Tailoring Highly Branched Poly(ß-amino ester)s for Efficient and Organ-Selective mRNA Delivery.

Development of mRNA therapeutics necessitates targeted delivery technology, while the clinically advanced lipid nanoparticles face difficulty for extrahepatic delivery. Herein, we design highly branched poly(ß-amino ester)s (HPAEs) for efficacious organ-selective mRNA delivery through tailoring their chemical compositions and topological structures. Using an "A2+B3+C2" Michael addition platform, a combinatorial library of 219 HPAEs with varied backbone structures, terminal groups, and branching degrees are synthesized. The branched topological structures of HPAEs provide enhanced serum resistance and significantly higher mRNA expression in vivo. The terminal amine structures of HPAEs determine the organ-selectivity of mRNA delivery following systemic administration: morpholine facilitates liver targeting, ethylenediamine favors spleen delivery, while methylpentane enables mRNA delivery to the liver, spleen, and lungs simultaneously. This study represents a comprehensive exploration of the structure-activity relationship governing both the efficiency and organ-selectivity of mRNA delivery by HPAEs, suggesting promising candidates for treating various organ-related diseases.

Enhancing gene transfection of poly(ß-amino ester)s through modulation of amphiphilicity and chain sequence.

Poly(ß-amino ester)s (PAEs) have emerged as a type of highly safe and efficient non-viral DNA delivery vectors. However, the influence of amphiphilicity and chain sequence on DNA transfection efficiency and safety profile remain largely unexplored. In this study, four PAEs with distinct amphiphilicity and chain sequences were synthesized. Results show that both amphiphilicity and chain sequence significantly affect the DNA binding and condensation ability of PAEs, as well as size, zeta potential and cellular uptake of PAE/DNA polyplexes. PAEs with different amphiphilicity and chain sequence exhibit cell type-dependent transfection capabilities: in human bladder transitional cell carcinoma (UM-UC-3), hydrophilic PAE (P-Philic) and amphiphilic PAE random copolymer (R-Amphilic) exhibit relatively higher gene transfection efficiency, while in human bladder epithelial immortalized cells (SV-HUC-1), hydrophobic PAE (P-Phobic), R-Amphilic, and amphiphilic PAE block copolymer (B-Amphilic) demonstrate higher transfection capability. Regardless of cell types, amphiphilic PAE block copolymer (B-Amphilic) always exhibits much lower gene transfection efficiency. In addition, in human colon cancer cells (HCT-116), P-Philic and R-Amphilic achieved superior gene transfection efficiency at high and low polymer/DNA weight ratios, respectively. Importantly, R-Amphilic can effectively deliver the gene encoding tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) to human chondrosarcoma cells SW1353 to induce their apoptosis, highlighting its potential application in cancer gene therapy. This study not only establishes a new paradigm for enhancing the gene transfection efficiency of PAEs by modulating their amphiphilicity and chain sequence but also identifies R-Amphilic as a potential candidate for the effective delivery of TRAIL gene in cancer gene therapy.

Enhanced gene transfection and induction of apoptosis in melanoma cells by branched poly(ß-amino ester)s with uniformly distributed branching units.

Melanoma, one of the most devastating forms of skin cancer, currently lacks effective clinical treatments. Delivery of functional genes to modulate specific protein expression to induce melanoma cell apoptosis could be a promising therapeutic approach. However, transfecting melanoma cells using non-viral methods, particularly with cationic polymers, presents significant challenges. In this study, we synthesized three branched poly(ß-amino ester)s (HPAEs) with evenly distributed branching units but varying space lengths through a two-step "oligomer combination" strategy. The unique topological structure enables HPAEs to condense DNA to form nano-sized polyplexes with favorable physiochemical properties. Notably, HPAEs, especially HPAE-2 with intermediate branching unit space length, demonstrated significantly higher gene transfection efficiency than the leading commercial gene transfection reagent, jetPRIME, in human melanoma cells. Furthermore, HPAE-2 efficiently delivered the Bax-encoding plasmid into melanoma cells, leading to a pronounced pro-apoptotic effect without causing noticeable cytotoxicity. This study establishes a potent non-viral platform for gene transfection of melanoma cells by harnessing the distribution of branching units, paving the way for potential clinical applications of gene therapy in melanoma treatment.

Recent progress of non-linear topological structure polymers: synthesis, and gene delivery.

Currently, many types of non-linear topological structure polymers, such as brush-shaped, star, branched and dendritic structures, have captured much attention in the field of gene delivery and nanomedicine. Compared with linear polymers, non-linear topological structural polymers offer many advantages, including multiple terminal groups, broad and complicated spatial architecture and multi-functionality sites to enhance gene delivery efficiency and targeting capabilities. Nevertheless, the complexity of their synthesis process severely hampers the development and applications of nonlinear topological polymers. This review aims to highlight various synthetic approaches of non-linear topological architecture polymers, including reversible-deactivation radical polymerization (RDRP) including atom-transfer radical polymerization (ATRP), nitroxide-mediated polymerization (NMP), reversible addition-fragmentation chain transfer (RAFT) polymerization, click chemistry reactions and Michael addition, and thoroughly discuss their advantages and disadvantages, as well as analyze their further application potential. Finally, we comprehensively discuss and summarize different non-linear topological structure polymers for genetic materials delivering performance both in vitro and in vivo, which indicated that topological effects and nonlinear topologies play a crucial role in enhancing the transfection performance of polymeric vectors. This review offered a promising guideline for the design and development of novel nonlinear polymers and facilitated the development of a new generation of polymer-based gene vectors.

Self-reporting and Biodegradable Thermosetting Solid Polymer Electrolyte.

To date, significant efforts have been dedicated to improve their ionic conductivity, thermal stability, and mechanical strength of solid polymer electrolytes (SPEs). However, direct monitoring of physical and chemical changes in SPEs is still lacking. Moreover, existing thermosetting SPEs are hardly degradable. Herein, by overcoming the limitation predicted by Flory theory, self-reporting and biodegradable thermosetting hyperbranched poly(ß-amino ester)-based SPEs (HPAE-SPEs) are reported. HPAE is successfully synthesized through a well-controlled "A2+B4" Michael addition strategy and then crosslinked it in situ to produce HPAE-SPEs. The multiple tertiary aliphatic amines at the branching sites confer multicolour luminescence to HPAE-SPEs, enabling direct observation of its physical and chemical damage. After use, HPAE-SPEs can be rapidly hydrolysed into non-hazardous ß-amino acids and polyols via self-catalysis. Optimized HPAE-SPE exhibits an ionic conductivity of 1.3×10-4  S/cm at 60 °C, a Na+ transference number ( t N a + ${{t}_{Na}^{+}}$ ) of 0.67, a highly stable sodium plating-stripping behaviour and a low overpotential of ≈190 mV. This study establishes a new paradigm for developing SPEs by engineering multifunctional polymers. The self-reporting and biodegradable properties would greatly expand the scope of applications for SPEs.

Enzymatically Activatable Polymers for Disease Diagnosis and Treatment.

The irregular expression or activity of enzymes in the human body leads to various pathological disorders and can therefore be used as an intrinsic trigger for more precise identification of disease foci and controlled release of diagnostics and therapeutics, leading to improved diagnostic accuracy, sensitivity, and therapeutic efficacy while reducing systemic toxicity. Advanced synthesis strategies enable the preparation of polymers with enzymatically activatable skeletons or side chains, while understanding enzymatically responsive mechanisms promotes rational incorporation of activatable units and predictions of the release profile of diagnostics and therapeutics, ultimately leading to promising applications in disease diagnosis and treatment with superior biocompatibility and efficiency. By overcoming the challenges, new opportunities will emerge to inspire researchers to develop more efficient, safer, and clinically reliable enzymatically activatable polymeric carriers as well as prodrugs.

Enhancing the Gene Transfection of Poly(ß-amino ester)/DNA Polyplexes by Modular Manipulation of Amphiphilicity.

Poly(ß-amino ester)s (PAEs) have been widely developed for gene delivery, and hydrophobic modification can further enhance their gene transfection efficiency. However, systematic manipulation of amphiphilicity of PAEs through copolymerization with hydrophobic monomers is time-consuming and, to some extent, uncontrollable. Here, a modular strategy is developed to manipulate the amphiphilicity of the PAE/DNA polyplexes. A hydrophobic polymer (DD-C12-122) and a hydrophilic polymer (DD-90-122) are synthesized separately and used as a hydrophobic module and a hydrophilic module, respectively. The amphiphilicity of polyplexes could be manipulated by changing the ratio of the hydrophobic module and hydrophilic module. Using the modular strategy, the PAE/DNA polyplexes with the highest gene transfection efficiency and safety profile as well as possible mechanisms are identified. The modular strategy provides a novel way to engineer the hydrophobicity of PAEs to improve their gene transfection and can be easily generalized and potentially extended to other polymeric gene delivery systems.

Emerging non-viral vectors for gene delivery.

Gene therapy holds great promise for treating a multitude of inherited and acquired diseases by delivering functional genes, comprising DNA or RNA, into targeted cells or tissues to elicit manipulation of gene expression. However, the clinical implementation of gene therapy remains substantially impeded by the lack of safe and efficient gene delivery vehicles. This review comprehensively outlines the novel fastest-growing and efficient non-viral gene delivery vectors, which include liposomes and lipid nanoparticles (LNPs), highly branched poly(ß-amino ester) (HPAE), single-chain cyclic polymer (SCKP), poly(amidoamine) (PAMAM) dendrimers, and polyethyleneimine (PEI). Particularly, we discuss the research progress, potential development directions, and remaining challenges. Additionally, we provide a comprehensive overview of the currently approved non-viral gene therapeutics, as well as ongoing clinical trials. With advances in biomedicine, molecular biology, materials science, non-viral gene vectors play an ever-expanding and noteworthy role in clinical gene therapy.

Phosphocholine-Functionalized Zwitterionic Highly Branched Poly(ß-amino ester)s for Cytoplasmic Protein Delivery.

Proteins have tremendous potential for vaccine development and disease treatment, but multiple extracellular and intracellular biological barriers must be overcome before they can exert specific biological functions in the target tissue. The use of polymers as carriers would greatly improve their bioavailability and therapeutic efficiency. Nevertheless, effective protein packaging and cell membrane penetration without causing cytotoxicity is particularly challenging, due largely to the simultaneous distribution of positive and negative charges on protein surface. Here, phosphocholine-functionalized zwitterionic poly(ß-amino ester)s, HPAE-D-(±), are developed for cytoplasmic protein delivery. The zwitterionic phosphocholine is capable of binding to both proteins and the cell membrane to facilitate protein packaging and nanoparticle cellular uptake. Compared to amine-functionalized HPAE-E-(+) and carboxylic acid-functionalized HPAE-C-(-), HPAE-D-(±) exhibits much higher cytoplasmic protein delivery efficiency and lower cytotoxicity. In addition, HPAE-D-(±) are readily degraded in aqueous solution. This strategy may be extended to other zwitterions and polymers, thus having profound implications for the development of safe and efficient protein delivery systems.

(Controlled) Free radical (co)polymerization of multivinyl monomers: strategies, topological structures and biomedical applications.

Free radical (co)polymerization (FRP/FRcP) of multivinyl monomers (MVMs) has emerged as a powerful strategy for the synthesis of chemically and topologically complex polymers due to its unique reaction kinetics, which enables the preparation of polymers with multiple functional groups and novel macromolecular structures. However, conventional FRP/FRcP of MVMs inevitably leads to insoluble crosslinked materials. Therefore, the development of advanced strategies for the controlled polymerization of MVMs is essential for the preparation of chemically and topologically complex polymers. In this review, we introduce the gelation mechanism of conventional FRP of MVMs and present the strategies of controlled polymerization of MVMs for the preparation of chemically and topologically complex polymers. We also discuss polymers with unique topologies synthesized by controlled polymerization of MVMs, such as crosslinked networks, (hyper)branched, star, cyclic, and single-chain cyclized/knotted structures. Finally, biomedical applications of various advanced polymeric materials prepared by controlled polymerization of MVMs are highlighted and the challenges is this field are discussed.

Genetic Engineering of Adipose-Derived Stem Cells Using Biodegradable and Lipid-Like Highly Branched Poly(ß-amino ester)s.

Biodegradable and lipid-like highly branched poly(ß-amino ester)s, HPAESA, were developed to enhance the biological functions of adipose-derived stem cells by gene transfection. Biodegradability reduces the cytotoxicity of HPAESA and enables controlled DNA release. Lipid mimicry enhances cellular uptake and endosomal escape of HPAESA/DNA polyplexes. HPAESA are able to transfect rat adipose-derived stem cells (rADSs) and human ADSCs (hADSCs) with orders of magnitude higher efficiency than commercial gene transfection reagents, with cell viability exceeding 90%. Most importantly, HPAESA can effectively transfer the nerve growth factor (NGF)-encoding plasmid to rADSCs and induce high NGF secretion, which significantly promotes neurite outgrowth of PC12 cells.

Cyclic poly(ß-amino ester)s with enhanced gene transfection activity synthesized through intra-molecular cyclization.

Topological structure plays a critical role in gene delivery of cationic polymers. Cyclic poly(ß-amino ester)s (CPAEs) are successfully synthesized via sequential Michael addition and free radical initiating ring-closure reaction. The CPAEs exhibit superior gene transfection efficiency and safety profile compared to their linear counterparts.