Investigation of Low Molecular Weight Chitosan Nanoparticles for CpG ODN 1826 Delivery

• Author / Creator

  Babii, Oksana

• Synthetic oligodeoxynucleotides containing unmethylated CpG motifs (CpG ODNs) are powerful stimulators of innate and adaptive immune responses, exerting their activity through the interaction with endolysosomal Toll-like receptor 9 (TLR9) expressed by antigen presenting cells (APCs). The strong immunopotency and a wide range of activity support the use of CpG ODNs as an effective treatment and prevention strategy to combat various infectious diseases. Recent evidence suggested that CpG ODNs might be a potent candidate for Bovine respiratory disease immunotherapy in the livestock. Nevertheless, their stimulatory activity is often transient due to the high susceptibility of free CpG ODNs to the serum nuclease degradation, poor targeting capacity, and inefficient cellular uptake. The overall objective of this research was to develop a vector system based on chitosan nanoparticles suitable for the efficient CpG ODN delivery to the target APCs, with minimal cellular toxicity. In particular, the influence of molecular structure of chitosan, such as the molecular weight, the degree of deacetylation and mannose grafting on the properties of CpG ODN-loaded nanoparticles was investigated. Chitosan samples with the molecular weights of 5 and 15 kDa and the degree of deacetylation of 50 and 80 % were prepared. Additionally, mannosylated chitosans with a substitution degree of 15% were synthesized. The self-assembled chitosan nanoparticles were produced by ionic gelation method using poly (L‐glutamic acid) as a cross-linking agent. The CpG ODN – loaded nanoparticles had the encapsulation efficiency over 88%, average hydrodynamic diameters ranging from 101.8 to 184.5 nm, and zeta potential values from +20.1 to +30.1 mV, providing desirable size and charge for targeting APCs.It was found that physicochemical properties and in vitro immunostimulatory effect of CpG ODN-loaded nanoparticles were strongly dependent on the chitosan molecular weight and degree of deacetylation. The size of nanoparticles was significantly reduced by lowering the chitosans molecular weight from 15 to 5 kDa. Moreover, chitosan with the molecular weight of 15 kDa formed more stable nanoparticles compared to 5 kDa sample due to the stronger chain entanglement effect. Chitosans with the molecular weight of 5 kDa tent to dissociate at pH 5.9 and prematurely release their cargo, which compromised their efficacy as a vector for the CpG ODN delivery. At the same time, increasing the degree of deacetylation facilitated the formation of nanoparticles with smaller sizes and higher surface charges due to the increased cationic charge density on the chitosan backbone. Samples with a higher degree of deacetylation exhibited a better CpG ODNs binding ability and were able to assemble into more stable nanoparticles when cross-linked by poly (L‐glutamic acid).There was no evidence of cellular toxicity of chitosan nanoparticles regardless of the molecular weight, degree of deacetylation or mannose grafting towards the RAW 264.7 cells. Furthermore, CpG ODN-loaded nanoparticles were biologically active, showing successful stimulation of IL-6 secretion in RAW 264.7 cells. The most efficient immunostimulatory effect was observed while using 50% acetylated and mannosylated chitosan samples with the molecular weight of 15 kDa. The decreased charge density of the chitosan backbone resulted in enhanced intracellular CPG ODN release, which promoted cytokine secretion in vitro. Overall, these finding extended the application of chitosan-based nanoparticles as efficient vectors for the intracellular delivery of CpG ODNs and revealed their potential use for the Bovine respiratory disease immunotherapy.

• Subjects / Keywords

  • Gene delivery
  • immunostimulatory activity
  • Nanoparticles
  • Chitosan
  • CpG ODN

• Graduation date

  Spring 2019

• Type of Item

  Thesis

• Degree

  Master of Science

• DOI

  https://doi.org/10.7939/r3-k83j-fc09

• License

  Permission is hereby granted to the University of Alberta Libraries to reproduce single copies of this thesis and to lend or sell such copies for private, scholarly or scientific research purposes only. Where the thesis is converted to, or otherwise made available in digital form, the University of Alberta will advise potential users of the thesis of these terms. The author reserves all other publication and other rights in association with the copyright in the thesis and, except as herein before provided, neither the thesis nor any substantial portion thereof may be printed or otherwise reproduced in any material form whatsoever without the author's prior written permission.