RESUMEN
Gene therapy provides a promising treatment for glioblastoma multiforme, which mainly depends on two key aspects, crossing the blood brain barrier (BBB) effectively and transfecting target cells selectively. In this work, we reported a series of peptide-based vectors for transfecting glioma cells specifically consisting of several functional segments including a cell-penetrating peptide, targeting segment substance P (SP), an endosomal escape segment, a PEG linker and a stearyl moiety. The conformations and DNA-loading capacities of peptide vectors and the self-assembly behaviors of peptide/pGL3 complexes were characterized. The in vitro gene transfection was evaluated in U87, 293T-NK1R, and normal 293T cell lines. The transfection efficiency ratio of P-02 (SP-PEG4-K(C18)-(LLHH)3-R9) to Lipo2000 in the U87 cell line was about 36% higher than that in the 293T cell line. The neurokinin-1 receptor (NK1R) in U87 cells mediated the transfection process via interactions with the ligand SP in peptide vectors. The mechanism of NK1R mediated transfection was demonstrated by the use of gene-modified 293T cells expressing NK1R, as well as the gene transfection in the presence of free SP. Besides, P-02 could promote the pGL3 plasmids to cross the BBB model in vitro and achieved the EGFP gene transfection in the brain of zebrafish successfully. The designed peptide vectors, owing to their specific transfection capacity in glioma cells, provide a potential approach for glioblastoma multiforme gene therapy.
Asunto(s)
Técnicas de Transferencia de Gen , Glioma/tratamiento farmacológico , Receptores de Neuroquinina-1/metabolismo , Sustancia P/uso terapéutico , Animales , Barrera Hematoencefálica , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Neurotransmisores/química , Neurotransmisores/uso terapéutico , Receptores de Neuroquinina-1/genética , Sustancia P/química , Pez CebraRESUMEN
Gene therapy offers an alternative approach to malignant glioma; however, glioma cells are difficult to transfect. Peptides, as nonviral vectors, can achieve efficient gene transfection in glioma cells due to their good biocompatibility and easy functionalization. In this article, we reported a series of peptide vectors, which were composed of amphiphilic α-helical segments, cationic cell-penetrating segments, and cysteine and glycine residues. The physicochemical properties of peptide vectors or peptide/pGL3 complexes, including conformation, DNA-loading capacity, size, zeta potential, and morphology, were characterized. Their gene delivery abilities were evaluated in U373, U87, and C6 glioma cell lines and a normal cell line 293 T. Compared with Lipo 2000 and other peptide vectors, the efficiency of P-03 (CLLHHLLHHLLHHGGRKKRRQRRR) to transfect glioma cells was higher. While in 293 T cells, the transfection efficiency of P-03 was much lower than that of Lipo 2000 and another positive control P-07. Furthermore, P-03 could facilitate the pGL3 plasmids crossing a blood-brain barrier model in vitro and achieved the expression of EGFP gene in the brain sites of zebrafish.
Asunto(s)
Glioma , Pez Cebra , Animales , Terapia Genética , Glioma/genética , Humanos , Péptidos/genética , TransfecciónRESUMEN
Cell penetrating peptides (CPPs) play a crucial role in the transportation of bioactive molecules. Although CPPs have been used widely in various delivery systems, further applications of CPPs are hampered by several drawbacks, such as high toxicity, low delivery efficiency, proteolytic instability and poor specificity. To design CPPs with great cell-penetrating ability, physicochemical properties and safety, researchers have tried to develop new methods to overcome the defects of CPPs. Briefly, (1) the side chain of arginine containing the guanidinium group is essential for the facilitation of cellular uptake; (2) the hydrophobic counterion complex around the guanidinium-rich backbone can "coat" the highly cationic structure with lipophilic moieties and act as an activator; (3) the conformation-constrained strategy was pursued to shield the peptide, thereby impeding access of the proteolytic enzyme; (4) targeting strategies can increase cell-type specificity of CPPs. In this review, the above four aspects were discussed in detail.