Two new anticancer polymer therapeutics were designed for tumour cell targeting. The bioconjugate... more Two new anticancer polymer therapeutics were designed for tumour cell targeting. The bioconjugates were synthesised by pullulan derivatisation with either doxorubicin or doxorubicin and folic acid. Pullulan was activated by periodate oxidation and functionalised by reductive conjugation with cysteamine and 1.9 kDa PEG(NH 2 ) 2 . The cysteamine thiol groups were conjugated to doxorubicin through a pH-sensitive hydrazone spacer while the pending PEG-NH 2 functions of one derivatised pullulan batch were conjugated to folic acid to obtain one of the two polymer therapeutics. The reaction intermediates and the final products were characterised by mass spectrometry, UV-vis analysis and reverse phase and gel permeation chromatography. The folic acid-free derivative [(NH 2 PEG)-Pull-(Cyst-Dox)] contained 6.3% (w/w) doxorubicin while the folic acid-doxorubicin-coupled derivative [(FA-PEG)-Pull-(Cyst-Dox)] contained 6% (w/w) doxorubicin and 4.3% (w/w) folic acid. Photon correlation spectroscopy showed that (NH 2 PEG)-Pull-(Cyst-Dox) and (FA-PEG)-Pull-(Cyst-Dox) assembled into particles of about 150 and 100 nm diameter, respectively. The two bioconjugates displayed similar drug release profiles either at pH 7.4 buffer or in plasma, where less than 20% of doxorubicin was released within three days. At pH 5.5, both conjugates underwent complete drug release in about 40 h. In vitro studies carried out with KB tumour cells over-expressing folic acid receptor showed that both free doxorubicin and (FA-PEG)-Pull-(Cyst-Dox) were rapidly taken up by the cells, while the internalisation of the non-folated derivative was significantly slower. Cell viability studies did not show relevant difference between the two bioconjugates. After 72 h of incubation with folic acid receptor non-expressing MCF7 cells, the IC 50 values of doxorubicin, (NH 2 PEG)-Pull-(Cyst-Dox) and (FA-PEG)-Pull-(Cyst-Dox) were 0.3 M, 1.2 M and 3.1 M, respectively. After incubation with KB cells over-expressing folic acid receptor, the IC 50 values were 0.4 M, 1.8 M and 1.1 M, respectively. Pharmacokinetic studies showed that 4 h after intravenous administration of the conjugates to Balb/c mice about 40% of the administered drug equivalent dose was present in the bloodstream while in the case of unconjugated doxorubicin, 80% of the drug was cleared within 30 min.
A new end-tailored monomethoxypoly(ethylene glycol) (PEG) for site-directed protein conjugation w... more A new end-tailored monomethoxypoly(ethylene glycol) (PEG) for site-directed protein conjugation was synthesized according to a three-step procedure: (1) linear 20 kDa PEG-NH 2 was conjugated to 12-(Boc-amino)dodecanoic acid; (2) PEG-NHCO(CH 2 ) 11 -Boc was deprotected by TFA treatment; (3) PEG-NHCO(CH 2 ) 11 -NH 2 was conjugated to 6-maleimidohexanoic acid to yield PEG-NHCO-(CH 2 ) 11 -NHCO(CH 2 ) 5 -Mal (PEG-C 18 -Mal). The chemical intermediates as well as the final product were purified by solvent precipitation/extraction and characterized by 1 H NMR spectroscopy and colorimetric analysis. The synthesis procedure yielded over 90% activated product [PEG-NHCO-(CH 2 ) 11 -NHCO(CH 2 ) 5 -Mal/PEG-NH 2 molar ratio, %]. Both PEG-C 18 -Mal and the commercial maleimido activated 20 kDa linear PEG (PEG-Mal) were used for conjugation to 17 Cys of recombinant human granulocyte colony stimulating factor (rh-G-CSF). Under denaturing conditions, at pH 7.0, both activated PEGs yielded over 90% protein conjugation. Under native conditions, about 55% and 7% PEGylated protein were obtained with PEG-C 18 -Mal and PEG-Mal, respectively. Circular dichroism analysis showed that the PEGylation does not induce detectable alteration of the protein secondary structure. On the other hand, the PEGylation conditions were found to affect significantly the protein stability. The derivatives obtained either with the two polymers by unfolding/refolding process or with PEG-Mal under native conditions displayed rapid aggregation with half-life ranging from 30 to 90 min. The derivative obtained with PEG-NHCO-(CH 2 ) 11 -NHCO(CH 2 ) 5 -Mal in the absence of guanidinium chloride displayed remarkably higher stability with aggregation half-life of about 60 h.
A two step protocol has been set up to selectively conjugate PEG to buried amino acids of protein... more A two step protocol has been set up to selectively conjugate PEG to buried amino acids of proteins. The process involves site-specific glycation followed by PEGylation of the oxidized glycosides. Aimed at glycating the cysteine groups of proteins, two maleimide-glycosylic linkers have been synthesised: galactosyl-glucono-CO-NH-(CH 2 ) 12 -NH-CO-(CH 2 ) 2 -maleimide and maltosyl-glucono-CO-NH-(CH 2 ) 12 -NH-CO-(CH 2 ) 2 -maleimide. The linkers were extensively characterized by 1 H NMR, FT-IR, ESI-TOF mass spectrometry and colorimetric assays. Complete conjugation of the activated linkers to Cys 34 of human serum albumin was obtained in about 2 h. The selective oxidation of the galactosyl and maltosyl moieties by periodate treatment yielded two and three available aldehyde groups, respectively. The PEGhydrazide conjugation to the aldehyde groups was found to be 100% in about 40 h, whereas less than 30% protein modification was obtained by direct conjugation of commercial PEG-maleimide to Cys 34 . The pH dependent PEG-glycosyl hydrazone bond hydrolysis at various pH values was verified. PEG release was faster under mild acidic and basic conditions than at neutral pH. Furthermore, the maltosyl derivatives, by virtue of the higher number of coupled PEG chains, showed a slower protein release as compared to the galactosyl counterpart, indicating that the choice of the glycosylic linker allows for control of protein release kinetics.
Supramolecular conjugation techniques have been developed to produce novel nanosized systems by a... more Supramolecular conjugation techniques have been developed to produce novel nanosized systems by assembling materials with diverse physicochemical and biological features. These techniques have been adapted to obtain innovative bioconjugates to deliver drugs with poor biopharmaceutical properties and nano-devices with potential “theranostic” activity. Supramolecular drug delivery systems include polymer therapeutics such as drug–polymer bioconjugates, and colloidal carriers such as micelles, liposomes, polyplexes, and organic and inorganic nanoparticles. By virtue of their wide array of chemical composition and properties, polymers represent key elements for the construction of novel supramoelcular formulations.Polymer bioconjugation is a fledged technique for fabrication of protein–polymer conjugates. PEGylation, in particular, produces derivatives with enhanced pharmacokinetic, immunological, and stability properties as compared to the parent protein. Over the years, new methods have been set up to obtain site-directed polymer conjugation. In this review we report few grafting to and growing from PEGylation examples for the preparation of therapeutically effective protein bioconjugates.Supramolecular formulations with unique properties can be also obtained by assembling functional polymers, targeting agents, physicochemical modifiers, and biomodulators. These systems may be designed for disease tissue disposition and cell recognition/penetration. Cyclodextrins, for example, have been functionalized with polyethylene glycol and folic acid to produce tumor-targeted drug carriers. Interesting results have been obtained with this novel class of drug delivery systems. In addition, responsive polymers have been conjugated to gold nanoparticles to endow a new colloidal platform with triggerable cell disposition properties, which can be exploited either in biomedicine or diagnosis.
Two new anticancer polymer therapeutics were designed for tumour cell targeting. The bioconjugate... more Two new anticancer polymer therapeutics were designed for tumour cell targeting. The bioconjugates were synthesised by pullulan derivatisation with either doxorubicin or doxorubicin and folic acid. Pullulan was activated by periodate oxidation and functionalised by reductive conjugation with cysteamine and 1.9 kDa PEG(NH 2 ) 2 . The cysteamine thiol groups were conjugated to doxorubicin through a pH-sensitive hydrazone spacer while the pending PEG-NH 2 functions of one derivatised pullulan batch were conjugated to folic acid to obtain one of the two polymer therapeutics. The reaction intermediates and the final products were characterised by mass spectrometry, UV-vis analysis and reverse phase and gel permeation chromatography. The folic acid-free derivative [(NH 2 PEG)-Pull-(Cyst-Dox)] contained 6.3% (w/w) doxorubicin while the folic acid-doxorubicin-coupled derivative [(FA-PEG)-Pull-(Cyst-Dox)] contained 6% (w/w) doxorubicin and 4.3% (w/w) folic acid. Photon correlation spectroscopy showed that (NH 2 PEG)-Pull-(Cyst-Dox) and (FA-PEG)-Pull-(Cyst-Dox) assembled into particles of about 150 and 100 nm diameter, respectively. The two bioconjugates displayed similar drug release profiles either at pH 7.4 buffer or in plasma, where less than 20% of doxorubicin was released within three days. At pH 5.5, both conjugates underwent complete drug release in about 40 h. In vitro studies carried out with KB tumour cells over-expressing folic acid receptor showed that both free doxorubicin and (FA-PEG)-Pull-(Cyst-Dox) were rapidly taken up by the cells, while the internalisation of the non-folated derivative was significantly slower. Cell viability studies did not show relevant difference between the two bioconjugates. After 72 h of incubation with folic acid receptor non-expressing MCF7 cells, the IC 50 values of doxorubicin, (NH 2 PEG)-Pull-(Cyst-Dox) and (FA-PEG)-Pull-(Cyst-Dox) were 0.3 M, 1.2 M and 3.1 M, respectively. After incubation with KB cells over-expressing folic acid receptor, the IC 50 values were 0.4 M, 1.8 M and 1.1 M, respectively. Pharmacokinetic studies showed that 4 h after intravenous administration of the conjugates to Balb/c mice about 40% of the administered drug equivalent dose was present in the bloodstream while in the case of unconjugated doxorubicin, 80% of the drug was cleared within 30 min.
A new end-tailored monomethoxypoly(ethylene glycol) (PEG) for site-directed protein conjugation w... more A new end-tailored monomethoxypoly(ethylene glycol) (PEG) for site-directed protein conjugation was synthesized according to a three-step procedure: (1) linear 20 kDa PEG-NH 2 was conjugated to 12-(Boc-amino)dodecanoic acid; (2) PEG-NHCO(CH 2 ) 11 -Boc was deprotected by TFA treatment; (3) PEG-NHCO(CH 2 ) 11 -NH 2 was conjugated to 6-maleimidohexanoic acid to yield PEG-NHCO-(CH 2 ) 11 -NHCO(CH 2 ) 5 -Mal (PEG-C 18 -Mal). The chemical intermediates as well as the final product were purified by solvent precipitation/extraction and characterized by 1 H NMR spectroscopy and colorimetric analysis. The synthesis procedure yielded over 90% activated product [PEG-NHCO-(CH 2 ) 11 -NHCO(CH 2 ) 5 -Mal/PEG-NH 2 molar ratio, %]. Both PEG-C 18 -Mal and the commercial maleimido activated 20 kDa linear PEG (PEG-Mal) were used for conjugation to 17 Cys of recombinant human granulocyte colony stimulating factor (rh-G-CSF). Under denaturing conditions, at pH 7.0, both activated PEGs yielded over 90% protein conjugation. Under native conditions, about 55% and 7% PEGylated protein were obtained with PEG-C 18 -Mal and PEG-Mal, respectively. Circular dichroism analysis showed that the PEGylation does not induce detectable alteration of the protein secondary structure. On the other hand, the PEGylation conditions were found to affect significantly the protein stability. The derivatives obtained either with the two polymers by unfolding/refolding process or with PEG-Mal under native conditions displayed rapid aggregation with half-life ranging from 30 to 90 min. The derivative obtained with PEG-NHCO-(CH 2 ) 11 -NHCO(CH 2 ) 5 -Mal in the absence of guanidinium chloride displayed remarkably higher stability with aggregation half-life of about 60 h.
A two step protocol has been set up to selectively conjugate PEG to buried amino acids of protein... more A two step protocol has been set up to selectively conjugate PEG to buried amino acids of proteins. The process involves site-specific glycation followed by PEGylation of the oxidized glycosides. Aimed at glycating the cysteine groups of proteins, two maleimide-glycosylic linkers have been synthesised: galactosyl-glucono-CO-NH-(CH 2 ) 12 -NH-CO-(CH 2 ) 2 -maleimide and maltosyl-glucono-CO-NH-(CH 2 ) 12 -NH-CO-(CH 2 ) 2 -maleimide. The linkers were extensively characterized by 1 H NMR, FT-IR, ESI-TOF mass spectrometry and colorimetric assays. Complete conjugation of the activated linkers to Cys 34 of human serum albumin was obtained in about 2 h. The selective oxidation of the galactosyl and maltosyl moieties by periodate treatment yielded two and three available aldehyde groups, respectively. The PEGhydrazide conjugation to the aldehyde groups was found to be 100% in about 40 h, whereas less than 30% protein modification was obtained by direct conjugation of commercial PEG-maleimide to Cys 34 . The pH dependent PEG-glycosyl hydrazone bond hydrolysis at various pH values was verified. PEG release was faster under mild acidic and basic conditions than at neutral pH. Furthermore, the maltosyl derivatives, by virtue of the higher number of coupled PEG chains, showed a slower protein release as compared to the galactosyl counterpart, indicating that the choice of the glycosylic linker allows for control of protein release kinetics.
Supramolecular conjugation techniques have been developed to produce novel nanosized systems by a... more Supramolecular conjugation techniques have been developed to produce novel nanosized systems by assembling materials with diverse physicochemical and biological features. These techniques have been adapted to obtain innovative bioconjugates to deliver drugs with poor biopharmaceutical properties and nano-devices with potential “theranostic” activity. Supramolecular drug delivery systems include polymer therapeutics such as drug–polymer bioconjugates, and colloidal carriers such as micelles, liposomes, polyplexes, and organic and inorganic nanoparticles. By virtue of their wide array of chemical composition and properties, polymers represent key elements for the construction of novel supramoelcular formulations.Polymer bioconjugation is a fledged technique for fabrication of protein–polymer conjugates. PEGylation, in particular, produces derivatives with enhanced pharmacokinetic, immunological, and stability properties as compared to the parent protein. Over the years, new methods have been set up to obtain site-directed polymer conjugation. In this review we report few grafting to and growing from PEGylation examples for the preparation of therapeutically effective protein bioconjugates.Supramolecular formulations with unique properties can be also obtained by assembling functional polymers, targeting agents, physicochemical modifiers, and biomodulators. These systems may be designed for disease tissue disposition and cell recognition/penetration. Cyclodextrins, for example, have been functionalized with polyethylene glycol and folic acid to produce tumor-targeted drug carriers. Interesting results have been obtained with this novel class of drug delivery systems. In addition, responsive polymers have been conjugated to gold nanoparticles to endow a new colloidal platform with triggerable cell disposition properties, which can be exploited either in biomedicine or diagnosis.
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