Self-immolative nanoparticles for simultaneous delivery of microRNA and targeting of polyamine metabolism in combination cancer therapy

Abstract

Combination of anticancer drugs with therapeutic microRNA (miRNA) has emerged as a promising anticancer strategy. However, the promise is hampered by a lack of desirable delivery systems. We report on the development of self-immolative nanoparticles capable of simultaneously delivering miR-34a mimic and targeting dysregulated polyamine metabolism in cancer. The nanoparticles were prepared from a biodegradable polycationic prodrug, named DSS-BEN, which was synthesized from a polyamine analog N¹,N¹¹-bisethylnorspermine (BENSpm). The nanoparticles were selectively disassembled in the cytoplasm where they released miRNA. Glutathione (GSH)-induced degradation of self-immolative linkers released BENSpm from the DSS-BEN polymers. MiR-34a mimic was effectively delivered to cancer cells as evidenced by upregulation of intracellular miR-34a and downregulation of Bcl-2 as one of the downstream targets of miR-34a. Intracellular BENSpm generated from the degraded nanoparticles induced the expression of rate-limiting enzymes in polyamine catabolism (SMOX, SSAT) and depleted cellular natural polyamines. Simultaneous regulation of polyamine metabolism and miR-34a expression by DSS-BEN/miR-34a not only enhanced cancer cell killing in cultured human colon cancer cells, but also improved antitumor activity in vivo. The reported findings validate the self-immolative nanoparticles as delivery vectors of therapeutic miRNA capable of simultaneously targeting dysregulated polyamine metabolism in cancer, thereby providing an elegant and efficient approach to combination nanomedicines.

Keywords: Bisethylnorspermine; Cancer; Combination delivery; Nanoparticles; Polyamine metabolism; Polyplexes; Self-immolative polymer; miRNA.

Figure 1

Physicochemical characterization of DSS-BEN/miRNA nanoparticles. (A) miRNA condensation by DSS-BEN in 10 mM HEPES buffer (pH 7.4) using agarose gel electrophoresis. (B) Hydrodynamic size of DSS-BEN/miRNA nanoparticles at various w/w ratios (n=3). (C) Size distribution and TEM image (inset) of DSS-BEN/miRNA (w/w=10). Scale bar = 50 nm. (D) Zeta-potential of DSS-BEN/miRNA nanoparticles at various w/w ratios (n=3). (E) Zeta-potential distribution of DSS-BEN/miRNA (w/w=10). (F) Size distribution of nanoparticles in the presence of 20 mM GSH. (G) Morphological changes in nanoparticles in the presence of 20 mM GSH determined by TEM. (Scale bar = 200 nm). (H) Heparin- and GSH-induced miRNA release from DSS-BEN/miRNA. Nanoparticles were prepared at w/w = 10 and incubated with increasing concentrations of heparin either with or without 20 mM GSH for 30 min. **P < 0.01, ***P < 0.001.

Figure 2

Cellular uptake and intracellular trafficking of DSS-BEN/FAM-miRNA nanoparticles in HCT116 cells. (A) Overlayed histogram of flow cytometry analysis of cells treated with DSS-BEN/FAM-miRNA nanoparticles at various w/w ratios (200 nM FAM-miRNA) after a 4-h incubation. Quantification of cellular uptake is shown by (B) mean fluorescence intensity (MFI) and (C) % of cells that had taken up the particles. Data are shown as the mean ± SD (n = 3). **P < 0.01, ***P < 0.001. (D) Intracellular trafficking of DSS-BEN/FAM-miRNA in HCT116 cells by CLSM after a 4-h incubation. (Scale bar 20 μm)

Figure 3

Effect of DSS-BEN/miRNA treatment on cellular polyamine metabolism. Cells were incubated with 12.3 μg/mL BENSpm or DSS-BEN/miRNA (w/w=10, 28 μg/mL DSS-BEN) for 24 h or 48 h. (A) Intracellular concentration of BENSpm was determined by HPLC (n = 3). (B) Relative changes in the expression of SMOX and SSAT mRNA in HCT116 cells. mRNA levels were measured by qRT-PCR. Results are expressed as the fold induction of specific mRNA in treated cells relative to that in the PBS- treated group (n = 3). (C) Intracellular polyamine concentration determined by HPLC (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 4

The ability of DSS-BEN nanoparticles to deliver functional miRNA. (A) miR-34a was measured by qRT-PCR in HCT116 cells following incubation with DSS-BEN/miR-34a or control DSS-BEN/miR-NC. (B) Effect of miR-34a delivery on the expression of Bcl-2. (C) Quantification of Western blot bands performed using ImageJ software. The results are expressed as relative Bcl-2 levels relative to untreated cells. (means ± SD, n = 3). *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 5

Anticancer activity of DSS-BEN/miRNA nanoparticles in HCT116 cells. (A) Cells were treated for 48 h with DSS-BEN/miR-NC or DSS-BEN/miR-34a prepared at different w/w ratios. Cell killing was measured by Cell Titer Blue assay. Results are normalized to the viability of untreated cells and shown as mean relative cell killing (%) ± SD (n = 3). *P < 0.05, ***P < 0.001. (B) Morphology of cells observed under light microscope (× 40) after treatment with DSS-BEN/miR-NC or DSS-BEN/miR-34a nanoparticles (w/w = 10, 200 nM).

Figure 6

Anticancer activity of DSS-BEN nanoparticles in vivo. (A) Representative images of HCT116 xenograft tumors at day 8 after intratumoral injection of various formulations. Mice were administered DSS-BEN/miRNA (w/w = 10) at doses of 5 mg/kg DSS-BEN and 0.5 mg/kg miRNA once every two days. (B) Tumor growth curve. Injections are represented by black arrows. (C) Body weight during the treatment period. (D) Tumor tissues were extracted from mice and photographed. (E) Weights of tumors collected from treated mice. Data are shown as the means ± SD (n = 5). *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 7

(A) Relative changes in the expression of polyamine catabolism enzymes SMOX and SSAT in HCT116 tumor tissues. (B) miR-34a level was detected by qRT-PCR in HCT116 tumor tissues. (C) The expression of Bcl-2 in tumor tissues determined by Western blot. (D) Quantification of Western blot bands performed using ImageJ software. (E) Immunohistochemistry analyses of tumor tissues after various treatments. The percentage (%) of Ki-67 positive cells in tumor tissues. (F) The images were taken under a light microscope (× 40). Data are shown as the means ± SD (n = 3). *P < 0.05, ***P < 0.001.

Scheme 1

Mechanism of action of DSS-BEN/miR-34a nanoparticles. (A) DSS-BEN condenses miRNA into nanoparticles by electrostatic interactions. (only linear form of DSS-BEN is shown but branched forms are also present) (B) Upon endocytosis and endosomal escape, the particles are subjected to cytoplasmic reduction by GSH, followed by disassembly and release of both BENSpm and miR-34a mimic. BENSpm induces expression of enzymes involved in polyamine catabolism, which reduces intracellular polyamine levels. MiR-34a mimic increases cellular miR-34a levels, which leads to Bcl-2 downregulation.