Mauro Di Stefano

New self-assembling polyaspartylhydrazide copolymer micelles for anticancer drug delivery.

A new amphiphilic copolymer have been synthesized starting from the hydrosoluble polyaspartylhydrazide (PAHy) polymer, by grafting both hydrophilic PEG(2000) chains and hydrophobic palmitic acid (C(16)) moieties on polymer backbone, and the structure of obtained PAHy-PEG(2000)-C(16) copolymer have been characterized by 2D (1)H/(13)C NMR experiments. PAHy-PEG(2000)-C(16) copolymer showed the ability of self-assembling in aqueous media giving a core-shell structure and resulted potentially useful for encapsulating and dissolving hydrophobic drug. The formation of micellar core-shell structure has been investigated by 2D (1)H NMR NOESY experiments. The presence of cross-peaks for protons of C(16) and PAHy portions, indicated that the two domains are in close proximity forming micelle core. The critical aggregation concentration (CAC) values of PAHy-PEG(2000)-C(16) amphiphilic graft copolymer was determined in water by fluorescence technique, and it was demonstrated that PAHy-PEG(2000)-C(16) micelles are well suited to be micellar vehicle of highly hydrophobic molecules. Therefore, anticancer drug tamoxifen, used as a model hydrophobic molecule, was loaded into PAHy-PEG(2000)-C(16) micelles obtaining an increase of drug solubility of about 3000 times. Transmission electron microscopy (TEM) observations showed the spherical morphology of micelles formed by PAHy-PEG(2000)-C(16) copolymer with a mean diameter of about 30nm, as confirmed also by dynamic light scattering (DLS) studies. Finally, in vitro cell viability studies were carried out on human breast cancer cells (MCF-7) testing the pharmacological activity of tamoxifen-loaded PAHy-PEG(2000)-C(16) micelles, in comparison with free tamoxifen at different drug concentrations, demonstrating that tamoxifen-loaded PAHy-PEG(2000)-C(16) micelles exhibited a concentration-dependent cytotoxic activity.

In situ forming hydrogels of hyaluronic acid and inulin derivatives for cartilage regeneration.

An in situ forming hydrogel obtained by crosslinking of amino functionalized hyaluronic acid derivatives with divinylsulfone functionalized inulin (INU-DV) has been here designed and characterized. In particular two hyaluronic acid derivatives bearing respectively a pendant ethylenediamino (EDA) portion (HA-EDA) and both EDA and octadecyl pendant groups (HA-EDA-C18) were crosslinked through an azo-Michael reaction with INU-DV. Gelation time and consumption of DV portions have been evaluated on hydrogel obtained using HA-EDA and HA-EDA-C18 derivatives with a concentration of 3% w/v and a ratio 80/20 w/w respect to the crosslinker INU-DV. The presence of pendant C18 chains improves mechanical performances of hydrogels and decreases the susceptibility to hyaluronidase hydrolysis. Bovine chondrocytes, encapsulated during crosslinking, sufficiently survive and efficiently proliferate until 28 days of analysis.

Phea-graft-polybutylmethacrylate copolymer microparticles for delivery of hydrophobic drugs

Polymeric microparticles encapsulating two model hydrophobic drugs, beclomethasone dipropionate (BDP) and flutamide (FLU) were prepared by using the high pressure homogenization-solvent evaporation method starting from a oil-in-water emulsion. For the preparation of polymeric microparticles a α,β-poly(N-2-hydroxyethyl)-D,L-aspartamide (PHEA) graft copolymer with comb like structure was properly synthesized via grafting from atom transfer radical polymerization (ATRP) technique, by using two subsequent synthetic steps. In the first step a polymeric multifunctional macroinitiator was obtained by the conjugation of a proper number of 2-bromoisobutyryl bromide (BIB) residues to the PHEA side chains, obtaining the PHEA-BIB copolymer. PHEA-BIB copolymer was then used as macroinitiator for the polymerization via ATRP of the hydrophobic monomer such as butyl methacrylate (BMA) to obtain the α,β-poly(N-2-hydroxyethyl)-D,L-aspartamide-co-(N-2-ethylen-isobutyrate)-graft-poly(butyl methacrylate) copolymer (PHEA-IB-p(BMA)). Spherical microparticles with 1-3 microns diameter were prepared. Microparticles loaded with BDP or FLU were also prepared. In vitro mucoadhesion and enzymatic degradation studies evidenced bioadhesive properties and biodegradability of prepared microparticles, while release studies showed a different release profiles for the two loaded drugs: BDP was totally released from nanoparticles until 24h in pulmonary mimicking conditions; differently a slower FLU release rate was observed in gastro-intestinal mimicking conditions. The in vitro cytotoxicity activity was assessed using 16HBE and Caco-2 cell lines. Results showed that exposure of both cell lines to BDP-loaded microparticles do not inhibited the cell growth; on the contrary FLU-loaded microparticles inhibited the cell growth, in particular of the Caco-2 cancer cell line, in a concentration- and time-dependent manner. Finally, uptake studies demonstrated that BDP-loaded microparticles and FLU-loaded microparticles effectively increased uptake of loaded drugs in a time-dependent manner, respectively on 16HBE and Caco-2 cell lines.

Medicated hydrogels of hyaluronic acid derivatives for use in orthopedic field.

Physical hydrogels have been obtained from hyaluronic acid derivatized with polylactic acid in the presence or in the absence of polyethylene glycol chains. They have been extemporarily loaded with antibacterial agents, such as vancomycin and tobramycin. These medicated hydrogels have been used to coat titanium disks (chosen as simple model of orthopedic prosthesis) and in vitro studies in simulated physiological fluid have been performed as a function of time and for different drug loading and polymer concentration values. Sterilization process performed on the hydrogels does not change their rheological behavior and release properties as well as the chemical structure of starting copolymers. A preliminary test has been performed by coating with the hydrogel a prosthesis that has been inserted in a seat of a lyophilized human femur, to confirm the ability of the hydrogel to adhere to the prosthesis surface also after its insertion in the implant seat. Cell compatibility of obtained hydrogels has been confirmed in vitro by using human dermal fibroblasts chosen as a model cell line. Obtained results suggest the potential use of these hydrogels in the orthopedic field, in particular for the production of antibacterial coatings of prostheses for implant in the human or animal body in the prevention and/or treatment of post surgical infections.

Polyaspartamide‐graft‐Polymethacrylate Nanoparticles for Doxorubicin Delivery

A new PHEA-IB-PMANa(+) copolymer has been synthesized and its pH-induced self-assembly has been investigated in an aqueous medium. PHEA-IB-PMANa+ formed nanoparticles with diameters from 25 to 50 nm upon protonation of the carboxylic acid moieties dislocated along the grafted polymethacrylate sodium salt side chains. The physico-chemical characterization of the nanoparticles was performed using light scattering, zeta-potential measurements, SEM, and AFM. Doxorubicin-loaded nanoparticles were prepared and drug release profiles were evaluated under conditions mimicking physiological media. A biological characterization was carried out by testing the cytotoxicity on Caco-2 cells, and cellular uptake on mouse monocyte macrophage (J774 A.1) and Caco-2 cells.

PEG-benzofulvene copolymer hydrogels for antibody delivery

Poly[monomethylnona(ethylene glycol) 1-methylene-3-(4-methylphenyl)-1H-indene-2-carboxylate] (poly-1b) a new polymer based on a PEG-functionalized benzofulvene macromonomer have been investigated as hydrogel-based material for complexation and release of immunoglobulin (IgG) at physiological mimicking conditions. The polymer ability to complex human IgG has been studied by preparing copolymer/protein complexes obtained by spontaneous protein interactions onto polymer hydrogel aggregates, and the protein release rate has been evaluated at physiological conditions. SEM analysis was used to visualize the copolymer/IgG aggregates and its microstructured deposition. Moreover, rheological studies performed at 37 degrees C allowed determining hydrogel mechanical properties. On the basis of these information and NMR transverse relaxation measurements, the estimation of hydrogel mesh size distribution was possible. Finally, biological studies performed with poly-1b aqueous dispersions showed no cytotoxic effect on MCF-7 cell line, suggesting potential biocompatibility features for this polymer and making this new polymer a good potential candidate for the production of drug delivery systems.

Dexamethasone dipropionate loaded nanoparticles of α-elastin-g-PLGA for potential treatment of restenosis.

A graft copolymer of α-elastin with poly(lactic-co-glycolic) acid (PLGA) has been synthesized and successfully employed to produce nanoparticles. Exploiting the known biological activity of α-elastin to promote the maintenance of smooth muscle cells (SMCs) contractile phenotype and the antiproliferative effect of glucocorticoids, the aim of this research was to produce drug-loaded nanoparticles suitable for potential treatment of restenosis. In particular, nanoparticles of α-elastin-g-PLGA with a mean size of 200 nm have been produced and loaded with dexamethasone dipropionate (10% w/w), chosen as a model drug that inhibits proliferation of vascular SMCs. These nanoparticles are able to prolong the drug release and show a pronounced sensibility to elastase. Drug unloaded nanoparticles stimulate the differentiation of human umbilical artery smooth muscle cells (HUASMCs) toward the contractile phenotype as demonstrated by immunofluorescence, flow cytofluorimetric, and western blotting analyses. Finally, drug-loaded nanoparticles efficiently reduce viability of HUASMCs as evidenced by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2- (4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) assay.