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A 3D SPHEROID MODEL AS A PLATFORM TO STUDY NON-VIRAL GENE DELIVERY SYSTEM

Chen, Yuzhe (2015) A 3D SPHEROID MODEL AS A PLATFORM TO STUDY NON-VIRAL GENE DELIVERY SYSTEM. Master's Thesis, University of Pittsburgh. (Unpublished)

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Abstract

Non-viral gene delivery has emerged as a promising strategy in the past two decades for gene therapy. However, it is difficult to translate non-viral gene delivery into clinical trials. This is because of the low transfection efficiency of non-viral vectors and little knowledge about transfection mechanism in 3D tissues. The complex microenvironment of 3D tissues results in the poor relevance of non-viral transfection in conventional 2D monolayer cells and 3D tissues. Here, we develop a 3D multicellular spheroid model to increase the understanding of the gene delivery process by non-viral vectors in 3D tissues. We synthesized Spermine-conjugated-Dextran (SD), non-viral vector, for gene delivery to 3D tissues. Then, we utilized micro-fabrication technique to prepare 3D spheroids with uniform size in a high throughput manner. We compared the transfection of SD/DNA nanocomplex in 2D cells and 3D spheroids. Our results revealed that the transfection efficiency and cytotoxicity of non-viral vectors were lower in 3D spheroids than those in 2D monolayer cells. We used confocal microscopy to visualize the cellular uptake of SD/DNA complex in monolayer cells and spheroids. The results demonstrated that SD vector was limited to the external layers of cells in 3D spheroids and couldn’t penetrate into the core of spheroids. Taken together, our study indicates that the non-viral transfection behavior in 3D spheroid model is different from that in 2D cells, and this model may be more relevant to in vivo setting than that of conventional 2D monolayer cell models. This system also provides a useful high throughput in vitro screening platform for the novel non-viral gene delivery system testing in the future.


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Details

Item Type: University of Pittsburgh ETD
Status: Unpublished
Creators/Authors:
CreatorsEmailPitt UsernameORCID
Chen, Yuzheyuc67@pitt.eduYUC67
ETD Committee:
TitleMemberEmail AddressPitt UsernameORCID
Thesis AdvisorSant, Shilpashs149@pitt.eduSHS149
Committee MemberLi, Jiangjil35@pitt.eduJIL35
Committee MemberSant, Vinayakvis45@pitt.eduVIS45
Date: 30 July 2015
Date Type: Publication
Defense Date: 23 July 2015
Approval Date: 30 July 2015
Submission Date: 28 July 2015
Access Restriction: 5 year -- Restrict access to University of Pittsburgh for a period of 5 years.
Number of Pages: 44
Institution: University of Pittsburgh
Schools and Programs: School of Pharmacy > Pharmaceutical Sciences
Degree: MS - Master of Science
Thesis Type: Master's Thesis
Refereed: Yes
Uncontrolled Keywords: Non-viral gene delivery, 3D spheroids, cationic polysaccharide vectors.
Date Deposited: 30 Jul 2015 14:46
Last Modified: 15 Nov 2016 14:29
URI: http://d-scholarship.pitt.edu/id/eprint/25829

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