Poly(β-amino ester)-based gene delivery systems From discovery to therapeutic applications

Purpose: Gene therapy requires safe and effective delivery vectors. PBAEs are biodegradable cationic polymers with excellent gene delivery properties, but a comprehensive and critical overview of their use as gene carriers is lacking. This review provides the first comprehensive and critical overview of PBAE-based gene delivery systems, from discovery to therapeutic applications, with emphasis on rational design and structure–performance relationships.

Hypothesis: This is a review article and does not test a single formal hypothesis. Its central premise is that PBAEs are one of the most promising non-viral gene delivery vectors because their chemical structure can be easily tuned — via monomer selection, molecular weight, architecture, and end-group modification — to achieve high transfection efficiency, biodegradability, and low toxicity, and that very small structural changes have an impressive impact on transfection efficiency.

Aims: Provide a brief historical background of PBAEs. - Review PBAE synthesis, properties, and advantages for gene delivery. - Summarize combinatorial library approaches for PBAE screening and structure–function relationships. - Review linear, star-shaped, branched, and hyperbranched PBAE architectures. - Describe PBAE-based copolymers and hybrid nanovectors. - Discuss cellular uptake and intracellular fate of PBAE-based vectors. - Review therapeutic applications, including brain and brain-cancer therapies, other cancers, bone regeneration, genetic vaccines, and immune-mediated therapies. - Discuss concluding remarks and future directions.

Delivery system: Polymer class: Poly(β-amino ester)s (PBAEs), biodegradable cationic polymers synthesized by Michael addition of diacrylates and primary or bis(secondary) amines. - Architectures: Linear homopolymers, copolymers (PEGylated, PEI-PBAE-PEI, PDMAEMA-PBAE-PDMAEMA, PBAE-pullulan), star-shaped, branched, and hyperbranched. - Payloads: Plasmid DNA, mRNA, siRNA, miRNA, shRNA, minicircle DNA, immunostimulatory RNA, cyclic dinucleotides, and CRISPR-Cas9 components. - Modifications: End-capping with amine-containing small molecules, PEGylation, disulfide (bioreducible) linkages, ketal groups, fluorination, mannosylation, folate, lactobionic acid, pullulan, RGD, and cell-penetrating peptides. - Hybrid systems: Lipopolyplexes with DOTAP or lipid-PEG, PLGA blends, gold nanoparticles, iron oxide nanoparticles, E. coli membrane hybrids, and layer-by-layer films/microneedles. - Targeting ligands: Mannose, folate, lactobionic acid, pullulan, CD3 antibody, MUC1 aptamer, and VCAM-1 targeting peptides. - Key properties: pKa between 5.5 and 7.4, good buffer capacity, hydrolyzable ester bonds, nontoxic degradation products, and easily tunable hydrophobicity.

Approach: Review of in vitro, in vivo, and preclinical literature. In vitro models include COS-7, HepG2, HUVEC, HeLa, HEK293, ARPE-19, primary human glioblastoma, and many other cell lines. In vivo models include mice, rats, and non-human primates for cancer, brain tumors, bone regeneration, genetic vaccines, and immune-mediated therapies. Clinical translation status is discussed. No new primary experiments are reported.

Key methods: Transfection efficiency and gene silencing assays. - Particle size, zeta potential, and polyplex characterization. - Cellular uptake and intracellular trafficking (flow cytometry, confocal microscopy). - Endosomal escape and pH environment measurements. - Cytotoxicity and cell viability assays. - In vivo biodistribution and gene expression. - High-throughput combinatorial library screening. - Gel electrophoresis for DNA binding. - Lyophilization and storage stability testing.

Key results: First library screening (2001): 140 PBAEs from 7 diacrylates and 20 amines; C93 and G28 gave 4–8 times higher transfection than PEI. - Large library (2003): 2,350 structurally unique PBAEs synthesized in a single day; 46 polymers transfected COS-7 cells as well as or better than PEI. - Lead polymer C32: Top performer; 4-fold better than jetPEI by intratumoral injection; diphtheria toxin A delivery suppressed tumor growth in 40% of treated animals. - Second-generation library (2005): 486 PBAEs; 20 polymers had transfection activity at least comparable to Lipofectamine 2000; most effective polyplexes had sizes <150 nm and positive surface charge. - End-modified C32-103 and C32-117: Delivered DNA approximately two orders of magnitude higher than unmodified C32, PEI 25 kDa, or Lipofectamine 2000; comparable to adenovirus at MOI 100. - C32-103 and C32-117 cellular uptake: Up to 5-fold higher than unmodified C32. - C32-103 vs C32-102: C32-103 transfection activity 130-fold (COS-7) and 300-fold (HepG2) higher than C32-102. - In vivo organ-specific expression: C32-117 transfected 5- to 65-fold more efficiently in bladder, spleen, and kidney than C32-103 and C32-116; C32-103 and C32-116 transfected 7-fold more in prostate. - Stem cell transfection: End-modified PBAEs achieved 24–56% transfected cells with 87–97% viability. - ARPE-19 cells: JJ32-based polyplexes achieved 44% transfection vs 8% for bPEI 25000, 26% for Lipofectamine 2000, and 22% for X-tremeGENE HP. - Brain tumor therapy: C28 end-modified with 1-(3-aminopropyl)-4-methylpiperazine achieved >80% transfection of T cells when combined with T-cell targeting and NLS; in vivo leukemia regression comparable to lentivirus-transduced T cells. - Hyperbranched PBAEs: 2–126-fold higher transfection activity than linear counterparts in various cell lines. - siRNA knockdown: Bioreducible PBAEs achieved near-complete gene knockdown in primary human glioblastoma cells. - HSV-tk/GCV therapy: PBAE/pDNA-HSV-tk polyplexes killed approximately 100% of cancer cells in two rat glioma cell lines and significantly increased animal survival. - Bone regeneration: Cystamine-terminated PBAEs achieved 91% knockdown 20 days post-transfection in hMSC. - Influenza vaccine: Mannosylated PBAE hybrid vectors elicited effective humoral immune responses.

Interpretation: PBAEs are a very promising class of non-viral gene delivery vectors. Their ease of synthesis, structural diversity, biodegradability, low toxicity, and tunable properties allow precise optimization for specific applications. More than 2,000 PBAEs have been assessed, with lead candidates such as C32 and C28 showing high potential in preclinical studies for cancer, brain therapies, bone regeneration, and genetic vaccines. Although PBAEs have not yet reached clinical practice, their high potential could promote the development of new gene delivery platforms for future clinical application.

Limitations: This is a review, not a primary study; no new experimental data or meta-analysis. - PBAEs have not yet reached clinical practice; less than 20 years since first in vitro studies. - Optimal PBAE structure is highly dependent on cell type and nucleic acid cargo, limiting universal design rules. - Some PBAEs show poor transfection in serum-containing media or certain hard-to-transfect cells. - The relationship between polymer structure and in vivo behavior is not fully understood. - Long-term safety, large-scale manufacturing, and regulatory issues are not comprehensively addressed. - No large-animal validation or clinical trial results are presented.