A multitude of microparticles and nanoparticles is developed to improve the delivery of different small drugs and large biomolecules, which are subject to several hindering biological barriers that limit their optimal biodistribution and therapeutic effects. Here, a soft, reliable, and scalable method based on compressed CO2 is reported for obtaining nanoconjugates of recombinant human epidermal growth factor and nanovesicles called quatsomes, where the latter consists of cholesterol and cetyltrimethylammonium bromide. These nanoconjugates exhibit appropriate values of the major critical quality attributes of colloidal nanomedicines, such as controlled and narrow nanoscopic particle size distribution (which play important roles in determining their stability), drug loading, drug release, drug protection, targeting ability, and bioactivity. Also, they exhibit a dual action by 1) inbuilt antimicrobial activity preventing infections and 2) promoting regeneration of granulation tissue and re‐epithelialization with complete closure of complex wounds. Therefore, such nanoconjugates are a potential nanomedicine for the topical treatment of complex wounds, particularly diabetic foot ulcers and venous leg ulcers.
A simple, reliable procedure for practically quantitative (90-98%) and fast (< 30 min) elution of proteins from SDS-PA gels is described with reproducible recoveries in the range from 100 to 1 pmol per band, which does not require the inclusion of detergents in the elution buffer. It consists in the combination of (1) highly sensitive on-gel protein detection (50 mol per band) with imidazole-SDS-zinc (reverse staining), (2) crushing of the protein band to produce 32-micron gel particles, and (3) vortexing of the slurry in a solution of a zinc-complexing agent, e.g. glycine 0.5 M or EDTA 100 mM (100 microliters for a 100-pmol BSA band), at room temperature. Eluted proteins can be directly analyzed by RP-HPLC, quantitatively loaded onto a PVDF membrane, or, provided that they are previously renatured on-gel, analyzed by biological activity tests. The application of the procedure to in-solution enrichment of scarce proteins for N-terminal analysis is shown.
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