- Email: [email protected]
Synthesis and characterization of new poly(ethylene glycol) derivatives
S96
381
383
SYNTHESIS AND CHARACTERIZATION OF NEW POLY(ETHYLENE GLYCOL) DERIVATIVES S, Aroicca, P. Brusa, F. Dosio, P. Crosasso and L. Cattel Dipartimcnto di Scienza e Tccnologia de.1Farmaw, Via P. Giuria, 9, 10125 Torino, Italy.
PER LIGAND BINDING SITES: cYsTEINF. RFSILWE MODIFICATIONBY NEM ilxQsf& 0. Gi4lMaw A Lacacchiai, c. Martini 1 Dknrtimmto dl wichiatria, Neumbiologia. Farmawlogla e Biotccnologje, Universita dcgli Studi di Plsa, via Bonanno, 6-56100 Pint.
One of the most cxtcnsivcly studied drug-delivciy systems involves the covalent linkage of the polymer monomcthoxypoly(cthylcnc glycol) (mPEG) to the surf& of proteins, mPEG-modified proteins exhibit increased stability and resistance to protcolitic inactivation, reduced immtmogenicity, increased ciroulating lives and low toxicity. In this study we describe the synthesis, the charactcriaation, and the rcaotivity of new mPEG derivatives comaming a thioimidoestcr ma&-c group. This cmsslinking agents arc able to react with the lysyl s-amino groups of suitable protcin generating an amidinatcd linkage and, thereby, prescwing the positive charge of the protein. mPEG of molecular weight of 2000 and 5000 Da wcrc used and two kinds of linkage wcrc prcpared introducing spacers of differ& length between the hydmxyl group of the polymer and the thioimidatc group. The derivatives wcrc cbamcterized by ‘H- and ‘%-NMR. To cvahtatc the rcaotivity toward lysyl a-ammo groups, proteins with dit%cm molaular weight wcrc modified, and the dcrivatization degree was estimated by TNBS mcthod. The different conjugates wcrc analyscd by gel filtration and by elcctrophorcsis. We observed that the dcrivatization degree incrcascs in proportion to the molar excess of mPEG used and in function of the spacer length. WC will extend this work to the derivatisation of proteins with pharmacologi~l properties usually damaged in their activity by charge modification. We thanks Prof. F. M. Vcronese for the helpful suggwtions. This work was supported by MURST 40%-60%.
The pu.iphcml bcnmdiascpinc recquer (PBR) exhibits higli atlinity both for the bcnmdlampinc 4’cblorodiizepsm (Ro5-4864) and isequinoline catboxamidc d&at& (m
382
384
CATIONIC! LIPOSOME-?LASMID DNA STRUCTURES AND TRANSFECTION EFFICIENCY J.C.Birchall, 1.W. Kc&way and S.N. Mills* Welsh School of Pharmacy, University of Wales. Cardiff. UK. *Glaxo Wellcome PLC. Park Road, Ware, Hettfordshire, UK.
SITE SPECIFIC DRUG CARRIER SYSTEMS - A CRlTICAL STUDY OF ALBUMIN MICRO f NAN0 SPHERES USING FOUR ANTI-TUMOR AGENTS SADhanarai. BSurcsh, K.Santhi, MRavichandran R.Raghu, G.S.Sariths and P.Vijayan. Ccntre for P.G. Studies & Raearch J.S.S. Collcgc of Pharmacy, Cotaeamund-643 001, Tamil Nadu, India.
Cationic liposomes are being developed as non-viral vectors for gene &livery. The cationlc vesicles spontaneously intcraot with negatively charged DNA to form a transfcetion competent lipidDNA complex capable of promoting the expression of a therapeutic We aimed to improve the understanding of the poorly $?zed mechanism of interaction between the cationic liposomcs Dioleoyl 1,2-diacyld-trimethylammonium and plasmid DNA. propane (DGTAP) and dimcthyldiootndeeylammonium bromide (DDAB):diolmylphosphatidylethanolamine (DOPE) (0.4: 1w/w) lasmid DNA to cationic Ii somes were formulated and mixed with form lipt.$-DNA complexes. The size and charge oP the complexes as determined by photon correlation spectmswpy and microelcctmphoresis were found to bc depcndant on the lipid:DNA ratio with both DDAB:DGPB-DNA and DOTAP-DNA complexes aggregating immediately at. neutral charge ratios. Negative stain transmission electron micmscouv demonstmted the structural Gel heterogeneity of the neutral ‘IjOTAPDNA aggregates. electmphorcsis was used to determine the efficiency and extent of lipid-DNA complex formation. Resulta showed that only DOTAP liposomes wen capable of protecting tbe enclosed plasmid DNA from nuclease digestion. Isothermal micmcalotimetry showed that the thermal instability of tbe supercoiled DNA was also delayed by complexation with DtYfAP liposomes. A range of complexes were tmnsfeetcd into A549 and COS-7 in vitro cell lines to establish the efficiency of cationic lipid mediated gene delivery, The efficiency of reporter gene (S-gala&s&se) expression depended on the type of liposome used in the complex, the ratio of IipidDNA and the transfceted cell line. Acknowledgements: This study was tinancially BBSRC and Glaxo Wellcome PLC.
supported
by the
Drug carrier systems have bcm recently investigated and introduced to regulate the rcleasc and targeting of drugs particularly anti-tumor drugs for their high cell and i or site specific activity. Bio-degmdable synthetic and natural polymers have been investigated as car&r systems irrespective of cost of formulation. Since Albumin microspheres have shown numerous advantages over other biodcgmdahle pol~ymers.we have decided to investigate the Albumin micro / nano spheres drug oanier system for the delivery of anti-tumor drugs. We have studied various process variables during the formulation of this carrier system from Egg Bovine and Human Serum Albumin Having selected the suitable albumin oarrier system, the drugs Etoposidc, Tamoxifen Citrate, 5-Fluro uracil(5Fu) Mcthotmxatc (MTX) were loaded into the system which were evaluated invitro and invivo for passive targeting efficiency. MTX Albumin microspheres were conjugated with Mono Clonal Antibodies raised against non specific tumor antigens and the efficiency was studied in mice model induced with Dalton’s ascites lymphoma. The MTX Lmmtmo microsphcrcs showed better protection against tumor than Free MIX and MTX nnnosphcrcs without monoclonal antibodies. The efficiency of the carrier system using anti tumor agents in relation with physical parameters will also be discussed.
381
383
SYNTHESIS AND CHARACTERIZATION OF NEW POLY(ETHYLENE GLYCOL) DERIVATIVES S, Aroicca, P. Brusa, F. Dosio, P. Crosasso and L. Cattel Dipartimcnto di Scienza e Tccnologia de.1Farmaw, Via P. Giuria, 9, 10125 Torino, Italy.
PER LIGAND BINDING SITES: cYsTEINF. RFSILWE MODIFICATIONBY NEM ilxQsf& 0. Gi4lMaw A Lacacchiai, c. Martini 1 Dknrtimmto dl wichiatria, Neumbiologia. Farmawlogla e Biotccnologje, Universita dcgli Studi di Plsa, via Bonanno, 6-56100 Pint.
One of the most cxtcnsivcly studied drug-delivciy systems involves the covalent linkage of the polymer monomcthoxypoly(cthylcnc glycol) (mPEG) to the surf& of proteins, mPEG-modified proteins exhibit increased stability and resistance to protcolitic inactivation, reduced immtmogenicity, increased ciroulating lives and low toxicity. In this study we describe the synthesis, the charactcriaation, and the rcaotivity of new mPEG derivatives comaming a thioimidoestcr ma&-c group. This cmsslinking agents arc able to react with the lysyl s-amino groups of suitable protcin generating an amidinatcd linkage and, thereby, prescwing the positive charge of the protein. mPEG of molecular weight of 2000 and 5000 Da wcrc used and two kinds of linkage wcrc prcpared introducing spacers of differ& length between the hydmxyl group of the polymer and the thioimidatc group. The derivatives wcrc cbamcterized by ‘H- and ‘%-NMR. To cvahtatc the rcaotivity toward lysyl a-ammo groups, proteins with dit%cm molaular weight wcrc modified, and the dcrivatization degree was estimated by TNBS mcthod. The different conjugates wcrc analyscd by gel filtration and by elcctrophorcsis. We observed that the dcrivatization degree incrcascs in proportion to the molar excess of mPEG used and in function of the spacer length. WC will extend this work to the derivatisation of proteins with pharmacologi~l properties usually damaged in their activity by charge modification. We thanks Prof. F. M. Vcronese for the helpful suggwtions. This work was supported by MURST 40%-60%.
The pu.iphcml bcnmdiascpinc recquer (PBR) exhibits higli atlinity both for the bcnmdlampinc 4’cblorodiizepsm (Ro5-4864) and isequinoline catboxamidc d&at& (m
382
384
CATIONIC! LIPOSOME-?LASMID DNA STRUCTURES AND TRANSFECTION EFFICIENCY J.C.Birchall, 1.W. Kc&way and S.N. Mills* Welsh School of Pharmacy, University of Wales. Cardiff. UK. *Glaxo Wellcome PLC. Park Road, Ware, Hettfordshire, UK.
SITE SPECIFIC DRUG CARRIER SYSTEMS - A CRlTICAL STUDY OF ALBUMIN MICRO f NAN0 SPHERES USING FOUR ANTI-TUMOR AGENTS SADhanarai. BSurcsh, K.Santhi, MRavichandran R.Raghu, G.S.Sariths and P.Vijayan. Ccntre for P.G. Studies & Raearch J.S.S. Collcgc of Pharmacy, Cotaeamund-643 001, Tamil Nadu, India.
Cationic liposomes are being developed as non-viral vectors for gene &livery. The cationlc vesicles spontaneously intcraot with negatively charged DNA to form a transfcetion competent lipidDNA complex capable of promoting the expression of a therapeutic We aimed to improve the understanding of the poorly $?zed mechanism of interaction between the cationic liposomcs Dioleoyl 1,2-diacyld-trimethylammonium and plasmid DNA. propane (DGTAP) and dimcthyldiootndeeylammonium bromide (DDAB):diolmylphosphatidylethanolamine (DOPE) (0.4: 1w/w) lasmid DNA to cationic Ii somes were formulated and mixed with form lipt.$-DNA complexes. The size and charge oP the complexes as determined by photon correlation spectmswpy and microelcctmphoresis were found to bc depcndant on the lipid:DNA ratio with both DDAB:DGPB-DNA and DOTAP-DNA complexes aggregating immediately at. neutral charge ratios. Negative stain transmission electron micmscouv demonstmted the structural Gel heterogeneity of the neutral ‘IjOTAPDNA aggregates. electmphorcsis was used to determine the efficiency and extent of lipid-DNA complex formation. Resulta showed that only DOTAP liposomes wen capable of protecting tbe enclosed plasmid DNA from nuclease digestion. Isothermal micmcalotimetry showed that the thermal instability of tbe supercoiled DNA was also delayed by complexation with DtYfAP liposomes. A range of complexes were tmnsfeetcd into A549 and COS-7 in vitro cell lines to establish the efficiency of cationic lipid mediated gene delivery, The efficiency of reporter gene (S-gala&s&se) expression depended on the type of liposome used in the complex, the ratio of IipidDNA and the transfceted cell line. Acknowledgements: This study was tinancially BBSRC and Glaxo Wellcome PLC.
supported
by the
Drug carrier systems have bcm recently investigated and introduced to regulate the rcleasc and targeting of drugs particularly anti-tumor drugs for their high cell and i or site specific activity. Bio-degmdable synthetic and natural polymers have been investigated as car&r systems irrespective of cost of formulation. Since Albumin microspheres have shown numerous advantages over other biodcgmdahle pol~ymers.we have decided to investigate the Albumin micro / nano spheres drug oanier system for the delivery of anti-tumor drugs. We have studied various process variables during the formulation of this carrier system from Egg Bovine and Human Serum Albumin Having selected the suitable albumin oarrier system, the drugs Etoposidc, Tamoxifen Citrate, 5-Fluro uracil(5Fu) Mcthotmxatc (MTX) were loaded into the system which were evaluated invitro and invivo for passive targeting efficiency. MTX Albumin microspheres were conjugated with Mono Clonal Antibodies raised against non specific tumor antigens and the efficiency was studied in mice model induced with Dalton’s ascites lymphoma. The MTX Lmmtmo microsphcrcs showed better protection against tumor than Free MIX and MTX nnnosphcrcs without monoclonal antibodies. The efficiency of the carrier system using anti tumor agents in relation with physical parameters will also be discussed.