title: Controlled release of cell-permeable gene complex from poly(L-lactide) scaffold for enhanced stem cell tissue engineering.
abstract: The use of tissue engineering to deliver genes to stem cells has been impeded by low transfection efficiency of the inserted gene and poor retention at the target site. Herein, we describe the use of non-viral gene transfer by cell-permeable peptide (CPP) to increase the transfection efficiency. The combination of this technique with the use of a controlled release concept using a poly (l-lactide) scaffold allowed for prolonged uptake in stem cells. High transfection efficiency was obtained using a human-derived arginine-rich peptide denoted as Hph-1 (YARVRRRGPRR). The formation of complex between pDNA and Hph-1 was monitored using gel retardation tests to measure size and zeta potential. Complex formation was further assessed using a DNase I protection assay. A sustained gene delivery system was developed using a fibrous 3-D scaffold coated with pDNA/Hph-1 complexes. Transfection efficiency and the mean fluorescence intensity of human adipose-derived stem cells (hASCs) on the sustained delivery scaffold were compared to those of cells transfected via bolus delivery. Plasmid DNA completely bound Hph-1 at a negative-to-positive (N/P) charge ratio of 10. After complex formation, Hph-1 appeared to effectively protect pDNA against DNase I attack and exhibited cytotoxicity markedly lower than that of the pDNA/PEI complex. Plasmid DNA/Hph-1 complexes were released from the scaffolds over 14days and were successfully transfected hASCs seeded on the scaffolds. Flow cytometry revealed that the transfection efficiency in hASCs treated with pDNA/Hph-1 complex was approximately 5-fold higher than that in cells transfected using Lipofectamine. The sustained delivery system showed a significantly higher transfection efficiency and remained able to transfect cells for a longer period of time than bolus delivery. These results suggest that cell-scaffold-based tissue regeneration can be further improved by transduction concept using CPP and controlled release using polymeric scaffold.
authors.affiliation: Department of Pharmacy, Ewha Womans University, Daehyun-dong, Seodaemun-gu, Seoul 120-750, Republic of Korea.
authors.forename: Mi-Ra
authors.initials: MR
authors.lastname: Jung
authors.forename: In-Kyong
authors.initials: IK
authors.lastname: Shim
authors.forename: Eun-Sung
authors.initials: ES
authors.lastname: Kim
authors.forename: Yoon-Jeong
authors.initials: YJ
authors.lastname: Park
authors.forename: Young-Il
authors.initials: YI
authors.lastname: Yang
authors.forename: Sang-Kyou
authors.initials: SK
authors.lastname: Lee
authors.forename: Seung-Jin
authors.initials: SJ
authors.lastname: Lee
mesh_terms: D000595:Amino Acid Sequence; D002470:Cell Survival; D057846:Cell-Penetrating Peptides / Q000378:metabolism*; D002478:Cells, Cultured; D004247:DNA / Q000008:administration & dosage* / Q000235:genetics; D003692:Delayed-Action Preparations / Q000378:metabolism*; D003851:Deoxyribonucleases / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics; D006801:Humans; D010957:Plasmids / Q000008:administration & dosage* / Q000235:genetics; D011091:Polyesters / Q000378:metabolism*; D013234:Stem Cells / Q000166:cytology / Q000378:metabolism*; D023822:Tissue Engineering; D014162:Transfection / Q000379:methods
chemical_list: D057846:Cell-Penetrating Peptides; D003692:Delayed-Action Preparations; D011091:Polyesters; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; C033616:poly(lactide); D004247:DNA; D003851:Deoxyribonucleases