In situ analysis of osmolyte mechanisms of proteome thermal stabilization.

Organisms use organic molecules called osmolytes to adapt to environmental conditions. In vitro studies indicate that osmolytes thermally stabilize proteins, but mechanisms are controversial, and systematic studies within the cellular milieu are lacking. We analyzed Escherichia coli and human protein thermal stabilization by osmolytes in situ and across the proteome. Using structural proteomics, we probed osmolyte effects on protein thermal stability, structure and aggregation, revealing common mechanisms but also osmolyte- and protein-specific effects. All tested osmolytes (trimethylamine N-oxide, betaine, glycerol, proline, trehalose and glucose) stabilized many proteins, predominantly via a preferential exclusion mechanism, and caused an upward shift in temperatures at which most proteins aggregated. Thermal profiling of the human proteome provided evidence for intrinsic disorder in situ but also identified potential structure in predicted disordered regions. Our analysis provides mechanistic insight into osmolyte function within a complex biological matrix and sheds light on the in situ prevalence of intrinsically disordered regions.

Systemic delivery of mRNA and DNA to the lung using polymer-lipid nanoparticles.

Non-viral vectors offer the potential to deliver nucleic acids including mRNA and DNA into cells in vivo. However, designing materials that effectively deliver to target organs and then to desired compartments within the cell remains a challenge. Here we develop polymeric materials that can be optimized for either DNA transcription in the nucleus or mRNA translation in the cytosol. We synthesized poly(beta amino ester) terpolymers (PBAEs) with modular changes to monomer chemistry to investigate influence on nucleic acid delivery. We identified two PBAEs with a single monomer change as being effective for either DNA (D-90-C12-103) or mRNA (DD-90-C12-103) delivery to lung endothelium following intravenous injection in mice. Physical properties such as particle size or charge did not account for the difference in transfection efficacy. However, endosome co-localization studies revealed that D-90-C12-103 nanoparticles resided in late endosomes to a greater extent than DD-90-C12-103. We compared luciferase expression in vivo and observed that, even with nucleic acid optimized vectors, peak luminescence using mRNA was two orders of magnitude greater than pDNA in the lungs of mice following systemic delivery. This study indicates that different nucleic acids require tailored delivery vectors, and further support the potential of PBAEs as intracellular delivery materials.

Hydrazone linked doxorubicin-PLA prodrug nanoparticles with high drug loading.

An optimal drug delivery system should be characterized by biocompatibility, biodegradability, high drug loading and favorable drug release profile. To achieve this goal a hydrazone linked doxorubicin-poly(lactic acid) prodrug (PLA-DOX) was synthesized by the functionalization of a short polymer chain produced by ring opening polymerization. The hydrophobic prodrug generated in this way was nanoprecipitated using a block copolymer to form polymeric nanoparticles (NPs) with a quantitative loading efficiency and a high and tunable drug loading. The effects of the concentration of the PLA-DOX prodrug and surfactant were studied by dynamic light scattering showing a range of NP size between 50 and 90 nm and monodispersed size distributions with polydispersity indexes lower then 0.27 up to a maximum DOX concentration of 27% w/w. The release profile of DOX from these NPs, tested at different pH conditions, showed a higher release rate in acidic conditions, consistent with the nature of the hydrazone bond which was used to conjugate the drug to the polymer. In vitro cytotoxicity studies performed on BV2 microglia-like cell line highlighted a specific cytotoxic effect of these NPs suggesting the maintenance of the drug efficacy and a modified release profile upon encapsulation of DOX in the NPs.