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.

Highlights Robust preparation of liposomal formulation by DELOS-susp method. Implementation of Quality by Design methodology to liposomes preparation. Influence of critical parameters on quality was studied through DoE analysis. Design Space was obtained for GLA-loaded liposomes formulation.

MicroRNAs (miRNAs) are small non‐coding endogenous RNAs, which are attracting a growing interest as therapeutic molecules due to their central role in major diseases. However, the transformation of these biomolecules into drugs is limited due to their unstability in the bloodstream, caused by nucleases abundantly present in the blood, and poor capacity to enter cells. The conjugation of miRNAs to nanoparticles (NPs) could be an effective strategy for their clinical delivery. Herein, the engineering of non‐liposomal lipid nanovesicles, named quatsomes (QS), for the delivery of miRNAs and other small RNAs into the cytosol of tumor cells, triggering a tumor‐suppressive response is reported. The engineered pH‐sensitive nanovesicles have controlled structure (unilamellar), size (<150 nm) and composition. These nanovesicles are colloidal stable (>24 weeks), and are prepared by a green, GMP compliant, and scalable one‐step procedure, which are all unavoidable requirements for the arrival to the clinical practice of NP based miRNA therapeutics. Furthermore, QS protect miRNAs from RNAses and when injected intravenously, deliver them into liver, lung, and neuroblastoma xenografts tumors. These stable nanovesicles with tunable pH sensitiveness constitute an attractive platform for the efficient delivery of miRNAs and other small RNAs with therapeutic activity and their exploitation in the clinics.

Supporting Information. This material is available free of charge via the Internet at http://pubs.acs.org. Equipment configuration and DELOS-susp methodology; Separation of free GLA by diafiltration; Concentration of the sample for in vivo testing; Stability of the sample in human serum; Composition of nanovesicles; Theoretical number of GLA per vesicle; Morphology of liposomes using SAXS; Enzymatic activity assay conditions; In vitro cell viability in endothelial HMEC-1; Plasma coagulation times (PDF).

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