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Development of a Cationic Mucic Acid Polymer-Based Nanoparticle siRNA Delivery System

Citation

Pan, Dorothy Weichi (2016) Development of a Cationic Mucic Acid Polymer-Based Nanoparticle siRNA Delivery System. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/Z9VM497J . http://resolver.caltech.edu/CaltechTHESIS:05092016-155820873

Abstract

Cancer chemotherapy has advanced from highly toxic drugs to more targeted treatments in the last 70 years. Chapter 1 opens with an introduction to targeted therapy for cancer. The benefits of using a nanoparticle to deliver therapeutics are discussed. We move on to siRNA in particular, and why it would be advantageous as a therapy. Specific to siRNA delivery are some challenges, such as nuclease degradation, quick clearance from circulation, needing to enter cells, and getting to the cytosol. We propose the development of a nanoparticle delivery system to tackle these challenges so that siRNA can be effective.

Chapter 2 of this thesis discusses the synthesis and analysis of a cationic mucic acid polymer (cMAP) which condenses siRNA to form a nanoparticle. Various methods to add polyethylene glycol (PEG) for stabilizing the nanoparticle in physiologic solutions, including using a boronic acid binding to diols on mucic acid, forming a copolymer of cMAP with PEG, and creating a triblock with mPEG on both ends of cMAP. The goal of these various pegylation strategies was to increase the circulation time of the siRNA nanoparticle in the bloodstream to allow more of the nanoparticle to reach tumor tissue by the enhanced permeation and retention effect. We found that the triblock mPEG-cMAP-PEGm polymer condensed siRNA to form very stable 30-40 nm particles that circulated for the longest time – almost 10% of the formulation remained in the bloodstream of mice 1 h after intravenous injection.

Chapter 3 explores the use of an antibody as a targeting agent for nanoparticles. Some antibodies of the IgG1 subtype are able to recruit natural killer cells that effect antibody dependent cellular cytotoxicity (ADCC) to kill the targeted cell to which the antibody is bound. There is evidence that the ADCC effect remains in antibody-drug conjugates, so we wanted to know whether the ADCC effect is preserved when the antibody is bound to a nanoparticle, which is a much larger and complex entity. We utilized antibodies against epidermal growth factor receptor with similar binding and pharmacokinetics, cetuximab and panitumumab, which differ in that cetuximab is an IgG1 and panitumumab is an IgG2 (which does not cause ADCC). Although a natural killer cell culture model showed that gold nanoparticles with a full antibody targeting agent can elicit target cell lysis, we found that this effect was not preserved in vivo. Whether this is due to the antibody not being accessible to immune cells or whether the natural killer cells are inactivated in a tumor xenograft remains unknown. It is possible that using a full antibody still has value if there are immune functions which are altered in a complex in vivo environment that are intact in an in vitro system, so the value of using a full antibody as a targeting agent versus using an antibody fragment or a protein such as transferrin is still open to further exploration.

In chapter 4, nanoparticle targeting and endosomal escape are further discussed with respect to the cMAP nanoparticle system. A diboronic acid entity, which gives an order of magnitude greater binding (than boronic acid) to cMAP due to the vicinal diols in mucic acid, was synthesized, attached to 5kD or 10kD PEG, and conjugated to either transferrin or cetuximab. A histidine was incorporated into the triblock polymer between cMAP and the PEG blocks to allow for siRNA endosomal escape. Nanoparticle size remained 30-40 nm with a slightly negative ca. -3 mV zeta potential with the triblock polymer containing histidine and when targeting agents were added. Greater mRNA knockdown was seen with the endosomal escape mechanism than without. The nanoparticle formulations were able to knock down the targeted mRNA in vitro. Mixed effects suggesting function were seen in vivo.

Chapter 5 summarizes the project and provides an outlook on siRNA delivery as well as targeted combination therapies for the future of personalized medicine in cancer treatment.

Item Type:Thesis (Dissertation (Ph.D.))
Subject Keywords:nanoparticle; siRNA; polymer;
Degree Grantor:California Institute of Technology
Division:Chemistry and Chemical Engineering
Major Option:Chemistry
Awards:Graduate Deans’ Award for Outstanding Community Service, 2016
Thesis Availability:Public (worldwide access)
Research Advisor(s):
  • Davis, Mark E.
Thesis Committee:
  • Heath, James R. (chair)
  • Davis, Mark E.
  • Campbell, Judith L.
  • Tirrell, David A.
Defense Date:5 May 2016
Record Number:CaltechTHESIS:05092016-155820873
Persistent URL:http://resolver.caltech.edu/CaltechTHESIS:05092016-155820873
DOI:10.7907/Z9VM497J
Related URLs:
URLURL TypeDescription
http://dx.doi.org/10.1021/acs.bioconjchem.5b00324DOIArticle adapted for ch. 2
http://dx.doi.org/10.1021/acs.bioconjchem.5b00139DOIArticle adapted for ch. 3
ORCID:
AuthorORCID
Pan, Dorothy Weichi0000-0003-4066-7750
Default Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:9714
Collection:CaltechTHESIS
Deposited By: Dorothy Pan
Deposited On:11 May 2016 16:27
Last Modified:14 Jun 2016 18:55

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