Biodegradable Polymers for Gene Delivery

Purpose: Gene therapy requires efficient delivery of genetic material, but viral vectors carry risks of immunogenicity, toxicity, and cancer formation. Cationic polymers such as polyethyleneimine (PEI) can condense DNA and facilitate delivery, but their lack of biodegradation causes significant toxicity due to polymer accumulation in tissues. This review addresses the need for biodegradable polymeric gene delivery vehicles developed over the past decade by modifying existing polymers and/or using natural biodegradable polymers.

Hypothesis: This is a review article and does not test a single formal hypothesis. Its central premise is that biodegradable polymers — developed by introducing hydrolytically or enzymatically cleavable linkages into synthetic polymers (e.g., PEI, PBAE, PLL) or by using natural carbohydrate polymers (chitosan, pullulan, dextran, hyaluronic acid) — can maintain or improve transfection efficiency while reducing cytotoxicity and enabling safe elimination from the body.

Aims: Summarize mechanistic aspects of gene delivery, including DNA condensation to nanoparticles and cellular uptake pathways. - Review the development of biodegradable polymers for gene delivery, focusing on advances during the past decade. - Describe modifications to synthetic polymers (PEI, PBAE, PLL) that confer biodegradability and reduce toxicity. - Summarize natural carbohydrate polymers (chitosan, pullulan, dextran, hyaluronic acid) used as biodegradable gene delivery vehicles. - Highlight polymer modifications that facilitate DNA condensation, cellular uptake, endosomal escape, nuclear entry, and gene expression.

Delivery system: Vector type: Cationic polymer/DNA polyplexes; nanoparticles of 50–200 nm diameter (toroids, rods, spheroids). - Synthetic polymers: Polyethyleneimine (PEI; linear and branched), poly(β-amino esters) (PBAEs), poly-L-lysine (PLL), poly(amine-co-ester) (PACE) terpolymers, branched ester-amine quadpolymers (BEQAs, rBEQAs). - Natural polymers: Chitosan, pullulan, dextran, hyaluronic acid (HA); also heparin, chondroitin sulfate, and alginate under investigation. - Payloads: Plasmid DNA (pDNA), siRNA, miRNA, oligonucleotides, minicircle DNA; also CRISPR/Cas9 components (Cas9 DNA + siRNA codelivery). - Targeting ligands: Folic acid (folate receptor), RGD peptide (integrins), hyaluronic acid (CD44 receptor), transferrin, cell-penetrating peptides (CPPs), substance P. - Stealth/functional modifications: PEGylation for prolonged circulation; disulfide linkages for redox-responsive degradation; boric acid ester for ROS-responsive degradation; histidine, imidazole, guanidinium, and quaternary ammonium groups for endosomal escape and DNA binding.

Approach: Review of in vitro and in vivo literature from the past decade. Model systems include numerous cancer cell lines (MCF-7, SK-BR3, T-47D, HeLa, A549, HepG2, SKOV-3, U87-MG, 9L, CHO, HEK293, etc.) and animal models (mice, rats) for cancer, lung disease, and brain tumors. Clinical trial data are referenced but not the focus. No new primary experiments are reported.

Key methods: DNA condensation: Electron microscopy, atomic force microscopy (AFM), laser light scattering, zeta potential. - Transfection efficiency: Luciferase reporter gene expression, GFP expression. - Gene silencing: siRNA-mediated knockdown. - Cytotoxicity: MTT assay, cell viability, hemocompatibility. - Cellular uptake: Fluorescent-labeled PEI/DNA tracking, confocal microscopy. - In vivo safety: Lung histopathology, bronchoalveolar lavage fluid (BALF) cell counts and neutrophil percentage. - In vivo efficacy: Tumor growth inhibition, survival time, transgene expression duration.

Key results: PEI: ~20% of nitrogen protonated at physiological pH; 22 kDa linear PEI showed more facile nuclear delivery than branched PEI. Non-degradable PEI causes severe cytotoxicity. - Biodegradable PEI derivatives: Reducible disulfide-linked linear PEI had transfection efficiency comparable to PEI with high cell viability. Zn-coordinated PEI (Zn-DDAC) induced up to 2 orders of magnitude higher luciferase activity than commercial reagents. Heparin-PEI (HPEI) nanogels had excellent transfection, low cytotoxicity, and better blood compatibility than 25 kDa PEI; degradation products excreted through urine. - PBAEs: Non-cytotoxic, biodegradable with half-life of 1–7 h; form 100–200 nm nanoparticles. PBAE-mucus-penetrating DNA nanoparticles provided uniform transgene expression throughout mouse lungs, superior to gold-standard systems, with expression lasting >4 months after a single administration. Safety profile excellent by intratracheal administration. PEG-coated PBAE nanoparticles penetrated brain parenchyma and orthotopic brain tumors in rats, improving survival in two aggressive brain tumor models. - PLL: Oligolysines and PLL collapse DNA into toroids, spheroids, cubes, and rods. PLL-modified PEI enhanced transfection in HeLa cells and significantly reduced toxicity. - Chitosan: 50 kDa chitosan with 94% deacetylation and N/P = 5 showed excellent transfection in HeLa cells at pH 6.5 with no cytotoxicity; nanoparticles ~50 nm. Folic acid-conjugated quaternized chitosan formed ~140 nm nanoparticles, less toxic and hemocompatible than PEI, with excellent transfection in KB epidermoid cells. Histidine-modified chitosan had transfection efficiency comparable to PEI. - Pullulan: Cationic pullulan derivatives (spermine, protamine) protected DNA and had excellent hemocompatibility. Pullulan-PEI conjugates decreased mortality and lung damage vs. PEI alone; high MW pullulan was more efficient for serum stability and gene expression. - Dextran: Dextran-PEI conjugates were less toxic than unmodified PEI. Histidine-containing peptide-grafted dextran showed 6–8-fold higher luciferase expression than 25 kDa branched PEI. - Hyaluronic acid: HA-PEI nanoparticles produced excellent gene transfection with negligible cytotoxicity in HeLa and A549 cells. HA targets CD44-overexpressing cancer cells.

Interpretation: Polymer-based non-viral gene carriers have safety advantages: avoidance of immunogenicity and toxicity, possibility of repeated administration, and ease of GMP manufacturing. Structural modifications (guanidinium, carboxyl, disulfide, alkyl chains, branching, acetyl, benzoyl, quaternary nicotinamide) facilitate DNA condensation, cellular uptake, endosomal escape, nuclear entry, and gene expression. Although significant progress has been made, an ideal biodegradable delivery vehicle has not yet been discovered; modification of existing natural and synthetic polymers with hydrolytically and enzymatically degradable linkers is making significant progress.

Limitations: This is a review, not a primary study; no new experimental data or meta-analysis. - Mechanistic aspects of cellular DNA transport are not clearly understood. - An ideal biodegradable delivery vehicle has not been accomplished. - Many systems still require PEGylation or other modifications for serum stability. - Clinical translation remains limited; most discussed systems are preclinical. - Long-term fate of degradation products and chronic toxicity are not fully characterized. - No large-animal validation or detailed clinical trial data are presented.