| Summary: | 碩士 === 國立清華大學 === 生醫工程與環境科學系 === 103 === Gene transfection is the technique capable of directly manipulating cellular gene expression, which can be applied for disease treatment via regulating cell physiology. In the past decade, human mesenchymal stem cells (hMSCs) have received great attention for their enormous potential on cancer therapy and regenerative medicine. Applications of gene transfection techniques on hMSCs further advanced their medical applications. One of the key tasks for such integration is the development of safe and efficient gene delivery tools for hMSCs.
In this study, we proposed a polymer/superparamagnetic iron oxide nanoparticles (SPIONs) polyplex (PNT) system, comprising of γ-poly (glutamic acid) (γPGA)-modified SPIONs (γPGA-SPIONs), poly (β-amino esters) (PAE), and plasmid DNA (pDNA) for efficient magnetically-assisted gene delivery (magnetofection) to hMSCs. SPIONs were prepared using thermal decomposition method, and γPGA-SPIONs were synthesized using a ligand exchange process. The magnetization of γPGA-SPIONs was up to 39.8 emμ/g measured by SQUID, which showed significant contrast enhancement on MRI T2-weighed imaging (r2 value = 334.7 mM-1s-1) of hMSCs. To prepare PNT system, PAE was used to fully condensed pDNA at or above the weight ratio (PAE pDNA) of 20. Afterwards, the polyplexes were combined with γPGA-SPIONs via electrostatic interactions to form PNT. PNT-mediated magnetofection efficiency was optimized by studying several key transfection parameters, including: polymer/pDNA weight ratio, polymer dilution factor and amount of γPGA-SPIONs. The transfection efficiency of PNT was greatly enhanced by applying with an external magnetic attraction. Under optimized magnetofection conditions, comparing to PAE polyplexes, PNT increased cellular uptake of pDNA up to 3-fold under serum-free condition. Additionally, PNT showed low cytotoxicity (viability ~ 90%) and exhibited excellent magnetofection efficiency (> 70%) on hMSCs compared to other commercial transfection agents. Additionally, PNT-mediated magnetofection did not cause detrimental effects on the osteogenic differentiation and tumor tropism of hMSCs.
In this study, we investigated the application potential of PNT magnetofection system on two important bioengineering aspects: (1) Cancer therapy and (2) Directing stem cell differentiation.
1. Cancer therapy: hMSCs were transfected to express tumor necrosis factor-related apoptosis inducing ligand (TRAIL) by using PNT magnetofection technique. TRAIL protein was successfully detected from the transfected hMSCs by ELISA assay. 5-fold increased on TRAIL expression was attained by PNT magnetofection compared to Lipofectamine 2000 (18.0 ng/mg → 86.3 ng/mL). The therapeutic potential of TRAIL-expressing hMSCs (TRAILhMSCs) for cancer therapy was explored on HeLa cells using an in vitro co-culture model. The results demonstrated that TRAILhMSCs could induce significant apoptosis on HeLa cells.
2. Directing stem cell chondrogenic differentiation: The genes known to promote stem cell chondrogensis, such as TGF-β and Sox9, were separately delivered using the PNT technique. Expression of TGF-β and Sox9 by PNT magnetofection was 5- and 4-fold higher than gene delivery by Lipofectamine 2000 respectively. After magnetofection for 4 weeks, the enhanced expression of chondrocyte-specific biomacromolecules, such as glycosaminoglycans (GAGs) and collagen II was observed from TGF-β- or Sox9-transfected hMSCs using Alcian blue staining and immunofluorescence staining. Finally, the expression amount of GAGs and collagen were quantitated by biochemical assay, showing around 2-fold increase of GAGs and 4-fold increase of collagen expression from TGF-β- or Sox9-transfected hMSCs compared to the control groups.
Taken together, we’ve successfully created a novel PNT Magnetofection system with excellent gene delivery efficiency and negligible cytotoxicity on hMSCs. Our current results suggest that the PNT-mediated genetically-engineered hMSCs possesses great potential on both cancer therapy and tissue regeneration.
|
|---|
