Biopolymer-Based Nanoparticles for Cystic Fibrosis Lung Gene Therapy Studies.

Lung gene therapy for cystic fibrosis disease has not been successful due to several challenges such as the absence of an appropriate vector. Therefore, optimal delivery of emerging therapeutics to airway epithelial cells demands suitable non-viral systems. In this work, we describe the formulation and the physicochemical investigation of biocompatible and biodegradable polymeric nanoparticles (NPs), including PLGA and chitosan (animal and non-animal), as novel methods for the safe and efficient delivery of CFTR-specific locked nucleic acids (LNAs).

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

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 (Mw 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.

A Chitosan-Based Liposome Formulation Enhances the In Vitro Wound Healing Efficacy of Substance P Neuropeptide.

Currently, there is considerable interest in developing innovative biodegradable nanoformulations for controlled administration of therapeutic proteins and peptides. Substance P (SP) is a neuropeptide of 11 amino acids that belongs to the tachykinins family and it plays an important role in wound healing. However, SP is easily degradable in vivo and has a very short half-life, so the use of chitosan-based nanocarriers could enhance its pharmaceutical properties. In light of the above, the aim of this work was to produce and characterize chitosan-coated liposomes loaded with SP (SP-CH-LP) as novel biomaterials with potential application in mucosal wound healing. The loaded system's biophysical properties were characterized by dynamic light scattering with non-invasive back scattering (DLS-NIBS), mixed mode measurements and phase analysis light scattering (M3-PALS) and high performance liquid chromatography with ultraviolet/visible light detection (HPLC-UV/VIS). Then, the efficacy of the obtained nanoformulations was examined via proof-of-principle experiments using in vitro cell assays. These assays showed an increment on cell motility and proliferation after treatment with free and encapsulated neuropeptides. Additionally, the effect of SP on wound healing was enhanced by the entrapment on CH-LP. Overall, the amenability of chitosan-based nanomaterials to encapsulate peptides and proteins constitutes a promising approach towards potential novel therapies to treat difficult wounds.

Chitosan as a non-viral co-transfection system in a cystic fibrosis cell line.

Successful gene therapy requires the development of suitable vehicles for the selective and efficient delivery of genes to specific target cells at the expense of minimal toxicity. In this work, we investigated a non-viral gene delivery system based on chitosan (CS) to specifically address cystic fibrosis (CF). Thus, electrostatic self-assembled CS-pEGFP and CS-pEGFP-siRNA complexes were prepared from high-pure fully characterized CS (Mw ∼ 20 kDa and degree of acetylation ∼ 30%). The average diameter of positively-charged complexes (i.e. ζ ∼+25 mV) was ∼ 200 nm. The complexes were found relatively stable over 14h in Opti-MEM. Cell viability study did not show any significant cytotoxic effect of the CS-based complexes in a human bronchial cystic fibrosis cell line (CFBE41o-). We evaluated the transfection efficiency of this cell line with both CS-pEGFP and co-transfected with CS-pEGFP-siRNA complexes at (N/P) charge ratio of 12. We reported an increase in the fluorescence intensity of CS-pEGFP and a reduction in the cells co-transfected with CS-pEGFP-siRNA. This study shows proof-of-principle that co-transfection with chitosan might be an effective delivery system in a human CF cell line. It also offers a potential alternative to further develop therapeutic strategies for inherited disease treatments, such as CF.

SYBR Gold Fluorescence Quenching is a Sensitive Probe of Chitosan-microRNA Interactions.

Competitive dye displacement titration has previously been used to characterize chitosan-DNA interactions using ethidium bromide. In this work, we aim to develop a fast and reliable method using SYBR Gold as a fluorescent probe to evaluate the binding affinity between ssRNA and chitosan. The interaction of chitosan with ssRNA was investigated as a function of temperature, molecular weight and degree of acetylation of chitosan, using competitive dye displacement titrations with fluorescence quenching. Affinity constants are reported, showing the high sensitivity of the interaction to the degree of acetylation of chitosan and barely dependent on the molecular weight. We propose that the mechanism of SYBR Gold fluorescence quenching is governed by both static and dynamic quenching.