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.

Acrylate-Based PEG Hydrogels with Ultrafast Biodegradability for 3D Cell Culture.

Poly(ethylene glycol) (PEG)-based hydrogels are particularly challenging to degrade, which hinders efficient cell harvesting within the gel matrix. Here, highly branched copolymers of PEG methyl ether acrylate (PEGMA) and disulfide diacrylate (DSDA) (PEG-DS) with short primary chains and multiple pendent vinyl groups were synthesized by a "vinyl oligomer combination" approach. PEG-DS readily cross-links with thiolated gelatin (Gel-SH) to form hydrogels. Results demonstrate that shortening the primary chains of PEG-DS significantly enhances the viability of bone marrow mesenchymal stem cells (BMSCs) by up to 193.2%. Importantly, DS junctions can be easily cleaved into short primary chains using dithiothreitol (DTT), triggering ultrafast degradation of PEG-DS/Gel-SH hydrogels within 2 min under mild conditions and release of the encapsulated BMSCs. This study establishes a novel strategy to enhance the degradation of acrylate-based PEG hydrogels for three-dimensional (3D) cell culture and harvesting. These findings expand the potential applications of such hydrogels in various biomedical fields.

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.

Controllable C-H Alkylation of Polyethers via Iron Photocatalysis.

The efficient conversion of a C-H bond in the polyether chain to other functional groups provides great opportunities for development of novel applications in many research fields. However, this field is quite underdeveloped due to the key challenge on controlling the selectivity of the C-H bond functionalization over the chain cleavage. In this work, we report a controllable C-H bond alkylation of polyethers under mild conditions via photoinduced iron catalysis. The level of functionalization could be controlled by using different amounts of alkenes and various reaction times, while the molecular weight distributions were maintained narrow. A broad scope of electron-deficient alkenes containing nitrile, ester, epoxide, terminal alkynyl, 2,5-dioxotetrafuranyl, and 2,5-dioxopyrrolidinyl groups could be utilized to functionalize the different polyethers with great efficiencies. The potential applications of the modified polyethylene glycols and polyethylene oxides were explored by the preparation of novel hydrogels and solid-state electrolytes with enhancement of lithium ion conductivities. Moreover, the density functional theory calculation disclosed the plausible mechanism and explained the high selectivity for the C-H alkylation.

Engineering siRNA therapeutics: challenges and strategies.

Small interfering RNA (siRNA) is a potential method of gene silencing to target specific genes. Although the U.S. Food and Drug Administration (FDA) has approved multiple siRNA-based therapeutics, many biological barriers limit their use for treating diseases. Such limitations include challenges concerning systemic or local administration, short half-life, rapid clearance rates, nonspecific binding, cell membrane penetration inability, ineffective endosomal escape, pH sensitivity, endonuclease degradation, immunological responses, and intracellular trafficking. To overcome these barriers, various strategies have been developed to stabilize siRNA, ensuring their delivery to the target site. Chemical modifications implemented with nucleotides or the phosphate backbone can reduce off-target binding and immune stimulation. Encapsulation or formulation can protect siRNA from endonuclease degradation and enhance cellular uptake while promoting endosomal escape. Additionally, various techniques such as viral vectors, aptamers, cell-penetrating peptides, liposomes, and polymers have been developed for delivering siRNA, greatly improving their bioavailability and therapeutic potential.

Non-viral delivery of CRISPR-Cas9 complexes for targeted gene editing via a polymer delivery system.

Recent advances in molecular biology have led to the CRISPR revolution, but the lack of an efficient and safe delivery system into cells and tissues continues to hinder clinical translation of CRISPR approaches. Polymeric vectors offer an attractive alternative to viruses as delivery vectors due to their large packaging capacity and safety profile. In this paper, we have demonstrated the potential use of a highly branched poly(ß-amino ester) polymer, HPAE-EB, to enable genomic editing via CRISPRCas9-targeted genomic excision of exon 80 in the COL7A1 gene, through a dual-guide RNA sequence system. The biophysical properties of HPAE-EB were screened in a human embryonic 293 cell line (HEK293), to elucidate optimal conditions for efficient and cytocompatible delivery of a DNA construct encoding Cas9 along with two RNA guides, obtaining 15-20% target genomic excision. When translated to human recessive dystrophic epidermolysis bullosa (RDEB) keratinocytes, transfection efficiency and targeted genomic excision dropped. However, upon delivery of CRISPR-Cas9 as a ribonucleoprotein complex, targeted genomic deletion of exon 80 was increased to over 40%. Our study provides renewed perspective for the further development of polymer delivery systems for application in the gene editing field in general, and specifically for the treatment of RDEB.

Manipulation of Transgene Expression in Fibroblast Cells by a Multifunctional Linear-Branched Hybrid Poly(ß-Amino Ester) Synthesized through an Oligomer Combination Approach.

Delivery of functional genetic materials into fibroblast cells to manipulate the transgene expression is of great significance in skin gene therapy. Despite numerous polymeric gene delivery systems having been developed, highly safe and efficient fibroblast gene transfection has not yet been achieved. Here, through a new linear oligomer combination strategy, linear poly(ß-amino ester) oligomers are connected by the branching units, forming a new type of poly(ß-amino ester). This new multifunctional linear-branched hybrid poly(ß-amino ester) (LBPAE) shows high-performance fibroblast gene transfection. In human primary dermal fibroblasts (HPDFs) and mouse embryo fibroblasts (3T3s), ultrahigh transgene expression is achieved by LBPAE: up to 3292-fold enhancement in Gaussia luciferase (Gluc) expression and nearly 100% of green fluorescence protein expression are detected. Concurrently, LBPAE is of high in vitro biocompatibility. In depth mechanistic studies reveal that versatile LBPAE can navigate multiple extra- and intracellular barriers involved in the fibroblast gene transfection. More importantly, LBPAE can effectively deliver minicircle DNA encoding  COL7A1 gene (a large and functional gene construct) to substantially upregulate the expression of type VII collagen (C7) in HPDFs, demonstrating its great potential in the treatment of C7-deficiency related genodermatoses such as recessive dystrophic epidermolysis bullosa.

Bacteria-Resistant Single Chain Cyclized/Knotted Polymer Coatings.

Further to conventional linear, branched, crosslinked, and dendritic polymers, single chain cyclized/knotted polymers (SCKPs) have emerged as a new class of polymer structure with unique properties. Herein, the development of bacteria-resistant SCKPs is reported and the effect of this structure on the resistance of polymer materials to bacteria is investigated. Four SCKPs were synthesized by reversible addition fragmentation chain transfer (RAFT) homopolymerization of multivinyl monomers (MVMs) and then crosslinked by UV light to form SCKP films. Regardless of MVM type used, the resulting SCKP films showed much higher resistance to bacteria, and up to 75 % less bacterial attachment and biofilm formation, in comparison with the corresponding non-SCKP films. This is due to the altered surface morphology and hydrophobicity of the SCKP films. These results highlight the critical role of the SCKP structure in enhancing the resistance of polymeric materials to bacteria.

Brushlike Cationic Polymers with Low Charge Density for Gene Delivery.

Using a combined synthesis approach comprising reversible addition-fragmentation transfer polymerization and ring opening reaction, a series of poly glycidyl methacrylate (polyGMA) polymers were designed and synthesized for gene delivery. These polymers characterized by low cationic charge respective to established gene delivery vectors such as PEI were studied to further elucidate the key structure-activity parameters that mediate efficient and biocompatible gene delivery. Compared to PEI, these brushlike polymers facilitated markedly improved safety and gene delivery efficiency.

Bioreducible Zinc(II)-Coordinative Polyethylenimine with Low Molecular Weight for Robust Gene Delivery of Primary and Stem Cells.

To transform common low-molecular-weight (LMW) cationic polymers, such as polyethylenimine (PEI), to highly efficient gene vectors would be of great significance but remains challenging. Because LMW cationic polymers perform far less efficiently than their high-molecular-weight counterparts, mainly due to weaker nucleic acid encapsulation, herein we report the design and synthesis of a dipicolylamine-based disulfide-containing zinc(II) coordinative module (Zn-DDAC), which is used to functionalize LMW PEI (Mw ≈ 1800 Da) to give a non-viral vector (Zn-PD) with high efficiency and safety in primary and stem cells. Given its high phosphate binding affinity, Zn-DDAC can significantly promote the DNA packaging functionality of PEI1.8k and improve the cellular uptake of formulated polyplexes, which is particularly critical for hard-to-transfect cell types. Furthermore, Zn-PD polymer can be cleaved by glutathione in cytoplasm to facilitate DNA release post internalization and diminish the cytotoxicity. Consequently, the optimal Zn-PD mediates 1-2 orders of magnitude higher gluciferase activity than commercial transfection reagents, Xfect and PEI25k, across diverse cell types, including primary and stem cells. Our findings provide a valuable insight into the exploitation of LMW cationic polymers for gene delivery and demonstrate great promise for the development of next-generation non-viral vectors for clinically viable gene therapy.

Controlled Polymerization of Multivinyl Monomers: Formation of Cyclized/Knotted Single-Chain Polymer Architectures.

Seventy years ago, Flory and Stockmayer predicted that the polymerization of multivinyl monomers (MVMs) would inevitably lead to insoluble cross-linked gel networks. Since then, the use of MVMs has largely been limited to as cross-linking agents. More recently, however, polymerization strategies such as reversible deactivation radical polymerization (RDRP) have paved the way for the exploration of new possibilities in terms of both polymer architectures and functional capabilities. This Minireview provides historical context to the problem of polymerizing MVMs, before highlighting how RDRP has led to the formation of new cyclized/knotted polymer structures. Although the potential of such cyclized/knot polymer architectures is far from being fulfilled, some emerging applications are discussed.

Highly Branched Poly(ß-amino esters) for Non-Viral Gene Delivery: High Transfection Efficiency and Low Toxicity Achieved by Increasing Molecular Weight.

A successful polymeric gene delivery vector is denoted by both transfection efficiency and biocompatibility. However, the existing vectors with combined high efficacy and minimal toxicity still fall short. The most widely used polyethylene imine (PEI), polyamidoamine (PAMAM) and poly(dimethylaminoethyl methacrylate) (PDMAEMA) suffer from the correlation: either too toxic or little effective. Here, we demonstrate that with highly branched poly(ß-amino esters) (HPAEs), a type of recently developed gene delivery vector, the high gene transfection efficiency and low cytotoxicity can be achieved simultaneously at high molecular weight (MW). The interactions of HPAE/DNA polyplexes with cell membrane account for the favorable correlation between molecular weight and biocompatibility. In addition to the effect of molecular weight, the molecular configuration of linear and branched segments in HPAEs is also pivotal to endow high transfection efficiency and low cytotoxicity. These findings provide renewed perspective for the further development of clinically viable gene delivery vectors.

Highly Branched Poly(ß-Amino Esters): Synthesis and Application in Gene Delivery.

Highly branched poly(ß-amino esters) (HPAEs) are developed via a facile and controllable "A2+B3/B2" strategy successfully. As nonviral gene delivery vectors, the performance of HPAEs is superior to the well-studied linear counterpart as well as the leading commercial reagent Superfect. When combined with minicircle DNA construct, HPAEs can achieve ultrahigh gene transfection efficiency, especially in keratinocytes.

Significance of branching for transfection: synthesis of highly branched degradable functional poly(dimethylaminoethyl methacrylate) by vinyl oligomer combination.

A series of degradable branched PDMAEMA copolymers were investigated with the linear PDMAEMA counterpart as gene-delivery vectors. The branched PDMAEMA copolymers were synthesized by controlled radical cross-linking copolymerization based on the "vinyl oligomer combination" approach. Efficient degradation properties were observed for all of the copolymers. The degree of branching was found to have a big impact on performance in transfection when tested on different cell types. The product with the highest degree of branching and highest degree of functionality had a superior transfection profile in terms of both transfection capability and the preservation of cell viability. These branched PDMAEMA copolymers show high potential for gene-delivery applications through a combination of the simplicity of their synthesis, their low toxicity, and their high performance.

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.

In-situ noncovalent interaction of ammonium ion enabled C-H bond functionalization of polyethylene glycols.

The noncovalent interactions of ammonium ion with multidentate oxygen-based host has never been reported as a reacting center in catalytic reactions. In this work, we report a reactivity enhancement process enabled by non-covalent interaction of ammonium ion, achieving the C-H functionalization of polyethylene glycols with acrylates by utilizing photoinduced co-catalysis of iridium and quinuclidine. A broad scope of alkenes can be tolerated without observing significant degradation. Moreover, this cyano-free condition respectively allows the incorporation of bioactive molecules and the PEGylation of dithiothreitol-treated bovine serum albumin, showing great potentials in drug delivery and protein modification. DFT calculations disclose that the formed α-carbon radical adjacent to oxygen-atom is reduced directly by iridium before acrylate addition. And preliminary mechanistic experiments reveal that the noncovalent interaction of PEG chain with the formed quinuclidinium species plays a unique role as a catalytic site by facilitating the proton transfer and ultimately enabling the transformation efficiently.

Enhancing Gene Transfection Efficiency of Star-Shaped Poly(ß-amino ester)s via "Top-down" Hydrolysis.

Gene therapy has emerged as a potent tool for treating a wide range of hereditary and acquired disorders. However, the development of high-performance nonviral gene delivery vectors remains a significant challenge. Here we report the development of a new type of star-shaped poly(ß-amino ester) (SPAE) through a "top-down" hydrolysis approach and demonstrate its exceptional DNA transfection efficiency and safety profiles. Two SPAEs with different monomer combinations are first synthesized using an "arm first" strategy and then hydrolyzed sequentially to produce h-SPAEs with varied chemical compositions and molecular weights. Results demonstrate that hydrolysis significantly influences the physiological characteristics of the resulting h-SPAEs and h-SPAE/DNA polyplexes. Dependent on the chemical composition, h-SPAEs with low to moderate hydrolysis degrees exhibit superior gene transfection efficiency and cell viability across various cell types. Notably, the leading candidate, h-SPAE-1-5h, achieves up to 88.8% gene transfection efficiency, which was 154-257% higher compared to SPAE-1. This study not only establishes an easy-to-operate "top-down" approach for reshaping the topological structure and chemical composition of SPAEs, but also identifies promising candidates for effective gene transfection. This strategy can be applied to other cationic polymers to enhance their gene transfection performance.

(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.

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.

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.