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Cyclodextrin-PEI-Tat Polymer as a Vector for Plasmid DNA Delivery to Placenta Mesenchymal Stem Cells.

ABSTRACT: This study aims to modify a cyclodextrin-PEI-based polymer, PEI-?-CyD, with the TAT peptide for plasmid DNA delivery to placenta mesenchymal stem cells (PMSCs). By using the disulfide exchange between the SPDP-activated PEI-?-CyD and TAT peptide, the TAT-PEI-?-CyD polymer was fabricated and the success of this was confirmed by the presence of characteristic peaks for PEI (at ? 2.8-3.2 ppm), CyD (at ? 5.2, 3.8-4.0 and 3.4-3.6 ppm) and TAT (at ? 1.6-1.9 and 6.8-7.2 ppm) in the (1)H NMR spectrum of TAT-PEI-?-CyD. The polymer-plasmid-DNA polyplex could condense DNA at an N/P ratio of 7.0-8.0, and form nanoparticles with the size of 150.6±5.6 nm at its optimal N/P ratio (20/1). By examining the transfection efficiency and cytotoxicity of TAT-PEI-?-CyD, conjugation of the TAT peptide onto PEI-?-CyD was demonstrated to improve the transfection efficiency of PEI-?-CyD in PMSCs after 48 and 96 hours of post-transfection incubation. The viability of PEI-?-CyD-treated PMSCs was shown to be over 80% after 5 h of treatment and 24 h of post-treatment incubation. In summary, this study showed that the TAT-PEI-?-CyD polymer as a vector for plasmid DNA delivery to PMSCs and other cells warrants further investigations. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s12668-011-0010-9) contains supplementary material, which is available to authorized users.

SUBMITTER: Lai WF

PROVIDER: S-EPMC3460531 | biostudies-literature | 2011 Sep

REPOSITORIES: biostudies-literature

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Cyclodextrin-PEI-Tat Polymer as a Vector for Plasmid DNA Delivery to Placenta Mesenchymal Stem Cells.

Lai Wing-Fu WF Tang Gu-Ping GP Wang Xin X Li Guo G Yao Hong H Shen Zan Z Lu Gang G Poon Wai Sang WS Kung Hsiang-Fu HF Lin Marie C M MC

BioNanoScience 20110618 3

This study aims to modify a cyclodextrin-PEI-based polymer, PEI-β-CyD, with the TAT peptide for plasmid DNA delivery to placenta mesenchymal stem cells (PMSCs). By using the disulfide exchange between the SPDP-activated PEI-β-CyD and TAT peptide, the TAT-PEI-β-CyD polymer was fabricated and the success of this was confirmed by the presence of characteristic peaks for PEI (at δ 2.8-3.2 ppm), CyD (at δ 5.2, 3.8-4.0 and 3.4-3.6 ppm) and TAT (at δ 1.6-1.9 and 6.8-7.2 ppm) in the (1)H NMR spectrum of ...[more]

PMID: 23024930

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Project description:siRNA treatment has great promise to specifically control gene expression and select cell behaviors but has delivery challenges limiting its use. Particularly for applications in regenerative medicine, uniform and consistent delivery of siRNA to control gene expression and subsequent stem cell functions, such as differentiation, is paramount. Therefore, a diblock copolymer was examined for its ability to effectively deliver siRNA to mesenchymal stem cells (MSCs). The diblock copolymers, which are composed of cationic blocks for siRNA complexation, protection, and uptake and pH-responsive blocks for endosomal escape, were shown to facilitate nearly 100% MSC uptake of siRNA. This is vastly superior to a commercially available control, DharmaFECT, which resulted in only ~60% siRNA positive MSCs. Moreover, the diblock copolymer, at conditions that result in excellent knockdown (down to ~10% of control gene expression), was cytocompatible, causing no negative effects on MSC survivability. In contrast, DharmaFECT/siRNA treatment resulted in only ~60% survivability of MSCs. Longitudinal knockdown after siRNA treatment was examined and protein knockdown persists for ~6 days regardless of delivery system (diblock copolymer or DharmaFECT). Finally, MSC phenotype and differentiation capacity was examined after treatment with control siRNA. There was no statistically significant differences on cell surface markers of diblock copolymer/siRNA or DharmaFECT/siRNA-treated or cells measured 2 weeks after siRNA delivery compared to untreated cells. Upon differentiation with typical media/culture conditions to adipogenic, chondrogenic, and osteogenic lineages and examination of histological staining markers, there was no discernible differences between treated and untreated cells, regardless of delivery mechanism. Thus, diblock copolymers examined herein facilitated uniform siRNA treatment of MSCs, inducing siRNA-specific gene and protein knockdown without adversely affecting MSC survival or differentiation capacity and therefore show great promise for use within regenerative medicine applications.

Project description:BackgroundThe therapeutic potential of mesenchymal stem cells (MSCs) may be attributed partly to humoral factors such as growth factors, cytokines, and chemokines. Human term placental tissue-derived MSCs (PlaMSCs), or conditioned medium left over from cultures of these cells, have been reported to enhance angiogenesis. Recently, the exosome, which can transport a diverse suite of macromolecules, has gained attention as a novel intercellular communication tool. However, the potential role of the exosome in PlaMSC therapeutic action is not well understood. The purpose of this study was to evaluate PlaMSC-derived exosome angiogenesis promotion in vitro and in vivo.MethodsMSCs were isolated from human term placental tissue by enzymatic digestion. Conditioned medium was collected after 48-h incubation in serum-free medium (PlaMSC-CM). Angiogenic factors present in PlaMSC-CM were screened by a growth factor array. Exosomes were prepared by ultracentrifugation of PlaMSC-CM, and confirmed by transmission electron microscopy, dynamic light scattering, and western blot analyses. The proangiogenic activity of PlaMSC-derived exosomes (PlaMSC-exo) was assessed using an endothelial tube formation assay, a cell migration assay, and reverse transcription-PCR analysis. The in-vivo angiogenic activity of PlaMSC-exo was evaluated using a murine auricle ischemic injury model.ResultsPlaMSC-CM contained both angiogenic and angiostatic factors, which enhanced endothelial tube formation. PlaMSC-exo were incorporated into endothelial cells; these exosomes stimulated both endothelial tube formation and migration, and enhanced angiogenesis-related gene expression. Laser Doppler blood flow analysis showed that PlaMSC-exo infusion also enhanced angiogenesis in an in-vivo murine auricle ischemic injury model.ConclusionsPlaMSC-exo enhanced angiogenesis in vitro and in vivo, suggesting that exosomes play a role in the proangiogenic activity of PlaMSCs. PlaMSC-exo may be a novel therapeutic approach for treating ischemic diseases.

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Cyclodextrin-PEI-Tat Polymer as a Vector for Plasmid DNA Delivery to Placenta Mesenchymal Stem Cells.

Publications

Cyclodextrin-PEI-Tat Polymer as a Vector for Plasmid DNA Delivery to Placenta Mesenchymal Stem Cells.

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