Formulation of plumbagin loaded long circulating pegylated liposomes: in vivo evaluation in C57BL/6J mice bearing B16F1 melanoma

Figures & data

Table 1.  Composition of liposomes and their respective particle size with zeta potential values.

Formulation	Plumbagin: Lipid	SPC: Chol: mPEG2000-DSPE	Average particle size (nm) ± SD	PDI	Zeta potential (mV)
	Molar Ratio
F1	1:10	9:2	78.51 ± 0.84	0.274	−56.6
F2	1:15	9:2	88.48 ± 0.70	0.271	−58.5
F3a	1:20	9:2	118.25 ± 1.06	0.228	−56.0
Plain conventional liposomes	−	9:3	101.77 ± 3.21	0.230	−58.8
F3b	1:20	9:3	114.75 ± 4.45	0.256	−58.5
F4	1:30	9:2	159.71 ± 1.55	0.260	−67.4
F5	1:40	9:2	174.55 ± 6.01	0.298	−67.5
Plain pegylated liposomes	−	9:3:0.5	120.16 ± 1.42	0.205	−51.85
C1	1:20	9:3:0.5	115.20 ± 7.07	0.265	−63.4

Note: F1 to F5 - Conventional liposomes; C1- Long circulating liposomes; PDI- Poly dispersity index

Figure 1.  (A) Transmission electron micrograph of pegylated liposomes (C1) of PLB. (B) Encapsulation efficiency (%) of PLB. F1-F5 represents conventional, whereas C1 represents long circulating liposomes. (C) Effect of cholesterol on in vitro release profile of conventional (CH1-CH4) and long circulating liposomes (C1) in PBS at 37 °C (n = 3).

Table 2.  Effect of cholesterol on physicochemical characteristics of liposomes.

Formulation	SPC:Chol Molar Ratio	EE% ± SD	Particle size (nm) ± SD	PDI	Zeta potential (mV)
CH1	9:1	52.51 ± 0.47	123.95 ± 4.03	0.253	−23.4
CH2	9:2	67.34 ± 1.70	123.40 ± 1.27	0.221	−61.4
CH3	9:3	66.00 ± 1.44	123.45 ± 2.19	0.229	−57.6
CH4	9:5	67.21 ± 7.72	141.95 ± 10.11	0.223	−54.3

Note: CH1 to CH4 represent formulation with varying molar ratios of SPC/Chol where the drug to lipid molar ratio remained1:20 for all the formulations; EE% - entrapment efficiency; PDI- Poly dispersity index.

Figure 2.  Physical stability of conventional (F3b) and long circulating liposomes (C1) in fetal bovine serum as a function of particle size up to 24 h incubation. Significant values: **p < 0.01 in comparison to particle size at initial time point.

Table 3.  Pharmacokinetic parameters of PLB, free and encapsulated as conventional and pegylated liposomes, after intravenous bolus administration at a dose of 6 mg/kg to tumor bearing mice (n = 4).

	Free PLB	Conventional liposomes (F3b)	Pegylated liposomes (C1)
AUC 0-∞ µg.min/ml	152.89 ± 10.05	262.66 ± 19.12	479.73 ± 80.11**
MRT (min)	49.96 ± 6.41	467.16 ± 19.75***	1455.96 ± 95.05***
CL (L/min)	0.03 ± 0.002	0.02 ± 0.001***	0.01 ± 0.001***
Vd (L)	2.02 ± 0.36	11.42 ± 0.33	27.40 ± 11.79**
T1/2 (min)	35.89 ± 7.95	346.87 ± 33.82***	1305.76 ± 278.16***

Note: AUC _0-∞, area under the curve; MRT, mean residence time; CL, clearance; T_1/2, half-life time. Significant values **p<0.01; *** p<0.001 compared to free PLB. The data represents mean ± SD values.

Figure 3.  Plasma concentration of PLB obtained from mice after intravenous administration of free PLB, conventional (F3b) and pegylated liposomes (C1) at a dose of 6 mg/kg. The data represents mean ± SD of four animals.

Table 4.  Tumor growth parameters for free PLB and liposomal formulations (n = 8).

	VDT ± SD	20X ± SD	GD at 20X ± SD
Vehicle treated	1.63 ± 0.42	8.12 ± 0.72
Free PLB	2.35 ± 0.17*	11.78 ± 0.51**	3.68 ± 0.51
Conventional liposomes (F3b)	3.77 ± 0.61**^a	13.09 ± 2.03**	5.17 ± 2.23
Pegylated liposomes (C1)	4.15 ± 0.67**^a	15.72 ± 2.02 **^a	7.01 ± 2.12**

The free plumbagin / liposomes administered at a dose of 2 mg/kg on 1, 3, 5, 7 and 10^th day after the attainment of 100 ± 10 mm³ tumor volume. VDT- volume doubling time; GD (growth delay at 20 X) = Days required for the tumor to reach 20 times its initial volume of 100 ± 10 mm³ (20 X) in treated group - Days required for the tumor to reach 20 times its initial volume of 100 ± 10 mm³(20 X) in vehicle treated group. Significant values: * p < 0.01, ** p < 0.001 compared with vehicle treated group; ^a p < 0.01 compared with free PLB.

Figure 4.  Effect of free PLB and formulated liposomes as conventional or pegylated (A) on tumor growth inhibition and (B) survival of C57BL/6J mice inoculated with B16F1 melanoma cells. Significant levels - ** p < 0.001 compared with vehicle treated group; ^a p < 0.01, ^b p < 0.001 compared to free plumbagin treated group. When tumor volume became about 100 ± 10 mm³, free PLB, conventional liposomes (F3b) or pegylated liposomes was intravenously injected (containing 2 mg/kg PLB each time on days 1, 3, 5, 7 and 10 (total dose of 10 mg/kg)). Control animals were injected with the vehicle (PBS containing 25% PEG-200). Each data represents the mean ± SD (n = 8).

Table 5.  Hematological changes after intravenous administration of free PLB and pegylated liposomes (C1) for 5 consecutive days.

Parameters	Vehicle treated	Free PLB	Pegylated Liposomes (C1)
Total WBC (x 10^3/µl)	7.75 ± 2.49	13.95 ± 1.28 ^b	12.70 ± 0.45 ^a
Lymphocytes (%)	91.11 ± 3.49	78.62 ± 10.34 ^a	83.65 ± 18.31
Monocytes (%)	5.05 ± 2.47	9.85 ± 4.20	6.73 ± 5.33
Granulocytes (%)	6.08 ± 1.02	11.52 ± 6.16	9.62 ± 12.99
Total RBC (x 10^6/µl)	9.70 ± 0.94	8.89 ± 2.69	10.08 ± 0.06
Hemoglobin (g/dl)	13.15 ± 1.92	14.52 ± 1.84	13.23 ± 0.44
Hematocrit (%)	46.77 ± 5.01	42.57 ± 13.60	47.28 ± 1.20
Platelets (x 10^6/µl)	0.72 ± 0.21	0.52 ± 0.28	0.71 ± 0.12

Significant levels: ^a p < 0.05; ^b p < 0.01 when compared to vehicle treated group. The data represents the mean ± SD (n = 4)

Figure 5.  Toxicity assessment of free PLB and long circulating liposomes (C1) in C57BL/6J mice. The histopathologic examination of tissue sections of (A) heart and kidney, (B) liver and spleen was made after mice were sacrificed and staining with H&E stain. All the treatment groups showed no signs of toxic pathological change; magnification 400X; 100X for liver.