Figure 1.
A, The hydrophobicity of PTX and gPTX was evaluated by reverse-phase HPLC using a C18 column at a flow rate of 1 mL/min with 55% (v/v) methanol under an isocratic condition. B, The ability of 3 µM PTX (open circle) and gPTX (open triangle) to stabilize porcine tubulin polymerization was evaluated using a Tubulin Polymerization Assay Kit (Cytoskeleton). The fluorescent reporter was detected with excitation at 360 nm and emission at 420 nm. Each dot represents the mean ± S.D. (n = 3).
Table 1.
Solubility of PTX and gPTX in different solvents.
Figure 2.
Schematic illustration of remote loading with a solubility gradient.
CEP-encapsulated liposomes (CEP-L) and gPTX dissolved in 40% EG were mixed and incubated. gPTX was encapsulated into liposomes under a solubility gradient.
Figure 3.
Influence of the lipid composition and incubation time for drug encapsulation by the solubility gradient method.
A, The encapsulation efficiency (EE) of gPTX-L with various lipid compositions was evaluated after 30 min of incubation for gPTX encapsulation. B, The drug retention of gPTX-L with different lipid compositions was evaluated in medium supplied with 10% FBS at 37°C. C, The efficiency of drug encapsulation under different durations of incubation was evaluated with a DPPC to Chol ratio of 3∶1.
Figure 4.
Comparison of the encapsulation efficiency (EE), loading efficiency (LE), and particle size.
A, EE. B, LE. C, particle size. #Control indicated that gPTX-L was prepared with liposomes encapsulating 40% EG in 1 mg/mL gPTX. Comparison of stability at 4°C in PBS with PTX-L and gPTX-L prepared by the solubility gradient method. D, The retention rates of PTX-L (open circle) and gPTX-L (open triangle) were evaluated by HPLC. E, TEM images of PTX-L and gPTX-L. Each formulation was observed after 4 days of incubation. Each data point is presented as the mean ± S.D. (n = 3). *, P<0.05.
Figure 5.
Cytotoxicity of different gPTX formulations by the MTT assay.
A, The IC50 values after drug exposure for 72 h are shown. B, The IT50 values at IC100 are shown. The data are presented as the mean ± S.D. for three independent experiments. *, P<0.05. **, P<0.01. ***, P<0.005. ****, P<0.001. NSD, no significant difference.
Figure 6.
Acute toxicity of the drugs in vivo.
gPTX (open circle), gPTX-L (open triangle) or gPTX-IL (open diamond) at a concentration of 150 mg/kg, CEP (open square), or PBS (cross) was intravenously injected into 6-week old female BALB/c mice. A, Changes in body weight. B, Survival rate. Data are presented as the mean ± S.D. (n = 4). gPTX is not shown S.D. after day 3. NSD, no significant difference.
Figure 7.
Anticancer efficacy of different gPTX formulations with single administration in HT-29 cells tumor-bearing mice.
When the tumor volume reached 50–200 mm3, gPTX-IL at the dose of 150 (open circle with solid line), 100 (open circle with broken line), 50 (brown circle with solid line), or 10 mg/kg (brown circle with broken line), gPTX-L at the dose of 150 mg/kg (open triangle), gPTX-L at the dose of 150 mg/kg with 150 mg/kg trastuzumab (gPTX-L + Tras, open square), naked gPTX at the dose of 100 mg/kg (open diamond), trastuzumab (Tras, closed square), CEP-IL (closed circle), or PBS (cross) was intravenously injected. A, Tumor weights at day 30. B, Changes in body weight. Data are presented as the mean ± S.D. *, P<0.05 compared with PBS.
Figure 8.
Anticancer efficacy of different gPTX formulations with repeated administration in HT-29 cells tumor-bearing mice.
gPTX-IL (open circle with line), gPTX-L (open triangle), gPTX-L with trastuzumab (open square), trastuzumab (closed square), CEP-IL (closed circle), CEP-L (closed triangle), or PBS (cross) was intravenously injected at day 0, 10, and 20. The dose of each administration was 150 mg/kg gPTX. A and B, Changes in tumor volume. C, Changes in body weight. D, Survival curves. Data are presented as the mean ± S.D. The changes of tumor volume and body weight in the mice administered with gPTX-L do not show S.D. after day 10. The P value shown compared with gPTX-IL and gPTX-L with trastuzumab treated group at day 43 (n = 4). *, P<0.05.