Lactosylated liposomes for targeted delivery of doxorubicin to hepatocellular carcinoma

Figures & data

Figure 1 Synthesis of Lac-DOPE.

Figure 2 Cellular uptake of liposomes in HepG2 cells.

Notes: Cells were incubated with L-calcein, Lac-L-calcein, and Lac-L-calcein plus 20 mM lactose as a blocking agent for one hour before being subjected to analysis. The fluorescence intensity in HepG2 cells treated with Lac-L-calcein was significantly higher than that of nontargeted liposomes. Meanwhile, the fluorescence of Lac-L-calcein in HepG2 cells was decreased by treatment of 20 mM lactose as an asialoglycoprotein receptor blocking agent.

Figure 3 Cellular uptake of Lac-L-calcein in HepG2 cells detected by confocal microscopy. Cells were incubated with phosphate-buffered saline, L-calcein, Lac-L-calcein, and Lac-L-calcein plus 20 mM lactose at the same concentrations of calcein for one hour. (A) Cells treated with L-calcein. (B) Cells treated with Lac-L-calcein. (C) Cells treated with Lac-L-calcein plus 20 mM lactose.

Table 1 Cytotoxicity of L-DOX formulations to HepG2 cells

	IC50 (μM)
Free DOX	5.3 ± 1.4
L-DOX	7.5 ± 2.8
Lac-L-DOX	2.7 ± 1.2a,b
Lac-L-DOX plus 20 mM Lactose	9.0 ± 2.0

Notes: Cytotoxicity was determined using the MTT assay as described in the Methods section. Data are represented as the mean ± standard deviation (n = 3).

a P < 0.05 compared with L-DOX treatment;

b P < 0.01 compared with Lac-L-DOX plus 20 mM lactose treatment.

Abbreviations: DOX, doxorubicin; L-DOX, liposomal doxorubicin; Lac-L-DOX, lactosylated liposomes encapsulating doxorubicin; IC₅₀, half-maximal inhibitory concentration.

Table 2 Pharmacokinetic parameters in different formulations after intravenous injection at a doxorubicin dose of 5 mg/kg body weight (n = 3)

	Free DOX	L-DOX	Lac-L-DOX
AUC (μg hours/mL)	16.83 ± 2.7	582.3 ± 27.3	350.2 ± 20.2a,b
t1/2 (hours)	1.96 ± 0.7	12.08 ± 0.8	8.73 ± 0.8a,b
CL (mL/hour)	11.33 ± 1.0	0.086 ± 0.0	0.014 ± 0.0a,b
MRT (hours)	2.56 ± 0.8	16.9 ± 1.1	11.8 ± 1.1a,b

Notes:

a P < 0.01 compared with free doxorubicin;

b P < 0.01 compared with L-DOX.

Abbreviations: AUC, the area under the concentration-time curve; t_1/2, half life; CL, total body clearance in mice; MRT, mean residence time; DOX, doxorubicin; L-DOX, liposomal doxorubicin; Lac-L-DOX, lactosylated liposomes encapsulating doxorubicin.

Figure 4 Plasma concentrations of DOX in different formulations.

Notes: DOX in different formulations (5 mg/kg) was injected via the tail veins of normal mice. Data are expressed as the mean ± standard deviation (n = 3). (●) Free DOX, (○) L-DOX, (▲) Lac-L-DOX.

Abbreviations: DOX, doxorubicin; L-DOX, liposomal doxorubicin; Lac-L-DOX, lactosylated liposomes encapsulating doxorubicin.

Figure 5 Tissue distribution of DOX in different formulations at 4 hours after intravenous injection in female tumor-bearing nude mice.

Notes: Data are expressed as the mean ± standard deviation (n = 5). ***P < 0.005, significant difference compared with free DOX; **P < 0.005, significant difference compared with L-DOX; *P < 0.05, significant difference compared with L-DOX.

Abbreviations: DOX, doxorubicin; L-DOX, liposomal doxorubicin; Lac-L-DOX, lactosylated liposomes encapsulating doxorubicin.

Figure 6 Confocal micrographs of tissue from female tumor-bearing nude mice injected with different formulations of DOX, 4 hours prior to euthanasia. The tissue section was labeled for cytoskeletal filamentous actin with Alexa Fluor 488^® conjugated with phalloidin. (A) Lac-L-DOX in liver, (B) L-DOX in liver, (C) free DOX in liver, (D) Lac-L-DOX in tumor, (E) L-DOX in tumor, and (F) free DOX in tumor.

Note: The white arrows denote Kupffer cells.

Abbreviations: DOX, doxorubicin; L-DOX, liposomal doxorubicin; Lac-L-DOX, lactosylated liposomes encapsulating doxorubicin.

Figure 7 Tumor growth inhibition after intravenous injection of free DOX, L-DOX, or Lac-L-DOX in tumor-bearing mice at a DOX dose of 5 mg/kg body weight.

Notes: Data are shown as the mean ± standard deviation (n = 10). ***P < 0.05, statistically significant compared with phosphate-buffered saline control; **P < 0.05, statistically significant compared with free DOX; *P < 0.05, statistically significant compared with L-DOX.

Abbreviations: DOX, doxorubicin; L-DOX, liposomal doxorubicin; Lac-L-DOX, lactosylated liposomes encapsulating doxorubicin; PBS, phosphate-buffered solution.

Table 3 Survival of mice after treatment with free DOX, L-DOX, and Lac-L-DOX in HepG2 tumor-bearing micea

Treatment	Tumor inhibition ratiob (%)	MSTc (days)	ILSd (%)	Log-rank P value
PBS	0	28	0	–
Free DOX	38.7	34	21.4	0.0612
L-DOX	60.8	42	50	0.00178
Lac-L-DOX	80.9	70	150	0.000557 (0.0205)e

Notes:

a n = 10 for each treatment group;

b tumor inhibition ratio was calculated by (C − T)/C × 100%;

c MST represents the mean survival time;

d ILS (%) was calculated by (MST of the treated mice/MST of control mice − 1) × 100%;

e P value was calculated in comparison with L-DOX.

Abbreviations: MRT, mean residence time; DOX, doxorubicin; L-DOX, liposomal doxorubicin; Lac-L-DOX, lactosylated liposomes encapsulating doxorubicin; MST, mean survival time; ILS, increased life span; PBS, phosphate-buffered saline.

Figure 8 Kaplan–Meier survival curve of mice bearing HepG2 xenografts.

Notes: Animals (10 per group) were treated with 5 mg/kg of free DOX, L-DOX or Lac-L-DOX. The treatment was continued every 4 days for four times.

Abbreviations: DOX, doxorubicin; L-DOX, liposomal doxorubicin; Lac-L-DOX, lactosylated liposomes encapsulating doxorubicin; PBS, phosphate-buffered solution.