Campus Access Only

All rights reserved. This publication is intended for use solely by faculty, students, and staff of University of the Pacific. No part of this publication may be reproduced, distributed, or transmitted in any form or by any means, now known or later developed, including but not limited to photocopying, recording, or other electronic or mechanical methods, without the prior written permission of the author or the publisher.

Modified alginates as a matrix for gene transfection in a HeLa cell model

Author

Kiran Padmanabhan, University of the Pacific

Date of Award

2000

Document Type

Thesis - Pacific Access Restricted

Degree Name

Master of Science (M.S.)

Department

Pharmaceutical and Chemical Sciences

First Advisor

Timothy J. Smith

First Committee Member

Paul A. Richmond

Second Committee Member

James W. Blankenship

Third Committee Member

Denis J. Meerdink

Abstract

The development of safe, efficient and well-characterized vector systems is one of the greatest limiting factors in gene therapy and transfection methodology. A number of synthetic vectors such as cationic polymers and liposomes have been developed for introducing 'therapeutic' genetic material into the cell. These approaches have the distinct advantage over viral vectors in being relatively non-immunogenic and noninfective. Though the efficiency of this procedure is poor compared to viruses, developing an effective and safe delivery system is a continuing challenge in pharmaceutical research and development.

Five novel polymer matrices were developed by chemically modifying alginic acid. Alginic acid is a linear co-polymer consisting of ~-(1-4)-D-mannuronic acid and a- (1-4)-L-guluronic acid. The carbodiimide coupling method was used to couple aromatic amines to the carboxylic acid functional groups on the alginate backbone, thus giving it a cationic character. Diaminoacridine hydrochloride, thionin, basic fuchsin, acridine yellow and diaminomethyl triazine were the aromatic amines used. The polymers were then tested for their efficiency in transfecting the pSV -P-Galactosidase control vector into HeLa cells. Expression of the reporter protein, P-Galactosidase, was quantified using a colorimetric assay. Compared to the controls, the modified compounds showed moderate to significant improvements in transfection capability, with one exception. The matrix prepared by modifying alginate with diaminoacridine hydrochloride proved lethal to the growth of cells in culture. The use of the alginate basic fuchsin matrix resultes in a significant increase in transfection compared to the controls. The polymers prepared using thionin and acridine yellow showed moderate improvements in transfection, when compared to controls. The complex formation between the plasmid and the alginate-basic fuchsin polymer matrix was evaluated using Transmission Electron Microscopy (TEM). Although there was a significant improvement in transfection with basic fuchsin-alginate polymer, the levels of protein expression appeared to be low in this system. Nevertheless, these results demonstrate the potential of modified alginates as vectors for gene delivery.

Pages

81

Recommended Citation

Padmanabhan, Kiran. (2000). Modified alginates as a matrix for gene transfection in a HeLa cell model. University of the Pacific, Thesis - Pacific Access Restricted. https://scholarlycommons.pacific.edu/uop_etds/547