Poly(beta-amino ester)s as gene delivery vehicles challenges and opportunities

Purpose: Non-viral gene delivery needs safe and efficient carriers. PBAEs are biodegradable cationic polymers with promising transfection activity, but they face challenges such as instability in physiological fluids, positive-charge toxicity, and limited systemic delivery. This review summarizes PBAE properties, advantages, limitations, and strategies to customize them for diverse gene delivery applications.

Hypothesis: This is a review article and does not test a single formal hypothesis. Its central premise is that PBAEs possess ideal gene delivery properties — reversible positive charge, nucleic acid binding, buffering capacity, and rapid hydrolytic degradability — but also have limitations that can be overcome by chemical modification, branching, end-capping, PEGylation, and hybrid formulation with other materials.

Aims: Summarize key properties and development of PBAEs for gene delivery. - Discuss advantages and disadvantages of PBAEs as nucleic acid carriers. - Review strategies to overcome intracellular and in vivo barriers, including endosomal escape, cargo release, colloidal stability, and targeting. - Describe PBAE-based hybrid materials, including polymer blends, lipid-coated particles, and PBAE coatings. - Discuss clinical translation challenges and opportunities.

Delivery system: Polymer class: Poly(beta-amino ester)s (PBAEs), synthetic biodegradable cationic polymers. - Synthesis: Michael addition of diacrylate backbone monomers and amine side-chain monomers, followed by end-capping; also ring-opening polymerization. - Payloads: Plasmid DNA, mRNA, siRNA, miRNA, immunostimulatory RNA, cyclic dinucleotides, CRISPR-Cas9 plasmids, sgRNA, Cas9 RNP. - Modifications: PEGylation, disulfide bonds (bioreducible), ketal groups, light-sensitive 2-nitrobenzene, branched/hyperbranched structures, carboxylated end groups. - Targeting ligands: Mannose, RGD peptide, CD3 antibody, retinol, oligopeptides, T-cell targeting moieties, nuclear localization signals. - Hybrid systems: PLGA/PBAE blends, lipid-coated PBAE polyplexes, PBAE coatings on gold nanoparticles, E. coli, layer-by-layer films/microneedles, hydrogels.

Approach: Review of in vitro and in vivo literature. In vitro models include HeLa, COS-7, HEK293T, ARPE-19, DC2.4, RAW264.7, B16F10, patient-derived glioblastoma and hepatocellular carcinoma cells. In vivo models include mice and rats for cancer, genetic vaccines, lung mRNA delivery, brain tumors, and in situ CAR T-cell generation. No new primary experiments are reported.

Key methods: Polymer characterization: GPC, NMR. - Nanoparticle characterization: DLS, TEM, nanoparticle tracking analysis, gel retardation. - Transfection/knockdown assays: luciferase, GFP, siRNA-mediated knockdown. - Cellular uptake and endosomal escape: fluorescence imaging, Gal8 recruitment assay. - In vivo: biodistribution, gene expression, tumor growth, immune responses. - Stability: lyophilization, freeze-thaw, serum incubation.

Key results: PBAE/DNA nanoparticles functionalized with T-cell targeting and nuclear localization signal achieved >80% transfection of T cells. - Bioreducible PBAEs achieved near-complete siRNA knockdown in patient-derived glioblastoma cells and <25% knockdown in non-cancerous counterparts. - Mannosylated PBAEs elicited higher antibody titers than protein-plus-adjuvant control. - PLGA/PBAE blends with 75–85% PLGA and 15–25% PBAE were optimal for antigen-presenting cell transfection. - Lyophilized PBAE/DNA nanoparticles showed no loss in transfection efficacy after at least two years of cold storage. - Lipid-coated PBAE/mRNA achieved 100% transfection in DC2.4 cells, while uncoated polyplexes did not detectably transfect. - PBAE/CDN nanoparticles at 500 w/w generated the same immunogenicity as 100-fold higher extracellular CDN. - PBAEs degrade hydrolytically with a half-life of several hours.

Interpretation: PBAEs are promising biodegradable, tunable non-viral gene delivery vehicles. Their positive charge, pH buffering, and degradability enable nucleic acid binding, cellular uptake, endosomal escape, and cargo release. However, excessive positive charge can cause toxicity and poor tissue mobility, and unmodified PBAEs have low colloidal stability in physiological fluids. Chemical modification and hybrid formulation can overcome these challenges. The approval of Onpattro encourages non-viral nucleic acid delivery, and PBAEs have potential for clinical translation if manufacturing, storage stability, and systemic delivery design criteria are addressed.

Limitations: This is a review, not a primary study; no new experimental data or meta-analysis. - PBAEs remain mostly preclinical; clinical data are lacking. - Systemic in vivo delivery is still limited by instability in physiological fluids, serum interactions, and clearance. - Rapid degradation may limit sustained delivery. - Positive charge can cause toxicity and immunogenicity. - Large-scale manufacturing, shelf-life, and regulatory issues remain unresolved. - No large-animal validation or clinical trial results are presented.