Development of Covalent Chitosan-Polyethylenimine Derivatives as Gene Delivery Vehicle: Synthesis, Characterization, and Evaluation.

There is an increasing interest in cationic polymers as important constituents of non-viral gene delivery vectors. In the present study, we developed a versatile synthetic route for the production of covalent polymeric conjugates consisting of water-soluble depolymerized chitosan (dCS; MW 6-9 kDa) and low molecular weight polyethylenimine (PEI; 2.5 kDa linear, 1.8 kDa branched). dCS-PEI derivatives were evaluated based on their physicochemical properties, including purity, covalent bonding, solubility in aqueous media, ability for DNA condensation, and colloidal stability of the resulting polyplexes. They were complexed with non-integrating DNA vectors coding for reporter genes by simple admixing and assessed in vitro using liver-derived HuH-7 cells for their transfection efficiency and cytotoxicity. Using a rational screening cascade, a lead compound was selected (dCS-Suc-LPEI-14) displaying the best balance of biocompatibility, cytotoxicity, and transfection efficiency. Scale-up and in vivo evaluation in wild-type mice allowed for a direct comparison with a commercially available non-viral delivery vector (in vivo-jetPEI). Hepatic expression of the reporter gene luciferase resulted in liver-specific bioluminescence, upon intrabiliary infusion of the chitosan-based polyplexes, which exceeded the signal of the in vivo jetPEI reference formulation by a factor of 10. We conclude that the novel chitosan-derivative dCS-Suc-LPEI-14 shows promise and potential as an efficient polymeric conjugate for non-viral in vivo gene therapy.

A DNA biosensors-based microfluidic platform for attomolar real-time detection of unamplified SARS-CoV-2 virus.

The emergence of the coronavirus 2019 (COVID-19) arose the need for rapid, accurate and massive virus detection methods to control the spread of infectious diseases. In this work, a device, deployable in non-medical environments, has been developed for the detection of non-amplified SARS-CoV-2 RNA. A SARS-CoV-2 specific probe was designed and covalently immobilized at the surface of glass slides to fabricate a DNA biosensor. The resulting system was integrated in a microfluidic platform, in which viral RNA was extracted from non-treated human saliva, before hybridizing at the surface of the sensor. The formed DNA/RNA duplex was detected in presence of SYBR Green I using an opto-electronic system, based on a high-power LED and a photo multiplier tube, which convert the emitted fluorescence into an electrical signal that can be processed in less than 10 min. The limit of detection of the resulting microfluidic platform reached six copies of viral RNA per microliter of sample (equal to 10 aM) and satisfied the safety margin. The absence of non-specific adsorption and the selectivity for SARS-CoV-2 RNA were established. In addition, the designed device could be applicable for the detection of a variety of viruses by simple modification of the immobilized probe.

Chitosan Functionalization: Covalent and Non-Covalent Interactions and Their Characterization.

Chitosan (CS) is a natural biopolymer that has gained great interest in many research fields due to its promising biocompatibility, biodegradability, and favorable mechanical properties. The versatility of this low-cost polymer allows for a variety of chemical modifications via covalent conjugation and non-covalent interactions, which are designed to further improve the properties of interest. This review aims at presenting the broad range of functionalization strategies reported over the last five years to reflect the state-of-the art of CS derivatization. We start by describing covalent modifications performed on the CS backbone, followed by non-covalent CS modifications involving small molecules, proteins, and metal adjuvants. An overview of CS-based systems involving both covalent and electrostatic modification patterns is then presented. Finally, a special focus will be given on the characterization techniques commonly used to qualify the composition and physical properties of CS derivatives.

Selection of Water-Soluble Chitosan by Microwave-Assisted Degradation and pH-Controlled Precipitation.

In the field of gene therapy, chitosan (CS) gained interest for its promise as a non-viral DNA vector. However, commercial sources of CS lack precise characterization and do not generally reach sufficient solubility in aqueous media for in vitro and in vivo evaluation. As low molecular weight CS showed improved solubility, we investigated the process of CS depolymerization by acidic hydrolysis, using either long time heating at 80 °C or short time microwave-enhanced heating. The resulting depolymerized chitosan (dCS) were analyzed by gel permeation chromatography (GPC) and 1H nuclear magnetic resonance (NMR) to determine their average molecular weight (Mn, Mp and Mw), polydispersity index (PD) and degree of deacetylation (DD). We emphasized the production of water-soluble CS (solubility > 5 mg/mL), obtained in reproducible yield and characteristics, and suitable for downstream functionalization. Optimal microwave-assisted conditions provided dCS with a molecular weight (MW) = 12.6 ± 0.6 kDa, PD = 1.41 ± 0.05 and DD = 85%. While almost never discussed in the literature, we observed the partial post-production aggregation of dCS when exposed to phase changes (from liquid to solid). Repeated cycles of freezing/thawing allowed the selection of dCS fractions which were exempt of crystalline particles formation upon solubilization from frozen samples.

Modifying Membrane Morphology and Interactions with DNA Origami Clathrin-Mimic Networks.

We describe the triggered assembly of a bioinspired DNA origami meshwork on a lipid membrane. DNA triskelia, three-armed DNA origami nanostructures inspired by the membrane-modifying protein clathrin, are bound to lipid mono- and bilayers using cholesterol anchors. Polymerization of triskelia, triggered by the addition of DNA staples, links triskelion arms to form a mesh. Using transmission electron microscopy, we observe nanoscale local deformation of a lipid monolayer induced by triskelion polymerization that is reminiscent of the formation of clathrin-coated pits. We also show that the polymerization of triskelia bound to lipid bilayers modifies interactions between them, inhibiting the formation of a synapse between giant unilamellar vesicles and a supported lipid bilayer.