The aim of the present study is to investigate the interactions between liposomes and proteins and to evaluate the role of liposomal lipid composition and concentration in the formation of protein corona. Liposomes composed of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) or hydrogenated soybean phosphatidylcholine (HSPC) with 1,2-dipalmitoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (sodium salt) (DPPG), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-3000] (DPPE-PEG 3000), cholesterol (CH) or mixtures of these lipids, were prepared at different concentrations by the thin-film hydration method. After liposomes were dispersed in HPLC-grade water and fetal bovine serum (FBS), their physicochemical characteristics, such as size, size distribution and ζ-potential, were determined using dynamic and electrophoretic light scattering. Aggregation of DPPC, HSPC, DPPC:CH (9:1 molar ratio) and HSPC:CH (9:1 molar ratio) in FBS was observed. On the contrary, liposomes incorporating DPPG lipids and cholesterol both in a molar ratio of 11% were found to be stable over time, while their size did not alter dramatically in biological medium. Liposomes containing cholesterol and PEGylated lipids retain their size in the presence of serum as well as their physical stability. In addition, our results indicate that the protein binding depends on the presence of polyethylene glycol (PEG), cholesterol, concentration and surface charge. In this paper, we introduce a new parameter, fraction of stealthiness (Fs), for investigating the extent of protein binding to liposomes. This parameter depends on the changes in size of liposomes after serum incubation, while liposomes have stealth properties when Fs is close to 1. Thus, we conclude that lipid composition and concentration affect the adsorption of proteins and the liposomal stabilization.

Rindera graeca is a Greek endemic plant of the Boraginaceae family which has never been studied before. Consequently, this study attempted to phytochemically examine the aerial parts of this species. Nine phenolic secondary metabolites were identified, consisting of seven caffeic acid derivatives and two flavonol glucosides, namely rutin and quercetin-3-rutinoside-7-rhamnoside. These flavonoids, together with rosmarinic acid, were isolated via column chromatography and structurally determined through spectral analysis. Quercetin-3-rutinoside-7-rhamnoside is an unusual triglycoside, which is identified for the first time in Rindera genus and among Boraginaceae plants. This metabolite was further examined with thermal analysis and its 3D structure was simulated, revealing some intriguing information on its interaction with biological membrane models, which might have potential applications in microcirculation-related conditions. R. graeca was also analyzed for its pyrrolizidine alkaloids content, and it was found to contain echinatine together with echinatine N-oxide and rinderine N-oxide. Additionally, the total phenolic and flavonoid contents of R. graeca methanol extract were determined, along with free radical inhibition assays. High total phenolic content and almost complete inhibition at experimental doses at the free radical assays indicate a potent antioxidant profile for this plant. Overall, through phytochemical analysis and biological activity assays, insight was gained on an endemic Greek species of the little-studied Rindera genus, while its potential for further applications has been assessed.

Liposomes are considered to be one of the most extensively investigated drug delivery nanosystems. Each drug can be loaded either in the liposomal hydrophilic core or within the lipidic bilayer and delivered eventually to the proper site into the organism. There are already many marketed approved liposomal products. The development of a liposomal product is a quite complicated process, while many critical parameters have to be investigated during the preparation process. The present study deals with the drug-to-lipid ratio (D/L ratio), which is a critical process parameter, expresses the actual capacity of the liposome to accommodate the drug and can play a key role at the optimization of every liposomal formulation. D/L ratio is affected by the composition, the different biomaterials and the loading method being used, so the improvement of D/L ratio can optimize the liposomal formulation. D/L ratio can be used as an index of the effectiveness of the preparation method too. Furthermore, D/L ratio influences the therapeutic efficacy of the liposomal product, expressing the actual dose of the drug being administrated. There is a variety of analytical methods, quantifying the drug and the lipids and estimating eventually the D/L ratio. According to the regulatory framework of nanomedicine, about the development of nanosimilars, D/L ratio is a necessary element for the nanosimilar product description and the statement of product comparability.

Quercetin (Que) is a flavonoid associated with high oxygen radical scavenging activity and potential neuroprotective activity against Alzheimer's disease. Que's oral bioavailability is limited by its low water solubility and extended peripheral metabolism; thus, nasal administration may be a promising alternative to achieve effective Que concentrations in the brain. The formation of Que−2-hydroxypropylated−β-cyclodextrin (Que/HP-β-CD) complexes was previously found to increase the molecule's solubility and stability in aqueous media. Que−methyl−β-cyclodextrin (Que/Me-β-CD) inclusion complexes were prepared, characterized, and compared with the Que/HP-β-CD complex using biophysical and computational methods (phase solubility, fluorescence and NMR spectroscopy, differential scanning calorimetry (DSC), and molecular dynamics simulations (MDS)) as candidates for the preparation of nose-to-brain Que's delivery systems. DSC thermograms, NMR, fluorescence spectroscopy, and MDS confirmed the inclusion complex formation of Que with both CDs. Differences between the two preparations were observed regarding their thermodynamic stability and inclusion mode governing the details of molecular interactions. Que's solubility in aqueous media at pH 1.2 and 4.5 was similar and linearly increased with both CD concentrations. At pH 6.8, Que's solubility was higher and positively deviated from linearity in the presence of HP-β-CD more than with Me-β-CD, possibly revealing the presence of more than one HP-β-CD molecule involved in the complex. Overall, water solubility of lyophilized Que/Me-β-CD and Que/HP-β-CD products was approximately 7−40 times and 14−50 times as high as for pure Que at pH 1.2−6.8. In addition, the proof of concept experiment on ex vivo permeation across rabbit nasal mucosa revealed measurable and similar Que permeability profiles with both CDs and negligible permeation of pure Que. These results are quite encouraging for further ex vivo and in vivo evaluation toward nasal administration and nose-to-brain delivery of Que.

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