Abstract
Cationic polypeptides and cationic polymers have cell-penetrating capacities and have been used in gene transfer studies. In this study, we investigate the capability of a polymer of d-lysine (PDL), a chiral form of α–Poly-lysine, as a possible nonviral vector for releasing genetic materials to neuroblastoma cells and evaluate its stability against proteases. We tested and compared its transfection effectiveness in vitro as a vehicle for the EGFP plasmid DNA (pDNA) reporter in the SH-SY5Y human neuroblastoma, HeLa, and 3T3 cell lines. Using fluorescent microscopy and flow cytometry, we demonstrated high transfection efficiencies based on EGFP fluorescence in SH-SY5Y cells, compared with HeLa and 3T3. Our results reveal PDL as an efficient vector for gene delivery specifically in the SH-SY5Y cell line and suggest that PDL can be used as a synthetic cell-penetrating polypeptide for gene therapy in neuroblastoma cells.
Highlights
Gene therapy is a technique that alters gene expression to change the biological properties of cells for therapeutic purposes [1] and has proven to be a promising tool.There are several methods to achieve this objective, some of which include changing pathologic genes for functional genetic sequences, inactivating a defective gene, or treating a pathological condition by inserting a new gene [1]; many challenges remain [2,3].One of the main problems is the difficulty in delivering the nucleic acids through the cell membrane
We tested several DNA–polymer of D-lysine (PDL) complexes and show that PDL has a high transfection efficiency in the SH-SY5Y neuroblastoma cell line when compared to HeLa and 3T3 cell lines. These results suggest that PDL can be used as a synthetic cell-penetrating polypeptide for gene therapy in neuroblastoma cells [42]
To analyze PDL’s ability to protect the plasmid DNA (pDNA) from enzymatic degradation, we carried out a DNase I (Sigma-Aldrich, Darmstadt, Germany) protection assay. pDNA–PDL complexes were prepared at w/w ratios of 1:2, 1:4, and control naked pDNA was incubated at 37 ◦ C for 60 min with DNase I (1 U/μg of DNA), 1 μL MgCl2 25 mM (Sigma-Aldrich, Darmstadt, Germany), at which point 1 μL EDTA 50 mM (Sigma-Aldrich, Darmstadt, Germany) was added and samples were heated at 65 ◦ C for 10 min to inactivates DNase
Summary
Gene therapy is a technique that alters gene expression to change the biological properties of cells for therapeutic purposes [1] and has proven to be a promising tool. An example of a synthetic custom-designed CPPs are the POD “peptide for ocular delivery,” a GGG(ARKKAAKA) chain used for drug delivery treatment related to eye diseases They showed that POD is capable of condensing siRNA and pDNA and can pass through the cell membrane of the neural retina, photoreceptors, ganglion cells, as well as enter in the sclera, choroids, and into the dura of the optic nerve via topical application [23]. Its use has been described in the systemic delivery of a PDL–plasmid complex in vivo [31] and in clinical trials to treat cystic fibrosis using conjugated PDL–peg [34] In this context, PDL induces a low immune response [35] and can be used to increase cell-type specificity [36]. These results suggest that PDL can be used as a synthetic cell-penetrating polypeptide for gene therapy in neuroblastoma cells [42]
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