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Fundamental study of silicate substituted nanostructured calcium phosphates (NanoSiCaPs) and 3-D scaffolds for non-viral gene delivery

Shekhar, Sudhanshu (2019) Fundamental study of silicate substituted nanostructured calcium phosphates (NanoSiCaPs) and 3-D scaffolds for non-viral gene delivery. Doctoral Dissertation, University of Pittsburgh. (Unpublished)

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Nanostructured calcium phosphate (NanoCaPs) particles are biocompatible and non-toxic bioceramics widely studied owing to their structural and compositional similarity to the mineralized tissue architecture of native bone. They are therefore considered as an ideal choice for gene delivery applications in bone tissue engineering. However, NanoCaPs are typically characterized by variable transfection, short shelf life due to particle aggregation, and difficulties associated with endosomal escape. The objectives of this dissertation were therefore crafted to develop strategies to circumvent, if not eliminate, some of the limitations that have previously stymied the success of NanoCaPs as a non-viral gene delivery vector for bone tissue engineering applications.
A modified version of NanoCaPs containing critical concentration of silicate ions substitutions were synthesized, aptly called NanoSiCaPs. Fundamental understanding of the influence of silicate ion substitutions in the NanoCaPs lattice structure was conducted using various materials characterization techniques. In-vitro transfection results indicated, a two-fold increase in transfection levels exhibited by NanoSiCaPs, owing to the enhanced dissolution kinetics and ability to limit particle aggregation. Subsequently, two different strategies were developed to achieve scaffold mediated gene delivery via generation of plasmid DNA bound to NanoSiCaPs (NanoSiCaPs complexes, NC). First, a novel and simple coating methodology was developed using NCs adsorbed on Ti-surfaces coated with polyelectrolyte. Surface characterization results indicated successful generation of the nanoceramic coating on the Ti-surfaces. Additionally, it was demonstrated that the Ti-polyelectrolyte-NC assemblies contribute to surface mediated gene transfection, without eliciting any cytotoxicity. Second, a lyophilization technique was developed that enabled long-term storage of the NCs under ambient conditions, without inducing either a significant change in particle size or loss in gene transfection efficiency. Subsequently, a 3-D gene delivery system comprising fibrin hydrogels and lyophilized NCs was developed. In-vitro transfection results indicated that gene expression mediated via synthesized gels can be meticulously controlled by modulating the amounts of fibrinogen and NCs utilized in the synthesis of the gels. In conclusion, the studies demonstrate creation of next generation NanoCaP vectors, NCs and their implementation in the development of 3-D scaffolds serving as effective gene delivery agents as well as functional scaffolds for bone tissue repair and regeneration.


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Item Type: University of Pittsburgh ETD
Status: Unpublished
CreatorsEmailPitt UsernameORCID
Shekhar, Sudhanshusus72@pitt.edusus72
ETD Committee:
TitleMemberEmail AddressPitt UsernameORCID
Committee ChairKumta,
Committee MemberLittle,
Committee MemberBrown,
Committee MemberPhillippi,
Date: 23 January 2019
Date Type: Publication
Defense Date: 16 October 2018
Approval Date: 23 January 2019
Submission Date: 9 November 2018
Access Restriction: 1 year -- Restrict access to University of Pittsburgh for a period of 1 year.
Number of Pages: 172
Institution: University of Pittsburgh
Schools and Programs: Swanson School of Engineering > Bioengineering
Degree: PhD - Doctor of Philosophy
Thesis Type: Doctoral Dissertation
Refereed: Yes
Uncontrolled Keywords: Calcium phosphate, non-viral gene delivery, tissue engineering, bone tissue engineering, scaffolds, gene transfection, nanoparticles, hydrogels, coatings
Date Deposited: 23 Jan 2019 19:47
Last Modified: 23 Jan 2020 06:15


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