Obtaining and Characterizing Composite Biomaterials of Animal Resources with Potential Applications in Regenerative Medicine.

Raw materials, such as collagen and chitosan, obtained from by-products from the food industry (beef hides and crustacean exoskeletons), can be used to obtain collagen-chitosan composite biomaterials, with potential applications in regenerative medicine. Functionalization of these composite biomaterials is a possibility, thus, resulting in a molecule with potential applications in regenerative medicine, namely clotrimazole (a molecule with antibacterial, antifungal, and antitumor activity), at a mass ratio (collagen-chitosan-clotrimazole) of 1:1:0.1. This functionalized composite biomaterial has great potential for application in regenerative medicine, due to the following properties: (1) it is porous, and the pores formed are interconnected, due to the use of a mass ratio between collagen and chitosan of 1:1; (2) the size of the formed pores is between 500-50 µm; (3) between collagen and chitosan, hydrogen bonds are formed, which ensure the unity of composite biomaterial; (4) the functionalized bio-composite exhibits in vitro antimicrobial activity for Candida albicans, Staphylococcus aureus, and Staphylococcus aureus MRSA; for the latter microorganism, the antimicrobial activity is equivalent to that of the antibiotic Minocycline; (5) the proliferation tests performed on a standardized line of normal human cells with simple or composite materials obtained by lyophilization do not show cytotoxicity in the concentration range studied (10-500) µg/mL.

3D hybrid structures based on biomimetic membranes and Caryophyllus aromaticus - "green" synthesized nano-silver with improved bioperformances.

The paper describes an innovative bio-design of some hybrid nanoarchitectures containing bioartificial membranes and silver nanoparticles phytogenerated by using a natural extract Caryophyllus aromaticus (cloves) that contains many bioactive compounds. Two kinds of liposomes with and without chlorophyll a (Chla) obtained through thin film hydration method were used to achieve bio-green-generated hybrids by a simple, cost effective bottom-up approach. The characteristic peaks of CE-nAg monitored by UV-Vis absorption have firstly demonstrated the biohybrids formation. The slightly blue shift and fluorescence quenching observed by fluorescence emission spectra highlighted the formation of hybrid systems by biointeraction between lipid vesicles and silver nanoparticles. The incorporation of silver nanoparticles in lipid vesicles resulted in significant changes of FT-IR spectra of liposomes, indicating a reorganization of biomimetic membranes. All the microscopic methods (SEM, AFM and TEM) confirmed the biosynthesis of "green" AgNPs together with associated biohybrids, their spherical and quasi-spherical shapes with nano-scaled size. By TEM assay it was shown that CE-nAg are surrounded by petal like cloud structures that consist of biopolymers like proteins or polysaccharides and other phytochemicals arising from clove extract. EDS spectra confirmed the formation of phyto-nanoAg and also the presence of silver in the biohybrids. In addition, Selected Area Electron Diffraction showed characteristic polycrystalline ring patterns for a cubic structure of the clove-generated AgNPs. The hybrid materials showed efficient physical stability,i.e. ξ value of -28.0 mV (for biohybrids without Chla, BH) and of -31.7 mV (for biohybrids labelled with Chla, Chla-BH), assured by strong electrostatic repulsive forces between particles. The "green" nano-silver particles (CE-nAg) showed remarkable antioxidant activity (AA = 90.2%). The biohybrids loaded with clove-AgNPs proved to be more effective, scavenging about 98.8% of free radicals (in case of Chla-BH), and of 92.6% (in case of BH). The antibacterial effectiveness showed that green AgNPs combine in a synergistic manner the antibacterial properties of clove extract with those of silver, resulting in an enhancement of inhibition diameter, by 20%. Chla-BH proved to be more potent against Escherichia coli, than BH, exhibiting an inhibition diameter of 42 mm. Regarding the in vitro cytotoxicity against tumour cells, the CE-nAg concentration significantly influenced the cell viability, i.e. IC50 was 3.6% (v/v) for HT-29 cells. Chla-BH was more effective against HT-29 cancer cells at the concentrations ranging from 0 to 18% (v/v), when the normal cells were not affected. Clove-generated AgNPs exhibited haemolytic activity against hRBCs, while the biohybrids were haemocompatible. The action mechanism on the two cell lines (mouse fibroblast L929 cells and human colorectal adenocarcinoma HT-29 cells) investigated by fluorescence microscopy demonstrated that CE-nAg killed almost all the cells (94%) through necrosis at a concentration of 33.4% (v/v). The treatment of HT-29 cells with BH resulted in: 71.5% viable cells, 19.5% apoptotic and only 9% necrotic cells, while in the case of Chla-BH treatment, only 77.5% cells were viable, 16% cells were apoptotic and 6.5% were necrotic. In this way, the developed silver-based nanoparticles can represent viable promoters to develop new biohybrids with improved features, e.g. antioxidant and antibacterial effectiveness, haemolytic activity and greater specificity towards tumour cells.