Folate-targeted docetaxel-lipid-based-nanosuspensions for active-targeted cancer therapy

Figures & data

Figure 1 Schematics of the synthetics of distearoylphosphatidylethanolamine-poly(ethylene glycol)2000-folate.

Abbreviations: DCC, dicyclohexylcarbodiimide; DMSO, dimethyl sulfoxide; DPSE, distearoylphosphatidylethanolamine; PEG, poly(ethylene glycol).

Figure 2 ¹H nuclear magnetic resonance spectra of synthesized distearoylphosphatidylethanolamine-poly(ethylene glycol)2000-folate in dimethyl sulfoxide-d₆.

Abbreviation: ppm, parts per millon.

Figure 3 Photographs of (A) coarse poly(ethylene glycol)-mediated docetaxel-lipid-based- nanosuspension; (B) poly(ethylene glycol)-mediated docetaxel-lipid-based-nanosuspension; (C) coarse targeted docetaxel-lipid-based-nanosuspension; and (D) targeted docetaxel-lipid-based-nanosuspension.

Figure 4 Transmission electron photomicrographs of freshly prepared (A) poly(ethylene glycol)-mediated docetaxel-lipid-based-nanosuspension; and (B) targeted docetaxel-lipid-based-nanosuspension.

Figure 5 Transmission electron photomicrographs of freeze dried (A) poly(ethylene glycol)-mediated docetaxel-lipid-based-nanosuspension; and (B) targeted docetaxel-lipid- based-nanosuspension.

Figure 6 In vitro release profile of docetaxel from poly(ethylene glycol)- mediated docetaxel-lipid-based-nanosuspension, targeted docetaxel-lipid-based-nanosuspension, and Duopafei^® in phosphate buffered saline (0.5% of Tween 80^® in phosphate buffered saline, pH 7.4) at 37°C ± 0.5°C (n = 3).

Abbreviations: pLNS, poly(ethylene glycol)-mediated docetaxel-lipid-based- nanosuspension; t, time; tLNS, targeted docetaxel-lipid-based-nanosuspension.

Table 1 The half maximal inhibitory concentration of HepG2 and B16 cells incubated with Duopafei^®, poly(ethylene glycol)-mediated docetaxel-lipid-based-nanosuspension, targeted docetaxel-lipid-based-nanosuspension, blank poly(ethylene glycol)-mediated docetaxel-lipid- based-nanosuspension, and blank targeted docetaxel-lipid-based-nanosuspension at 48 hours

Cell line	Duopafei	pLNS	tLNS	Blank pLNS	Blank tLNS
B16	1.03 ± 0.15	0.70 ± 0.06**	0.43 ± 0.05##	26.18 ± 2.75	30.79 ± 1.43
HepG2	1.08 ± 0.06	0.65 ± 0.05**	0.69 ± 0.07	24.80 ± 0.67	30.45 ± 1.44

Notes: The range of concentrations of docetaxel used was from 0.01–10 μM (n = 3). Data are presented as mean ± standard deviation;

## P < 0.01 versus the poly(ethylene glycol)-mediated docetaxel-lipid-based-nanosuspension group;

** P < 0.01 versus the Duopafei group.

Abbreviations: pLNS, poly(ethylene glycol)-mediated docetaxel-lipid-based-nanosuspension; tLNS, targeted docetaxel-lipid-based-nanosuspension.

Figure 7 Cytotoxic effects of blank poly(ethylene glycol)-mediated docetaxel-lipid-based-nanosuspension, blank targeted docetaxel-lipid-based-nanosuspension, Duopafei^®, poly(ethylene glycol)-mediated docetaxel-lipid-based-nanosuspension, and targeted docetaxel-lipid-based-nanosuspension against (A) B16 and (B) HepG2 cells.

Note: Data represent mean ± standard deviation (n = 3).

Abbreviations: DTX, docetaxel; pLNS, poly(ethylene glycol)-mediated docetaxel-lipid-based-nanosuspension; tLNS, targeted docetaxel-lipid-based-nanosuspension.

Figure 8 Antitumor effects of poly(ethylene glycol)-mediated docetaxel-lipid-based-nanosuspension, targeted docetaxel-lipid-based-nanosuspension, Duopafei^®, blank poly(ethylene glycol)-mediated docetaxel-lipid-based-nanosuspension, blank targeted docetaxel-lipid-based-nanosuspension, and NS on B16 tumor-bearing mice after intravenous administration. Data represent mean ± standard deviation (n = 6). (A) Tumor volume; (B) tumor weight; (C) photographs of tumors excised on day 21; and (D) body weight change.

Notes: **P < 0.01 versus the Duopafei group; ^#P < 0.05 versus the poly(ethylene glycol)-mediated docetaxel-lipid-based-nanosuspension group; ^##P < 0.01 versus the poly(ethylene glycol)-mediated docetaxel-lipid-based-nanosuspension group.

Abbreviations: pLNS, poly(ethylene glycol)-mediated docetaxel-lipid-based-nanosuspension; t, time; tLNS, targeted docetaxel-lipid-based-nanosuspension.

Table 2 The tumor inhibition ratio of the Duopafei^®, poly(ethylene glycol)-mediated docetaxel-lipid-based-nanosuspension, and targeted docetaxel-lipid-based-nanosuspension groups

Group	Dose (mg/kg)	Number of animals (start/end)	Average tumor weight (g)	TIR (100%)	P
NS	–	10/10	5.31 ± 0.19	–	–
Blank pLNS	–	10/10	4.24 ± 0.73	–	–
Blank tLNS	–	10/10	3.97 ± 0.30	–	–
Duopafei	20	10/7	1.15 ± 0.12	78.44 ± 5.16	<0.01$$
pLNS	20	10/10	0.64 ± 0.04	87.93 ± 2.55	<0.01**
tLNS	20	10/10	0.42 ± 0.02	92.09 ± 2.72	<0.01##

Notes:

$$ P < 0.01 versus the NS group;

** P < 0.01 versus the Duopafei group;

## P < 0.01 versus the poly(ethylene glycol)-mediated docetaxel-lipid-based-nanosuspension group.

Abbreviations: NS, normal saline; pLNS, poly(ethylene glycol)-mediated docetaxel-lipid-based-nanosuspension; TIR, tumor inhibition rate; tLNS, targeted docetaxel-lipid-based- nanosuspension.

Table 3 Plasma pharmacokinetic parameters after intravenous injection of Duopafei^®, poly(ethylene glycol)-mediated docetaxel-lipid- based-nanosuspension, and targeted docetaxel-lipid-based-nanosuspension at a dose of 60 mg/kg of docetaxel (n = 5)

Pharmacokinetic parameters	Formulations
	Duopafei	pLNS	tLNS
Cmax (μg/mL)	481.62 ± 13.23	217.92 ± 10.54	213.45 ± 13.16
T1/2α (hours)	0.31 ± 0.06	0.230 ± 0.19	0.230 ± 0.08
T1/2β (hours)	1.78 ± 0.11	3.68 ± 0.23**	3.70 ± 0.17**
Tmax (hours)	0.083	0.083	0.083
AUC0–∞ (mg/L/hour)	308.42 ± 20.23	489.65 ± 11.19**	511.30 ± 19.81**
MRT (hours)	1.76 ± 0.15	4.229 ± 0.17*	4.230 ± 0.67*
CL (L/hour/kg)	0.195	0.100**	0.097**

Notes: Data are presented as mean ± standard deviation.

* P < 0.05 versus the Duopafei group;

** P < 0.01 versus the Duopafei group.

Abbreviations: AUC_0–∞, area under the plasma concentration-time profiles; CL, total plasma clearance; C_max, maximum concentration; MRT, mean residence time; pLNS, poly(ethylene glycol)-mediated docetaxel-lipid-based-nanosuspension; tLNS, targeted docetaxel-lipid-based-nanosuspension; T_1/2α, distribution; T_1/2β, elimination half-life; T_max, time to maximum concentration.

Table 4 Targeting disposition of docetaxel after intravenous administration of Duopafei^®, poly(ethylene glycol)-mediated docetaxel-lipid-based-nanosuspension, and targeted docetaxel-lipid- based-nanosuspension to mice at a dose of 60 mg/kg of docetaxel (n = 5)

Tissue	Duopafei	pLNS			tLNS
	Te (%)	Te (%)	re	Ce	Te (%)	re	Ce
Heart	24.03	4.92	0.97	0.51	4.84	0.96	0.49
Liver	8.58	32.50	17.86	2.80	30.96	17.14	2.79
Spleen	18.91	39.31	9.80	2.40	39.70	9.97	2.32
Lung	19.23	13.19	3.23	1.79	13.63	3.37	1.77
Kidney	22.31	4.40	0.93	0.41	4.47	0.95	0.38
Tumor	5.22	5.67	5.22	1.02	6.40	5.92	1.17

Abbreviations: C_e, maximum concentration; pLNS, poly(ethylene glycol)-mediated docetaxel-lipid-based-nanosuspension; r_e, relative efficiency; T_e, targeting efficiency; tLNS, targeted docetaxel-lipid-based-nanosuspension.

Figure 9 Mean plasma concentration of docetaxel after intravenous administration of Duopafei^®, poly(ethylene glycol)-mediated docetaxel-lipid-based-nanosuspension, and targeted docetaxel-lipid-based-nanosuspension.

Note: Data represent mean ± standard deviation (n = 5).

Abbreviations: C, mean concentration; pLNS, poly(ethylene glycol)-mediated docetaxel-lipid-based-nanosuspension; t, time; tLNS, targeted docetaxel-lipid-based-nanosuspension.

Figure 10 The distribution of docetaxel in mice organs at different time points after intravenous administration of (A) Duopafei^®; (B) poly(ethylene glycol)-mediated docetaxel-lipid-based-nanosuspension; and (C) targeted docetaxel-lipid-based-nanosuspension.

Note: Data represent mean ± standard deviation (n = 5).

Abbreviations: C, concentration; h, hours.

Figure 11 Distribution of docetaxel in tumor tissues of mice after intravenous administration of Duopafei^®, poly(ethylene glycol)-mediated docetaxel-lipid-based-nanosuspension, and targeted docetaxel-lipid-based-nanosuspension.

Notes: Data represent mean ± standard deviation (n = 5). *P < 0.05 versus the Duopafei group; **P < 0.01 versus the Duopafei group; ^#P < 0.05 versus the poly(ethylene glycol)-mediated docetaxel-lipid-based-nanosuspension group; ^##P < 0.01 versus the poly(ethylene glycol)-mediated docetaxel-lipid-based-nanosuspension group.

Abbreviations: C, concentration; pLNS, poly(ethylene glycol)-mediated docetaxel-lipid-based-nanosuspension; t, time; tLNS, targeted docetaxel-lipid-based-nanosuspension.