Margherita Morpurgo

Smart delivery of antitumoral platinum complexes from biomimetic hydroxyapatite nanocrystals

This study widens the role of biomimetic hydroxyapatite (HA) nanocrystals as bone substitutes and describes how they can be used as bone-specific drug delivery devices for in situ treatment of bone tumors upon local implantation. The adsorption and release kinetics of bis-{ethylenediamineplatinum(II)}-2-amino-1-hydroxyethane-1,1-diyl-bisphosphonate and bis-{ethylenediamineplatinum(II)}medronate on two kinds of HA nanocrystals having different morphologies, crystallinity degrees and surface areas have been investigated. The different chemical structures of the two Pt complexes appreciably affect not only the affinity towards the two kinds of HA, but also their release. The Pt complex loading is slightly greater for the HA characterized by lower crystallinity and higher surface area, with respect to the more crystalline one. The cytotoxicity of Pt complexes released from the HA were tested against human cervix carcinoma cells and, interestingly, were found to be more cytotoxic than the unmodified complexes. The released Pt species are therefore the active dichloridoethylenediamineplatinum(II) or related solvato species formed by Pt-bisphosphonate bond breaking.

N-hydroxysuccinimide carbonates and carbamates are useful reactive reagents for coupling ligands to lysines on proteins.

Ligands containing amino or hydroxyl groups were converted to their corresponding activated N-hydroxysuccinimidyl carbamate and carbonate by reaction with disuccinimidyl carbonate (DSC). The latter reagents can be used for the group-specific modification of primary amines as an alternative to the widespread usage of N-hydroxysuccinimide esters. Biotin and 2,4-dinitrophenyl (DNP) derivatives were used as examples to demonstrate the approach. Biotin and DNP were each extended by attaching two different spacer arms, carrying either a hydroxyl group or a primary amine as terminal functions. The latter were then activated via their conversion to N-hydroxysuccinimide carbonates and carbamates, respectively. The usefulness of these reagents for protein modification was investigated. The modified proteins obtained exhibited similar stability and activity characteristics compared to those modified with active N-hydroxysuccinimdyl esters. The activation of hydroxy- or amino-terminating compounds with DSC represents a general method that can be applied to any ligand which contains these functional groups for its covalent coupling to amines.

In vitro and in vivo evaluation of acellular diaphragmatic matrices seeded with muscle precursors cells and coated with VEGF silica gels to repair muscle defect of the diaphragm.

In this work, a bioartificial system consisting of VEGF-loaded porous silica gel and myoblasts cultured on acellular diaphragmatic matrix (ADM) has been implanted to repair a surgically created diaphragmatic defect in Lewis rats. ADMs exerted a strong angiogenic response on chorio-allantoic membrane. Cytotoxicity, VEGF release and matrix erodibility in vitro tests demonstrated that the silica support was nontoxic and that the VEGF bioactivity was maintained after matrix entrapment and it was released within a timeframe that can be modulated by synthesis parameters. Different grafts composed by ADMs with and without autologous male myoblasts or/and VEGF-loaded porous silica gel have been implanted to repair previously created diaphragmatic defects in female Lewis rats. Patches composed of ADMs and myoblasts appeared well preserved until 8 weeks, and contained multinucleated cells and cholinergic fibers. At 8 weeks, the implanted cells were still present inside the patches. The disappointing results obtained when VEGF was delivered by porous silica gel were probably due to an abnormal angiogenic response following an excess of local growth factor concentration. Taken together, these results confirmed that our matrices contained biologically active angiogenic factors which were per se sufficient to induce neo-vessels formation, thus allowing the survival of implanted myoblasts.

Covalent modification of mushroom tyrosinase with different amphiphic polymers for pharmaceutical and biocatalysis applications.

Two different poly(ethylene glycol) derivatives (linear, mol wt 5000 and a branched form, mol wt 10000) and a new polymer (poly [acryloylmorfoline], mol wt 5500) were covalently bound to the enzyme tyrosinase. The polymer-protein conjugates were studied with a view to their potential pharmaceutical application and to their use for the bioconversion of phenolic substrates in organic solvents.Vmax andKm for the dopa-dopaquinone conversion, thermostability, stability toward inactivation by dopa oxidation products, half-life in blood circulation, and behavior in organic solvents for the different adducts were investi gated. Arrhenius plots for the dopa-dopaquinone conversion were also obtained in order to study the effects of temperature on the different enzyme forms. Covalent attachment of the polymers increased enzyme stability in aqueous solution and the solubility in organic solvents. However, organic solvent solubilization brought about loss of enzyme conformation as assessed by CD measurements, which is accompanied by a nonreversible loss of catalytic activity.

Protective effect of superoxide dismutase and polyethylene glycol-linked superoxide dismutase against renal warm ischemia/reperfusion injury

The protective effect of oxygen free radical scavenger superoxide dismutase (SOD) against the warm ischemic damage that occurs in kidneys harvested from non-heart-beating donors is controversial because of its short half-life. In this model, we compared the protective effect of SOD and two longer lasting polyethylene glycol (PEG)-linked forms of SOD in a model of renal ischemia induced by 60 min of arterial clamping in rats. Rats treated with PEG1-SOD and PEG2-SOD had a better renal function than controls, with significantly lower serum creatinine levels throughout the follow-up period and a significantly higher creatinine clearance on postoperative days 1, 2, and 4. In native SOD treated-rats, serum creatinine was lower than in controls, though not significantly so, and creatinine clearance was significantly higher on postoperative day 4. Our results indicate that the protective effect of SOD against renal warm ischemia can be enhanced by prolonging its half-life by binding the enzyme to PEG.

Conjugates of Peptides and Proteins to Polyethylene Glycols

This chapter provides a critical overview of the technology presently available in the field of protein PEGylation. The chemistry of the polymer and of its reactive derivatives is discussed and presented together with several protocols used to obtain PEG-protein conjugates. The coupling protocols are critically discussed on the basis of the properties of the protein to be modified and those desired for the final product. Methods for product purification and characterization are also provided. The overall information provided will guide the reader toward all of the critical steps involved in the preparation of PEG-protein adducts.

In Vivo Fate of Avidin-Nucleic Acid Nanoassemblies as Multifunctional Diagnostic Tools

This study describes the formulation optimization and body-cell distribution and clearance in mice of a dually fluorescent biodegradable poly avidin nanoassembly based on the novel Avidin-Nucleic-Acid-Nano-ASsembly (ANANAS) platform as a potential advancement of classic avidin/biotin-based targeted delivery. The nanoformulation circulates freely in the bloodstream; it is slowly captured by filter organs; it is efficiently cleared within 24-48 h, and it is poorly immunogenic. The system displays more favorable properties than its parent monomeric avidin and it is a promising tool for diagnostic purposes for future translational aims, for which free circulation in the bloodstream, safety, multifunctionality and high composition definition are all necessary requirements. In addition, the assembly shows a time-dependent cell penetration capability, suggesting it may also function as a NP-dependent drug delivery tool. The ease of preparation together with the possibility to fine-tune the surface composition makes it also an ideal candidate to understand if and how nanoparticle composition affects its localization.

An approach to increased polyplex gene delivery by peptides selected from a phage display library

Phage display libraries were screened for peptides to be incorporated in nonviral gene delivery vehicles. Cells in culture were incubated with heptamer random peptide libraries displayed on M13 bacteriophages in three to five copies per phage. Surface-adherent phages were removed or inactivated and the cells were fractionated in a nuclear pellet and supernatant. Bacteriophages from each of the two fractions were amplified and reincubated with the cells. Three successive rounds of selection were performed. Eighteen sequenced clones revealed 14 different sequences. Two sequences were homologous to segments of the HIV gp120 protein. For three sequences, the corresponding synthetic peptides were generated and attached via avidin-biotin to polylysine-condensed plasmid DNA containing a reporter gene. The addition of the peptides led to 8-14 times increase in the expression of the reporter.

Bisphosphonate complexation and calcium doping in silica xerogels as a combined strategy for local and controlled release of active platinum antitumor compounds

The production of bone substitute biomimetic materials which could also act as antitumoral drug release agents is of enormous interest. We report in this paper the synthesis and characterization of a novel platinum dinuclear complex containing a geminal bisphosphonate and its embodiment into xerogels prepared by the sol-gel method. Our goal was to obtain a hybrid inorganic matrix that could release a platinum species active against bone tumors or metastases, upon local implant. Two silica xerogels were considered: one was composed of pure silica, while the other contained also some calcium as potential release-modulating agent thanks to its high affinity for bisphophonates. The platinum-complex loading capacity of the inorganic matrices, the release kinetics in buffer simulating physiological conditions, and the stability upon storage were investigated as a function of Pt-complex concentration and calcium addition. We found that the presence of calcium in the composites deeply influences not only the stability of the formulations but also the nature of the platinum complex liberated in solution.

Preservation of Thrombolytic Activity of Urokinase Modified with Monomethoxypoly(ethylene glycol

A method is described to modify urokinase by covalent binding of monomethoxypoly(ethylene glycol) (mPEG) without impairing its catalytic ac tivity towards high molecular weight substrates. The urokinase active site is protected by an inhibitor, benzamidine, bound to Sepharose during the mPEG modification in order to avoid binding mPEG chains to the active site or to the surrounding area. The mPEG modified urokinase had increased activity towards small molecular weight substrates (acetyl-Gly-methyl ester) as com pared to the unmodified enzyme, while the activity towards the high molecular weight plasminogen and the insoluble substrate fibrin clot was preserved. This did not occur when the enzyme was modified in the absence of active site pro tection. The polymer modification increased the enzymes thermostability and the stability in plasma in vitro and prolonged in vivo retention after in travenous injection in rats.