Cationic nano-copolymers mediated IKKbeta targeting siRNA inhibit the proliferation of human Tenon's capsule fibroblasts in vitro

Abstract

Purpose: To synthesize a ternary cationic copolymer called CS-g-(PEI-b-mPEG) and characterize its features as a non-viral siRNA carrier; in turn, to investigate the influence of small interfering RNA (siRNA) targeting IkappaB kinase subunit beta (IKKbeta) on the proliferation of human Tenon's capsule fibroblasts (HTFs) in vitro.

Methods: First, a novel cationic copolymer composed of low molecular weight, linear poly(ethyleneimine) [PEI] blocked with polyethylene glycol (PEG) and grafted onto a chitosan (CS) molecule was synthesized. CS-g-(PEI-b-mPEG) was then compacted with 21nt siRNA at various copolymer/siRNA charge (N/P) ratios, and the resulting complexes were characterized by dynamic light scattering, gel electrophoresis, and serum incubation. Cell Titer 96 AQ(ueous) One Solution cell proliferation assay was used to investigate the cytotoxicity of this cationic copolymer. Second, siRNAs targeting IKKbeta (IKKBeta-siRNAs) were delivered into the HTFs using CS-g-(PEI-b-mPEG) as the vehicle. Real-time reverse transcription polymerase chain reaction (RT-PCR) subsequently assessed the mRNA level of IKKbeta, and western blot assay was used to determine protein expression. After IKKB-siRNA transfection, Cell Titer 96 AQ(ueous) One Solution cell proliferation assay was used to evaluate the proliferation of HTFs.

Results: The diameter of the CS-g-(PEI-b-mPEG)/siRNA complexes tended to decrease whereas their zeta potential tended to increase as the N/P ratio increased. The CS-g-(PEI-b-mPEG) copolymer showed good siRNA binding ability and high siRNA protection capacity. Furthermore, the copolymer presented remarkable transfection efficiency and showed much less cytotoxicity than 25 kDa PEI. IKKB-siRNAs were successfully delivered into HTFs using CS-g-(PEI-b-mPEG) as a vector. As a result, the expression of IKKbeta was downregulated at both the mRNA and protein levels, and the activation of nuclear factor-kappaB (NF-kappaB) in the HTFs was subsequently inhibited. Most impressively, the proliferation of HTFs was also effectively suppressed through the blocking of the NF-kappaB pathway.

Conclusions: All the results demonstrate that CS-g-(PEI-b-mPEG) is a promising candidate for siRNA delivery, featuring excellent biocompatibility, biodegradability, and transfection efficiency. The RNA interference (RNAi) strategy using cationic copolymers as siRNA carriers will be a safe and efficient anti-scarring method following glaucoma filtration surgery.

Figure 1

The synthesis schedule of CS-g-(PEI-b-mPEG) copolymer.

Figure 2

The ¹H NMR spectra of CS and its derivatives. Spectra were obtained with 32 scans, and delay of 2 s between pulses. A: CS in D₂O and CF3COOD at 293 K, B: CS-CHO in D₂O at 293 K; C: CS-g-(PEI-b-mPEG) in D₂O at 293 K.

Figure 3

The effects of N/P ratio on the hydrodynamic diameter and zeta-potential of CS-g-(PEI-b-mPEG)/siRNA complexes and 25 kDa PEI/siRNA complexes. The diameter of complexes decreased while the N/P ratios increased from 1 to 10 and then remained constant up to N/P of 20; the zeta-potential of complexes increased in parallel with the rinsing N/P ratio. A: hydrodynamic diameter (mean±SD, n=3); B: zeta-potential (mean±SD, n=3).

Figure 4

The electrophoretic mobility of CS-g-(PEI-b-mPEG)/siRNA complexes. The complete retardation is indicated by the white arrow head.

Figure 5

The protection ability of CS-g-(PEI-b-mPEG) for siRNA against serum degradation. The images of intact siRNAs at determined time intervals are shown (A), and the percentage of intact siRNAs in reference to the non-FBS treated control is also calculated (B, mean±SD, n=3).

Figure 6

The transfection efficiency of CS-g-(PEI-b-mPEG) and 25 kDa PEI at different N/P ratios in HeLa cells and HTFs. Data are presented as the percentage of HeLa cells or HTFs containing FITC-conjuncted siRNA, respectively, and data of 25 kDa PEI/siRNA complexes at N/P ratio of 20 can’t be measured (not available, NA). A: transfection efficiency in HeLa cells (mean±SD, n=3); B: transfection efficiency in HTFs (mean±SD, n=3).

Figure 7

Cytotoxicity of CS-g-(PEI-b-mPEG) and 25 kDa PEI at various concentrations of pure polymers and complexes with siRNA at determined N/P ratios. Pure CS-g-(PEI-b-mPEG) copolymer did not exhibit significant cytotoxicity at any determined concentration, and after compacting with siRNAs, both CS-g-(PEI-b-mPEG)/siRNA and 25 kDa PEI/siRNA complexes showed much lower cytotoxicity. A: Cytotoxicity of various concentrations of pure CS-g-(PEI-b-mPEG) or 25 kDa PEI polymers; B: cytotoxicity of CS-g-(PEI-b-mPEG)/siRNA or 25 kDa PEI/siRNA complexes at determined N/P ratios. Data are presented as the percentage of viable cells compared with the untreated (control) cells (The asterisk indicates a p<0.05, mean ± SD, n=6).

Figure 8

IKKΒ-siRNA inhibits the expression of IKKβ on both the mRNA and protein level. A: mRNA transcription of IKKβ in HTFs assessed by real-time RT-PCR 24 h after 5-100 nM IKKΒ-siRNA was transfected. The normalized IKKβ mRNA level of non-transfected HTFs is taken as 1.0 (the asterisk indicates a p<0.05, mean±SD, n=3). B: Protein levels of IKKβ demonstrated by western blot. C-N: Confocal laser scanning microscopy images shows the intracellular distribution of NF-κB in HTFs. Green fluorescence indicates the intracellular distribution of phosphated NF-κB, and blue fluorescence represents the DAPI counterstained cell nuclei.

Figure 9

The inhibition effect of blocking NF-κB pathway on the proliferation of HTFs through RNAi. Data are presented as the percentage of viable cells compared with the untreated (control) cells (mean ± SD, n=6). An asterisk indicates that p<0.05.