Evaluation of dendrimer type bio-reducible polymer as a siRNA delivery carrier for cancer therapy

Abstract

Small interfering ribonucleic acid (siRNA), 20-25 base pairs in length, can interfere with the expression of specific genes. Recently, many groups reported the therapeutic intervention of siRNA in various cancer cells. In this study, dendrimer type bio-reducible polymer (PAM-ABP) which was synthesized using arginine grafted bio-reducible poly(cystaminebisacrylamide-diaminohexane) (ABP) and polyamidoamine (PAMAM) was used to deliver anti-VEGF siRNA into cancer cell lines including human hepatocarcinoma (Huh-7), human lung adenocarcinoma (A549), and human fibrosarcoma (HT1080) cells and access their potential as a siRNA delivery carrier for cancer therapy. PAM-ABP and siRNA formed polyplexes with average diameter of 116 nm and charge of around +24.6 mV. The siRNA in the PAM-ABP/siRNA polyplex was released by 5mM DTT and heparin. VEGF gene silencing efficiency of PAM-ABP/siRNA polyplexes was shown to be more effective than PEI/siRNA polyplexes in three cell lines with the following order HT1080>A549>Huh-7.

Keywords: Bio-reducible polymer; Dendrimer; VEGF; siRNA.

Fig. 1

Agarose gel electrophoresis retardation assay of PAM-ABP/siRNA and PEI/siRNA polyplexes at weight ratios ranging from 0.2 to 5 based on siRNA (300 μg). (A) siRNA condensation ability, (B) siRNA releasing behavior from polyplexes by 5 mM DTT, and (C) siRNA releasing behavior from PAM-ABP/siRNA polyplex (weight ratio 1:5, siRNA (300 μg)) by increasing unit of heparin.

Fig. 2

(A) Particle size and zeta potential of PAM-ABP/siRNA polyplexes at weight ratios ranging from 0.2 to 10 based on siRNA (4 μg). (B) Particle size and zeta potential of PAM-ABP/siRNA polyplex (weight ratio 1:5, siRNA(4 μg)) and PEI/siRNA polyplex (weight ratio 1:1, siRNA (4 μg)) with 5 mM DTT or without DTT. The results are presented as mean ± SD (n=3).

Fig. 3

Cytotoxicity of PAM-ABP at various amounts ranging from 5 to 10 μg in (A) Huh-7, (B) A549, and (C) HT1080 cells (3 × 10⁴ cell/well) without anti-VEGF siRNA. PEI was used the compared group at various amounts ranging from 1 to 2 μg. The results are presented as mean ± SD (n=3).

Fig. 4

Cellular uptake by FACs in (A) Huh-7, (B) A549, and (C) HT1080 cells (6 × 10⁴ cell/well). PAM-ABP/YOYO-1 labeled siRNA poloyplex was formed at weight ratio 1:5 and PEI/YOYO-l labeled siRNA polyplex was formed at weight ratio 1:1 based on YOYO-1 labeled siRNA (2 μg). (D) Bar graph representing the mean percentages of cellular uptake by M region gating. The results are presented as mean ± SD (n=3).

Fig. 5

Expression of VEGF determined using ELISA kit in (A) Huh-7, (B) A549, and (C) HT1080 cells (6 × 10⁴ cell/well). PAM-ABP/siRNA poloyplex was formed at weight ratio 1:5 and PEI/siRNA polyplex was formed at weight ratio 1:1 based on siRNA (2 μg). (D) Quantification of VEGF expression is shown by relative VEGF expression %. Relative VEGF expression (%) = Amount of VEGF (Treated)/Amonut of VEGF (Control) × 100. The results are presented as mean ± SD (n=3).