Due to their biodegradability and biocompatibility, chitosan-based hydrogels have great potential in regenerative medicine, with applications such as bacteriostasis, hemostasis, and wound healing. However, toxicity and high cost are problems that must be solved for chitosan-based hydrogel crosslinking agents such as formaldehyde, glutaraldehyde, and genipin. Therefore, we developed a biocompatible yet cost-effective chitosan-based hydrogel system as a candidate biomaterial to prevent infection during wound healing. The hydrogel was fabricated by crosslinking chitosan with dialdehyde chitosan (CTS–CHO) via dynamic Schiff-base reactions, resulting in a self-healable and injectable system. The rheological properties, degradation profile, and self-healable properties of the chitosan-based hydrogel were evaluated. The excellent antibacterial activity of the hydrogel was validated by a spread plate experiment. The use of Live/Dead assay on HEK 293 cells showed that the hydrogel exhibited excellent biocompatibility. The results demonstrate that the newly designed chitosan-based hydrogel is an excellent antibacterial wound dressing candidate with good biocompatibility.

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(β-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.

Synthetic reactive oxygen species (ROS)-responsive biomaterials have emerged as a useful platform for regulating critical aspects of ROS-induced pathologies and can improve such hostile microenvironments. Here, we report a series of new hyperbranched poly(β-hydrazide ester) macromers (HB-PBHEs) with disulfide moieties synthesized via an “A2 + B4” Michael addition approach. The three-dimensional structure of HB-PBHEs with multiacrylate end groups endows the macromers with rapid gelation capabilities to form (1) injectable hydrogels via cross-linking with thiolated hyaluronic acid and (2) robust UV-cross-linked hydrogels. The disulfide-containing macromers and hydrogels exhibit H₂O₂-responsive degradation compared with the counterparts synthesized by a dihydrazide monomer without disulfide moieties. The cell viability under a high ROS environment can be well-maintained under the protection of the disulfide containing hydrogels.

The topological structures of polymers play a critical role in determining their gene delivery efficiency. Exploring novel polymeric structures as gene delivery vectors is thus of great interest. In this work, a new generation of multi-cyclic poly(β-amino ester)s (CPAEs) with unique topology structure was synthesized for the first time via step growth polymerization. Through controlling the occurrence stage of cyclization, three types of CPAEs with rings of different sizes and topologies were obtained. In vitro experiments demonstrated that the CPAEs with macro rings (MCPAEs) significantly boosted the transgene expression comparing to their branched counterparts. Moreover, the MCPAE vector with optimized terminal group efficiently delivered the CRISPR plasmid coding both Staphylococcus aureus Cas9 nuclease and dual guide sgRNAs for gene editing therapy.

Protein therapeutics are highly promising for complex disease treatment. However, the lack of ideal delivery vectors impedes their clinical use, especially the carriers for in vivo delivery of functional cytosolic protein. In this study, we modified poly(β amino ester)s (PAEs) with a phenyl guanidine (PG) group to enhance their suitability for cytosolic protein delivery. The effects of the PG group on protein binding, cell internalization, protein function protection, and endo/lysosomal escape were systematically evaluated. Compared to the unmodified PAEs (L3), guanidyl rich PAEs (L3PG) presented superior efficiency of protein binding and protein internalization, mainly via clathrin-mediated endocytosis. In addition, both PAEs showed robust capabilities to deliver cytosolic proteins with different molecular weight (ranging from 30 to 464 kDa) and isoelectric points (ranging from 4.3 to 9), which were significantly improved in comparison with the commercial reagents of PULsin and Pierce Protein Transection Reagent. Moreover, L3PG successfully delivered Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) Cas9 ribonucleoprotein (RNP) into HeLa cells expressing green fluorescent protein (GFP) and achieved more than 80% GFP expression knockout. These results demonstrated that guanidyl modification on PAEs can enhance its capabilities for intracellular delivery of cytosolic functional proteins and CRISPR/Cas9 ribonucleoprotein. The guanidyl-rich PAEs are promising nonviral vectors for functional protein delivery and potential use in protein and nuclease-based gene editing therapies.