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The development of nonviral gene delivery vectors offers the potential to provide effective treatment for glioblastoma in the form of gene therapy. Here, we report the use of retro-inverso C-end rule (CendR) peptide _D(RPPREGR) as a targeting ligand to prepare a _D(RPPREGR)-PEG-PEI gene vector. _D(RPPREGR) peptide specifically recognized the neuropilin-1 receptor that was overexpressed on U87 glioma cells, and showed enhanced tumor spheroid penetration ability. Compared with parental RGERPPR, _D(RPPREGR) possessed improved biological stability and had a higher affinity for U87 glioma cells; it also showed enhanced penetration of the tumor spheroid. mPEG-PEI/pDNA and _D(RPPREGR)-PEG-PEI/pDNA complexes were prepared and MTT assay results revealed that the cytotoxicity of _D(RPPREGR)-PEG-PEI complexes was significantly lower than that of PEI complexes, with cell survival rates …