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.

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).

The synthesis and study of the tripeptide Arg-Gly-Asp (RGD), the binding site of different extracellular matrix proteins, e.g., fibronectin and vitronectin, has allowed the production of a wide range of cell adhesive surfaces. Although the surface density and spacing of the RGD peptide at the nanoscale have already shown a significant influence on cell adhesion, the impact of its hierarchical nanostructure is still rather unexplored. Accordingly, a versatile colloidal system named quatsomes, based on fluid nanovesicles formed by the self-assembling of cholesterol and surfactant molecules, has been devised as a novel template to achieve hierarchical nanostructures of the RGD peptide. To this end, RGD was anchored on the vesicle’s fluid membrane of quatsomes, and the RGD-functionalized nanovesicles were covalently anchored to planar gold surfaces, forming a state of quasi-suspension, through a long poly(ethylene glycol) (PEG) chain with a thiol termination. An underlying self-assembled monolayer (SAM) of a shorter PEG was introduced for vesicle stabilization and to avoid unspecific cell adhesion. In comparison with substrates featuring a homogeneous distribution of RGD peptides, the resulting hierarchical nanoarchitectonic dramatically enhanced cell adhesion, despite lower overall RGD molecules on the surface. The new versatile platform was thoroughly characterized using a multitechnique approach, proving its enhanced performance. These findings open new methods for the hierarchical immobilization of biomolecules on surfaces using quatsomes as a robust and novel tissue engineering strategy.