PolyMetformin combines carrier and anticancer activities for in vivo siRNA delivery

Abstract

Metformin, a widely implemented anti-diabetic drug, exhibits potent anticancer efficacies. Herein a polymeric construction of Metformin, PolyMetformin (PolyMet) is successfully synthesized through conjugation of linear polyethylenimine (PEI) with dicyandiamide. The delocalization of cationic charges in the biguanide groups of PolyMet reduces the toxicity of PEI both in vitro and in vivo. Furthermore, the polycationic properties of PolyMet permits capture of siRNA into a core-membrane structured lipid-polycation-hyaluronic acid (LPH) nanoparticle for systemic gene delivery. Advances herein permit LPH-PolyMet nanoparticles to facilitate VEGF siRNA delivery for VEGF knockdown in a human lung cancer xenograft, leading to enhanced tumour suppressive efficacy. Even in the absence of RNAi, LPH-PolyMet nanoparticles act similarly to Metformin and induce antitumour efficacy through activation of the AMPK and inhibition of the mTOR. In essence, PolyMet successfully combines the intrinsic anticancer efficacy of Metformin with the capacity to carry siRNA to enhance the therapeutic activity of an anticancer gene therapy.

Figure 1. Synthesis and characterization of PolyMet.

(a) Synthesis scheme of PolyMet polymer. (b) Ultraviolet spectra of Metformin, PEI and PolyMet in the range of 220–300 nm. (c) Colour test of Metformin, PEI and PolyMet. Test reagents were prepared by mixing equal volumes of 10% w/v sodium nitroprusside with 10% w/v potassium hexacyanoferrate (III) and 10% sodium hydroxide. Equal amounts of Metformin, PEI unimer or PolyMet unimer in aqueous solution were mixed with 100 μl of the test reagent. The image was taken 30 min after mixing. (d) Cytotoxicity of Metformin, PEI and PolyMet. H460 cell availability was measured using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay after 24 h of exposure to Metformin, PolyMet and PEI solutions. Data are mean±s.d. (n=8). Data are representative of b and c or combined (d) from three independent experiments.

Figure 2. Schematic illustration and representative TEM images of PolyMet nanoparticles.

Anionic HA+siRNA mixture was condensed by cationic PolyMet into a negatively charged PolyMet/(HA+siRNA) complex (a,c). DOTAP/cholesterol cationic liposomes were added to the complex to form lipid coating, then DSPE-PEG and DSPE-PEG-anisamide were used to liposome by the post-insertion method to form LPH-PolyMet final nanoparticles (b,d).

Figure 3. LPH nanoparticles composed of PolyMet can systemically deliver VEGF siRNA to the tumour site and inhibit tumour growth.

(a) H460 tumour-bearing mice were injected i.v. every other day and tumour volumes were measured every day. (b) H460 tumour VEGF protein levels after two injections were measured by western blot analysis. Bar chart in b represent quantitative analysis of relative normalized VEGF band intensity (Image J). (c) TUNEL staining (green) in H460 tumour cells after treatment with siRNA in different formulations in vivo. Nucleic acid was stained with 4,6-diamidino-2-phenylindole (blue). Bar chart in c is a quantitative analysis of % of TUNEL-positive cells. Five randomly selected microscopic fields were quantitatively analysed on Image J. Data are mean±s.e.m. (n=5 per group) analysed by two-way analysis of variance with Tukey's correction. Data are representative of b and c or combined from (a) three independent experiments. NS, not significant; *P<0.05, **P<0.01, ***P<0.005. Scale bar, 200 μm.

Figure 4. Metformin and PolyMet inhibit H460 tumour growth.

PBS, Metformin, LPH-PEI and LPH-PolyMet were administered i.v. every other day, and mice were killed 24 h after the final injection. (a) Tumour volumes were measured every day. (b) Tumour weights were measured on day after final injection and compared with body weights to determine the percentage of tumour burden. (c) Visual observations of the H460 tumour sizes in each treatment group at the end time point; Data are mean±s.e.m. (n=5 per group) analysed by two-way analysis of variance with Tukey's correction. Data are combined from a and b or representative of (c) three independent experiments. NS, not significant; *P<0.05, **P<0.01, ***P<0.005.

Figure 5. Metformin and PolyMet inhibit tumour growth by activating the AMPK and inhibiting the mTOR pathways, inducing autophagy and apoptosis mechanisms.

Mice bearing H460 tumours were given i.v. injections every other day and tumour proteins was prepared 24 h after the second injection for analysis. Bar charts in (a) represent quantitative analysis of normalized p-AMPK and p-mTOR band intensity using Image J. Data are shown as mean±s.d. (n=3). Cells under autophagy were stained by LC3b antibody (red) and apoptosis of cells was indicated by TUNEL assay (green). Nuclei were stained blue. Bar charts in (b) are quantitative analysis of percentage of LC3b-positive cells and the percentage of TUNEL-positive cells, respectively. Five randomly selected microscopic fields were analysed using Image J. Data are analyzed by two-way analysis of variance (ANOVA) with Tukey's correction. Data are shown as mean±s.d. (n=5) and represent three independent experiments; NS, not significant; *P<0.05, **P<0.01, ***P<0.005. Scale bar, 200 μm.