Chitosan in Non-Viral Gene Delivery: Role of Structure, Characterization Methods, and Insights in Cancer and Rare Diseases Therapies

Abstract

Non-viral gene delivery vectors have lagged far behind viral ones in the current pipeline of clinical trials of gene therapy nanomedicines. Even when non-viral nanovectors pose less safety risks than do viruses, their efficacy is much lower. Since the early studies to deliver pDNA, chitosan has been regarded as a highly attractive biopolymer to deliver nucleic acids intracellularly and induce a transgenic response resulting in either upregulation of protein expression (for pDNA, mRNA) or its downregulation (for siRNA or microRNA). This is explained as the consequence of a multi-step process involving condensation of nucleic acids, protection against degradation, stabilization in physiological conditions, cellular internalization, release from the endolysosome ("proton sponge" effect), unpacking and enabling the trafficking of pDNA to the nucleus or the siRNA to the RNA interference silencing complex (RISC). Given the multiple steps and complexity involved in the gene transfection process, there is a dearth of understanding of the role of chitosan's structural features (M_w and degree of acetylation, DA%) on each step that dictates the net transfection efficiency and its kinetics. The use of fully characterized chitosan samples along with the utilization of complementary biophysical and biological techniques is key to bridging this gap of knowledge and identifying the optimal chitosans for delivering a specific gene. Other aspects such as cell type and administration route are also at play. At the same time, the role of chitosan structural features on the morphology, size and surface composition of synthetic virus-like particles has barely been addressed. The ongoing revolution brought about by the recent discovery of CRISPR-Cas9 technology will undoubtedly be a game changer in this field in the short term. In the field of rare diseases, gene therapy is perhaps where the greatest potential lies and we anticipate that chitosans will be key players in the translation of research to the clinic.

Keywords: chitosan structure; gene delivery; non-viral vectors; pDNA; siRNA.

Figure 1

Barriers to successful in vivo delivery of nucleic acids.

Figure 2

Schematic representation of the “proton sponge” hypothesis in which the endosomes containing the complexes with protonable polymers (A) evolve to late endosomes where protons are introduced by ATPase proton pumps, producing protonation of the polymer and a reduction in the pH (B); subsequently, chloride ions will be introduced in a non-active way causing a water inflow due to the osmotic pressure (C). Swelling of the endosomes leads to their rupture and finally release of their content into the cytoplasm (D).

Figure 3

Preparation of chitosan-based DNA/siRNA nanoparticles following different strategies.

Figure 4

Transfection efficiency expressed as downregulation of JAM-A mRNA in MCF-7 cells: (A) complexes containing CS HDP-12 at (N/P) charge ratio = 1.5; and (B) complexes containing CS HDP-1.9, HDP-12, HDP-29 and HDP-49 at (N/P) charge ratio = 8. Duplex miRNA (dose 1× = 0.05 nmol/well), DharmaFECT (5 µL/well) and Novafect O 25 were used as controls. Data represent mean values (± SD) of three independent biological experiments and three technical replicates. Statistical comparisons were between each treatment and the control of untreated cells using non-parametric Kruskal–Wallis test (* p < 0.1; **; p < 0.01***; p < 0.001; **** p < 0.0001). Source Santos-Carballal et al. Scientific Reports 5, Article number: 13567 (2015) doi:10.1038/srep13567 [81], licensed under a Creative Commons Attribution 4.0 International License.

Figure 5

Representative TEM images of complexes containing: (A) CS HDP-29 N/P = 8; and (B) CS LDP-25 N/P = 0.6 stained with uranyl acetate. The embedded table shows the measured diameter of the complexes using ImageJ v1.49n (n = 8; mean average ± SD). Source Santos-Carballal et al. Scientific Reports 5, Article number: 13567 (2015) doi:10.1038/srep13567 [81], licensed under a Creative Commons Attribution 4.0 International License.

Figure 6

Tapping mode AFM height topographs of: uncomplexed pBR322 (A); and linear DNA (C); alongside with complexes of these formed when mixed with the chitosan C (0.01,162) (B, D) cDNA 4 µg/mL and N/P = 1. Reprinted with permission from Danielsen et al. (2004) Biomacromolecules 5, 928–936 [120]. Copyright 2004 American Chemical Society.

Figure 7

General principle of SPR, where n2 is the refractive index of medium with lower refractive index, E is the evanescent field amplitude, k_sp is the wavevector of surface plasmons, and k_x is the wavevector of photon.

Figure 8

Saturation curve for hsa-miR-145-5p with HDP-12. Acetate buffer (35 mM, pH 5.1/10 mM NaCl) (n = 2). Source Santos-Carballal et al. Scientific Reports 5, Article number: 13567 (2015) doi:10.1038/srep13567 [81], licensed under a Creative Commons Attribution 4.0 International License.

Figure 9

Schematic view of an isothermal titration calorimeter.

Figure 10

Simplified scheme of RNA interference mechanism in mammalian cells. Only processes mentioned in the text are illustrated.