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Production of anticancerous ice cream
New Biotechnology · Volume 25S · September 2009
ABSTRACTS
interaction with SF. The experiments proved that the interaction mode between SF and CT-DNA was intercalation. doi:10.1016/j.nbt.2009.06.088
1.5.06 Hyper branched polylysines: optimization of DNA bonding, endosomalytic properties and DNA stabilization in the complexes against enzyme hydrolysis G. Vlasov 1,∗ , A. Egorova 2 , A. Kiselev 3 , P. Ilyina 2 , G. Pankova 1 , I. Tarasenko 1 , I. Ilyina 1 , V. Baranov 3 1 Institute of Macromolecular Compounds RAS, Sankt-Petersburg, Russian Federation 2 Sankt-Petersburg State University, Sankt-Petersburg, Russian Federation 3 Institute of Obstetrics and Gynecology, RAMN, Sankt-Petersburg, Russian Federation
Conjugation of biologically active compounds to polymer carriers guarantees their targeting delivery, controls allotment in organism, diminishes their toxicity (if any) and defends them from neutralization action of enzymes, inhibitors and immune system of organism. Lysine dendrimers was found to be the most perspective polymer carrier. However their synthesis is complicated and time-withdrawing. In attempt to overcome the problems we have proposed to use hyper branched polylysines (HbpK) as polymer carriers for targeted delivery of biologically active compounds. Now we communicate on possibility of promotion of DNA-bonding and transfection properties of the new kind of biodegradable and biocompatible carriers of DNA on the basis of HbpK. It was shown that HbpK itself does not possess any endosomalytic properties and as a result of that does not guarantee high level of transfection. However optimization of endosomalytic properties of HbpK, as a result of their amino groups modification with histidine, introducing in complex consisting of HbpK and plasmid DNA of endosomalytic peptide JTS-1 and realization of transfection experiments in the presence of glycerin results in the rise of transfection efficacy of HbpK—plasmid DNA complexes. It was found that modification of HbpK with histidine increases as DNA bonding so as transfection efficacy of complex consisting of HbpK and DNA. Transfection efficacy of complexes can be also extended as result of realization of transfection experiments in the presence of glycerin or endosomalytic peptide JTS-1. However realization of transfection experiments with usage of complex histidine modified HbpK in the joint presence of glycerin and endosomalytic peptide JTS-1 results in sinking of transfection efficacy. Influence of HbpK structure on stabilization plasmid DNA against enzymatic hydrolysis will be also presented and discussed. doi:10.1016/j.nbt.2009.06.089
1.5.07 Low molecular weight hyper branched polylysines for DNA binding and delivery: investigation of peculiar properties S. Gorbunova 1,∗ , G. Pankova 1 , I. Tarasenko 1 , V. Vorobév 2 , G.P. Vlasov 1 1
2
Institute of Macromolecular Compounds RAS, Riga, Latvia Institute of Cytology RAS, Riga, Latvia
Recently we have devised a method of synthesis and application of hyper branched polylysines (hbpK) as a new type of biocompatible and biodegradable polymer carriers on the basis of lysine for drug delivery, particularly in Gene Therapy. Their ability to bind DNA, form stable complexes and transfect certain cell lines was shown. The synthesis of hbpK was realized under the condition of reducing catalytic removing of carbobenzoxy blocking groups from N-carboxyanhydride; carbobenzoxylysine (N-CA) by the action of gaseous hydrogen or formic acid in the presence of activated palladium. Now we report the investigation of structure features of low molecular weight hbpK formed in the presence of large excess of reducing agent. Polymer properties were studied using gel permeation chromatography, circular dichroism and capillary electrophoresis as well as enzymatic hydrolysis under the presence of trypsin. It was found that low molecular weight hbpK with molar masses in the range from 2000 to 6000 Da contain hyper branched polylysine ‘core’: low ␣-helix conformation contribution and presence of -sheet and random coil structures is relevant for it. Their structure reminds that of lysine dendrimer of different number of generation. Michaelis—Menten constant values characterizing the enzyme—substrate affinity were found to exceed that for linear polylysine and to decrease with the increase of polymer molar mass, which indicates the beginning of linear peptide chain formation. HbpKs’ DNA binding capacity was shown to increase with the increase in polymer molar mass; nucleotide/-aminogroup of lysine molar ratio in complexes stuck in the start wells, where the neutralization of DNA charge occurs was about 1:1, what is in good agreement with literature data. doi:10.1016/j.nbt.2009.06.090
1.5.08 Production of anticancerous ice cream D. Nalage 1,∗ , S. Shinde 2 , C. Khedkar 1 1
2
College of Dairy Technology, Pusad, India Yeshwant Mahavidalaya, Nanded, India
Ice cream is a frozen dessert usually made from dairy products, such as cream and milk, combined with fruits or other ingredients. Most varieties contain sugar, although some are made with other sweeteners. In some cases, artificial flavourings and colouring are used in addition to the natural ingredients. In the colouring agent,
S36
www.elsevier.com/locate/nbt
New Biotechnology · Volume 25S · September 2009
ABSTRACTS
different types of chemical substance are used for the colouring of ice cream. Various types of bacteria produce various types of pigment. Purified pigment also used as drug. Prodigiosin is a natural red pigment, characterized by a common pyrrolylpyrromethene skeleton. It has immunosuppressive and apoptotic effects in vitro and also shows antitumor activity in vivo. When that compound is added into the ice cream, it gives pink colour. That pink colour ice cream possesses anticancer activity. Prodigiosin shows dual role in ice one is colouring and second is anticancer activity.
The results of the study suggest that PHB is a good candidate for fabricating prolonged-action drugs in the form of microparticles.
doi:10.1016/j.nbt.2009.06.091
F. Guzman ∗ , G. Fernando, V. Henriquez, J. Olivares, C. Cardenas, S. Marshall
1.5.09 Biodegradable microspheres based on polyhydroxyalkanoates (PHA) as drug carriers A. Goreva 1,∗ , E. Shishatskaya 1 , T. Volova 2 1
2
Institute of Biophysics SB RAS, Krasnoyarsk, Russian Federation Siberian Federal University, Russian Federation
Designing of controlled-release drug delivery systems is a promising and rapidly developing line biotechnology and pharmacology. Encapsulation of therapeutic agents into polymeric carriers provides several benefits over traditional formulations. The main advantage of using drug delivery systems is that drug concentration in a blood and tissues can be maintained at a target level for an extended time. The principal requirement for fabricating controlled-release drug delivery systems is availability of an appropriate material, which must be absolutely harmless to an organism and possess the necessary physical—mechanical and biomedical properties. Bioresorbable polyesters of microbiological origin — polyhydroxyalkanoates (PHAs) — are considered to be promising materials for drug delivery. The purpose of this study was to prepare microspheres from poly(3-hydroxybutyrate) (PHB) and to test their in vitro and in vivo. Microspheres were prepared from the resorbable linear polyester of hydroxybutyric acid (PHB) by the solvent evaporation technique and investigated in vitro and in vivo. It was shown that the fabrication technique (the type of emulsion, the technique of dispersion and the temperature of the medium) affects the yield, structure and size of microspheres. Microspheres loaded with different antibiotics (rubomycin and tienam) were prepared and the kinetics of the in vitro drug release was studied. Biocompatibility of the microspheres was proved in tests in the culture of mouse fibroblast cell line and in experiments on intramuscular implantation of the microspheres to Wistar rats for three months. Tissue response to the implantation of polymeric microspheres was found to consist in a mild inflammatory reaction, pronounced macrophage infiltration that increases over time, involving mono- and poly-nuclear foreign body giant cells that resorb the polymeric matrix. No fibrous capsules were formed around polymeric microparticles; neither necrosis nor any other adverse morphological changes and tissue transformation in response to the implantation of the PHB microparticles were recorded.
doi:10.1016/j.nbt.2009.06.092
1.5.10 Evaluation of immunogenic peptides derived from a highly protective antigenic protein of Piscirickettsia salmonis as a potential source for vaccine development
Universidad Catolica de Valparaiso, Valparaiso, Chile
The salmon industry in Chile has been seriously threatened by several aggressive pathogens among which undoubtedly outstands the Gram negative bacteria Piscirickettsia salmonis, described as the etiological agent of a devastating salmonid Rickettsial septicaemia known as SRS. Efforts to develop a protective SRS vaccine are absolutely necessary to control this detrimental disease of high economical impact in Chile. A new antigen, named ChaPs, endowed with immunogenic capacity has been recently described by our group. This immunogen has been identified as a member of the HSP60 family, and it appears as an ideal candidate for the development of potential vaccines against this bacterium. ChaPs have been already assayed on vaccination trials, high protection level being attained against the disease even six months post-challenge. Using this protein as a framework, a recombinant protein was designed based upon its putative immunogenic relevant domains. Simultaneously, 20 amino acid residue epitopes were designed and synthesized by solid-phase peptide synthesis. ChaPs protein variants from different P. salmonis strains, the designed recombinant protein as well as the newly designed and synthesized peptides were tested by ELISA immunoassay against sera of P. salmonis naturally infected fish. Two of the synthesized peptides and the designed recombinant protein showed an encouraging recognition pattern over ChaPs protein variants. Additionally, heterologous antibodies generated against the two most reactive peptides exhibited a high cross-reactivity pattern against from naturally infected fish. Results showed that ChaPs from different P. salmonis strains display different levels of recognition where specific domains are considerably more immunoreactive than others. Thus, this characterization contributes to the selection of ChaPs immunogenic epitopes for the subsequent design and formulation of effective vaccines against P. salmonis. doi:10.1016/j.nbt.2009.06.093
www.elsevier.com/locate/nbt S37
ABSTRACTS
interaction with SF. The experiments proved that the interaction mode between SF and CT-DNA was intercalation. doi:10.1016/j.nbt.2009.06.088
1.5.06 Hyper branched polylysines: optimization of DNA bonding, endosomalytic properties and DNA stabilization in the complexes against enzyme hydrolysis G. Vlasov 1,∗ , A. Egorova 2 , A. Kiselev 3 , P. Ilyina 2 , G. Pankova 1 , I. Tarasenko 1 , I. Ilyina 1 , V. Baranov 3 1 Institute of Macromolecular Compounds RAS, Sankt-Petersburg, Russian Federation 2 Sankt-Petersburg State University, Sankt-Petersburg, Russian Federation 3 Institute of Obstetrics and Gynecology, RAMN, Sankt-Petersburg, Russian Federation
Conjugation of biologically active compounds to polymer carriers guarantees their targeting delivery, controls allotment in organism, diminishes their toxicity (if any) and defends them from neutralization action of enzymes, inhibitors and immune system of organism. Lysine dendrimers was found to be the most perspective polymer carrier. However their synthesis is complicated and time-withdrawing. In attempt to overcome the problems we have proposed to use hyper branched polylysines (HbpK) as polymer carriers for targeted delivery of biologically active compounds. Now we communicate on possibility of promotion of DNA-bonding and transfection properties of the new kind of biodegradable and biocompatible carriers of DNA on the basis of HbpK. It was shown that HbpK itself does not possess any endosomalytic properties and as a result of that does not guarantee high level of transfection. However optimization of endosomalytic properties of HbpK, as a result of their amino groups modification with histidine, introducing in complex consisting of HbpK and plasmid DNA of endosomalytic peptide JTS-1 and realization of transfection experiments in the presence of glycerin results in the rise of transfection efficacy of HbpK—plasmid DNA complexes. It was found that modification of HbpK with histidine increases as DNA bonding so as transfection efficacy of complex consisting of HbpK and DNA. Transfection efficacy of complexes can be also extended as result of realization of transfection experiments in the presence of glycerin or endosomalytic peptide JTS-1. However realization of transfection experiments with usage of complex histidine modified HbpK in the joint presence of glycerin and endosomalytic peptide JTS-1 results in sinking of transfection efficacy. Influence of HbpK structure on stabilization plasmid DNA against enzymatic hydrolysis will be also presented and discussed. doi:10.1016/j.nbt.2009.06.089
1.5.07 Low molecular weight hyper branched polylysines for DNA binding and delivery: investigation of peculiar properties S. Gorbunova 1,∗ , G. Pankova 1 , I. Tarasenko 1 , V. Vorobév 2 , G.P. Vlasov 1 1
2
Institute of Macromolecular Compounds RAS, Riga, Latvia Institute of Cytology RAS, Riga, Latvia
Recently we have devised a method of synthesis and application of hyper branched polylysines (hbpK) as a new type of biocompatible and biodegradable polymer carriers on the basis of lysine for drug delivery, particularly in Gene Therapy. Their ability to bind DNA, form stable complexes and transfect certain cell lines was shown. The synthesis of hbpK was realized under the condition of reducing catalytic removing of carbobenzoxy blocking groups from N-carboxyanhydride; carbobenzoxylysine (N-CA) by the action of gaseous hydrogen or formic acid in the presence of activated palladium. Now we report the investigation of structure features of low molecular weight hbpK formed in the presence of large excess of reducing agent. Polymer properties were studied using gel permeation chromatography, circular dichroism and capillary electrophoresis as well as enzymatic hydrolysis under the presence of trypsin. It was found that low molecular weight hbpK with molar masses in the range from 2000 to 6000 Da contain hyper branched polylysine ‘core’: low ␣-helix conformation contribution and presence of -sheet and random coil structures is relevant for it. Their structure reminds that of lysine dendrimer of different number of generation. Michaelis—Menten constant values characterizing the enzyme—substrate affinity were found to exceed that for linear polylysine and to decrease with the increase of polymer molar mass, which indicates the beginning of linear peptide chain formation. HbpKs’ DNA binding capacity was shown to increase with the increase in polymer molar mass; nucleotide/-aminogroup of lysine molar ratio in complexes stuck in the start wells, where the neutralization of DNA charge occurs was about 1:1, what is in good agreement with literature data. doi:10.1016/j.nbt.2009.06.090
1.5.08 Production of anticancerous ice cream D. Nalage 1,∗ , S. Shinde 2 , C. Khedkar 1 1
2
College of Dairy Technology, Pusad, India Yeshwant Mahavidalaya, Nanded, India
Ice cream is a frozen dessert usually made from dairy products, such as cream and milk, combined with fruits or other ingredients. Most varieties contain sugar, although some are made with other sweeteners. In some cases, artificial flavourings and colouring are used in addition to the natural ingredients. In the colouring agent,
S36
www.elsevier.com/locate/nbt
New Biotechnology · Volume 25S · September 2009
ABSTRACTS
different types of chemical substance are used for the colouring of ice cream. Various types of bacteria produce various types of pigment. Purified pigment also used as drug. Prodigiosin is a natural red pigment, characterized by a common pyrrolylpyrromethene skeleton. It has immunosuppressive and apoptotic effects in vitro and also shows antitumor activity in vivo. When that compound is added into the ice cream, it gives pink colour. That pink colour ice cream possesses anticancer activity. Prodigiosin shows dual role in ice one is colouring and second is anticancer activity.
The results of the study suggest that PHB is a good candidate for fabricating prolonged-action drugs in the form of microparticles.
doi:10.1016/j.nbt.2009.06.091
F. Guzman ∗ , G. Fernando, V. Henriquez, J. Olivares, C. Cardenas, S. Marshall
1.5.09 Biodegradable microspheres based on polyhydroxyalkanoates (PHA) as drug carriers A. Goreva 1,∗ , E. Shishatskaya 1 , T. Volova 2 1
2
Institute of Biophysics SB RAS, Krasnoyarsk, Russian Federation Siberian Federal University, Russian Federation
Designing of controlled-release drug delivery systems is a promising and rapidly developing line biotechnology and pharmacology. Encapsulation of therapeutic agents into polymeric carriers provides several benefits over traditional formulations. The main advantage of using drug delivery systems is that drug concentration in a blood and tissues can be maintained at a target level for an extended time. The principal requirement for fabricating controlled-release drug delivery systems is availability of an appropriate material, which must be absolutely harmless to an organism and possess the necessary physical—mechanical and biomedical properties. Bioresorbable polyesters of microbiological origin — polyhydroxyalkanoates (PHAs) — are considered to be promising materials for drug delivery. The purpose of this study was to prepare microspheres from poly(3-hydroxybutyrate) (PHB) and to test their in vitro and in vivo. Microspheres were prepared from the resorbable linear polyester of hydroxybutyric acid (PHB) by the solvent evaporation technique and investigated in vitro and in vivo. It was shown that the fabrication technique (the type of emulsion, the technique of dispersion and the temperature of the medium) affects the yield, structure and size of microspheres. Microspheres loaded with different antibiotics (rubomycin and tienam) were prepared and the kinetics of the in vitro drug release was studied. Biocompatibility of the microspheres was proved in tests in the culture of mouse fibroblast cell line and in experiments on intramuscular implantation of the microspheres to Wistar rats for three months. Tissue response to the implantation of polymeric microspheres was found to consist in a mild inflammatory reaction, pronounced macrophage infiltration that increases over time, involving mono- and poly-nuclear foreign body giant cells that resorb the polymeric matrix. No fibrous capsules were formed around polymeric microparticles; neither necrosis nor any other adverse morphological changes and tissue transformation in response to the implantation of the PHB microparticles were recorded.
doi:10.1016/j.nbt.2009.06.092
1.5.10 Evaluation of immunogenic peptides derived from a highly protective antigenic protein of Piscirickettsia salmonis as a potential source for vaccine development
Universidad Catolica de Valparaiso, Valparaiso, Chile
The salmon industry in Chile has been seriously threatened by several aggressive pathogens among which undoubtedly outstands the Gram negative bacteria Piscirickettsia salmonis, described as the etiological agent of a devastating salmonid Rickettsial septicaemia known as SRS. Efforts to develop a protective SRS vaccine are absolutely necessary to control this detrimental disease of high economical impact in Chile. A new antigen, named ChaPs, endowed with immunogenic capacity has been recently described by our group. This immunogen has been identified as a member of the HSP60 family, and it appears as an ideal candidate for the development of potential vaccines against this bacterium. ChaPs have been already assayed on vaccination trials, high protection level being attained against the disease even six months post-challenge. Using this protein as a framework, a recombinant protein was designed based upon its putative immunogenic relevant domains. Simultaneously, 20 amino acid residue epitopes were designed and synthesized by solid-phase peptide synthesis. ChaPs protein variants from different P. salmonis strains, the designed recombinant protein as well as the newly designed and synthesized peptides were tested by ELISA immunoassay against sera of P. salmonis naturally infected fish. Two of the synthesized peptides and the designed recombinant protein showed an encouraging recognition pattern over ChaPs protein variants. Additionally, heterologous antibodies generated against the two most reactive peptides exhibited a high cross-reactivity pattern against from naturally infected fish. Results showed that ChaPs from different P. salmonis strains display different levels of recognition where specific domains are considerably more immunoreactive than others. Thus, this characterization contributes to the selection of ChaPs immunogenic epitopes for the subsequent design and formulation of effective vaccines against P. salmonis. doi:10.1016/j.nbt.2009.06.093
www.elsevier.com/locate/nbt S37