Article Figures & Data
Figures
- Figure 1.
Binding and uptake of [3H]CHE-labeled doxorubicin-loaded immunoliposomes and nontargeted liposomes in EpCAM-positive (MCF-7 and SW2) and EpCAM-negative (RL) cells as a function of phospholipid concentration (phospholipid and [3H]CHE are in a known molar ratio). Binding and cellular uptake of EpCAM-targeted immunoliposomes encapsulating doxorubicin was measured after incubation of cells for 2 h at 4°C (Δ) or 37°C (▪). Nontargeted liposomes (•) were incubated with tumor cells for 2 h at 37°C. In competition experiments, EpCAM-positive tumor cells were incubated with a 50- to 60-fold excess of free scFv 4D5MOCB over liposome-bound scFv for 30 min before addition of the targeted immunoliposomes (□). Points, nmol phospholipids/106 cells (n = 3); bars, SD.
- Figure 2.
Blood clearance of EpCAM-targeted immunoliposomes versus nontargeted liposomes in CD-1 nude mice bearing SW2 tumor xenografts. Doxorubicin-containing sterically stabilized liposomes (EpCAM targeted or nontargeted) were radiolabeled with [3H]CHE. Mice (three per time point) were injected i.v. via the tail vein with anti-EpCAM immunoliposomal doxorubicin (SIL-Dox) or nontargeted liposomal doxorubicin (SL-Dox), with a single bolus dose of 4 mg doxorubicin/kg (0.66 μmol phospholipid per mouse). At selected time points after injection (0.5, 2, 12, 24, and 48 h), mice were euthanized and whole blood was analyzed for radioactivity. Results are expressed as the percent of the total injected phospholipid dose, set to 100 %, remaining in the blood at various time points after the injection of liposomes. Points, mean (n = 3); bars, SD.
- Figure 3.
Therapeutic efficacy of EpCAM-targeted immunoliposomal doxorubicin in a tumor xenograft model. CD-1 athymic mice (five to seven per group) bearing SW2 tumor xenografts (85–100 mm3) received i.v. injections of anti-EpCAM immunoliposomal doxorubicin (SIL-Dox) at a dose of either 4 or 7.5 mg/kg (MTD of the free doxorubicin equivalent). Other treatment groups included HEPES-buffered saline, nontargeted liposomal doxorubicin (SL-Dox) given at the same dose, and free doxorubicin given at its MTD of 7.5 mg/kg. All treatment groups received a total of three treatments at weekly intervals for 3 wk. Liposomal doxorubicin was injected to a total dose of 12 mg/kg and free doxorubicin was 22.5 mg/kg. Arrows in the tumor growth curve, treatment schedule. The median tumor size at the start of treatment was 90 mm3. Points, mean values of the treatment groups; bars, SE.
Tables
- Table 1.
Cytotoxicity of EpCAM-targeted immunoliposomes (SIL-Dox), nontargeted liposomes (SL-Dox), and free doxorubicin against EpCAM-positive (MCF-7 and SW2) and EpCAM-negative (RL) tumor cells
Cell line Formulation IC50 (μmol/L)* 2 h 24 h MCF-7 Free doxorubicin 0.6 ± 0.2 0.15 ± 0.1 SIL-Dox (anti-EpCAM) 15 ± 3† 5 ± 1† SL-Dox (nontargeted) >175 55 ± 10 SW2 Free doxorubicin 5 ± 2 1.5 ± 0.5 SIL-Dox (anti-EpCAM) 60 ± 10† 25 ± 5† SL-Dox (nontargeted) >175 80 ± 10 RL Free doxorubicin 9 ± 1 2 ± 1 SIL-Dox (anti-EpCAM) >175 100 ± 10 SL-Dox (nontargeted) >175 120 ± 10 - Table 2.
Pharmacokinetic and blood clearance of EpCAM-targeted immunoliposomes (SIL-Dox) and nontargeted liposomes (SL-Dox) in tumor-bearing mice
Liposome Half-life (h) Clearance (mL/h/kg) AUCt = 0→α (μg·h/mL) Vd (mL/kg) SL-Dox 11.2 ± 0.4 4.8 ± 0.2 3,526 ± 144 77.9 ± 3.6 SIL-Dox 11.4 ± 0.2 5 ± 0.3 3,358 ± 239 79.8 ± 2.0 NOTE: Mice bearing SW2 tumor xenografts (85–100 mm3) were injected i.v. with a single dose of 4 mg doxorubicin/kg (0.66 μmol phospholipid per mouse) encapsulated in liposomes labeled with [3H]CHE. Pharmacokinetic variables were determined using WinNonLin software. Data represent the mean ± SD (n = 3 mice per time point).
Abbreviations: AUC, area under the blood concentration versus time curve; Vd, volume of distribution.
- Table 3.
Biodistribution of EpCAM-targeted immunoliposomes (SIL-Dox) and nontargeted liposomes (SL-Dox) loaded with doxorubicin in mice bearing SW2 tumor xenografts
Tissue SIL-Dox (%ID/g) SL-Dox (%ID/g) 24 h 48 h 24 h 48 h Blood 11.8 ± 1.7* 1.6 ± 0.5 12.2 ± 0.9 2.6 ± 0.4 Liver 14.1 ± 0.6 14.5 ± 1.1 12.0 ± 1.3 14.7 ± 1.9 Spleen 19.3 ± 2.2 22.7 ± 2.1 18.3 ± 1.6 22.0 ± 1.6 Heart 2.4 ± 0.9 2.6 ± 0.4 1.5 ± 0.1 1.7 ± 0.4 Lung 2.5 ± 0.8 1.8 ± 0.6 1.4 ± 0.2 1.7 ± 0.3 Kidney 6.1 ± 1.4 10.3 ± 1.2 6.4 ± 1.5 9.1 ± 1.1 Tumor 13.0 ± 0.5† 15.0 ± 1.0† 7.7 ± 0.7 7.1 ± 1.7 ↵* Values were determined after i.v. injection of [3H]CHE-labeled liposomes. Data represent the mean %ID/g tissue ± SD (n = 3 mice per time point).
↵† P < 0.005, compared with SL-Dox. Statistical significance of the difference between SIL-Dox and SL-Dox was evaluated with a two-tailed, unpaired Student's t test.
Volume 6, Issue 11
