Nanoparticles targeted with NGR motif deliver c-myc siRNA and doxorubicin for anticancer therapy

Abstract

We have designed a PEGylated LPD (liposome-polycation-DNA) nanoparticle for systemic, specific, and efficient delivery of small interfering RNA (siRNA) into solid tumors in mice by modification with NGR (aspargine-glycine-arginine) peptide, targeting aminopeptidase N (CD13) expressed in the tumor cells or tumor vascular endothelium. LPD-PEG-NGR efficiently delivered siRNA to the cytoplasm and downregulated the target gene in the HT-1080 cells but not CD13(-) HT-29 cells, whereas nanoparticles containing a control peptide, LPD-PEG-ARA, showed only little siRNA uptake and gene silencing activity. LPD-PEG-NGR efficiently delivered siRNA into the cytoplasm of HT-1080 xenograft tumor 4 hours after intravenous injection. Three daily injections (1.2 mg/kg) of c-myc siRNA formulated in the LPD-PEG-NGR effectively suppressed c-myc expression and triggered cellular apoptosis in the tumor, resulting in a partial tumor growth inhibition. When doxorubicin (DOX) and siRNA were co-formulated in LPD-PEG-NGR, an enhanced therapeutic effect was observed.

Figure 1

Intracellular uptake of siRNA and c-myc expression inhibited by siRNA formulation in vitro. (a) The structure of DSPE-PEG-NGR. (b) Fluorescence photographs of HT-1080 and HT-29 cells after treatment with 5′-Cy3-labeled siRNA against an irrelevant target in LPD-PEG-NGR and LPD-PEG-ARA for 4 hours. Western blot analysis of c-myc and β-actin in (c) HT-1080 cells and (d) HT-29 after treatment with 250 nmol/l siRNA in different formulations for 24 hours. (e) Immunocytochemical staining of c-myc after treatment with 250 nmol/l siRNA in different formulations for 24 hours.

Figure 2

Tumor uptake of siRNA in different formulations. (a) Fluorescence signal of cy3-labeled siRNA in HT-1080 tumor observed by confocal microscopy. (b) Tissue distribution of FITC-siRNA in different formulations. Data = mean ± SD, n = 3. *P < 0.05 compared with free siRNA. ARA: LPD-PEG-ARA; c-myc: c-myc siRNA; Control: control siRNA; NGR: LPD-PEG-NGR.

Figure 3

c-myc expression and apoptosis induction in HT-1080 xenograft tumor. (a) Western blot analysis of c-myc in the HT-1080 xenograft tumor after treatment with different formulations. (b) c-myc expression and (c) TUNEL staining in HT-1080 tumor cells after treated with siRNA with different formulation in vivo. (d) Quantitative analysis of TUNEL positive staining in the tumors treated with different formulations. N = 3~5. **P < 0.001.

Figure 4

HT-1080 xenograft tumor growth inhibition by siRNA in different formulations. Solid arrows indicate the intravenous administrations of siRNA (1.2 mg/kg). Data = mean, n = 5–7. SD of the data points is not shown for clarity. *P < 0.05. PBS, phosphate-buffered saline.

Figure 5

Intracellular uptake of DOX in vitro. (a) Illustration of preparation of LPD-PEG-NGR containing siRNA and DOX. (b) Fluorescence photographs of HT-1080 and HT-29 cells after treatment with DOX in LPD-PEG-NGR (NGR), LPD-PEG-ARA (ARA), and LPD-PEG (PEG) for 1 hour. (c) Quantitative measurement of DOX uptake in HT-1080 cells by flow cytometry. (d) Uptake of siRNA and DOX by HT-1080 and HT-29 cells were compared. Cells were treated with different formulations containing DOX and FITC-siRNA for 1 hour and analyzed for fluorescence by flow cytometry. Data = mean ± SD, n = 3. **P < 0.001. DOX, doxorubicin.

Figure 6

Tumor uptake of DOX in different formulations. (a) Fluorescence signal of DOX in HT-1080 tumor observed by confocal microscopy. (b) Tissue distribution of DOX in different formulations. Data = mean ± SD, n = 3. *P < 0.05 compared with free DOX. DOX, doxorubicin.

Figure 7

HT-1080 xenograft tumor growth inhibition by siRNA and DOX in different formulations. Solid arrows indicate the intravenous administrations of siRNA (1.2 mg/kg) and DOX (0.3 mg/kg). Data = mean, n = 5–7. SD of the data points is not shown for clarity. **P < 0.01. DOX, doxorubicin; PBS, phosphate-buffered saline.