Bioreducible polymers for therapeutic gene delivery

Purpose: Cationic polymeric gene carriers are promising non-viral vectors, but their slow degradability and intracellular/tissue accumulation cause cytotoxicity. Bioreducible polymers containing disulfide linkages are attractive because they are stable extracellularly but degrade selectively in the reducing intracellular environment via thiol–disulfide exchange, triggered by high glutathione (GSH) levels. This review covers recent advances in bioreducible polymers for therapeutic delivery of pDNA, siRNA, cells, and adenovirus in cardiovascular disease, diabetes, and cancer.

Hypothesis: This is a review article and does not test a single formal hypothesis. Its central premise is that bioreducible polymers with disulfide bonds exploit the redox difference between oxidizing extracellular space and reducing intracellular cytosol (GSH 50–1000× higher inside cells), enabling extracellular stability, intracellular degradation, reduced cytotoxicity, and enhanced gene transfection.

Aims: Describe the structure and characteristics of bioreducible polymers, including SS-PAEI, bioreducible PEIs, poly(CBA-DAH), ABP/GBP, PAM-ABP dendrimer, and bioreducible PLL copolymers. - Review disease applications of bioreducible polymers in cardiovascular disease, type 1 and type 2 diabetes, and cancer. - Highlight hybrid and smart oncolytic gene carriers combining bioreducible polymers with adenovirus. - Discuss DNA and siRNA delivery, targeting strategies, PEGylation, and stimuli-sensitive modifications. - Emphasize the need for thorough biocompatibility evaluation before clinical application.

Delivery system: Polymer class: Bioreducible cationic polymers containing disulfide linkages that degrade via thiol–disulfide exchange in the reducing cytosol. - Specific polymers: Poly(amido ethylenimine) (SS-PAEI); bioreducible PEIs cross-linked with disulfide-containing linkers; poly(CBA-DAH); arginine-grafted bioreducible poly(disulfide amine) (ABP); guanidinylated bioreducible polymer (GBP); PAM-ABP dendrimer; bioreducible PEG-SS-PLL and PEG-SS-P[Asp(DET)] polyion complex micelles; PEG-SS-PLL cross-linked polyplex micelles. - Payloads: Plasmid DNA (pDNA), siRNA, adenovirus (Ad), VEGF plasmid, human EPO plasmid, GLP-1 plasmid, exendin-4 plasmid, Fas siRNA, SHP-1 siRNA, IL-8 shRNA, etc. - Targeting/functional ligands: RGD peptide, PGE2, primary cardiomyocyte-specific peptide (PCM), ischemic myocardium-targeted peptide (IMTP), Eph peptide, Tat, D-9-arginine (9R), cell-penetrating peptides. - Formulations: Polyplexes, polyion complex (PIC) micelles, hybrid adenovirus/polymer complexes, paclitaxel-conjugated micelles (APP).

Approach: Review of in vitro and in vivo literature. In vitro models include C2C12, HeLa, 293T, H9C2 cardiomyoblasts, PC-3 prostate cancer cells, A2780 ovarian cancer cells, HUVEC, primary skeletal myoblasts, etc. In vivo models include mouse tumor models, NOD mice for type 1 diabetes, ZDF rats/DIO mice for type 2 diabetes, rabbit myocardial infarct model, rat myocardial ischemia/reperfusion model, and tumor-bearing mice for adenovirus delivery. No new primary experiments, group sizes, or doses are reported.

Key methods: Polyplex characterization: size, zeta potential, gel electrophoresis, DLS. - Transfection/gene expression: luciferase, VEGF, EPO, GLP-1, exendin-4. - Gene silencing: siRNA-mediated knockdown of VEGF, Fas, SHP-1; qRT-PCR and ELISA. - Intracellular trafficking/degradation: confocal microscopy, YOYO-1-labeled pDNA, flow cytometry. - Reductive degradation: glutathione/DTT treatment, GSH depletion with buthionine sulfoximine (BSO). - In vivo efficacy: hematocrit, reticulocytosis, blood glucose, diabetes incidence, tumor regression, biodistribution, liver/tumor viral genome ratios. - Safety: cytotoxicity, IL-6/IFN-α immune response, histology.

Key results: SS-PAED/RTP-VEGF produced 67-fold and 76-fold increases in VEGF expression under hypoxia vs normoxia in H9C2 cells; in a rabbit infarct model, VEGF expression was about 4-fold higher than RTP-Luc control and 2-fold higher than WSLP positive control. - GBP polyplexes showed transfection efficiency almost 40× higher than bPEI25k and about 8× higher than poly(CBA-DAH) in C2C12 cells, with strong nuclear localization of pDNA. - PEG-SS-PLL cross-linked polyplex micelles showed approximately 50× higher transfection efficiency toward 293T cells than a Traut’s reagent-type control; after intravenous injection, uniform gene expression was observed in liver parenchymal cells. - PEG-SS-P[Asp(DET)] micelles showed 1–3 orders of magnitude higher gene transfection efficiency and more rapid onset than non-disulfide PEG-P[Asp(DET)] micelles. - PAM-ABP completely retarded pDNA electrophoretic mobility at a weight ratio of 2, compared with ABP requiring >5; at ratio 5, polyplexes had zeta potential 11.0 mV and size 126 nm, with superior cellular uptake and greater transfection than ABP. - VEGF siRNA/poly(CBA-DAH) silenced VEGF by 70.4%, compared with 49.7% for VEGF siRNA/bPEI, without detectable cytotoxicity. - ABP-coated adenovirus enhanced transduction in CAR-positive and CAR-negative cells, reduced IL-6 release, and was stable in serum. - Ad DNA/ABP or Ad DNA/ABPsk reduced liver accumulation by 99.1% and 98.4%, increased tumor accumulation 2.1-fold and 6.6-fold, and gave tumor-to-liver ratios 229-fold and 419-fold greater than naked Ad. - PNLG-coated oncolytic Ad increased tumor-to-liver ratio by 1229-fold compared with naked Ad. - sRAE-1y/Eph-PEG-p(CBA-DAH) delayed diabetes onset by up to 10 weeks; hyperglycemia occurred at 16 weeks vs 6–7 weeks for controls in NOD mice. - TSTA(SP-GLP-1)/ABP enhanced GLP-1 secretion; TSTA(SP-exendin-4)/ABP decreased blood glucose from 3 to 12 days after a single intravenous injection. - APP micelles had molecular weight 9.01 × 10³, CMC 0.062 mg/mL, size 2.61 ± 0.98 nm, and surface charge 13.5 ± 4.1 mV; they increased gene delivery, cellular uptake, and anticancer potency vs paclitaxel alone in vitro. - Combination siRNA/ABP targeting Bcl-2, VEGF, and Myc produced 35–50% tumor regression by area and weight vs untreated controls.

Interpretation: Bioreducible poly(disulfide amine)s are efficient gene vectors because multiple functional groups provide DNA condensation, protonation, charge density, buffering capacity, and intracellular biodegradability. They achieve enhanced gene transfection with minimal cytotoxicity. Smart chemical modifications — targeting peptides, PEGylation, and stimuli-sensitive systems — should accelerate clinical translation. Thorough and systematic biocompatibility evaluation is necessary before human application.

Limitations: This is a review, not a primary study; no new experimental data or meta-analysis. - Most systems remain preclinical; human clinical application has not yet been achieved. - Pharmacokinetics, long-term safety, and biocompatibility require thorough systematic evaluation. - Exact mechanisms and molecular effects of reductive disulfide cleavage are not fully understood. - Studies use heterogeneous cell lines, animal models, and endpoints, limiting direct comparison. - No large-animal validation or detailed clinical trial data are presented.