Oligohistidine and targeting peptide functionalized TAT-NLS for enhancing cellular uptake and promoting angiogenesis in vivo.

BACKGROUND: Gene therapy has been developed and used in medical treatment for many years, especially for the enhancement of endothelialization and angiogenesis. But slow endosomal escape rate is still one of the major barriers to successful gene delivery. In order to evaluate whether introducing oligohistidine (Hn) sequence into gene carriers can promote endosomal escape and gene transfection or not, we designed and synthesized Arg-Glu-Asp-Val (REDV) peptide functionalized TAT-NLS-Hn (TAT: typical cell-penetrating peptide, NLS: nuclear localization signals, Hn: oligohistidine sequence, n: 4, 8 and 12) peptides with different Hn sequence lengths. pEGFP-ZNF580 (pZNF580) was condensed by these peptides to form gene complexes, which were used to transfect human umbilical vein endothelial cells (HUVECs). RESULTS: MTT assay showed that the gene complexes exhibited low cytotoxicity for HUVECs. The results of cellular uptake and co-localization ratio demonstrated that the gene complexes prepared from TAT-NLS-Hn with long Hn sequence (n = 12) benefited for high internalization efficiency of pZNF580. In addition, the results of western blot analysis and PCR assay of REDV-TAT-NLS-H12/pZNF580 complexes showed significantly enhanced gene expression at protein and mRNA level. Wound healing assay and transwell migration assay also confirmed the improved proliferation and migration ability of the transfected HUVECs by these complexes. Furthermore, the in vitro and in vivo angiogenesis assay illustrated that these complexes could promote the tube formation ability of HUVECs. CONCLUSION: The above results indicated that the delivery efficiency of pZNF580 and its expression could be enhanced by introducing Hn sequence into gene carriers. The Hn sequence in REDV-TAT-NLS-Hn is beneficial for high gene transfection. These REDV and Hn functionalized TAT-NLS peptides are promising gene carriers in gene therapy.

POSS-cored and peptide functionalized ternary gene delivery systems with enhanced endosomal escape ability for efficient intracellular delivery of plasmid DNA.

Biocompatibility, stability and high efficiency profiles are critical points for promoting the practical applications of gene delivery systems. The incorporation of cell-penetrating peptides (CPPs), REDV, and a nuclear localization signal (NLS) peptide sequence has been considered to be a promising strategy for developing efficient gene carriers to transfect vascular endothelial cells (ECs). However, these integrated multifunctional peptide carriers are usually limited by their inefficient targeting function and weak endosomal escape ability. Aiming to develop more efficient gene carriers, the integrated multifunctional REDV-G-TAT-G-NLS-C sequence was conjugated to polyhedral oligomeric silsesquioxane (POSS) by heterobifunctional poly(ethylene glycol) in the current study. This star-shaped polymer carrier complexed with the pZNF580 plasmid to form gene complexes, and then the histidine-rich peptide of REDV-TAT-NLS-H12 (TP-H12) was incorporated into their surface to obtain ternary gene delivery systems with enhanced endosomal escape ability. These ternary gene delivery systems exhibited low cytotoxicity towards ECs and possessed high REDV-mediated cellular uptake, excellent internalization efficiency, rapid endosomal escape and high nucleus translocation capacity. The endosomal escape of the ternary complexes was improved due to the pH buffering capacity of the histidine residue in TP-H12 and the optimized macropinocytosis internalization pathway. Moreover, these CPP-based ternary gene delivery systems have high gene delivery efficiency and could improve the migration of ECs as demonstrated by gene expression and transwell assay. These systems may serve as a promising candidate for gene delivery and transfection in ECs, which is advantageous for EC migration and endothelialization on the biomaterial surface.

Multitargeting Peptide-Functionalized Star-Shaped Copolymers with Comblike Structure and a POSS-Core To Effectively Transfect Endothelial Cells.

Gene therapy meets one serious bottleneck in clinical application, namely, lack of safe and efficient gene delivery systems. In order to solve this problem, we designed a lowly cytotoxic and highly efficient gene delivery system for the transfection of endothelial cells. Octa(3-ammoniumpropyl)octasilsesquioxane octachloride reacted with 2-bromoisobutyryl bromide under alkaline condition, and sequentially initiated 2-(dimethylamino)ethyl methacrylate (DMAEMA) and poly(ethylene glycol) monomethacrylate (PEGMA) via atom transfer radical polymerization (ATRP) to prepare the eight-arm copolymer with a biocompatible polyhedral oligomericsilsesquioxane (POSS). The side chain ends of the comblike PPEGMA were double-bonded to facilitate the attachment of CAGW or CAG-TAT-NLS functional peptide, thereby enabling the star-shaped copolymers with multifunction. The peptide-functionalized star-shaped copolymers were self-assembled into nanoparticles (NPs) and used to condense pEGFP-ZNF580 (pDNA) to prepare the NPs/pDNA complexes. These complexes had low toxicity as confirmed by MTT. The results of fluorescence microscopy and flow cytometry showed that these multitargeting functionalized gene complexes could effectively transfect endothelial cells. Their transfection efficiency is higher than the positive control PEI 25 kDa group. Moreover, the Western blot test, wound healing assay, and in vitro tube formation assay also demonstrated that the transfected cells showed high migration and enhanced angiogenesis. The pDNA was effective delivered and expressed in endothelial cells by these multitargeting functionalized gene complexes, and promoted cell migration and tube formation. These star-shaped copolymers with comblike structure and a POSS core are a potential gene carrier for gene therapy.