Capsaicin-Loaded Chitosan Nanocapsules for wtCFTR-mRNA Delivery to a Cystic Fibrosis Cell Line

Abstract

Cystic fibrosis (CF), a lethal hereditary disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene coding for an epithelial chloride channel, is characterized by an imbalanced homeostasis of ion and water transports in secretory epithelia. As the disease is single-gene based, transcript therapy using therapeutic mRNA is a promising concept of treatment in order to correct many aspects of the fatal pathology on a cellular level. Hence, we developed chitosan nanocapsules surface-loaded with wtCFTR-mRNA to restore CFTR function. Furthermore, we loaded the nanocapsules with capsaicin, aiming to enhance the overall efficiency of transcript therapy by reducing sodium hyperabsorption by the epithelial sodium channel (ENaC). Dynamic light scattering with non-invasive back scattering (DLS-NIBS) revealed nanocapsules with an average hydrodynamic diameter of ~200 nm and a Zeta potential of ~+60 mV. The results of DLS-NIBS measurements were confirmed by asymmetric flow field-flow fractionation (AF4) with multidetection, while transmission electron microscopy (TEM) images confirmed the spherical morphology and size range. After stability measurements showed that the nanocapsules were highly stable in cell culture transfection medium, and cytotoxicity was ruled out, transfection experiments were performed with the CF cell line CFBE41o-. Finally, transepithelial measurements with a new state-of-the-art Ussing chamber confirmed successfully restored CFTR function in transfected cells. This study demonstrates that CS nanocapsules as a natural and non-toxic delivery system for mRNA to target cells could effectively replace risky vectors for gene delivery. The nanocapsules are not only suitable as a transcript therapy for treatment of CF, but open aspiring possibilities for safe gene delivery in general.

Microencapsulated Chitosan-Based Nanocapsules: A New Platform for Pulmonary Gene Delivery

“…In that study, the level of transfection was highly dependent on the excipient used for the preparation of the powders, being the MS prepared with HA the ones that gave the best results [ 80 ]—which could be expected taking into account the HA transfection enhancer effect. CS NCs have already been proposed for lung delivery of genes [ 37 ], but they had not been assessed in vivo. The main novelty of this work is that we devised the microencapsulation of the genetic material-loaded CS-based NCs in Ma MS to facilitate their pulmonary administration in dry powders form, leading to revealing results.…”

“…In addition, an interesting advantage of the CS-based NCs is that they can be used for a combined gene therapy, loading the genetic material on the coat and an adjuvant lipophilic molecule, in the core. This molecule can enhance the transfection, like capsaicin [ 37 ]—which decreases the thickness of the mucus layer [ 81 ] and opens the intercellular tight junctions in a reversible way [ 82 , 83 ]. The versatility of the simultaneous transport of HA and different active molecules in the same simple structure allows to carry out a synergistic transfection effect, being of enormous value for a more effective treatment of genetic lung diseases.…”

“…CS-based NCs were administered in vivo by the oral, nasal and ocular routes, but never before by the pulmonary route for gene therapy purposes [ 1 , 2 , 3 , 4 ], as in this study. In fact, they have recently been evaluated in vitro for gene therapy of CF—by administration of wtCFTR-mRNA in the CF cell line CFBE41o- [ 37 ]—confirming their interest in this field. However, their potential for lung gene therapy has not been investigated in vivo.…”

“…They have been assayed as carriers of lipophilic molecules—like anti-inflammatory, antibacterial, and antitumoral drugs (as docetaxel, for the treatment of breast, ovarian and non-small cells lung cancer)—and hydrosoluble AIs—such as peptides, proteins, vaccines (as tetanus toxoid), antigens (as of recombinant hepatitis B surface, influenza and IutA E. coli) and polynucleotides, among many others [ 30 , 31 , 36 ]. In this sense, CS-based NCs are very interesting in gene therapy because they allow the combined administration of genetic material (hydrophilic) on their surface, as well as an adjuvant molecule (lipophilic) in the oily nucleus [ 37 ]. CS-based NCs were administered in vivo by the oral, nasal and ocular routes, but never before by the pulmonary route for gene therapy purposes [ 1 , 2 , 3 , 4 ], as in this study.…”

Biotechnology-Derived Chitosans with Non-Random Patterns of Acetylation Differ from Conventional Chitosans in Their Properties and Activities

“…5). The two commercial chitosans have been used in biomedical research, especially for the transfection of human cells [54][55][56] . We therefore prepared polyplexes by mixing these chitosans with plasmid DNA encoding eGFP at different molar charge chitosan/DNA ratios.…”