Publications (14)52.22 Total impact
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Article: Relevance of folic acid/polymer ratio in targeted PEG-epirubicin conjugates.
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ABSTRACT: A series of PEG-epirubicin conjugates with different folic acid contents per polymer chain was synthesized in order to study the influence of polymer/targeting moiety ratio on selective cytotoxicity, cellular uptake and intracellular localization. Analogous carboxyl-terminated conjugates without folic acid were studied as control. The heterobifunctional HO-PEG-COOH was used as polymeric carrier, allowing the synthesis of conjugates with a good control over the chemical structure and the drug/polymer and polymer/targeting residue ratios. A dendron structure was synthesized at one end of the PEG chain with the aim to increase the number of folic acid molecules. L-2-aminoadipic acid was used as branching unit. The conjugates showed high stability under several physiological conditions. Biological evaluation was carried out in A549, HeLa and KB-3-1 human cell lines, as these cells have different levels of folate receptor (FR) expression. In particular A549 cells are FR negative (FR-), HeLa cells are FR positive (FR+) and KB-3-1 cells over-express FR (FR++). It was clearly shown that the biological activity of the conjugates was influenced by the presence and the number of folic acid molecules per polymer chain and by the level of FR expression on cell surface. Conjugates conformation in solution was also studied, as differences in size might well affect cell internalization. In the cell viability assay, conjugates without folic acid were unexpectedly more cytotoxic than the targeted conjugates, but their IC(50) values were similar in the three cell lines. Differently, the anti-proliferative activity of targeted derivatives markedly increased going from FR(-) to FR(++) cells. FACS and confocal microscopy studies showed greater cellular internalization with the targeted conjugates than with their non-targeted analogues; more importantly, this relationship is clearly dependent on folic acid content in the conjugates and FR expression level in the cell line used.Journal of Controlled Release 09/2010; 146(3):388-99. · 5.73 Impact Factor -
Article: Antitumoral activity of PEG-gemcitabine prodrugs targeted by folic acid.
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ABSTRACT: Gemcitabine, 2',2'-difluoro-2'-deoxycytidine (dFdC), is an antitumor agent effective in the treatment of several solid tumors but its use is hampered by short plasma half-life, rapid metabolism and low selectivity towards tumor tissue. To overcome these limits, bioconjugates of gemcitabine were studied using poly(ethylene glycol) as polymeric carrier. Two types of conjugates were prepared, non-targeted and folic acid targeted conjugates. The formers were obtained starting from mPEG-OH of 5 and 20 kDa with linear or branched structure. The folic acid targeted conjugates, differing for the drug loading, were prepared exploiting a heterobifunctional PEG that allowed a consecutive coupling of the targeting agent and the drug. Folic acid was chosen as targeting agent because its receptor is often over-expressed in many tumors. To increase the polymer drug payload, the bicarboxylic amino acid, aminoadipic acid, was used. All conjugates were able to release the drug in a pH-dependent manner with no role of enzymes. The pharmacokinetic profiles are strictly related to the polymer molecular weight and the folic acid targeting increased 2-3 times the affinity towards the cells over-expressing folic acid receptors. These results are promising and encourage in vivo studies on these conjugates that act as polymeric prodrugs.Journal of Controlled Release 06/2008; 127(3):239-48. · 5.73 Impact Factor -
Article: Immobilization of Purified Penicillin Acylase in a Polarized Ultrafiltration Membrane Reactor
Annals of the New York Academy of Sciences 12/2006; 434(1):127 - 130. · 3.15 Impact Factor -
Chapter: New Targetable Conjugates of Anticancer Drugs with Soluble Biodegradable Polymeric Carriers
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ABSTRACT: The main objective of this study is to prepare a new generation of tumour targeting polymer conjugates of anticancer drugs with improved therapeutic performance.The proposed polymeric systems will have the structure of multiblock copolymers in which polyester residues containing maleate or fumarate units are coupled with residues of polyethylenglycol.In particular, they will show the following features:a) high molecular weight, in order to improve selective accumulation at the level of tumour cells; b) biodegradable backbone, in order to permit elimination from the body of the macromolecular residues; c) water solubility, achieved by the presence of polyethylenglycol moieties; d) possibility of further functionalisation with introduction of drug residues and assisting moieties according to well established general principles.12/2005: pages 179 - 184; , ISBN: 9783527606146 -
Article: PEG-doxorubicin conjugates: influence of polymer structure on drug release, in vitro cytotoxicity, biodistribution, and antitumor activity.
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ABSTRACT: Polymer-drug conjugates (polymer therapeutics) are finding increasing use as novel anticancer agents. Here a series of poly(ethylene glycol) PEG-doxorubicin (Dox) conjugates were synthesized using polymers of linear or branched architecture (molecular weight 5000-20000 g/mol) and with different peptidyl linkers (GFLG, GLFG, GLG, GGRR, and RGLG). The resultant conjugates had a drug loading of 2.7-8.0 wt % Dox and contained <2.0% free drug (% total drug). All conjugates containing a GFLG linker showed approximately 30% release of Dox at 5 h irrespective of PEG molecular weight or architecture. The GLFG linker was degraded more quickly (approximately 57% Dox release at 5 h), and the other linkers more slowly (<16% release at 5 h), by lysosomal enzymes in vitro. In vitro there was no clear relationship between cytotoxicity toward B16F10 cells and the observed Dox release rate. All PEG conjugates were more than 10-fold less toxic (IC50 values > 2 microg/mL) than free Dox (IC50 value = 0.24 microg/mL). Biodistribution in mice bearing sc B16F10 tumors was assessed after administration of PEGs (5000, 10000, or 20000 g/mol) radioiodinated using the Bolton and Hunter reagent or PEG-Dox conjugates by HPLC. The 125I-labeled PEGs showed a clear relationship between Mw and blood clearance and tumor accumulation. The highest Mw PEG had the longest plasma residence time and consequently the greatest tumor targeting. The PEG-Dox conjugates showed a markedly prolonged plasma clearance and greater tumor targeting compared to free Dox, but there was no clear molecular weight-dependence on biodistribution. This was consistent with the observation that the PEG-Dox conjugates formed micelles in aqueous solution comprising 2-20 PEG-Dox molecules depending on polymer Mw and architecture. Although PEG-Dox showed greater tumor targeting than free Dox, PEG conjugation led to significantly lower anthracycline levels in heart. Preliminary experiments to assess antitumor activity against sc B16F10 in vivo showed the PEG5000linear (L)-GFLG-Dox and PEG10000branched (B)-GLFG-Dox (both 5 mg/kg Dox-equiv) to be the most active with T/C values of 146 and 143%, respectively. Free Dox did not show significant activity in this model (T/C = 121%). Dose escalation of PEG5000(L)-GFLG-Dox to 10 mg/kg Dox-equiv prolonged further animal survival (T/C = 161%). Using the Dox-sensitive model ip L1210 (where Dox displayed a T/C = 150% after single ip dose), the PEG5000(L)-GFLG-Dox displayed a maximum T/C of 141% (10 mg/kg Dox-equiv) using a once a day (x3) schedule. Further studies are warranted with PEG5000(L)-GFLG-Dox to determine its spectrum of antitumor activity and also the optimum dosing schedule before clinical testing.Bioconjugate Chemistry 05/2005; 16(4):775-84. · 4.93 Impact Factor -
Article: Poly(ethylene glycol)-poly(ester-carbonate) block copolymers carrying PEG-peptidyl-doxorubicin pendant side chains: synthesis and evaluation as anticancer conjugates.
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ABSTRACT: Water soluble polymer anticancer conjugates can improve the pharmacokinetics of covalently bound drugs by limiting cellular uptake to the endocytic route, thus prolonging plasma circulation time and consequently facilitating tumor targeting by the enhanced permeability and retention (EPR) effect. Many of the first generation antitumor polymer conjugates used nonbiodegradable polymeric carriers which limits the molecular weight that can be safely used to <40,000 g/mol. The aim of this ambitious study was to synthesize and evaluate a novel, prototype biodegradable polymeric system based on high molecular weight, water-soluble functionalized polyesters. The main polymeric platform was prepared from bis(4-hydroxy)butyl maleate (DBM) and poly(ethylene glycol) (PEG4000) blocks to give the polymer DBM2-PEG4000 containing biodegradable carbonate bonds and having a M(w) of 100,000-190,000 g/mol; M(n) of 37,000-53,000 g/mol, and M(w)/M(n) of 3.0-3.7. Using thioether linkages, this polymer was then grafted with HS-PEG3000-Gly-Phe-Lue-Gly doxorubicin (HS-PEG3000-GFLG-Dox) pendant side chains ( approximately 30 per DBM2-PEG chain). The final construct, DBM2-PEG4000-S-PEG3000-GFLG-Dox had a total Dox content of 3-4 wt % and a free Dox content of < or = 0.7% total Dox. During incubation with isolated lysosomal enzymes, the rate of Dox release from the polymer backbone was relatively slow (<5% release over 5 h) compared to that seen for PEG5000-GFLG-Dox alone (>20% over 5 h). The in vitro cytotoxicity was assessed using B16F10 murine melanoma (MTT assay). DBM2-PEG4000-S-PEG3000-GFLG-Dox was 10-20-fold less toxic than free Dox. In vivo antitumor activity of the DBM2-PEG4000-S-PEG3000-GFLG-Dox conjugates was assessed using a subcutaneous (s.c.) B16F10 murine melanoma model, and an intraperitoneal (i.p.) L1210 leukaemia model. The increased toxicity (attributed to poor solubility) and low antitumor activity of DBM2-PEG4000-S-PEG3000-GFLG-Dox conjugates compared to PEG5000-GFLG-Dox and HPMA copolymer-Dox conjugates was attributed to the slow rate of Dox release. The DBM2-PEG4000-S-PEG3000-GFLG-Dox conjugates were considered unfavorable as candidates for further development. However, the successful scale-up synthesis of DBM2-PEG4000-S-PEG3000 constructs suggest that they are worthy of further investigation as carriers for controlled release and targeting of less hydrophobic agents.Biomacromolecules 01/2005; 6(2):914-26. · 5.48 Impact Factor -
Article: Synthesis and molecular weight characterization of low molecular weight end‐functionalized poly(4‐acryloylmorpholine)
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ABSTRACT: New amphiphilic oligomers ending at one side with a reactive function were synthesized by radical polymerization of a vinyl monomer, N-acryloylmorpholine, in the presence of functionalized chain-transfer agents (CTAs). The oligomers were characterized in terms of molecular weight and molecular weight distribution, by means of analytical size-exclusion chromatography (SEC) working in buffered aqueous media. This has been accomplished by determining a calibration curve for a set of SEC columns, with poly(N-acryloylmorpholine) standards purposely obtained by means of preparative SEC. The transfer constant CT of some CTAs towards N-acryloylmorpholine has been estimated to be approximately 1.Macromolecular Chemistry and Physics 03/2003; 195(10):3469 - 3479. · 2.36 Impact Factor -
Article: Synthesis and molecular weight characterization of end‐functionalized N‐vinyl‐2‐pyrrolidone oligomers
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ABSTRACT: New oligomers of N-vinyl-2-pyrrolidone, functionalized at one end with hydroxy functions, were obtained by radical polymerization in the presence of a hydroxylated compound, namely 2-isopropoxyethanol, acting as chain-transfer agent. The oligomeric samples obtained were characterized in terms of molecular weight and molecular weight distribution by means of analytical size exclusion chromatography (SEC). To this purpose, a calibration curve for a set of SEC columns had been determined, making use of poly(N-vinyl-2-pyrrolidone) standards purposely prepared. The chain transfer constant CT of 2-isopropoxyethanol towards N-vinyl-2-pyrrolidone was determined from the knowledge of the cumulated number-average degree of polymerization X̄n and monomer conversion Yt of the samples during the reaction.Macromolecular Chemistry and Physics 03/2003; 196(3):763 - 774. · 2.36 Impact Factor -
Article: Polyethylene glycol-superoxide dismutase, a conjugate in search of exploitation.
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ABSTRACT: Without a doubt PEG-SOD has been the enzyme most studied in PEGylation. One can say that it represents the preferred model to assess chemistries for PEG activation, analytical procedures suitable for conjugate characterization, the influence of PEG size in conjugate removal from circulation and elimination of immunogenicity and antigenicity, and the effect of route of administration. The effect of PEG conjugation was studied in vitro and in vivo models in comparison with the free enzyme and the following conclusions may be drawn: (1) At the blood vessel level, PEG-SOD has been shown to provide a greater resistance to oxidant stress, to improve endothelium relaxation and inhibit lipid oxidation. (2) In the heart, PEG-SOD proved to be at least as effective as native SOD in treatment of reperfusion-induced arrhythmias and myocardial ischemia. (3) In the lung, PEG-SOD appeared to be able to reduce oxygen toxicity and E. coli-induced lung injury, but not in the treatment of lung physiopathology associated with endotoxin-induced acute respiratory failure and in the reduction of asbestos-induced cell damage. (4) On cerebral ischemia/reperfusion injuries the effect of PEG-SOD was uncertain, also due to the difficulty of cerebral cell penetration. (5) In kidney and liver ischemia both enzyme forms were found to ameliorate reperfusion damage. In view of so much positive research on PEG-SOD, it is surprising that no approved application in human therapy has been established and approved.Advanced Drug Delivery Reviews 07/2002; 54(4):587-606. · 11.50 Impact Factor -
Article: An improved procedure for the synthesis of branched polyethylene glycols (PEGs) with the reporter dipeptide Met-betaAla for protein conjugation.
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ABSTRACT: A new and more efficient route to the synthesis of branched PEG for protein conjugation, bearing a reporter dipeptide Met-betaAla, is described, which allows better purification of the final product by ion exchange chromatography. The product has the combined advantages of an 'umbrella-like' branched structure, which allows a better coverage of the protein surface, and the presence of the dipeptide Met-betaAla which has been used to detect the position of PEGylation within the peptide sequence.Bioorganic & Medicinal Chemistry Letters 02/2002; 12(2):177-80. · 2.55 Impact Factor -
Article: Preparation, physico-chemical and pharmacokinetic characterization of monomethoxypoly(ethylene glycol)-derivatized superoxide dismutase
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ABSTRACT: The surface modification of enzymes by polymers has recently been proposed in order to improve their use in therapy. It is found that the derivatization with monomethoxypoly(ethylene glycol) (MPEG) of Superoxide dismutase (SOD), an enzyme now used in inflammatory diseases, gives a heterogenous product. It is demonstrated that this heterogeneity depends on the presence of bifunctional PEG coming from non-methoxylated molecules. Covalent binding of PEG is accompanied by some loss in enzymatic activity, which has been demonstrated to be due to the decrease in the affinity of the Superoxide ion at the SOD surface, while the active site structure at the metals is not modified. The MPEG bound to the SOD surface modifies certain physico-chemical properties of the enzyme, such as solvent solubility, metal binding and structural stability. The pharmacokinetic parameters of the enzyme, which were evaluated in rats following i.V., i.m., i.p. and s.c. administration, has been found to be greatly modified by the polymer binding.Journal of Controlled Release 02/1989; 10:145-154. · 5.73 Impact Factor -
Article: Poly(N-acryloylmorpholine) as a new soluble support for the liquid-phase synthesis of oligonucleotides
Tetrahedron Letters 37(27):4761-4764. · 2.68 Impact Factor -
Article: Modification of physico-chemical and biopharmaceutical properties of superoxide dismutase by conjugation to the co-polymer of divinyl ether and maleic anhydride
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ABSTRACT: The co-polymer of divinyl ether and maleic anhydride (MW 30 000) was covalently conjugated to superoxide dismutase in the presence of the protecting agent 2,3-dimethylmaleic anhydride as previously reported in this journal (T. Hirano et al., Synthesis of the conjugate of superoxide dismutase with the copolymer of divinyl ether and maleic anhydride retaining enzymatic activity, J. Control. Release 28 (1994) 203–209). The polymer-enzyme conjugate obtained has been now characterised with respect to its physico-chemical, structural, pharmacokinetic and biological properties. Polymer conjugation was found not to modify the protein structure as revealed by circular dichroism measurements but did enhance the thermal stability of the enzyme. On the other hand, covalent coupling of the polymer on the protein surface was found to decrease the reversible capability of exchanging the metal co-factor Cu2+ of superoxide dismutase. The pharmacokinetic profile of the conjugate after intravenous administration in rats indicated that the conjugate presented a rapid distribution phase, while the elimination rate was significantly delayed with respect to the native form. Immunoprecipitation studies were performed by using anti-superoxide dismutase serum, which was enriched with polyclonal antibodies. Decreased antigenicity of the polymer conjugate with respect to the native form was observed.Journal of Controlled Release. -
Article: Improvement of pharmacokinetic, immunological and stability properties of asparaginase by conjugation to linear and branched monomethoxy poly( ethylene glycol)
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ABSTRACT: A comparative pre formulation study of native asparaginase and conjugated with linear monomethoxypoly(ethylene glycol) 5000 (mPEG) or branched monomethoxypoly(ethylene glycol) (mPEG2) is reported. The first polymer, mPEG, was obtained with norleucine as spacer between polymer chain and protein, while mPEG2 was obtained by linking of mPEG 5000 Mw to the α- and ϵ-lysine amino groups. The comparison regarded the enzymatic activity, stability, pharmacokinetic and immunological properties of the different enzyme forms. The enzyme modified by the branched polymer showed increased in vitro activity and proteolytic resistance, improved stability towards temperature and pH variations and enhanced half life. Furthermore, the conjugation with mPEG largely reduced recognition by polyclonal antibodies raised against native asparaginase, the effect being more marked in the mPEG2 conjugate. All these data are in favour of the use of asparaginase modified by the mPEG2 in the treatment of patients with tumour responding to this enzyme, in particular in case of intolerance to native asparaginase.Journal of Controlled Release.
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2003–2010
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University of Padua
Padova, Veneto, Italy
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