Bhavik Manocha - Academia.edu (original) (raw)

Papers by Bhavik Manocha

Manocha, Bhavik and Gaikar, Vilas G.(2006) 'Permeabilization of Aspergillus niger by Reverse MicellarSolutions and Simultaneous Purification of Catalase', Separation Science and Technology, 41: 14, 3279 — 3296

Separation Science and Technology

Reverse micellar solutions(RMS) of sodium bis- (2-ethylhexyl)-sulfosuccinate (AOT) in aliphatic o... more Reverse micellar solutions(RMS) of sodium bis- (2-ethylhexyl)-sulfosuccinate (AOT) in aliphatic organic solvents were used for permeabilization and protein removal directly from Aspergillus niger cells. Most of the cell wall proteins (�95– 100%) were solubilized into the reverse micelles solutions. A significant fraction of intracellular catalase (26–30%) permeates out of the cells and remains on the cell surface and is recovered in a cell wash. Because of it’s size the catalase in not solubilized in the RM water pools and thus is not detected in the stripping solution used to recover other proteins from the organic reverse micellar solutions. The remaining amount of catalase is recovered by breaking the cells in purer form. Multiple extractions were used for the extraction of the cell wall proteins followed by ultrasonication of the cells to recover intracellular catalase in a purified form. Therefore, catalase with 5 fold purification was recovered in 88% yield from the RMS- treat...

Biotechnology progress

Sterility of cell culture media is an important concern in biotherapeutic processing. In large sc... more Sterility of cell culture media is an important concern in biotherapeutic processing. In large scale biotherapeutic production, a unit contamination of cell culture media can have costly effects. Ultraviolet (UV) irradiation is a sterilization method effective against bacteria and viruses while being non-thermal and non-adulterating in its mechanism of action. This makes UV irradiation attractive for use in sterilization of cell culture media. The objective of this study was to evaluate the effect of UV irradiation of cell culture media in terms of chemical composition and the ability to grow cell cultures in the treated media. The results showed that UV irradiation of commercial cell culture media at relevant disinfection doses impacted the chemical composition of the media with respect to several carboxylic acids, and to a minimal extent, amino acids. The cumulative effect of these changes, however, did not negatively influence the ability to culture Chinese Hamster Ovary cells, a...

Production, purification and controlled release application of gamma-polyglutamic acid

Separation Science and Technology, 2006

Reverse micellar solutions(RMS) of sodium bis-(2-ethylhexyl)-sulfosuccinate (AOT) in aliphatic or... more Reverse micellar solutions(RMS) of sodium bis-(2-ethylhexyl)-sulfosuccinate (AOT) in aliphatic organic solvents were used for permeabilization and protein removal directly from Aspergillus niger cells. Most of the cell wall proteins (95 -100%) were solubilized into the reverse micelles solutions. A significant fraction of intracellular catalase (26-30%) permeates out of the cells and remains on the cell surface and is recovered in a cell wash. Because of it's size the catalase in not solubilized in the RM water pools and thus is not detected in the stripping solution used to recover other proteins from the organic reverse micellar solutions. The remaining amount of catalase is recovered by breaking the cells in purer form. Multiple extractions were used for the extraction of the cell wall proteins followed by ultrasonication of the cells to recover intracellular catalase in a purified form. Therefore, catalase with 5 fold purification was recovered in 88% yield from the RMS-treated cells.

Journal of Thrombosis and Haemostasis, 2011

Background: Platelet microparticles (PMPs) are a promising prognostic marker for thrombotic disor... more Background: Platelet microparticles (PMPs) are a promising prognostic marker for thrombotic disorders because of their release during platelet activation. The use of flow cytometry for the enumeration of PMPs in plasma has generated controversy due to their size, which is below the stated detection limits of conventional flow cytometry instruments. The potential impact of this is an underestimation of PMP counts. Objectives/Methods: To address this possibility, we used a combination of fluorescence-activated cell sorting (FACS) and atomic force microscopy (AFM) to determine the size distribution of PMPs present in plasma from acute myocardial infarction (AMI) patients and normal volunteers, and PMPs generated by expired platelet concentrates and washed platelets treated with agonists such as thrombin and calcium ionophore (A23187). Results: According to AFM image analysis, there was no statistically significant difference in height or volume distributions in PMPs from thrombinactivated, calcium ionophore-activated, expired platelet concentrates and plasma from healthy volunteers and AMI patients. Based on volume, expired platelets released the greatest proportion of exosomes (< 1.0 • 10)22 L 3 in volume) in relation to the entire PMP population (29.7%) and the smallest proportion of exosomes was observed in AMI patient plasma (1.8%). Moreover, AFM imaging revealed that PMPs from expired platelets exhibited smooth surfaces compared with other PMP types but this was not statistically significant. Conclusions: We confirm that flow cytometry is capable of analyzing PMPs from plasma by using AFM to perform nanoscale measurements of individual PMP events isolated by FACS. This method also provided the first quantitative nanoscale images of PMP ultrastructure.

Production and Characterization of γ-Polyglutamic Acid Nanoparticles for Controlled Anticancer Drug Release

Critical Reviews in Biotechnology, 2008

Gamma-polyglutamic acid (gamma-PGA) is a hydrophilic, biodegradable, and naturally available biop... more Gamma-polyglutamic acid (gamma-PGA) is a hydrophilic, biodegradable, and naturally available biopolymer produced by a number of microbial species, most commonly, the Bacillaceae family. Its biological properties such as nontoxicity, biocompatibility, and nonimmunogenicity qualify it as an important biomaterial in drug delivery applications. This review focuses mainly on the development of gamma-PGA nanoparticles as drug delivery carriers for anticancer therapeutics. We discuss various techniques for the production and characterization of gamma-PGA nanoparticles and controlled-release strategies. We also present a brief overview of the tumor physiology that forms the basis for the development of various targeted drug delivery approaches in cancer chemotherapy.

Biotechnology Progress, 2010

Microbially produced gamma-polyglutamic acid (c-PGA) is a commercially important biopolymer with ... more Microbially produced gamma-polyglutamic acid (c-PGA) is a commercially important biopolymer with many applications in biopharmaceutical, food, cosmetic and waste-water treatment industries. Owing to its increasing demand in various industries, production of c-PGA is well documented in the literature, however very few methods have been reported for its recovery. In this paper, we report a novel method for the selective recovery and purification of c-PGA from cell-free fermentation broth of Bacillus licheniformis. The cell-free fermentation broth was treated with divalent copper ions, resulting in the precipitation of c-PGA, which was collected as a pellet by centrifugation. The pellet was resolubilized and dialyzed against de-ionized water to obtain the purified c-PGA biopolymer. The efficiency and selectivity of c-PGA recovery was compared with ethanol precipitation method. We found that 85% of the original c-PGA content in the broth was recovered by copper sulfate-induced precipitation, compared to 82% recovery by ethanol precipitation method. Since ethanol is a commonly used solvent for protein precipitation, the purity of c-PGA precipitate was analyzed by measuring proteins that co-precipitated with c-PGA. Of the total proteins present in the broth, 48% proteins were found to be co-precipitated with c-PGA by ethanol precipitation, whereas in copper sulfate-induced precipitation, only 3% of proteins were detected in the final purified c-PGA, suggesting that copper sulfate-induced precipitation offers better selectivity than ethanol precipitation method. Total metal content analysis of the purified c-PGA revealed the undetectable amount of copper ions, whereas other metal ions detected were in low concentration range. The purified c-PGA was characterized using infrared spectroscopy. V

Journal of Nanomaterials, 2010

Formation of drug/polymer complexes through ionic interactions has proven to be very effective fo... more Formation of drug/polymer complexes through ionic interactions has proven to be very effective for the controlled release of drugs. The stability of such drug/polymer ionic complexes can be greatly influenced by solution pH and ionic strength. The aim of the current work was to evaluate the potential ofγ-polyglutamic acid (γ-PGA) as a carrier for the anticancer drug, Doxorubicin (DOX). We investigated the formation of ionic complexes betweenγ-PGA and DOX using scanning electron microscopy, spectroscopy, thermal analysis, and X-ray diffraction. Our studies demonstrate that DOX specifically interacts withγ-PGA forming random colloidal aggregates and results in almost 100% complexation efficiency. In vitro drug release studies illustrated that these complexes were relatively stable at neutral pH but dissociates slowly under acidic pH environments, facilitating a pH-triggered release of DOX from the complex. Hydrolytic degradation ofγ-PGA and DOX/γ-PGA complex was also evaluated in phys...

Separation Science and Technology

Permeabilization of Aspergillus niger by Reverse Micellar Solutions and Simultaneous Purification... more Permeabilization of Aspergillus niger by Reverse Micellar Solutions and Simultaneous Purification of Catalase

Small, 2011

Multivalent nanoparticles have several key advantages in terms of solubility, binding avidity, an... more Multivalent nanoparticles have several key advantages in terms of solubility, binding avidity, and uptake, making them particularly well suited to molecular imaging applications. Herein is reported the stepwise synthesis and characterization of NIR viral nanoparticles targeted to gastrin-releasing peptide receptors that are over-expressed in human prostate cancers. The pan-bombesin analogue, [β-Ala11, Phe13, Nle14]bombesin-(7-14), is conjugated to cowpea mosaic virus particles functionalized with an NIR dye (Alexa Fluor 647) and polyethylene glycol (PEG) using the copper(I)-catalyzed azide-alkyne cycloaddition reaction. Targeting and uptake in human PC-3 prostate cells is demonstrated in vitro. Tumor homing is observed using human prostate tumor xenografts on the chicken chorioallantoic membrane model using intravital imaging. Further

Manocha, Bhavik and Gaikar, Vilas G.(2006) 'Permeabilization of Aspergillus niger by Reverse MicellarSolutions and Simultaneous Purification of Catalase', Separation Science and Technology, 41: 14, 3279 — 3296

Separation Science and Technology

Reverse micellar solutions(RMS) of sodium bis- (2-ethylhexyl)-sulfosuccinate (AOT) in aliphatic o... more Reverse micellar solutions(RMS) of sodium bis- (2-ethylhexyl)-sulfosuccinate (AOT) in aliphatic organic solvents were used for permeabilization and protein removal directly from Aspergillus niger cells. Most of the cell wall proteins (�95– 100%) were solubilized into the reverse micelles solutions. A significant fraction of intracellular catalase (26–30%) permeates out of the cells and remains on the cell surface and is recovered in a cell wash. Because of it’s size the catalase in not solubilized in the RM water pools and thus is not detected in the stripping solution used to recover other proteins from the organic reverse micellar solutions. The remaining amount of catalase is recovered by breaking the cells in purer form. Multiple extractions were used for the extraction of the cell wall proteins followed by ultrasonication of the cells to recover intracellular catalase in a purified form. Therefore, catalase with 5 fold purification was recovered in 88% yield from the RMS- treat...

Biotechnology progress

Sterility of cell culture media is an important concern in biotherapeutic processing. In large sc... more Sterility of cell culture media is an important concern in biotherapeutic processing. In large scale biotherapeutic production, a unit contamination of cell culture media can have costly effects. Ultraviolet (UV) irradiation is a sterilization method effective against bacteria and viruses while being non-thermal and non-adulterating in its mechanism of action. This makes UV irradiation attractive for use in sterilization of cell culture media. The objective of this study was to evaluate the effect of UV irradiation of cell culture media in terms of chemical composition and the ability to grow cell cultures in the treated media. The results showed that UV irradiation of commercial cell culture media at relevant disinfection doses impacted the chemical composition of the media with respect to several carboxylic acids, and to a minimal extent, amino acids. The cumulative effect of these changes, however, did not negatively influence the ability to culture Chinese Hamster Ovary cells, a...

Production, purification and controlled release application of gamma-polyglutamic acid

Separation Science and Technology, 2006

Reverse micellar solutions(RMS) of sodium bis-(2-ethylhexyl)-sulfosuccinate (AOT) in aliphatic or... more Reverse micellar solutions(RMS) of sodium bis-(2-ethylhexyl)-sulfosuccinate (AOT) in aliphatic organic solvents were used for permeabilization and protein removal directly from Aspergillus niger cells. Most of the cell wall proteins (95 -100%) were solubilized into the reverse micelles solutions. A significant fraction of intracellular catalase (26-30%) permeates out of the cells and remains on the cell surface and is recovered in a cell wash. Because of it's size the catalase in not solubilized in the RM water pools and thus is not detected in the stripping solution used to recover other proteins from the organic reverse micellar solutions. The remaining amount of catalase is recovered by breaking the cells in purer form. Multiple extractions were used for the extraction of the cell wall proteins followed by ultrasonication of the cells to recover intracellular catalase in a purified form. Therefore, catalase with 5 fold purification was recovered in 88% yield from the RMS-treated cells.

Journal of Thrombosis and Haemostasis, 2011

Background: Platelet microparticles (PMPs) are a promising prognostic marker for thrombotic disor... more Background: Platelet microparticles (PMPs) are a promising prognostic marker for thrombotic disorders because of their release during platelet activation. The use of flow cytometry for the enumeration of PMPs in plasma has generated controversy due to their size, which is below the stated detection limits of conventional flow cytometry instruments. The potential impact of this is an underestimation of PMP counts. Objectives/Methods: To address this possibility, we used a combination of fluorescence-activated cell sorting (FACS) and atomic force microscopy (AFM) to determine the size distribution of PMPs present in plasma from acute myocardial infarction (AMI) patients and normal volunteers, and PMPs generated by expired platelet concentrates and washed platelets treated with agonists such as thrombin and calcium ionophore (A23187). Results: According to AFM image analysis, there was no statistically significant difference in height or volume distributions in PMPs from thrombinactivated, calcium ionophore-activated, expired platelet concentrates and plasma from healthy volunteers and AMI patients. Based on volume, expired platelets released the greatest proportion of exosomes (< 1.0 • 10)22 L 3 in volume) in relation to the entire PMP population (29.7%) and the smallest proportion of exosomes was observed in AMI patient plasma (1.8%). Moreover, AFM imaging revealed that PMPs from expired platelets exhibited smooth surfaces compared with other PMP types but this was not statistically significant. Conclusions: We confirm that flow cytometry is capable of analyzing PMPs from plasma by using AFM to perform nanoscale measurements of individual PMP events isolated by FACS. This method also provided the first quantitative nanoscale images of PMP ultrastructure.

Production and Characterization of γ-Polyglutamic Acid Nanoparticles for Controlled Anticancer Drug Release

Critical Reviews in Biotechnology, 2008

Gamma-polyglutamic acid (gamma-PGA) is a hydrophilic, biodegradable, and naturally available biop... more Gamma-polyglutamic acid (gamma-PGA) is a hydrophilic, biodegradable, and naturally available biopolymer produced by a number of microbial species, most commonly, the Bacillaceae family. Its biological properties such as nontoxicity, biocompatibility, and nonimmunogenicity qualify it as an important biomaterial in drug delivery applications. This review focuses mainly on the development of gamma-PGA nanoparticles as drug delivery carriers for anticancer therapeutics. We discuss various techniques for the production and characterization of gamma-PGA nanoparticles and controlled-release strategies. We also present a brief overview of the tumor physiology that forms the basis for the development of various targeted drug delivery approaches in cancer chemotherapy.

Biotechnology Progress, 2010

Microbially produced gamma-polyglutamic acid (c-PGA) is a commercially important biopolymer with ... more Microbially produced gamma-polyglutamic acid (c-PGA) is a commercially important biopolymer with many applications in biopharmaceutical, food, cosmetic and waste-water treatment industries. Owing to its increasing demand in various industries, production of c-PGA is well documented in the literature, however very few methods have been reported for its recovery. In this paper, we report a novel method for the selective recovery and purification of c-PGA from cell-free fermentation broth of Bacillus licheniformis. The cell-free fermentation broth was treated with divalent copper ions, resulting in the precipitation of c-PGA, which was collected as a pellet by centrifugation. The pellet was resolubilized and dialyzed against de-ionized water to obtain the purified c-PGA biopolymer. The efficiency and selectivity of c-PGA recovery was compared with ethanol precipitation method. We found that 85% of the original c-PGA content in the broth was recovered by copper sulfate-induced precipitation, compared to 82% recovery by ethanol precipitation method. Since ethanol is a commonly used solvent for protein precipitation, the purity of c-PGA precipitate was analyzed by measuring proteins that co-precipitated with c-PGA. Of the total proteins present in the broth, 48% proteins were found to be co-precipitated with c-PGA by ethanol precipitation, whereas in copper sulfate-induced precipitation, only 3% of proteins were detected in the final purified c-PGA, suggesting that copper sulfate-induced precipitation offers better selectivity than ethanol precipitation method. Total metal content analysis of the purified c-PGA revealed the undetectable amount of copper ions, whereas other metal ions detected were in low concentration range. The purified c-PGA was characterized using infrared spectroscopy. V

Journal of Nanomaterials, 2010

Formation of drug/polymer complexes through ionic interactions has proven to be very effective fo... more Formation of drug/polymer complexes through ionic interactions has proven to be very effective for the controlled release of drugs. The stability of such drug/polymer ionic complexes can be greatly influenced by solution pH and ionic strength. The aim of the current work was to evaluate the potential ofγ-polyglutamic acid (γ-PGA) as a carrier for the anticancer drug, Doxorubicin (DOX). We investigated the formation of ionic complexes betweenγ-PGA and DOX using scanning electron microscopy, spectroscopy, thermal analysis, and X-ray diffraction. Our studies demonstrate that DOX specifically interacts withγ-PGA forming random colloidal aggregates and results in almost 100% complexation efficiency. In vitro drug release studies illustrated that these complexes were relatively stable at neutral pH but dissociates slowly under acidic pH environments, facilitating a pH-triggered release of DOX from the complex. Hydrolytic degradation ofγ-PGA and DOX/γ-PGA complex was also evaluated in phys...

Separation Science and Technology

Permeabilization of Aspergillus niger by Reverse Micellar Solutions and Simultaneous Purification... more Permeabilization of Aspergillus niger by Reverse Micellar Solutions and Simultaneous Purification of Catalase

Small, 2011

Multivalent nanoparticles have several key advantages in terms of solubility, binding avidity, an... more Multivalent nanoparticles have several key advantages in terms of solubility, binding avidity, and uptake, making them particularly well suited to molecular imaging applications. Herein is reported the stepwise synthesis and characterization of NIR viral nanoparticles targeted to gastrin-releasing peptide receptors that are over-expressed in human prostate cancers. The pan-bombesin analogue, [β-Ala11, Phe13, Nle14]bombesin-(7-14), is conjugated to cowpea mosaic virus particles functionalized with an NIR dye (Alexa Fluor 647) and polyethylene glycol (PEG) using the copper(I)-catalyzed azide-alkyne cycloaddition reaction. Targeting and uptake in human PC-3 prostate cells is demonstrated in vitro. Tumor homing is observed using human prostate tumor xenografts on the chicken chorioallantoic membrane model using intravital imaging. Further