Peptide ligand and PEG-mediated long-circulating liposome targeted to FGFR overexpressing tumor in vivo

Figures & data

Figure 1 Synthetic scheme for preparation of targeted PEGylated liposomes.

Abbreviations: PEG, poly(ethylene glycol); EPC, egg phosphatidylcholine; Chol, cholesterol; DSPE, distearolyphosphatidyl ethanolamine; EDC, 1-ethyl-3-(3- dimethylaminopropyl)-carbodiimide hydrochloride; NHS, N-hydroxysuccinimide; tbFGF, truncated basic fibroblast growth factor.

Table 1 Particle size and zeta potential (mean ± SD, n = 3) of CL-PTX and TL-PTX

Formulation	Samples	Size (nm)	Polydispersity index	Zeta potential (mV)
CL-PTX	1	161.2	0.286	−8.94
	2	135.8	0.223	−9.16
	3	115	0.143	−9.55
	Mean ± SD	137.3 ± 23.1	0.217 ± 0.072	−9.20 ± 0.31
TL-PTX	1	186.3	0.346	−5.18
	2	146.5	0.245	−5.11
	3	93.8	0.175	−4.87
	Mean ± SD	142.2 ± 46.4	0.255 ± 0.086	−5.05 ± 0.16

Abbreviations: CL-PTX, paclitaxel-loaded conventional liposomes; TL-PTX, paclitaxel-loaded targeted PEGylated liposomes; SD, standard deviation.

Figure 2 Particle size distribution of paclitaxel-loaded conventional liposomes (A) and paclitaxel-loaded targeted PEGylated liposomes (B), and typical atomic force microscopy image of paclitaxel-loaded targeted PEGylated liposomes (C).

Table 2 Stability of formulated liposomes (mean ± SD, n = 3)

Formulation	Parameters	Day
		0	1	3	7
CL-PTX	Size (nm)	137.3 ± 23.1	138.1 ± 24.2	137.5 ± 23.6	138.1 ± 25.1
	Zeta potential (mV)	9.20 ± 0.31	9.24 ± 0.35	9.18 ± 0.29	9.25 ± 0.37
	Encapsulation efficiency (%)	89.7 ± 3.6	89.4 ± 3.2	88.9 ± 2.8	89.1 ± 3.0
TL-PTX	Size (nm)	142.3 ± 46.4	142.6 ± 46.8	144.1 ± 47.2	143.6 ± 46.9
	Zeta potential (mV)	5.05 ± 0.16	5.13 ± 0.19	4.98 ± 0.12	5.02 ± 0.18
	Encapsulation efficiency (%)	89.1 ± 2.3	89.3 ± 2.5	88.7 ± 1.8	88.9 ± 2.1

Abbreviations: CL-PTX, paclitaxel-loaded conventional liposomes; TL-PTX, paclitaxel-loaded targeted PEGylated liposomes; SD, standard deviation.

Figure 3 Time course of paclitaxel release from TL-PTX (▲), CL-PTX (■), and free PTX(◆) at 37°C and pH 7.0. The paclitaxel Released was separated by dialysis and quantified using high-performance liquid chromatography.

Notes: There were five mice per group per time point. Bars represent the mean ± standard deviation. Error bars correspond to 95% confidence intervals.

Abbreviations: CL-PTX, paclitaxel-loaded conventional liposomes; TL-PTX, paclitaxel-loaded targeted PEGylated liposomes; Free PTX, paclitaxel dissolved in Cremophor EL.

Table 3 Pharmacokinetic parameters for paclitaxel in plasma after intravenous administration of free PTX, CL-PTX, and TL-PTX (mean ± standard deviation)

Parameters	Units	Free-PTX	CL-PTX	TL-PTX
T1/2	h	8.562 ± 0.11	14.476 ± 0.07	29.122 ± 0.11
Tmax	h	0.5 ± 0.00	0.5 ± 0.00	0.5 ± 0.00
Cmax	mg L^−1	5.58 ± 0.31	7.93 ± 0.47	18.95 ± 1.03*
AUC0>t	mg L^−1 h	26.9 ± 1.97	39.9 ± 2.67	62.17 ± 4.72*
AUC0→∝	mg L^−1 h	30.01 ± 2.96	52.98 ± 2.49	120.00 ± 6.92*
AUMC0→∝		160.89 ± 7.15	281.93 ± 12.29	480.57 ± 16.92*
MRT0→∝	h	9.15 ± 0.27	16.42 ± 0.36	3.50 ± 0.93
Vd	L kg−1	4.12 ± 0.04	3.94 ± 0.08	3.50 ± 0.02
CL	L h^−1 kg^−1	0.33 ± 0.03	0.19 ± 0.04	0.08 ± 0.06*

Notes: Each data was from five mice.

* P values ≤ 0.05 were considered to be statistically significant compared with the free PTX group.

Abbreviations: CL-PTX, paclitaxel-loaded conventional liposomes; TL-PTX, paclitaxel-loaded targeted PEGylated liposomes; Free PTX, paclitaxel dissolved in Cremophor EL; T_max, time to maximum plasma concentration of drug; C_max, maximum plasma concentration of drug; T_1/2, elimination half life; CL, clearance; MRT, mean residence time; V_d, apparent volume of distribution; AUC_0→∝, area under the plasma concentration-time curve.

Figure 4 Areas under the curve over 24 hours for paclitaxel in serum after treatment with free PTX (◆), CL-PTX (■), or TL-PTX (▲).

Notes: There were five mice per group per time point. Bars represent the mean ± standard deviation.

Abbreviations: CL-PTX, paclitaxel-loaded conventional liposomes; TL-PTX, paclitaxel-loaded targeted PEGylated liposomes; Free PTX, paclitaxel dissolved in Cremophor EL.

Table 4 Increase versus time of distribution of TL-PTX in the tumors compared with CL-PTX and free PTX, respectively

Folds vs time	0.5 h	1 h	3 h	5 h	12 h
CL-PTX	1.89	2.04	1.69	1.58	2.29
Free-PTX	4.66	4.48	3.61	4.40	5.04

Abbreviations: CL-PTX, paclitaxel-loaded conventional liposomes; TL-PTX, paclitaxel-loaded targeted PEGylated liposomes; Free PTX, paclitaxel dissolved in Cremophor EL.

Figure 5 Mean paclitaxel concentration in tumor tissue at each time point after treatment with free PTX, CL-PTX, and TL-PTX. Paclitaxel was extracted from the tumors and measured by ultraperformance liquid chromatography.

Notes: There were five mice per group per time point. Bars represent the mean ± standard deviation.

Abbreviations: CL-PTX, paclitaxel-loaded conventional liposomes; TL-PTX, paclitaxel-loaded targeted PEGylated liposomes; Free PTX, paclitaxel dissolved in Cremophor EL.

Figure 6 Mean paclitaxel concentration in tumor, lungs, heart, spleen, liver, and kidneys of mice treated with free PTX, CL-PTX, and TL-PTX. Mice were sacrificed at the indicated time points of (A) 0.5, (B) 1, (C) 3, (D) 5, (E) 12, and (F) 24 hours after administration, and paclitaxel was extracted from the tissues and measured by ultraperformance liquid chromatography.

Notes: There were five mice per group per time point. Bars represent the mean ± standard deviation.

Abbreviations: CL-PTX, paclitaxel-loaded conventional liposomes; TL-PTX, paclitaxel-loaded targeted PEGylated liposomes; Free PTX, paclitaxel dissolved in Cremophor EL.