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Electrocatalytic quantification of gold nanoparticles based on their effect on hydrogen evolution and application for cell analysis
New Biotechnology · Volume 25S · September 2009
ABSTRACTS
1.4.07
1.4.08
Generation of recombinant metapneumovirus nucleocapsid protein as nucleocapsid-like nanoparticles and their application in serological tests
The use of nanoparticles conjugates as vaccine adjuvant and antigen delivery vehicles
K. Sasnauskas ∗ , R. Petraityte, D. Simaite, A. Zvirbliene
J. Reverter ICN-Institute Català de Nanotecnologia, Bellaterra-Cerdanyola, Spain
Institute of Biotechnology, Vilnius, Lithuania
Human metapneumovirus (HMPV) is a member of the Paramyxoviridae family and has been tentatively assigned to the Metapneumovirus genus of the Pneumovirus subfamily. HMPV has been isolated in several continents, suggesting that it is worldwide in prevalence, and resembles human respiratory syncytial virus with regard to disease signs and ability to infect and cause disease in the young infant as well as individuals of all ages. HMPV was first identified in respiratory specimens from young children hospitalized with mild to severe lower respiratory tract illness, and later studies indicate that HPMV may cause upper and lower respiratory tract illness in patients between the ages of 2 months and 87 years. Our aim was to construct efficient high-level yeast expression system for generation of HMPV nucleocapsid protein as nucleocapsid-like nanoparticles for use in HMPV serology. Genome of HMPV was isolated from oral fluid of patient by using specific primers and RT-PCR. ORF corresponding to nucleocapsid gene was inserted into yeast expression vector under inducible GAL7 promoter. SDS-PAGE analysis of crude lysates of yeast S. cerevisiae harboring recombinant plasmid revealed the presence of an additional protein band of approximately 43 kDa molecular weight. Electron microscopy analysis of purified protein revealed structures with typical herring-bone morphology: rods of 20 nm diameter with repeated serration along the edges and central core of 5 nm. Several hybridomas producing monoclonal antibodies against recombinant nucleocapsid protein were generated. Monoclonal antibodies reacted with the recombinant HMPV nucleocapsid protein in Western blots, and did not react with recombinant nucleoproteins from other paramyxoviruses used as negative control. Immunoreactivity of recombinant nucleocapsid protein was tested with a set of human sera. Some sera reacted specifically with recombinant nucleocapsid in Western blot. Recombinant HMPV nucleocapsid protein and corresponding monoclonal antibodies were used for developing of serological tests of HMPV-specific IgM and IgG antibodies in human serum/oral fluid specimens. In summary, we demonstrated that yeast produced recombinant HMPV nucleocapsid protein is suitable for the detection of virus-specific antibodies in human serum and oral fluid.
Although the notion that some materials improve immune responses was expounded a long time ago, the generation of new adjuvants remains one of the objectives of immunologists. Although our knowledge of the immune system has increased, we still lack effective vaccines for many diseases. Adjuvants can act in several ways to increase both native and adaptative immune responses that will generate an effective immunological memory. Ideally, an adjuvant should improve antigen presentation and increase co-stimulatory molecules and cytokine production. Currently, only aluminium salts have been used in licensed human vaccines. Despite maintaining a good safety profile for more than seven decades, safety concerns remain regarding the use of these salts. Therefore, there is an immediate need for alternative immuno-potentiators, and several studies have focused on the development of novel adjuvants with an emphasis on systems at the micro- and nano-scale. We have found that non-activating peptides which, in themselves, do not trigger any activation of the immune system, even when they are in an aggregated state, are capable to activate the immune system, in particular the innate immune system, when ordered onto the surface of a metallic nanoparticle. These conjugates are able to achieve activation of the innate immune system, in particular the innate immune system despite their small size. Thus, the use of the conjugates of the invention offers the possibility to render molecules, otherwise undetectable, visible to the immune system. The present research relates to a conjugate having colloidal stability in a medium comprising a metallic nanoparticle coated with a non-activating peptide which is ordered on the nanoparticle surface. It also relates to a pharmaceutical composition and to a process for the preparation thereof. The conjugates of the invention are used for the activation of the immune system, in particular the innate immune system, as well as for the modulation of immune responses, production of antibodies and detection of substances via antigen—antibody interactions. doi:10.1016/j.nbt.2009.06.072
1.4.09
doi:10.1016/j.nbt.2009.06.071 Electrocatalytic quantification of gold nanoparticles based on their effect on hydrogen evolution and application for cell analysis J. Reverter ICN-Institut Catala De Nanotecnologia, Bellaterra-Cerdanyola, Spain
The present invention relates to conjugates having colloidal stability in a medium comprising metallic nanoparticles coated with platinum containing compounds. It also relates to a process for www.elsevier.com/locate/nbt S29
New Biotechnology · Volume 25S · September 2009
ABSTRACTS
their preparation and to pharmaceutical compositions containing them. The conjugates of the invention are used for the treatment of cancer. Platinum compounds play an important role in cancer chemotherapy. CisPt, the first generation of Platinum based chemotherapy drug, is one of the most common anticancer agents and has a wide spectrum of anticancer activity. However, drawbacks such as the poor selectivity between malignant and normal cells, leading to severe toxic effects and the presence of intrinsic or acquired resistance, so that the doses must be increased, importantly limit its efficacy. We have found that when a CisPt is conjugated to a metallic nanoparticle through a linker via coordination bonds, the resulting delivery system is capable of delivering 10 times more Platinum to the tumor without increasing the toxicity to normal tissues. As a result, tumor resistance to Pt compounds is reduced and side effects are diminished. In addition, the conjugates of the invention are highly soluble in comparison with the currently used free Platinum compounds, whose solubility is low. doi:10.1016/j.nbt.2009.06.073
1.4.10 Investigation of carnuba wax in the formulation of solid lipid microparticles for controlled release M. Uwumagbe Uhumwangho ∗ , R.S. Okor, P. Adebayo Department of Pharmaceutics and Pharmaceutical Technology, Faculty of Pharmacy, University of Benin, Benin City, Nigeria
This study was carried out to investigate the drug entrapment efficiency, release potential and drug release mechanisms of solid lipid microparticles prepared with carnuba wax with different concentration of non-ionic surfactants. Solid lipid microparticles (SLMs) were prepared by melt dispersion technique, where the drug (theophylline)—carnuba wax was emulsified in heated aqueous phase with different ionic surfactants at varying concentrations to form primary o/w emulsion; this was followed by cooling to form the microparticles. The prepared SLMs were characterized for their packing, flow properties and drug entrapment efficiency. These were also encapsulated for dissolution test. The data were analysed using zero order, first order and Higuchi drug release model. It was observed that all the SLMs prepared had a high drug entrapment efficiency of >80%. Increase in surfactant concentration decreased the drug entrapment efficiency although the type of non-ionic surfactant used had no significant effect on the drug entrapment efficiency (P > 0.05). The drug release profile followed first order as well as Higuchi square root of time model. The results of this study showed that it is possible to formulate SLMs with high drug entrapment efficiency when carnuba wax is used as lipid matrix. These prepared SLMs can be used effectively to modulate drug release for prolonged action of theophylline which will in turn improve compliance. doi:10.1016/j.nbt.2009.06.074
S30
www.elsevier.com/locate/nbt
1.4.11 Molecular modeling of the melting temperature of a tuberculosis DNA nanobiosensor D. Glossman-Mitnik ∗ , M. Alvarado-González, E. Orrantia-Borunda Centro de Investigación en Materiales Avanzados, SC, Chihuahua, Mexico
Effective tuberculosis (TB) treatment requires accurate diagnosis, and this remains a challenge. Ideally, TB diagnostics for the developing world should quickly and accurately detect all forms of infection. Breakthroughs in chemical instrumentation and prudent design of diagnostics at the molecular level may contribute to future advancements in TB diagnosis. Although real-time PCR (Polymerase Chain Reaction) has been extensively evaluated for diagnosis of Mycobacterium tuberculosis (Mtb) infections, data are limited on molecular-beacon (MB) applications. An MB-based PCR protocol was designed and evaluated for direct M. tuberculosis detection and quantification in clinical specimens. MB are short nucleotide probes that can be used to interrogate polymerase chain reaction PCR amplified Mtb DNA from a patient’s sputum. MB is sensitive enough to detect single-nucleotide mutations in DNA gene. Beacons feature a fluorophore and a quencher on opposite ends. Mutated genes cannot bind to the beacon, which adopts a looped conformation with a quenched fluorophore. The normal DNA polymerase gene binds to the beacon, unfolding the loop and distancing the fluorophore from the quencher to give a light-based readout. In this work we modeled and analyzed the biosensor DNA molecular structure to detect Mtb strain that contains: 20 nucleotides fragment (loop) and the annealing of complementary arm 5 nucleotides sequences that are located on either side of the probe sequence (stem), using the Oligonucleotide Modeling Platform (OMP; DNA Software, Inc.) to obtain the theoretical values of Tm and G and predict the biosensor-target melting temperature, and we analyzed biosensor Dabcyl—Fluorescein complex (Flurophore—Quencher) interaction with Computational Chemistry theoretical tools. doi:10.1016/j.nbt.2009.06.075
1.4.12 Evaluation of thrombolytic efficacy and target to thrombus of RGDS—lipid nanoparticles carrying subtilisin FS33 in vivo C. Wang Beijing Technology & Business University, Beijing, China
A novel fibrinolytic enzyme subtilisin FS33, which exhibited much higher activity for decomposing fibrin than urokinase, was purified from Douchi, a traditional soybean-fermented food in China. In order to increase bio-utilization and target to thrombus part of subtilisin FS33 labeled by fluorescein isothiocyanate (FITC), surface modified liposomes encapsulating subtilisin FS33 and FITC with a synthetic peptide Arg-Gly-Asp-Ser (RGDS), being putatively a specific antagonist of fibrinogen receptor on platelet membrane, were prepared and used to evaluate therapeutic efficacy in a induced
ABSTRACTS
1.4.07
1.4.08
Generation of recombinant metapneumovirus nucleocapsid protein as nucleocapsid-like nanoparticles and their application in serological tests
The use of nanoparticles conjugates as vaccine adjuvant and antigen delivery vehicles
K. Sasnauskas ∗ , R. Petraityte, D. Simaite, A. Zvirbliene
J. Reverter ICN-Institute Català de Nanotecnologia, Bellaterra-Cerdanyola, Spain
Institute of Biotechnology, Vilnius, Lithuania
Human metapneumovirus (HMPV) is a member of the Paramyxoviridae family and has been tentatively assigned to the Metapneumovirus genus of the Pneumovirus subfamily. HMPV has been isolated in several continents, suggesting that it is worldwide in prevalence, and resembles human respiratory syncytial virus with regard to disease signs and ability to infect and cause disease in the young infant as well as individuals of all ages. HMPV was first identified in respiratory specimens from young children hospitalized with mild to severe lower respiratory tract illness, and later studies indicate that HPMV may cause upper and lower respiratory tract illness in patients between the ages of 2 months and 87 years. Our aim was to construct efficient high-level yeast expression system for generation of HMPV nucleocapsid protein as nucleocapsid-like nanoparticles for use in HMPV serology. Genome of HMPV was isolated from oral fluid of patient by using specific primers and RT-PCR. ORF corresponding to nucleocapsid gene was inserted into yeast expression vector under inducible GAL7 promoter. SDS-PAGE analysis of crude lysates of yeast S. cerevisiae harboring recombinant plasmid revealed the presence of an additional protein band of approximately 43 kDa molecular weight. Electron microscopy analysis of purified protein revealed structures with typical herring-bone morphology: rods of 20 nm diameter with repeated serration along the edges and central core of 5 nm. Several hybridomas producing monoclonal antibodies against recombinant nucleocapsid protein were generated. Monoclonal antibodies reacted with the recombinant HMPV nucleocapsid protein in Western blots, and did not react with recombinant nucleoproteins from other paramyxoviruses used as negative control. Immunoreactivity of recombinant nucleocapsid protein was tested with a set of human sera. Some sera reacted specifically with recombinant nucleocapsid in Western blot. Recombinant HMPV nucleocapsid protein and corresponding monoclonal antibodies were used for developing of serological tests of HMPV-specific IgM and IgG antibodies in human serum/oral fluid specimens. In summary, we demonstrated that yeast produced recombinant HMPV nucleocapsid protein is suitable for the detection of virus-specific antibodies in human serum and oral fluid.
Although the notion that some materials improve immune responses was expounded a long time ago, the generation of new adjuvants remains one of the objectives of immunologists. Although our knowledge of the immune system has increased, we still lack effective vaccines for many diseases. Adjuvants can act in several ways to increase both native and adaptative immune responses that will generate an effective immunological memory. Ideally, an adjuvant should improve antigen presentation and increase co-stimulatory molecules and cytokine production. Currently, only aluminium salts have been used in licensed human vaccines. Despite maintaining a good safety profile for more than seven decades, safety concerns remain regarding the use of these salts. Therefore, there is an immediate need for alternative immuno-potentiators, and several studies have focused on the development of novel adjuvants with an emphasis on systems at the micro- and nano-scale. We have found that non-activating peptides which, in themselves, do not trigger any activation of the immune system, even when they are in an aggregated state, are capable to activate the immune system, in particular the innate immune system, when ordered onto the surface of a metallic nanoparticle. These conjugates are able to achieve activation of the innate immune system, in particular the innate immune system despite their small size. Thus, the use of the conjugates of the invention offers the possibility to render molecules, otherwise undetectable, visible to the immune system. The present research relates to a conjugate having colloidal stability in a medium comprising a metallic nanoparticle coated with a non-activating peptide which is ordered on the nanoparticle surface. It also relates to a pharmaceutical composition and to a process for the preparation thereof. The conjugates of the invention are used for the activation of the immune system, in particular the innate immune system, as well as for the modulation of immune responses, production of antibodies and detection of substances via antigen—antibody interactions. doi:10.1016/j.nbt.2009.06.072
1.4.09
doi:10.1016/j.nbt.2009.06.071 Electrocatalytic quantification of gold nanoparticles based on their effect on hydrogen evolution and application for cell analysis J. Reverter ICN-Institut Catala De Nanotecnologia, Bellaterra-Cerdanyola, Spain
The present invention relates to conjugates having colloidal stability in a medium comprising metallic nanoparticles coated with platinum containing compounds. It also relates to a process for www.elsevier.com/locate/nbt S29
New Biotechnology · Volume 25S · September 2009
ABSTRACTS
their preparation and to pharmaceutical compositions containing them. The conjugates of the invention are used for the treatment of cancer. Platinum compounds play an important role in cancer chemotherapy. CisPt, the first generation of Platinum based chemotherapy drug, is one of the most common anticancer agents and has a wide spectrum of anticancer activity. However, drawbacks such as the poor selectivity between malignant and normal cells, leading to severe toxic effects and the presence of intrinsic or acquired resistance, so that the doses must be increased, importantly limit its efficacy. We have found that when a CisPt is conjugated to a metallic nanoparticle through a linker via coordination bonds, the resulting delivery system is capable of delivering 10 times more Platinum to the tumor without increasing the toxicity to normal tissues. As a result, tumor resistance to Pt compounds is reduced and side effects are diminished. In addition, the conjugates of the invention are highly soluble in comparison with the currently used free Platinum compounds, whose solubility is low. doi:10.1016/j.nbt.2009.06.073
1.4.10 Investigation of carnuba wax in the formulation of solid lipid microparticles for controlled release M. Uwumagbe Uhumwangho ∗ , R.S. Okor, P. Adebayo Department of Pharmaceutics and Pharmaceutical Technology, Faculty of Pharmacy, University of Benin, Benin City, Nigeria
This study was carried out to investigate the drug entrapment efficiency, release potential and drug release mechanisms of solid lipid microparticles prepared with carnuba wax with different concentration of non-ionic surfactants. Solid lipid microparticles (SLMs) were prepared by melt dispersion technique, where the drug (theophylline)—carnuba wax was emulsified in heated aqueous phase with different ionic surfactants at varying concentrations to form primary o/w emulsion; this was followed by cooling to form the microparticles. The prepared SLMs were characterized for their packing, flow properties and drug entrapment efficiency. These were also encapsulated for dissolution test. The data were analysed using zero order, first order and Higuchi drug release model. It was observed that all the SLMs prepared had a high drug entrapment efficiency of >80%. Increase in surfactant concentration decreased the drug entrapment efficiency although the type of non-ionic surfactant used had no significant effect on the drug entrapment efficiency (P > 0.05). The drug release profile followed first order as well as Higuchi square root of time model. The results of this study showed that it is possible to formulate SLMs with high drug entrapment efficiency when carnuba wax is used as lipid matrix. These prepared SLMs can be used effectively to modulate drug release for prolonged action of theophylline which will in turn improve compliance. doi:10.1016/j.nbt.2009.06.074
S30
www.elsevier.com/locate/nbt
1.4.11 Molecular modeling of the melting temperature of a tuberculosis DNA nanobiosensor D. Glossman-Mitnik ∗ , M. Alvarado-González, E. Orrantia-Borunda Centro de Investigación en Materiales Avanzados, SC, Chihuahua, Mexico
Effective tuberculosis (TB) treatment requires accurate diagnosis, and this remains a challenge. Ideally, TB diagnostics for the developing world should quickly and accurately detect all forms of infection. Breakthroughs in chemical instrumentation and prudent design of diagnostics at the molecular level may contribute to future advancements in TB diagnosis. Although real-time PCR (Polymerase Chain Reaction) has been extensively evaluated for diagnosis of Mycobacterium tuberculosis (Mtb) infections, data are limited on molecular-beacon (MB) applications. An MB-based PCR protocol was designed and evaluated for direct M. tuberculosis detection and quantification in clinical specimens. MB are short nucleotide probes that can be used to interrogate polymerase chain reaction PCR amplified Mtb DNA from a patient’s sputum. MB is sensitive enough to detect single-nucleotide mutations in DNA gene. Beacons feature a fluorophore and a quencher on opposite ends. Mutated genes cannot bind to the beacon, which adopts a looped conformation with a quenched fluorophore. The normal DNA polymerase gene binds to the beacon, unfolding the loop and distancing the fluorophore from the quencher to give a light-based readout. In this work we modeled and analyzed the biosensor DNA molecular structure to detect Mtb strain that contains: 20 nucleotides fragment (loop) and the annealing of complementary arm 5 nucleotides sequences that are located on either side of the probe sequence (stem), using the Oligonucleotide Modeling Platform (OMP; DNA Software, Inc.) to obtain the theoretical values of Tm and G and predict the biosensor-target melting temperature, and we analyzed biosensor Dabcyl—Fluorescein complex (Flurophore—Quencher) interaction with Computational Chemistry theoretical tools. doi:10.1016/j.nbt.2009.06.075
1.4.12 Evaluation of thrombolytic efficacy and target to thrombus of RGDS—lipid nanoparticles carrying subtilisin FS33 in vivo C. Wang Beijing Technology & Business University, Beijing, China
A novel fibrinolytic enzyme subtilisin FS33, which exhibited much higher activity for decomposing fibrin than urokinase, was purified from Douchi, a traditional soybean-fermented food in China. In order to increase bio-utilization and target to thrombus part of subtilisin FS33 labeled by fluorescein isothiocyanate (FITC), surface modified liposomes encapsulating subtilisin FS33 and FITC with a synthetic peptide Arg-Gly-Asp-Ser (RGDS), being putatively a specific antagonist of fibrinogen receptor on platelet membrane, were prepared and used to evaluate therapeutic efficacy in a induced