Purification and Surface Modification of Chitosan-based Polyplexes Using Tangential Flow Filtration and Coating by Hyaluronic Acid.

Chitosan (CS)-based polyplexes are produced by spontaneous electrostatic association with nucleic acids using CS in excess. Interactions of positively charged polyplexes, and the unbound CS, with negatively charged blood components limit the applicable dosage of such polymeric nanoparticles (NPs) and development of formulations with improved hemocompatibility and transfection efficiency is needed. Here, we introduce a strategy based on Tangential Flow Filtration (TFF) to remove unbound CS, concentrate polyplexes and subsequently coat with hyaluronic acid (HA) to improve hemocompatibility and bioactivity. Optimal TFF conditions were established. A library of HA with different molecular weights and degrees of sulfation was used at different carboxyl + sulfate to phosphate ratios for polyplex coating, bioactivity and hemocompatibility assessment. A systematic optimization of TFF conditions allowed for purification of polylpexes from excess unbound CS and subsequent coating with HA. Except for high molecular weight HA, for which macroscopic aggregation was observed, both sulfated and non-sulfated HAs resulted in small sized and homogenous coated complexes. However, sulfated HAs displayed higher stability during the second filtration process indicating their stronger binding affinity to polyplexes. Finally, we found that low molecular weight HA-coated polyplexes have equivalent silencing efficiency in vitro and improved hemocompatibility compared to uncoated polyplexes.

CAR T cells produced in vivo to treat cardiac injury.

Fibrosis affects millions of people with cardiac disease. We developed a therapeutic approach to generate transient antifibrotic chimeric antigen receptor (CAR) T cells in vivo by delivering modified messenger RNA (mRNA) in T cell­targeted lipid nanoparticles (LNPs). The efficacy of these in vivo­reprogrammed CAR T cells was evaluated by injecting CD5-targeted LNPs into a mouse model of heart failure. Efficient delivery of modified mRNA encoding the CAR to T lymphocytes was observed, which produced transient, effective CAR T cells in vivo. Antifibrotic CAR T cells exhibited trogocytosis and retained the target antigen as they accumulated in the spleen. Treatment with modified mRNA-targeted LNPs reduced fibrosis and restored cardiac function after injury. In vivo generation of CAR T cells may hold promise as a therapeutic platform to treat various diseases.

Efficiency of Chitosan/Hyaluronan-Based mRNA Delivery Systems In Vitro: Influence of Composition and Structure.

Messenger RNA (mRNA)-containing nanoparticles were produced by electrostatic complexation with a library of pharmaceutical grade chitosans with different degrees of deacetylation and hyaluronic acids (HAs) (native vs. sulfated). Polymer length (Mn), HA degree of sulfation (DS), and amine to phosphate to carboxyl + sulfate (from HA) ratio (N:P:C) were controlled. In vitro transfections were performed in the presence/absence of trehalose and at different pH. Particle size and ζ-potential were correlated with transfection efficiency. Polymer length and charge densities (degree of deacetylation, degree of sulfation) of both HA and chitosan had a direct influence on transfection efficiency through modulation of avidity to mRNA. N:P:C ratio, trehalose, mixing concentration, and nucleic acid dose influenced transfection efficiency with optimized formulations reaching ∼60%-65% transfection efficiency relative to commercially available lipid control with no apparent toxicity for transfection at slightly acidic pH 6.5.

Automated in-line mixing system for large scale production of chitosan-based polyplexes.

Chitosan (CS)-based polyplexes are efficient non-viral gene delivery systems that are most commonly prepared by manual mixing. However, manual mixing is not only poorly controlled but also restricted to relatively small preparation volumes, limiting clinical applications. In order to overcome these drawbacks and to produce clinical quantities of CS-based polyplexes, a fully automated in-line mixing platform was developed for production of large batches of small-size and homogeneous CS-based polyplexes. Operational conditions to produce small-sized homogeneous polyplexes were identified. Increasing mixing concentrations of CS and nucleic acid was directly associated with an increase in size and polydispersity of both CS/pDNA and CS/siRNA polyplexes. We also found that although the speed of mixing has a negligible impact on the properties of CS/pDNA polyplexes, the size and polydispersity of CS/siRNA polyplexes are strongly influenced by the mixing speed: the higher the speed, the smaller the size and polydispersity. While in-line and manual CS/pDNA polyplexes had similar size and PDI, CS/siRNA polyplexes were smaller and more homogenous when prepared in-line in the non-laminar flow regime compared to manual method. Finally, we found that in-line mixed CS/siRNA polyplexes have equivalent or higher silencing efficiency of ApoB in HepG2 cells, compared to manually prepared polyplexes.