Nanostructures for protection and vehiculation of bioactive molecules

Advances in nanostructured materials and nanotechnologies play a crucial role in engineering innovative systems useful in nanomedicine applications. For example, the most promising drug delivery systems belong to the colloidal domain and, particularly, to the soft matter. Indeed, a growing effort in the discovery of innovative therapies has led to an increasing demand for drug delivery vehicles whose capability should go far beyond the task for which they were originally designed for, that is the simple transport and indiscriminate release of the drug. In principle, every newly formulated nanocarrier should be able to selectively release the drug. Mainly, overcoming the biological barriers that prevent the drug to reach the pathologic site represents the strictest requirement. Thus, a major objective of formulation chemistry is to improve bioavailability, stability, and, last but not least, convenience to the patient. Lipids, along with proteins and nucleic acids, are essential biomolecules for the structure and function of living matter. Most lipids are fats and waxes, but many others have unique structural features due to their amphiphilic character. The choice of polar lipid based systems to build nanostructured architectures to entrap, protect, and release therapeutic agents with hydrophilic, lipophilic, or amphiphilic nature, appears a good strategy since polar lipids such as phospholipids or monoglycerides are generally friendly toward biological membranes and favor bioadhesion. The physico-chemical characterization of structure and stability of three different lipid based formulations intended for drug delivery is the central issue in this thesis. The main contents here discussed are outlined as follows. General features of amphiphiles and their behavior in aqueous solutions will be presented in Chapter 1. Chapter 2 is a theoretical review that describes the main techniques used throughout this study. Chapter 3 reports on two different formulations where monoolein, a natural monoglyceride, and lauroylcholine chloride are the main components. Finally, Chapter 4 presents a third formulation: a water-in-oil emulsion stabilized by a liquid crystalline lamellar phase consisting of a technical grade soy lecithin (LCT), triolein (GTO) and water (W). The first system investigated is a novel cationic liposome nanocarrier, having interesting performance in topical drug delivery. Both monoolein and lauroylcholine chloride, due to their high biocompatibility, can be regarded as strong penetration enhancers. They are combined to rapidly formulate (15 min) a cationic liposome nanostructure endowed of excellent stability ( > 6 months) and skin penetration ability, along with low short-term cytotoxicity, as evaluated via the MTT test. Cytotoxicity tests and lipid droplet analysis give a strong indication that monoolein and lauroylcholine synergistically endanger long-term cells viability. The physicochemical features, investigated through SAXS, DLS, and cryo-TEM techniques, reveal that the nanostructure is retained after loading with diclofenac in its acid (hydrophobic) form. The drug release performances are studied using intact newborn pig skin. Analysis of the different skin strata proves that the drug mainly accumulates into the viable epidermis with almost no deposition into the derma. Indeed, the flux of the drug across the skin is exceptionally low, with only 1% release after 24 h. These results validate the use of this novel formulation for topical drug release when the delivery to the systemic circulation should be avoided. The second formulation is strictly related to the first one, since it concerns an innovative vesicle-based gel essentially obtained by concentrating the system previously described. A number of vesicular formulations were prepared, in the range of 4–14 wt% of the dispersed phase, to investigate the system evolution from the dilute unilamellar vesicle dispersion (the first formulation) to a vesicle lipid gel. Morphology, thermal stability up to 55 °C, and viscoelastic properties, along with the effect of acid diclofenac inclusion within the formulation, were evaluated by cryo-TEM, SAXS, and rheological measurements. Moreover, the nanostructure of the vesicle dispersion obtained upon gel dilution in water was assessed by cryo-TEM and SAXS, while DLS was used to monitor the formulation stability (size and -potential). All the collected results lead to the conclusion that this new vesicle-based gel displays all the requirements needed for application in the pharmaceutical and cosmetic fields. The LCT/GTO/W partial phase diagram was investigated by visual inspection and optical microscopy in polarized light, SAXS, and 2H NMR spectroscopy to define the type of phases and characterize their structural parameters. Ternary LCT/GTO/W mixtures prepared with 85/15 and 90/10 constant mass ratio and increasing amounts of water were investigated. Particularly, it was found that the dominant hexagonal liquid crystalline phase identified at low water content evolves toward an emulsion region stabilized by a lamellar liquid crystalline phase for water content greater than 15% wt/wt. The emulsion having composition LCT/GTO/W = 58/11/31 was chosen to encapsulate the bioactive molecules lysozyme (LSZ) and caffeine. The thermal stability was assessed by means of SAXS in the temperature range 4-55 °C. The drug release performances were studied using Franz cell apparatus and a synthetic membrane made by cellulose acetate. After 24 h, the percentage of protein detected in the release media was equal to 10% of the nominal content of the formulation, while in the case of caffeine 80% of the initial content was released after 15 hours. In both cases, due to the use of a liquid crystalline phase as dispersing medium, burst release of the biomolecules from the formulations is prevented. Evaluation of the residual activity of the released protein was also carried out using the Micrococcus lysodeikticus assay. Approximately 30% of the released LSZ retains its enzymatic activity. The full biocompatibility of the formulation ingredients along with the absence of burst release for the investigated cases allow suggesting this formulation as a promising topical drug carrier for sustained release. Indeed, this formulation presents other advantages over conventional creams and ointments such as higher storage stability, good production feasibility, higher kinetic stability and, in particular, the absence of organic solvents in the preparation.