electrostatic interaction between quercetin and liposomes.

 

In-vitro diffusion study:

Release features of quercetin from liposome were assessed in-vitro and compared to that of pure drug. It was detected that the release of quercetin suspension was accomplished within 8 h while liposomal formulations exhibit84% release within 24 h. This result suggests that the layer of drug-encapsulated liposomes adjacent to the semi permeable membrane leaches its contents slowly and another layer exchanges the permeated vesicles. Controlled release of drug in liposomes can be anticipated over a prolonged period due to this mechanism.

 

Table 3.Checkpoint batch with their predicted and observed value of Responses

Independent Variables			Vesicle size(Y1)		Entrapment efficiency (Y2)
Batch	X1	X2	Observed	Predicted	Observed	Predicted
B10	-0.089 (1:9.5)	+1 (50:50)	475	463	76.14	73.43
Percentage prediction error (%)			+2.59		+3.69

 

 

Stability Study:

Stability study reveals substantial drug loss (approx. 15%). This loss took place with formulation stored at high temperature, i.e., 37±2 °C. On contrary, formulation stored at 4-8 °C and 25±2 °C, could retain 95% and 92% respectively of the entrapped drug. Significant loss of drug at high temperature may be due to change in gel to liquid transition of lipid bilayer. The outcomes of the study indicate that liposome can overcome the limitation of the molecule associated with deprived oral absorption and can improve the bioactivity of the quercetin.

 

CONCLUSION:

In this study, 3² full factorial designs were employed for predicting the finest condition for fabrication of liposome. The formulations were fruitfully prepared by thin film hydration method to perceive the consequences of drug: lipid and soyphosphatidylcholine: cholesterol ratio on vesicle dimensions and entrapment efficiency. Rise in lipid concentration was found to prepare liposome with maximum entrapment efficiency. In contrast, decrease in SPC concentration yield smaller vesicle. These responses were fed into polynomial model to identify the noteworthy effects of independent variables on response. The efficiency of experimental design was established by close agreement of experimental value with estimated value of optimized formulation prepared. Thus, 3² full factorial design with desirability function is a productive means to improve quercetin loaded liposome.

 

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Received on 15.09.2017          Modified on 05.10.2017

Accepted on 21.11.2017        © RJPT All right reserved