Prolonged in vivo gene silencing using multilayered nanoparticles as a sirna delivery system

Currently, there are various diseases without effective mode of cure. Many interventions are hampered by undesirable proteins produced over time. One example is the accumulation of excessive collagen protein at surgical site in post glaucoma surgery patients, rendering surgery failure. Excessive...

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Main Author: Tan, Yang Fei
Other Authors: School of Materials Science & Engineering
Format: Theses and Dissertations
Language:English
Published: 2018
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Online Access:http://hdl.handle.net/10356/75868
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Institution: Nanyang Technological University
Language: English
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spelling sg-ntu-dr.10356-758682023-03-04T16:44:10Z Prolonged in vivo gene silencing using multilayered nanoparticles as a sirna delivery system Tan, Yang Fei School of Materials Science & Engineering DRNTU::Engineering::Materials Currently, there are various diseases without effective mode of cure. Many interventions are hampered by undesirable proteins produced over time. One example is the accumulation of excessive collagen protein at surgical site in post glaucoma surgery patients, rendering surgery failure. Excessive collagen accumulation results in scarring.Current anti-proliferative agents used such as 5 Fluorouracil and Mitomycin C lack specificity, indiscriminately affecting tissues adversely when applied topically or subconjunctivally injected, causing complications. Thus, there is a need for an effective anti-scarring agent with localized, specific action that also gives a prolonged effect over time. siRNA has been explored to prevent fibrosis but its use was limited due to the lack of stable and sustained delivery systems. This work reports on the successful development of a new nanocarrier system for the management of fibrosis. The new carrier has a hydroxy apatite core, with alternating layers of siRNA and a cationic peptide. While the siRNA silences a fibroblast gene responsible for producing a key mediator of fibrosis called SPARC, the nanocarrier is formulated to protect the siRNA, improve cell uptake and ensure specific prolonged silencing of genes for 2 weeks. The Layer by Layer nanoparticle of HA|ARG|siRNA|ARG|siRNA|ARG configuration was 242 nm in hydrodynamic diameter with 49.3 mV surface charge. The nanoparticle has a positively charged outermost layer to protect the siRNA and to bind electrostatically to cellular membranes when added to cells, facilitating cell uptake. FESEM and TEM imaging showed that the NPs were spherical, with smooth surfaces, smaller than 200 nm in diameter and uniform in size. The designed nanoparticles (at various dosages) did not cause any toxic effects on MCF cells over 2 weeks and in vitro studies showed sustained SPARC knockdown without affecting cell growth and maintained siRNA presence in cells for 2 weeks with a single dose treatment. In addition, in vivo studies in the mouse model of conjunctival scarring showed that a single dose injection into the conjunctiva facilitated SPARC knockdown, leading to prolonged efficacy of fibrosis suppression. Moreover, the nanoparticle treatment did not cause tissue apoptosis or foreign body response. Mechanism studies were conducted to investigate the breakdown behaviour of the particles, focusing on the release patterns of siRNA. This is the first demonstration of a stabilized siRNA delivery system showing sustained action against fibrosis over 2 weeks with a single injection. Doctor of Philosophy 2018-06-27T14:06:55Z 2018-06-27T14:06:55Z 2018 Thesis Tan, Y. F. (2018). Prolonged in vivo gene silencing using multilayered nanoparticles as a sirna delivery system. Doctoral thesis, Nanyang Technological University, Singapore. http://hdl.handle.net/10356/75868 10.32657/10356/75868 en 128 p. application/pdf
institution Nanyang Technological University
building NTU Library
continent Asia
country Singapore
Singapore
content_provider NTU Library
collection DR-NTU
language English
topic DRNTU::Engineering::Materials
spellingShingle DRNTU::Engineering::Materials
Tan, Yang Fei
Prolonged in vivo gene silencing using multilayered nanoparticles as a sirna delivery system
description Currently, there are various diseases without effective mode of cure. Many interventions are hampered by undesirable proteins produced over time. One example is the accumulation of excessive collagen protein at surgical site in post glaucoma surgery patients, rendering surgery failure. Excessive collagen accumulation results in scarring.Current anti-proliferative agents used such as 5 Fluorouracil and Mitomycin C lack specificity, indiscriminately affecting tissues adversely when applied topically or subconjunctivally injected, causing complications. Thus, there is a need for an effective anti-scarring agent with localized, specific action that also gives a prolonged effect over time. siRNA has been explored to prevent fibrosis but its use was limited due to the lack of stable and sustained delivery systems. This work reports on the successful development of a new nanocarrier system for the management of fibrosis. The new carrier has a hydroxy apatite core, with alternating layers of siRNA and a cationic peptide. While the siRNA silences a fibroblast gene responsible for producing a key mediator of fibrosis called SPARC, the nanocarrier is formulated to protect the siRNA, improve cell uptake and ensure specific prolonged silencing of genes for 2 weeks. The Layer by Layer nanoparticle of HA|ARG|siRNA|ARG|siRNA|ARG configuration was 242 nm in hydrodynamic diameter with 49.3 mV surface charge. The nanoparticle has a positively charged outermost layer to protect the siRNA and to bind electrostatically to cellular membranes when added to cells, facilitating cell uptake. FESEM and TEM imaging showed that the NPs were spherical, with smooth surfaces, smaller than 200 nm in diameter and uniform in size. The designed nanoparticles (at various dosages) did not cause any toxic effects on MCF cells over 2 weeks and in vitro studies showed sustained SPARC knockdown without affecting cell growth and maintained siRNA presence in cells for 2 weeks with a single dose treatment. In addition, in vivo studies in the mouse model of conjunctival scarring showed that a single dose injection into the conjunctiva facilitated SPARC knockdown, leading to prolonged efficacy of fibrosis suppression. Moreover, the nanoparticle treatment did not cause tissue apoptosis or foreign body response. Mechanism studies were conducted to investigate the breakdown behaviour of the particles, focusing on the release patterns of siRNA. This is the first demonstration of a stabilized siRNA delivery system showing sustained action against fibrosis over 2 weeks with a single injection.
author2 School of Materials Science & Engineering
author_facet School of Materials Science & Engineering
Tan, Yang Fei
format Theses and Dissertations
author Tan, Yang Fei
author_sort Tan, Yang Fei
title Prolonged in vivo gene silencing using multilayered nanoparticles as a sirna delivery system
title_short Prolonged in vivo gene silencing using multilayered nanoparticles as a sirna delivery system
title_full Prolonged in vivo gene silencing using multilayered nanoparticles as a sirna delivery system
title_fullStr Prolonged in vivo gene silencing using multilayered nanoparticles as a sirna delivery system
title_full_unstemmed Prolonged in vivo gene silencing using multilayered nanoparticles as a sirna delivery system
title_sort prolonged in vivo gene silencing using multilayered nanoparticles as a sirna delivery system
publishDate 2018
url http://hdl.handle.net/10356/75868
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