Formulation, Characterization and Stability Study of Encapsulated Anticancer drug in multilayered PEGylated Tumor targeting stealth Liposomes

 

M Padmasree¹, B A Vishwanath¹, Shankarrao Patil², Mythili S¹, Kirupakar B R¹

¹Department of Pharmaceutics, Aditya Bangalore Institute of Pharmacy Education and Research,

Yelahanka Bangalore, 560064, India.

²Department of Biotechnology, Aditya Institute of Management Studies and Research, Yelahanka Bangalore, 560064, India.

 

ABSTRACT:

Present chemotherapy is low tumor selectivity with consequence undesirable side effects. Encapsulation of anticancer drug in to a pharmaceutical carrier such as liposome has been come with to overcome the difficulties developed by chemotherapeutic drugs. For this reason we formulated Capecitabine loaded stealth liposomes for anti-cancer therapy, in order to enhance bioavailability and to reduce dose frequency by reducing the toxicity and to target sites. Capecitabine is a prodrug of Fluorouracil (5 - FU), which is used to treat colorectal cancer, breast cancer and gastric cancer with improving drug concentrations through tumor-specific conversion to the active drug. PEGylated liposomes of capecitabine were prepared by thin film hydration Method from different combinations of phospholipids. Capectitabine stealth liposomes were prepared and evaluated for Particle size analysis, Zeta Potential, Entrapment Efficiency and drug release studies. Drug excipient compatibility was determined by FTIR. Percent entrapment efficiency of the formulations was found in the range of 54 % to 73%. The particle size analysis and zeta potential studies of Capecitabine stealth liposomes were analyzed by Malvern zetasizer ZX. Average size of Capecitabine loaded stealth liposomes was found to be in the range of 110-201 nm. Capecitabine loaded stealth liposomes have proved extended drug release and increased biological half life. After running ANOVA, the formulations of F3 CAP, F7 CAP were designated as the optimum formulations.

 

 

1. INTRODUCTION:

Cancer is defined as uncontrolled multiplication and spread of abnormal forms of body’s own cells. Cancer is a life-threatening disease and one of the major causes of death in the developed countries. The main aim of current research work is to reduce the undesirable side effects. The obstacles developed by chemotherapeutic drugs which can be minimized by using a pharmaceutical carrier such as liposome.

 

An improvement has been made with inclusion of the synthetic polymer; poly-ethylene glycol (PEG) in liposome compositions to reduce the mononuclear phagocyte system uptake (stealth liposomes). Stealth liposomes has been proved that high target efficiency with reduce side effects. [1,2]. Our study involved designing and development of stealth liposomes containing anticancer drug using different phospholipids. Among multiple drug-delivery systems, liposomes exhibit advanced technology to deliver active molecule to the site of action. Incorporation of PEG in to a liposomal carrier has been proved extend blood-circulation time. Capecitabine is a oral chemotherapeutic agent used in the treatment of advanced stage of colorectal cancers (CRC), breast cancer and gastric cancer. Capecitabine is an innovative chemotherapeutic drug and converted to the cytotoxic moiety 5-fluorouracil in target cancer tissue by thymidine phosphorylase. [3-7].

 

2. MATERIAL AND METHODS:

Chemicals and reagents:

Capecitabine was received as a gift sample from Mylon Laboratories, Bangalore, India. DSPC and PE- PEG 2000 were purchased from Lipoid Germany. Solvents and reagents used were of laboratory reagent (LR) grade. All other chemicals and reagents used were of analytical grade.

 

a.     Preparation of liposomal formulation and optimization by Design of Experiment:

Table 1. Factors (independent variables) Factor levels used: Low (-1), High (+1):

Factorial design was applied to prepare capecitabine loaded liposomes using DSPC and Cholesterol. Independent variables chosen were Lipid: Cholesterol ratio (X), Hydration time (Y) & Sonication time (Z), while dependent variables were; Vesicle size in nm (Y1), Drug release (Y2) & % Entrapment Efficiency (Y3).

 

Table 1 shows the independent factors and their levels studied in factorial design [08, 09].

Independent variables	Low	High
Lipid: Cholesterol	3:1	4:1
Hydration time (mins)	60	90
Sonication time (mins)	15	20

 

Among the various independent factors studied during the preliminary trials, which could affect the size of vesicles, drug release and entrapment efficiency of formulation, Lipid: Cholesterol ratio hydration time and sonication time were found to have a significant effect on the responses measured. So, three critical factors (Lipid: Cholesterol, Hydration time and sonication time) affecting size of vesicles, drug release and % EE, hence, full factorial design was chosen to optimize the liposomal formulations as shown in Table 2. Based upon the polynomial equation generated by model fitting and optimization, the effect of various independent factors can be analyzed on the responses measured [10,11].

 

2.2 Preparation Method for PEGylated liposomes of capecitabine:

 PEGylated liposomes of capecitabine were formulated by using thin film hydration method from various combinations of phospholipids. The weighed quantity of drug, phospholipids and cholesterol was dissolved in mixture of anhydrous Ethyl acetate & ethanol (2:1) in a sterile round bottom flask. Round bottom flask is connected to a rotary evaporator, which is immersed in water bath and vacuum is applied through a vacuum pump and rotated at 80 rpm and subjected to evaporation and the temperature is maintained above the phase transition temperature of the phospholipids to obtain a thin dry lipid film. The lipid film is thoroughly dried over night to remove residual organic solvent by using vacuum pump. The film was allowed to hydrate using PBS pH 7.4 above transition temperature by hand shaking for 15 minutes and further kept at room temperature. The constituted liposomes were subjected to sonication for size reduction. The non-entrapped drug was removed by centrifugation this step is called as liposome purification. Final liposomal dispersion was filled in sterile glass vials covered with special stoppers for lyophilization. The liposomal dispersions were preserved by addition of sodium azide 0.05 % w/v related to total aqueous phase [12-14].

 

Table 2. Full Factorial experimental layouts with the measured responses

Formulations	X	Y	Z	Y1	Y2	Y3
F1 CAP	3:1	60	20	170	79	61
F2 CAP	3:1	90	20	165	80	62
F3 CAP	4:1	90	20	133	95	70
F4 CAP	3:1	90	15	201	73	56
F5 CAP	4:1	90	15	120	92	67
F6 CAP	4:1	60	15	140	89	64
F7 CAP	4:1	60	20	110	94	73
F8 CAP	3:1	60	15	195	72	54

 

3. RESULTS:

3.1 Pre-formulation Studies:

Solubility Study: Capecitabine is freely soluble in ethanol, methanol, DMSO, DMF and sparingly soluble in water. Melting Point Determination: The melting point of Capecitabine was found to be 110 -121°C. This complied with IP and BP standards thus indicating the purity of the drug sample.

 

3.2.a Preparation of standard solution of capecitabine:

100 mg of Capecitabine was accurately weighed and quantitatively transferred into a volumetric flask and dissolved with minimum quantity of ethanol and made up to 100 ml. The solution was examined to contain 1000 mg/ml (stock solution). The stock solution was diluted to make aliquots of working solution containing 100 mg/ml.

 

3.2. b Preparation of Calibration curve:

From stock solution of Capecitabine (0.5 – 3.0 ml of 100 mg/ml) were pipette out and transferred into 10 ml volumetric flask and made up to the mark with phosphate buffer saline, pH 7.4. Different concentrations were measured at 295 nm with phosphate buffer saline as blank in UV-visible spectrophotometer, to obtain λ_max. Standard Calibration curve was plotted using concentration against absorbance. The standard calibration curve curve obtained was linear with the concentration range of 5 - 30 mg/ml [15, 16].

 

 

3.3 Drug Excipients Compatibility Study:

Fourier transform Infrared spectroscopy (FTIR) has been used to collect infrared spectrum of absorption and to study the compatibility between drug and the excipients used. Fourier transform infrared (FTIR) spectra of Capecitabine, DSPC, PE- PEG 2000, Cholesterol, PEG-4000, were recorded using an FT-IR spectrophotometer. The FTIR spectra were plotted as shown in Figure 1 (a) – 1 (e). [17,18].

 

 

Figure 1 (a)

 

 

Figure 1 (b)

 

 

Figure 1 (c)

 

 

Figure 1 (d)

 

Figure 1 (e)

Figure 1. Drug-Excipients compatibility studies by FT-IR. Figure 1 (a). FTIR spectra of pure drug Capecitabine; 1 (b). FTIR spectra of Lipid DSPC(Distearoyl phosphatidyl choline); 1 (c). FTIR spectra of Lipid PE 18:0/18:0‑PEG 2000; 1 (d). FTIR spectra of Cholesterol; 1 (e). FTIR spectra of physical mixture of Capecitabine +DSPC+ PE- PEG 2000+ Cholesterol.

 

3.4 Zeta potential and particle size distribution:

Particle size analysis and zeta potential of the various liposome’s are shown in Table 3. Particle size analysis of the various formulations was measured by using Malvern seta analyzer zx. The zeta potential of all the liposomal vesicles was measured in millivolts (mV). The poly index was measured in PDI and particle size was measured in nanometer (nm). F7 CAP showed the minimum average particle size of 110 ± 2.61 nm & mean zeta potential for the same was found to be ‒12.3 ± 0.83 mV which may be attributed due to the negative charge on the polymer [19-21].

 

Table 3. Particle size distribution of capecitabine loaded stealth liposomes

Formulation Code	Particle Size	Zeta Potential	Drug Entrapment	In-vitro drug release
F1 CAP	170 ± 9.24	-20.1 ± 0.17	61 ± 0.2	79.4±0.5
F2 CAP	165 ± 1.87	-17.2 ± 1.40	62 ± 0.01	80±0.5
F3 CAP	133 ± 1.52	-24.7 ± 0.12	70 ± 1.4	94±0.33
F4 CAP	201 ± 5.61	-37.6 ± 1.86	56 ± 0.6	63±0.1
F5 CAP	120 ± 0.48	-12.3 ± 2.6	67 ± 1.3	89±0.3
F6 CAP	140 ± 7.89	-28.3 ± 2.12	64 ± 2.4	85±0.04
F7 CAP	110 ± 2.61	-12.3 ± 0.83	73 ± 1.8	93±0.05
F8 CAP	195 ± 0.66	-40.26 ± 2.83	54 ± 0.8	69±0.01

 

3.5 In vitro release study:

The in vitro release of capecitabine from PEGylated liposomes was determined by dialysis method. The liposomal dispersion was placed in dialysis tube (donar compartment) (Himedia Laboratories Pvt. Ltd., Mumbai) with molecular weight cutoff 14000 Da. The dialysis tube was then immersed in a beaker containing release medium, i.e. PBS (pH 7.4) and stirred with magnetic stirrer at 100 rpm to maintain sink condition. The sample (1 ml) was taken at predetermined time intervals of 1 h, 2 h, 4 h, 6 h, 12 h, 24 h, 28 h, 30 h, 36 h from release medium. During every removal of sample equivalent volume of phosphate buffer saline (pH 7.4) was added to the cell to maintain a steady volume. Initial burst release was detected for all the formulations. PEGylated liposomes showed a more prolonged release because of presence of PEG coating on the surface, which release the drug slowly over a prolonged period of time. F3 formulation showed more prolonged release and in 36 hrs it gave 94 % release of drug. In vitro Drug release was determined by UV spectrophotometric methods shown in Table 3 and Figure 2. [22-25].

 

Figure 2. In - vitro drug release profile of capecitabine liposomes for selected PEGylated formulations (F3 CAP & F7 CAP). The PEGylated liposomes released maximum 94% and 93% of capecitabine within 36 h at room temperature, respectively.

 

3.6 Entrapment Efficiency:

Entrapment efficiency of the capecitabine loaded stealth liposomes was analyzed as shown in Table 3. F7CAP showed an average drug entrapment efficiency of 73 ± 1.8 which is higher amongst the other formulations. Different lipid combinations can influence the entrapment efficiency of liposomal formulation. [26-28].

 

3.7 Statistical analysis of the data and optimization (JMP13):

Statistical analysis of the experimental data and optimization of the formulation was done using JMP 13 statistical discovery software Version 13(SW). Based on p - value obtained, conclusion was drawn whether the model terms are significant or non significant. P - value less than 0.05 was considered statistically significant. ANOVA was also applied to test the significance of model terms. Model F - value and p - value were used to conclude the results as shown in Table 4a, 4b, 4c, 5a, 5b, 5c, 6a, 6b, 6c and Figures 3a, 3b, 4a, 4b and 5a, 5b.

 

Table 4. Full model for Particle Size (Y1)

Table 4 (a) Parameter Estimates

Term	Estimate	Std Error	t - Ratio	Prob > (t)
Intercept	83.5	0.75	111.33	<.0001*
Hydration time (60, 90 mins)	0	0.75	0.00	1.0000
Sonication Time (15, 20 mins)	2	0.75	2.67	0.0560
Lipid: Cholesterol ratio (3:1), (4:1)	-9	0.75	-12.00	0.0003*

 

Table 4 (b) Analysis of Variance

Source	DF	Sum of Squares	Mean Square	F Ratio
Model	3	7260.5000	2420.17	13.1710
Error	4	735.0000	183.75	Prob> F
C.Total	7	7995.5000		0.0154*

 

Table 4 (c) Summary of Fit

R Square	0.908073
R Square Adj	0.839128
Root Mean Square Error	13.55544
Mean of Response	154.25
Observations (Sum Wgts)	8

 

 

Figure 3 (a)

 

Figure 3 (b)

 

Figure 3(a). Actual by predicted plot of Drug Release; RMSE = 13.555, RSq = 0.91, p-value = 0.0154. Figure 3 (b). Response Surface Plot for showing the effect of independent variables on particle size of Capecitabine stealth Liposomes.

 

Table 5. Full model for Drug Release (Y2)

Table 5 (a) Parameter Estimates

Source	DF	Sum of Squares	Mean Square	F Ratio
Model	3	609.50000	203.167	135.4444
Error	4	6.00000	1.500	Prob>F
C.Total	7	615.50000		0.0002*

 

Table 5 (b) Analysis of Variance

R Square	0.990252
R Square Adj	0.982941
Root Mean Square Error	1.224745
Mean of Response	84.25
Observations (Sum Wgts)	8

 

Table 5 (c) Summary of Fit

Term	Estimate	Std Error	t Ratio	Prob>(t)
Intercept	84.25	0.433013	194.57	<.0001*
Hydration time (60,90)	0.75	0.433013	1.73	0.1583
Sonication Time (15,20)	2.75	0.433013	6.35	0.0031*
Lipid:Chol ratio (3:1), (4:1)	-8.25	0.433013	-19.05	<.0001*

 

 

Figure 4 (a)

 

Figure 4 (b)

 

Figure 4 (a). Actual by predicted plot of Drug Release; RMSE = 1.1547, RSq = 0.99, p-value = 0.0013 Figure 4 (b). Response Surface Plot for showing the effect of independent variables on Drug release of Capecitabine stealth Liposomes

 

Table 6 (a) Parameter Estimates

Source	DF	Sum of Squares	Mean Square	F Ratio
Model	3	289.37500	96.4583	36.7460
Error	4	10.50000	2.6250	Prob>F
C. Total	7	299.87500		0.0023*

 

Table 6 (b) Analysis of Variance

R Square	0.964985
R Square Adj	0.938724
Root Mean Square Error	1.620185
Mean of Response	63.375
Observations (Sum Wgts)	8

 

Table 6 (c) Summary of Fit

Term	Estimate	Std Error	t Ratio	Prob >(t)
Intercept	63.375	0.572822	110.64	<.0001*
Hydration time (60,90)	0.375	0.572822	0.65	0.5484
Sonication Time (15,20)	3.125	0.572822	5.46	0.0055*
Lipid: Chol ratio (3:1), (4:1)	-5.125	0.572822	-8.95	0.0009*

 

Figure 5 (a)

 

Figure 5 (b)

 

Figure 5 (a). Actual by predicted plot of Drug Entrapment; RMSE = 1.6202, RSq = 0.96, p-value = 0.0023 Figure 5 (b). Response Surface Plot for showing the effect of independent variables on Drug entrapment of Capecitabine stealth Liposome

 

4. DISCUSSION:

The capecitabine stealth liposomes was formulated and analyzed by using Facorial design JMP 13. For the optimization of capecitabine stealth liposomes formulation using JMP software, three parameters namely drug entrapment, drug diffusion and particle size were used. After running ANOVA, the formulations of F3 CAP and F7 CAP were designated as the optimum formulations. To further evaluate the effectiveness of this formulation, more studies have to be carried out. The studies include stability studies, In vitro and In vivo studies to assess the efficacy and bioavailability of the formulation.

 

5. CONCLUSIONS:

Stealth liposomes serve as a rising carriers in cancer therapy, increase permeability, inefficient and drug release at the target site. This capecitabine loaded stealth liposomes might be a suitable for sustained release drug carrier system for effective treatment of colorectal cancer.

 

6. ABBREVIATIONS:

F CAP Formulation code for Capecitabine stealth liposomes

(X) Lipid: Cholesterol ratio

(Y) Hydration time

(Z) Sonication time

(Y1) Particle size

(Y2) Drug release

(Y3) % Entrapment Efficiency

 

7. ACKNOWLEDGMENTS:

I would like to extend my thanks to Mr. Rahil M Patait, for generous gift of capecitabine (pure drug). I would like to thank Dr. B A Vishwanath for his continuous support and encouragement.

 

8. CONFLICTS OF INTEREST:

The authors declare no conflict of interest.

 

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Received on 21.02.2019           Modified on 21.04.2019

Accepted on 28.06.2019         © RJPT All right reserved