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Gene therapy is a promising strategy for treating ischemic disease by solving the dual dilemma of ischemia and inflammation. However, its development remains limited by inefficient gene transfection. Hence, we propose a “dual genes + all-adaptive carrier” idea. We have innovatively co-delivered eNOS gene and the ZNF580gene encoding its transcription factor to enhance the efficiency of eNOS expression. The overexpressed ZNF580protein significantly promotes angiogenesis viaregulating the transcription of multiple genes. This implies a potential synergistic effect of eNOS and ZNF580genes in anti-ischemic therapy. Additionally, we have designed an all-adaptive gene carrier with cascaded bio-responsive functions based on the characteristic bio-signals of the ischemic site (including extracellular excessive matrix metalloproteinase-2, the endo/lysosomal pH gradient and high cytoplasmic glutathione level). This carrier can sequentially overcome transfection bottlenecks and achieve high transfection. Excitingly, this cascaded bio-responsive delivery strategy remarkably enhanced blood perfusion, accelerated angiogenesis and alleviated inflammation in critical limb ischemia (CLI) mice, which was attributed to the combined effects of pro-angiogenic ZNF580expression and synergistically produced eNOS expression. Thereby, we believe that the co-delivery of eNOS and ZNF580genes assisted by a cascaded bio-responsive carrier is a powerful strategy to treat CLI.

Polymeric gene carriers have been developed to deliver therapeutic genes for treating various genetic diseases. They may diminish the problems related with viral vectors in terms of safety, immunogenicity and mutagenesis. But inefficient endosomal release, cytoplasmic transport and nuclear entry are the main limiting issues in the usage of polymeric carriers. Different strategies have been proposed to functionalize gene carriers for individually overcoming these barriers. Towards this aim, various polymeric carriers have been developed and further modified with certain ligands and peptides to achieve properties like good cargo DNA protection, excellent colloidal stability, high cellular uptake efficiency, efficient endo/lysosome escape, efficient import into the nucleus and specificity to target cells. These multifunctional polymeric carriers are beneficial for efficient gene delivery. In this review, we provide a comprehensive summary of the interaction between gene and polymeric carriers and the mechanism and challenges of gene carriers. We also highlight and discuss the recent developments and advancements in the synthesis of ligand targeting and peptide functionalized gene carriers.

Small interfering RNA (siRNA)-based therapy is an emerging treatment to address serious cardiovascular disease. It is essential to construct highly efficient vehicles for therapeutic siRNA intracellular delivery. Extracellular signal-regulated kinase-2 (ERK2) siRNA (abbreviated as ERK2-siRNA) is known as a type of siRNA to selectively silence the expression of ERK2. Herein, a type of ternary delivery system characterized by an endosome-selective-self-accelerating-escape ability was designed and prepared for the purpose of inhibiting the migration of vascular smooth muscle cells (VSMCs) in vitro. This system was called ternary ERK2-siRNA complexes (abbreviated as TRCs-Aco), which were fabricated viasequential electrostatic self-assembly of a star-shaped cell-penetrating peptide based on polyhedral oligomeric silsesquioxane (POSS-(C-G-R8-G-W)16), ERK2-siRNA and a pH-sensitive anionic polymer of cis-aconitic anhydride grafted -poly(l-lysine). Importantly, TRCs-Aco could break down the obstacle of biocompatibility-silencing efficiency. In comparison with the parent binary siRNA complexes (abbreviated as BRCs), which are composed of POSS-(C-G-R8-G-W)16and ERK2-siRNA, our designed TRCs-Aco revealed more excellent biocompatibility including hemocompatibility and cytocompatibility. Unexpectedly, TRCs-Aco exhibited stronger ERK2 silencing efficiency at the level of mRNA and protein, which was mainly due to its remarkable self-accelerating endosomal escape. Definitive evidence demonstrated that this ternary ERK2-siRNA delivery system significantly prevented the migration of VSMCs and decreased the dermal thickness in bleomycin-treated mice. In brief, this unique structured system could provide a valuable nanoplatform for highly efficient siRNA delivery in VSMCs, and it might hold great potential in guiding ERK2-siRNA-based proliferative disease therapy.

In this work, a biodegradable star-shaped copolymer poly(lactide-co-3(S)-methyl-morpholine-2,5-dione)6(Star-(PLMD)6) was synthesized viaring-opening polymerization (ROP), and subsequently a gene carrier Star-PLMD-g-PEI-g-PEG-CREDVW was prepared by grafting polyethyleneimine (PEI), polyethylene glycol (PEG) and targeting peptide REDV onto Star-(PLMD)6. This gene carrier could form stable micelles to condense pEGFP-ZNF580 through electrostatic interaction. The resulting complexes were biocompatible and showed high efficiency in gene delivery. In addition, these complexes exhibited high selectivity for endothelial cells (ECs), high transfection efficiency and enhanced migration of ECs. The protein level of ZNF580 expression was significantly high (up to 85%), while the control group was only 51%. This combination of degradability, targeting ligand and star-structure strategy exhibits a significant advantage in transfection efficiency and migration of ECs.