Poly (beta‐amino ester) as an in vivo nanocarrier for therapeutic nucleic acids

Purpose: Therapeutic nucleic acids require safe and effective in vivo delivery vectors. Most gene and cell therapies rely on ex vivo gene delivery, which is laborious, time-consuming, and costly. PBAE is a promising biodegradable synthetic cationic polymer for in vivo gene delivery due to its transfection efficiency, biodegradability, and structural tunability. This review addresses the need for a systematic understanding of PBAE components and how environmental and physicochemical factors affect its in vivo performance.

Hypothesis: This is a review article and does not test a single formal hypothesis. Its central premise is that PBAE nanoparticles possess unique properties — tunable charge density, structural diversity, high encapsulation capacity, stimuli-responsive release, and effective endosomal escape — that make them promising for in vivo delivery of therapeutic nucleic acids, but that every component of PBAE synthesis and every physiological barrier must be carefully considered for successful clinical translation.

Aims: Provide an overview of PBAE synthesis, including Michael addition and microfluidics approaches. - Discuss how each PBAE component (diacrylate backbone, amine monomer, end-capping group) affects gene delivery. - Review the impact of molecular weight, branching, end-group structure, pH/pKa, and cationic charge on PBAE performance. - Describe the barriers to in vivo delivery, including blood flow, MPS/RES clearance, protein corona, renal/hepatic excretion, and margination. - Summarize PBAE degradation, immunogenicity, endosomal escape, and nucleic acid release mechanisms. - Review in vivo applications of PBAE for systemic nucleic acid delivery and site-specific targeting. - Discuss storage/stability and future clinical perspectives.

Delivery system: Polymer class: Poly(beta-amino ester) (PBAE), a degradable cationic polymer synthesized by Michael addition of diacrylate monomers (B) and amine monomers (S), with optional end-capping groups (E). - Synthesis: Michael addition reaction at 60–90 °C for 20–70 h in DMSO or other solvents; base polymer precipitated and end-capped; microfluidics (hydrodynamic flow focusing) as an emerging controlled synthesis approach. - Nanoparticle type: PBAE/nucleic acid polyplexes formed in sodium acetate buffer (pH 5). - Payloads: Plasmid DNA (pDNA), mRNA, siRNA, miRNA, minicircle DNA, and CAR-encoding transgenes. - Targeting/functional ligands: Single-chain variable fragments (scFvs, e.g., CD3e scFv for T cells), VHPK peptide (targets VCAM-1), lysine/histidine oligopeptides, cell-penetrating peptides (mTAT, bPrPp, MPG). - Modifications: PEGylation, disulfide bonds (redox-responsive), light-sensitive 2-nitrobenzene moieties, bisphosphonate, β-cyclodextrin, carbon atoms, mannosylated groups, PLGA coating. - Key properties: Tunable charge density, branching, molecular weight, hydrophobicity, pH/pKa, zeta potential.

Approach: Review of in vitro and in vivo literature. In vitro models include HeLa, COS-7, 293T, human glioblastoma cells, human mesenchymal stem cells, adipose-derived stem cells, astrocytes, and endothelial cells. In vivo models include mice (lung mRNA delivery, CAR-T engineering, melanoma lung metastasis, inflamed endothelial cells) and rats. No new primary experiments, group sizes, or doses are reported.

Key methods: Gel permeation chromatography (GPC) for molecular weight characterization. - Transmission electron microscopy (TEM), dynamic light scattering (DLS), laser Doppler anemometry, and gel retardation for polyplex characterization. - Transfection efficiency and gene silencing assays. - Cellular uptake, endosomal colocalization, and intracellular trafficking. - In vivo biodistribution, gene expression, and tumor targeting. - Hemolysis, cytotoxicity, and immunogenicity assays. - Freeze-thaw stability testing.

Key results: PBAE half-life: 1–7 h in aqueous conditions. - Freeze-thaw stability: PBAE NPs stored at −20 °C are stable through 8 freeze/thaw cycles without significant efficacy changes. - Optimal aqueous incubation: <10 min before mixing with plasmid DNA and 5–20 min after mixing. - Highly branched PBAEs: Delivered minicircle DNA to multipotent adipose-derived stem cells with 77% transfection efficiency and astrocytes with 52%. - End-group library: Of 320 end-modified PBAEs, specific formulations (B3-S5-E1, B3-S5-E5, B3m-S5-E7, B4-S5-E3, B4-S5-E4, B4-S5-E7) demonstrated satisfactory transfection and low toxicity. - In vivo CAR-T engineering: CD3e scFv-modified PBAE NPs delivered CAR-encoding DNA and mRNA to circulating T cells in mice, providing proof of concept for in situ T-cell programming. - mRNA vs. DNA delivery: Subtle changes in the diacrylate backbone (DD vs. D) led to differential potency: D90-C12-103 PBAE favored DNA delivery with late endosomal localization, while DD90-C12-103 PBAE was more effective for mRNA delivery in vivo. - Targeted endothelial delivery: VHPK peptide-conjugated PBAE NPs successfully delivered anti-miR-712 to inflamed endothelial cells in mice. - Tissue specificity: Lysine/histidine oligopeptide-modified PBAE delivered siRNA to specific endothelial cells and tissues without accumulating in liver, heart, or thymus.

Interpretation: PBAE nanocarriers are an emerging class of gene delivery vehicles with the potential to manipulate and genetically engineer specific cells in vivo with robust efficacy. Systemic in vivo gene delivery could reduce the cost and complexity of ex vivo gene and cell therapies. However, each PBAE component — backbone, side chain, end-group, branching, molecular weight, and formulation — plays a critical role in delivery success. Clinical translation requires defined manufacturing protocols, quality control, in vivo imaging, immunogenicity prevention, controlled release, and comprehensive toxicology. The relationship between tumor pathophysiology and PBAE nanocarrier behavior in humans is poorly understood and needs further investigation.

Limitations: This is a review, not a primary study; no new experimental data or meta-analysis. - No defined approaches for large-scale PBAE nanoparticle production; microfluidics research on PBAE formation is negligible. - Most structure-function studies were in vitro only and not validated in vivo. - The link between PBAE degradation and intrinsic immunogenicity is not well-studied. - Anti-PEG antibodies can develop and induce clearance of PEGylated PBAE NPs. - The exact dosage for clinical purposes cannot be well-defined from animal studies. - No large-animal validation or clinical trial data are presented. - Regulatory, manufacturing, and long-term safety issues remain unresolved.