Gemma Leone

Novel carboxymethylcellulose-based microporous hydrogels suitable for drug delivery

Several materials capable of acting as structures for controlled release were analysed for the fabrication of matrices. Among those used, hydrophilic polysaccharides appeared to be the most suitable materials. Carboxymethylcellulose (a semi-synthetic polysaccharide) was chemically cross-linked with a 60% and 90% cross-linking degree in order to obtain hydrogels and utilised as matrix for the realisation of controlled drug release systems. The morphology of the gels was changed in order to obtain a microporous structure with different porosity (14, 30 and 40 μm). The obtained porous matrices were characterised in terms of pore density, dimension and swelling behaviour. The influence of both the pore dimension and technique of loading on the release kinetics was analysed. By increasing the pore dimension the release of ibuprofen-lysin was slower. Inducing the microporous structure after the loading of the hydrogel with the drug resulted in a slower release.

A PVA/PVP hydrogel for human lens substitution: Synthesis, rheological characterization, and in vitro biocompatibility

To overcome opacification and absence of accommodation of human lens substitutes a new poly(vinyl alcohol) (PVA)/poly(N-vinyl-2- pyrrolidinone) (PVP) based hydrogel (PPS31075) was realised. The Infrared Spectroscopy and the mechanical spectra confirmed the successful occurrence of crosslinking reaction. The rheological analysis pointed out a behavior comparable with that of young human lens in terms of complex shear modulus and accommodation capability. Further analysis in terms of optical properties, water content measurements, diffusion coefficient, cytotoxicity, and human capsular cell adhesion confirmed the applicability of such a hydrogel as potential human lens substitute.

Cu2+- and Ag+-complexes with a hyaluronane-based hydrogel

Copper(II) and silver(I) hyaluronane-based hydrogel complexes were synthesised. The amount of metal ion uptaken by hydrogel was determined by atomic absorption and the hydrogel–metal ion complexes were characterised by water-uptake measurements, SEM-EDAX and FT-IR analysis. The coordination sites were identified for both metal ions and the stability at various pH levels was determined. The cytotoxicity of hydrogel metal ions was evaluated by using fibroblast (3T3) cells. The copper(II) complex was “in vivo” tested and showed proangiogenic activity, stimulating the growth of new vessels without inducing an inflammatory reaction. The antibacterial activity of a silver complex was determined, showing that the presence of silver ions drastically reduced the adhesion and proliferation of Staphylococcus epidermidis.

In Vitro Biocompatibility of New PVA-Based Hydrogels as Vitreous Body Substitutes

In order to synthesize injectable hydrogels suitable as vitreous body substitutes, a new method based on the use of trisodium trimetaphosphate (STMP) to cross-link PVA was recently proposed. Hydrogels with different molar ratios between STMP and PVA were realised. The aim of the present study was the evaluation of the biocompatibility of the different STMP/PVA hydrogels synthesised by analysing the effects of their in vitro interaction with cultures of mouse fibroblasts NIH3T3, primary human microvascular endothelial cells adult (HMVECad) and human lens cells. Cytotoxicity of hydrogels was first evaluated by analysing cell density and proliferation. Morphological and morphometric analysis of cell in contact with hydrogels was then performed using light microscopy and scanning electron microscopy, respectively. Moreover, cell adhesion and growth onto the hydrogels surface was evaluated and correlated to the amount of adsorbed proteins. At last, the biocompatibility of the sheared STMP/PVA 1:8 hydrogel was tested. The in vitro data of all the STMP/PVA hydrogels demonstrated their good biocompatibility, and indicated that the 1:8 sample was the most promising as vitreous body substitute.

Continuous multilayered composite hydrogel as osteochondral substitute

Cartilage is a highly organized avascular soft tissue that assembles from nano-to macro-scale to produce a complex structural network. To mimic cartilage tissue, we developed a stable multilayered composite material, characterized by a tailored gradient of mechanical properties. The optimized procedure implies chemical crosslinking of each layer directly onto the previous one and ensures a drastic reduction of the material discontinuities and brittleness. The multilayered composite was characterized by infrared spectroscopy, differential scanning calorimetry, thermogravimetry, and scanning electron microscopy in order to compare its physico-chemical characteristics with those of cartilage tissue. The rheological behavior of the multilayered composite was similar to that of human cartilage. Finally its cytocompatibility toward chondrocytes and osteoblasts was evaluated.

The effects of spacer arms in cross-linked hyaluronan hydrogel on Fbg and HSA adsorption and conformation

Abstract Four hyaluronan based hydrogels with different cross-linking agents were synthesised. Compounds with an increasing hydrophobic character and length as spacer arms were chosen, i.e. O , O ′-bis(2 aminopropyl)polyethylen glicol, 1.3 diaminopropane, 1.6 diaminohexane, and 1.12 diaminedodecane. The cross-linking reaction involved 50% of the carboxylate groups present in the hyaluronan macromolecule chain. Hydrogels were characterised by water-uptake measurements and SEM analysis. The adsorption of two plasma proteins (HSA and Fbg) was analysed onto the four hydrogels in flow conditions by ATR-FTIR, evaluating the adsorption kinetics and the eventual protein conformational changes. The increasing hydrophobic character of the surface imposes a clear trend to the protein adsorption kinetics. The influence of the spacer arms on the protein conformation is evident on HSA, whereas Fbg does not seem to be significantly influenced by the change of the substrate hydrophobic properties.

Formulation of liposomes functionalized with Lotus lectin and effective in targeting highly proliferative cells

BACKGROUND Liposomes, used to improve the therapeutic index of new and established drugs, have advanced with the insertion of active targeting. The lectin from Lotus tetragonolobus (LTL), which binds glycans containing alpha-1,2-linked fucose, reveals surface regionalized glycoepitopes in highly proliferative cells not detectable in normally growing cells. In contrast, other lectins localize the corresponding glycoepitopes all over the cell surface. LTL also proved able to penetrate the cells by an unconventional uptake mechanism. METHODS We used confocal laser microscopy to detect and localize LTL-positive glycoepitopes and lectin uptake in two cancer cell lines. We then constructed doxorubicin-loaded liposomes functionalized with LTL. Intracellular delivery of the drug was determined in vitro and in vivo by confocal and electron microscopy. RESULTS We confirmed the specific localization of Lotus binding sites and the lectin uptake mechanism in the two cell lines and determined that LTL-functionalized liposomes loaded with doxorubicin greatly increased intracellular delivery of the drug, compared to unmodified doxorubicin-loaded liposomes. The LTL-Dox-L mechanism of entry and drug delivery was different to that of Dox-L and other liposomal preparations. LTL-Dox-L entered the cells one by one in tiny tubules that never fused with lysosomes. LTL-Dox-L injected in mice with melanoma specifically delivered loaded Dox to the cytoplasm of tumor cells. CONCLUSIONS Liposome functionalization with LTL promises to broaden the therapeutic potential of liposomal doxorubicin treatment, decreasing non-specific toxicity. GENERAL SIGNIFICANCE Doxorubicin-LTL functionalized liposomes promise to be useful in the development of new cancer chemotherapy protocols.

Polysaccharide Based Hydrogels for Biomedical Applications

Polysaccharide based hydrogels for their physico-chemical and biological properties can be used as scaffolds for soft tissue regneration and as vehicles for drug controlled release. For both these applications, Hyaluronan shows optimal characteristics even though its quick enzymatic degradability makes this natural polysaccharide unsuitable for applications which require prolonged presence in the human organism.

Effect of resveratrol on platelet aggregation by fibrinogen protection

The effect of resveratrol (RSV) in inhibiting platelet adhesion and aggregation, as well as fibrinogen (FBG) conformational changes promoted by epinephrine (EP), were studied, by using complementary experimental techniques. NMR and IR spectroscopies were used to investigate possible protective effects by RSV towards FBG, in presence of EP. The protective effect of RSV towards FBG was highlighted by spin nuclear relaxation experiments that were interpreted for determining the thermodynamic equilibrium constants of FBG-EP interaction, and by infrared measurements, that showed EP-induced conformational changes of FBG. The ability of RSV in inhibiting platelet adhesion and aggregation promoted by EP was evaluated by scanning electron microscopy (SEM), measuring the platelet adhesion and aggregation degree, in comparison to data obtained for platelet aggregation in platelet rich plasma (PRP). The experimental combined approach pointed out that RSV is able to protect both FBG and platelets from the denaturant and aggregating action of EP at stress level concentration.

Cartilage replacement implants using hydrogels

Abstract: Articular cartilage (AC), a highly specialised tissue that provides low friction and allows for efficient load bearing and distribution, can undergo disruption for traumatic or degenerative processes. Conservative treatment with various medications gives only temporary relief of symptoms rather than cure. Since 1960 several surgical procedures have been tested without significant improvement of AC regeneration. Actually, third generation tissue engineering applied to the cartilage tissue regeneration is giving promising results. The right combination of cell types, scaffolds and culture media can represent the only way to cure degenerated cartilage tissue. However, several critical issues need to be overcome.