Background Theranostic nanomedicines contain a nanovehicle that has fluorescent properties and ca... more Background Theranostic nanomedicines contain a nanovehicle that has fluorescent properties and can be used for diagnostic, therapeutic and prognostic purposes. The transferrin receptor expression is 1000-fold higher in rapidly growing cancer cells as compared to the normal cells and, therefore, can be used in targeted drug delivery systems. The objective of the present study was to design a novel targeted gold nanoparticle (GNPs)-based theranostic formulation for gastrointestinal (GI) tract-related cancers. The synthesized GNPs were conjugated to transferrin and doxorubicin both separately and collectively to check their cytotoxic properties. The in vitro cytotoxicity of nanocomposites was observed against colon cancer cell line HCT-116. The doxorubicin conjugated nanocomposites showed almost the same cytotoxicity, but more effect at later hours (h). The IC50 and IC100 were 50 µg/ml and 250 µg/ml, respectively, equivalent to the doxorubicin weight for GNP theranostic nanomedicine. R...
Exposure to carcinogenic chemicals, Helicobacter pylori infection, and high dietary salt are the ... more Exposure to carcinogenic chemicals, Helicobacter pylori infection, and high dietary salt are the risk factors associated with gastric cancer. Mice models of gastric cancer are key to understanding the cancer mechanism, to discerning the role played by different factors, and to determining therapeutic effects of different treatments. The goal has been to find targets which are only expressed with cancer so that they can be targeted specifically without harming normal cells. One such target could be the transferrin receptor, a glycoprotein receptor that is expressed many-folds on rapidly growing cells due to the greater demand of iron. In this study, gastric cancer was developed in mice (BALB/c) with human cancer-associated risk factors by feeding them with tumor-inducing concentration of methyl nitrosourea, dietary salt, and H. pylori along with normal feed and water. Three strategies were adopted to induce gastric cancer; (1) use of N-methyl-N-nitrosourea (MNU) with high dietary salt (NaCl), (2) infection with H. pylori (isolated from human gastric tissue), and (3) use of MNU along with high concentration of NaCl after H. pylori infection. Mice were dissected after induction, and histological study of gastric tissue was done with Hematoxylin and Eosin staining. A diagnostic probe comprising transferrin conjugated with cadmium sulfide quantum dots was prepared and characterized. It was used to study the transferrin receptor overexpression in gastric tissue of cancer-induced mice relative to the normal mice. Mice of group 3 showed the highest rate of the cancer incidence ratio (96%) along with a high expression of transferrin receptors among the three groups. Histochemical studies showed that different types of gastric cancer depend upon the cancer-induction conditions. The mouse model of group 3 has the potential to be used in the future to study the therapeutic effects of cancer medicines, and overexpression of transferrin receptors could be identified through the designed probe to be used as diagnostics.
Among glycosidases, β-mannosidase enzyme (EC 3.2.1.25) hydrolyze the non reducing end of Nlinked ... more Among glycosidases, β-mannosidase enzyme (EC 3.2.1.25) hydrolyze the non reducing end of Nlinked mannose residue conjugated to glycoprotein and galacto(gluco)mannans. In mammals, an impaired activity of β-mannosidase leads to β-mannosidosis, In this study, antibodies against purified human lysosomal β-mannosidase were developed and characterized. The reactivity of β-mannosidase antibodies with cell free homogenates of human placenta, B and T cells and livers of mouse, goat and bovine was detected. Total human leukocytes were extracted on histopaque density gradient whereas human B and T lymphocytes were separated by panning method using antihuman CD22 and anti-human CD4/CD8 receptors antibodies, respectively. β-mannosidase enzyme was immunodetected in cell free homogenates by ELISA and slot blot analysis. The western blot analysis revealed the 97 KDa protein band in all homogenates of cell and tissues tested. Immunohistochemical studies indicated the deposition of reaction product on blood vessels, chorionic villi of human placenta and on the B and T cells. β-mannosidase antibodies will be effective tool to detect the β-mannosidase activity during β-mannosidosis and other clinically associated diseases.
Therapeutic effects of anticancer medicines can be improved by targeting the specific receptors o... more Therapeutic effects of anticancer medicines can be improved by targeting the specific receptors on cancer cells. Folate receptor (FR) targeting with antibody (Ab) is an effective tool to deliver anticancer drugs to the cancer cell. In this research project, a novel formulation of targeting drug delivery was designed, and its anticancer effects were analyzed. Folic acid-conjugated magnetic nanoparticles (MNPs) were used for the purification of folate receptors through a novel magnetic affinity purification method. Antibodies against the folate receptors and methotrexate (MTX) were developed and characterized with enzyme-linked immunosorbent assay and Western blot. Targeting nanomedicines (MNP-MTX-FR Ab) were synthesized by engineering the MNP with methotrexate and anti-folate receptor antibody (anti-FR Ab). The cytotoxicity of nanomedicines on HeLa cells was analyzed by calculating the % age cell viability. A fluorescent study was performed with HeLa cells and tumor tissue sections t...
A Novel Method of Affinity Purification and Characterization of Polygalacturonase of Aspergillus flavus by Galacturonic Acid engineered Magnetic Nanoparticle
Food Chemistry, 2021
Pectinases hydrolyze pectin and make up 25% of global food processing enzyme sales. In this study... more Pectinases hydrolyze pectin and make up 25% of global food processing enzyme sales. In this study, we aimed to purify exo-polygalacturonase (Exo-PG) by using galacturonic acid conjugated magnetic nanoparticles (MNPs) and examined its application in juice purification. The submerged fermentation was carried out in the presence of apple pectin (1%) to promote production of exo-PG from Aspergillus flavus. Maximum exo-PG activity was observed after 4 days (30 °C and pH 5.0). A single protein band (66 kDa) of purified exo-PG was observed in SDS-PAGE. Purification of exo-PG enzyme was ∼ 10 fold with a yield of 29%. The enzyme retained 98% activity in the presence of 15 % glycerol at 4 °C. The purified exo-PG using MNPs yielded a 10-12% increase in juice production as compare to without treated fruit juice. To the best of our knowledge, this is the first report of affinity purification of exo-PG enzyme, using engineered magnetic nanoparticles.
Antibodies are good escorts for a therapeutic and diagnostic tool against many diseases as well a... more Antibodies are good escorts for a therapeutic and diagnostic tool against many diseases as well as for targeting drug delivery and in immunohistochemistry. Cysteine-capped cadmium sulfide (quantum dot) nanoparticles have wide applications in immunohistochemistry due to their unique physical, chemical, and fluorescent properties. In this study, polyclonal antibodies against transferrin (as ligand), doxorubicin (anticancer drug), and H. pylori (cancer-causing agent) were developed in rabbits and purified. Purified antibodies were labeled with cadmium sulfide (CdS) quantum dot (QD) and used as a fluorescent-labeled marker for one-step identification in diagnosis and therapeutics. Cysteine-capped CdS QDs were synthesized in the presence of taurine (antioxidant) and characterized by FTIR, VSM, DLS, and TEM. CdS nanoparticles are monodisperse with a narrow size range below 7 nm and showed an increase of almost 30 nm after conjugation to IgG. The binding of QD-labeled antibodies was observ...
An immunoassay based on immunochromatographic lateral flow is developed for qualitative determina... more An immunoassay based on immunochromatographic lateral flow is developed for qualitative determination of HBsAg in serum samples using anti-preS2 antibodies. The diagnostic test strip consists of four components i) sample application pad ii) conjugate pad iii) nitrocellulose membrane and iv) absorbent pad. Affinity purified anti-HBsAg antibodies and goat anti-rabbit IgG antibodies were coated onto nitrocellulose membrane as a test line and control line, respectively. The affinity purified anti-Pre-S2 antibodies conjugated to colloidal gold particles (20 nm) were coated on polyester conjugate pad. The conjugate pad was attached to nitrocellulose membrane and the sample application pad was attached to conjugate pad. The absorbent pad comprising 3mm Whatman paper was attached to the nitrocellulose strip opposite to the sample pad. Purified HBsAg (1.4µg/µl in PBS), 100 µl, was loaded on sample pad and allowed to flow over the strip for 10 minutes. Pink colour test line indicates the corr...
Résumé/Abstract Helicobacter pylorus is considered for chronic gastritis, gastric ulcers and aden... more Résumé/Abstract Helicobacter pylorus is considered for chronic gastritis, gastric ulcers and adenocarcinoma and its high infection rate is observed in overcrowded and lower socioeconomic groups in developing countries. This study was designed to identify the ...
This study was undertaken to detect immunochemically the PreS2 epitope of HBsAg of HBV present in... more This study was undertaken to detect immunochemically the PreS2 epitope of HBsAg of HBV present in serum samples. Polyclonal antibodies against synthetic peptide of PreS2 epitope of HbsAg were raised in rabbit, purified by affinity chromatography and characterized by ELISA and immunodotblot assay. Affinity resin was prepared by coupling the purified HbsAg with commercially availableyanogens bromide activated sepharose 4-B. Fifty clinically HBV positive human blood samples with medical history were collected from different hospitals of Lahore whereas five normal serum samples were collected as a negative control. The HBV in the sera samples was inactivated and used for immunochemical tests. An ELISA of all the serum samples was performed which revealed the presence of PreS2 epitope of HBV in positive sera. Immunodotblot and western blot analysis also showed the reactivity of PreS2 epitope towards the antibodies by giving purple blue color reaction product. So, in all these immunoassay...
Background: Herbs and spices are very important and useful as therapeutic agent against many path... more Background: Herbs and spices are very important and useful as therapeutic agent against many pathological infections. Increasing multidrug resistance of pathogens forces to find alternative compounds for treatment of infectious diseases. Methods: In the present study the antimicrobial potency of garlic and ginger has been investigated against eight local clinical bacterial isolates. Three types of extracts of each garlic and ginger including aqueous extract, methanol extract and ethanol extract had been assayed separately against drug resistant Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis, Staphylococcus aureus, Klebsiella pneumoniae, Shigella sonnei, Staphylococcus epidermidis and Salmonella typhi. The antibacterial activity was determined by disc diffusion method. Results: All tested bacterial strains were most susceptible to the garlic aqueous extract and showed poor susceptibility to the ginger aqueous extract. The (minimum inhibitory concentration) MIC of different bacterial species varied from 0.05 mg/ml to 1.0 mg/ml. Conclusion: In the light of several socioeconomic factors of Pakistan mainly poverty and poor hygienic condition, present study encourages the use of spices as alternative or supplementary medicine to reduce the burden of high cost, side effects and progressively increasing drug resistance of pathogens.
Background and Objectives: Molecular epidemiology of hepatitis C virus (HCV) is very important fo... more Background and Objectives: Molecular epidemiology of hepatitis C virus (HCV) is very important for the treatment of hepatitis C infection. The aim of this study was to determine the distribution of HCV genotypes in Bushehr province (South West of Iran). Materials and Methods: A total of 100 patients who were detected as positive for HCV antibody (by using ELISA method and RIBA test) referred to Arya Virology Laboratory between 2007-2009 in order to molecular diagnosis and furthermore virus genotyping. After detection of HCV, RNA genotyping of virus was done by using genotype specific primers. Results: Genotype 1a was found in 49% of the patients and genotype 3a was found in 40% of the patients and 1b in 5% of patients, while the genotype of the virus could not be identified in 5% of the patients. Finally, in 1% of patients coinfection due to 1a-3a genotypes was identified. Conclusion: The dominant genotype of HCV in Bushehr province, Iran, was determined as 1a.
Background Theranostic nanomedicines contain a nanovehicle that has fluorescent properties and ca... more Background Theranostic nanomedicines contain a nanovehicle that has fluorescent properties and can be used for diagnostic, therapeutic and prognostic purposes. The transferrin receptor expression is 1000-fold higher in rapidly growing cancer cells as compared to the normal cells and, therefore, can be used in targeted drug delivery systems. The objective of the present study was to design a novel targeted gold nanoparticle (GNPs)-based theranostic formulation for gastrointestinal (GI) tract-related cancers. The synthesized GNPs were conjugated to transferrin and doxorubicin both separately and collectively to check their cytotoxic properties. The in vitro cytotoxicity of nanocomposites was observed against colon cancer cell line HCT-116. The doxorubicin conjugated nanocomposites showed almost the same cytotoxicity, but more effect at later hours (h). The IC50 and IC100 were 50 µg/ml and 250 µg/ml, respectively, equivalent to the doxorubicin weight for GNP theranostic nanomedicine. R...
Exposure to carcinogenic chemicals, Helicobacter pylori infection, and high dietary salt are the ... more Exposure to carcinogenic chemicals, Helicobacter pylori infection, and high dietary salt are the risk factors associated with gastric cancer. Mice models of gastric cancer are key to understanding the cancer mechanism, to discerning the role played by different factors, and to determining therapeutic effects of different treatments. The goal has been to find targets which are only expressed with cancer so that they can be targeted specifically without harming normal cells. One such target could be the transferrin receptor, a glycoprotein receptor that is expressed many-folds on rapidly growing cells due to the greater demand of iron. In this study, gastric cancer was developed in mice (BALB/c) with human cancer-associated risk factors by feeding them with tumor-inducing concentration of methyl nitrosourea, dietary salt, and H. pylori along with normal feed and water. Three strategies were adopted to induce gastric cancer; (1) use of N-methyl-N-nitrosourea (MNU) with high dietary salt (NaCl), (2) infection with H. pylori (isolated from human gastric tissue), and (3) use of MNU along with high concentration of NaCl after H. pylori infection. Mice were dissected after induction, and histological study of gastric tissue was done with Hematoxylin and Eosin staining. A diagnostic probe comprising transferrin conjugated with cadmium sulfide quantum dots was prepared and characterized. It was used to study the transferrin receptor overexpression in gastric tissue of cancer-induced mice relative to the normal mice. Mice of group 3 showed the highest rate of the cancer incidence ratio (96%) along with a high expression of transferrin receptors among the three groups. Histochemical studies showed that different types of gastric cancer depend upon the cancer-induction conditions. The mouse model of group 3 has the potential to be used in the future to study the therapeutic effects of cancer medicines, and overexpression of transferrin receptors could be identified through the designed probe to be used as diagnostics.
Among glycosidases, β-mannosidase enzyme (EC 3.2.1.25) hydrolyze the non reducing end of Nlinked ... more Among glycosidases, β-mannosidase enzyme (EC 3.2.1.25) hydrolyze the non reducing end of Nlinked mannose residue conjugated to glycoprotein and galacto(gluco)mannans. In mammals, an impaired activity of β-mannosidase leads to β-mannosidosis, In this study, antibodies against purified human lysosomal β-mannosidase were developed and characterized. The reactivity of β-mannosidase antibodies with cell free homogenates of human placenta, B and T cells and livers of mouse, goat and bovine was detected. Total human leukocytes were extracted on histopaque density gradient whereas human B and T lymphocytes were separated by panning method using antihuman CD22 and anti-human CD4/CD8 receptors antibodies, respectively. β-mannosidase enzyme was immunodetected in cell free homogenates by ELISA and slot blot analysis. The western blot analysis revealed the 97 KDa protein band in all homogenates of cell and tissues tested. Immunohistochemical studies indicated the deposition of reaction product on blood vessels, chorionic villi of human placenta and on the B and T cells. β-mannosidase antibodies will be effective tool to detect the β-mannosidase activity during β-mannosidosis and other clinically associated diseases.
Therapeutic effects of anticancer medicines can be improved by targeting the specific receptors o... more Therapeutic effects of anticancer medicines can be improved by targeting the specific receptors on cancer cells. Folate receptor (FR) targeting with antibody (Ab) is an effective tool to deliver anticancer drugs to the cancer cell. In this research project, a novel formulation of targeting drug delivery was designed, and its anticancer effects were analyzed. Folic acid-conjugated magnetic nanoparticles (MNPs) were used for the purification of folate receptors through a novel magnetic affinity purification method. Antibodies against the folate receptors and methotrexate (MTX) were developed and characterized with enzyme-linked immunosorbent assay and Western blot. Targeting nanomedicines (MNP-MTX-FR Ab) were synthesized by engineering the MNP with methotrexate and anti-folate receptor antibody (anti-FR Ab). The cytotoxicity of nanomedicines on HeLa cells was analyzed by calculating the % age cell viability. A fluorescent study was performed with HeLa cells and tumor tissue sections t...
A Novel Method of Affinity Purification and Characterization of Polygalacturonase of Aspergillus flavus by Galacturonic Acid engineered Magnetic Nanoparticle
Food Chemistry, 2021
Pectinases hydrolyze pectin and make up 25% of global food processing enzyme sales. In this study... more Pectinases hydrolyze pectin and make up 25% of global food processing enzyme sales. In this study, we aimed to purify exo-polygalacturonase (Exo-PG) by using galacturonic acid conjugated magnetic nanoparticles (MNPs) and examined its application in juice purification. The submerged fermentation was carried out in the presence of apple pectin (1%) to promote production of exo-PG from Aspergillus flavus. Maximum exo-PG activity was observed after 4 days (30 °C and pH 5.0). A single protein band (66 kDa) of purified exo-PG was observed in SDS-PAGE. Purification of exo-PG enzyme was ∼ 10 fold with a yield of 29%. The enzyme retained 98% activity in the presence of 15 % glycerol at 4 °C. The purified exo-PG using MNPs yielded a 10-12% increase in juice production as compare to without treated fruit juice. To the best of our knowledge, this is the first report of affinity purification of exo-PG enzyme, using engineered magnetic nanoparticles.
Antibodies are good escorts for a therapeutic and diagnostic tool against many diseases as well a... more Antibodies are good escorts for a therapeutic and diagnostic tool against many diseases as well as for targeting drug delivery and in immunohistochemistry. Cysteine-capped cadmium sulfide (quantum dot) nanoparticles have wide applications in immunohistochemistry due to their unique physical, chemical, and fluorescent properties. In this study, polyclonal antibodies against transferrin (as ligand), doxorubicin (anticancer drug), and H. pylori (cancer-causing agent) were developed in rabbits and purified. Purified antibodies were labeled with cadmium sulfide (CdS) quantum dot (QD) and used as a fluorescent-labeled marker for one-step identification in diagnosis and therapeutics. Cysteine-capped CdS QDs were synthesized in the presence of taurine (antioxidant) and characterized by FTIR, VSM, DLS, and TEM. CdS nanoparticles are monodisperse with a narrow size range below 7 nm and showed an increase of almost 30 nm after conjugation to IgG. The binding of QD-labeled antibodies was observ...
An immunoassay based on immunochromatographic lateral flow is developed for qualitative determina... more An immunoassay based on immunochromatographic lateral flow is developed for qualitative determination of HBsAg in serum samples using anti-preS2 antibodies. The diagnostic test strip consists of four components i) sample application pad ii) conjugate pad iii) nitrocellulose membrane and iv) absorbent pad. Affinity purified anti-HBsAg antibodies and goat anti-rabbit IgG antibodies were coated onto nitrocellulose membrane as a test line and control line, respectively. The affinity purified anti-Pre-S2 antibodies conjugated to colloidal gold particles (20 nm) were coated on polyester conjugate pad. The conjugate pad was attached to nitrocellulose membrane and the sample application pad was attached to conjugate pad. The absorbent pad comprising 3mm Whatman paper was attached to the nitrocellulose strip opposite to the sample pad. Purified HBsAg (1.4µg/µl in PBS), 100 µl, was loaded on sample pad and allowed to flow over the strip for 10 minutes. Pink colour test line indicates the corr...
Résumé/Abstract Helicobacter pylorus is considered for chronic gastritis, gastric ulcers and aden... more Résumé/Abstract Helicobacter pylorus is considered for chronic gastritis, gastric ulcers and adenocarcinoma and its high infection rate is observed in overcrowded and lower socioeconomic groups in developing countries. This study was designed to identify the ...
This study was undertaken to detect immunochemically the PreS2 epitope of HBsAg of HBV present in... more This study was undertaken to detect immunochemically the PreS2 epitope of HBsAg of HBV present in serum samples. Polyclonal antibodies against synthetic peptide of PreS2 epitope of HbsAg were raised in rabbit, purified by affinity chromatography and characterized by ELISA and immunodotblot assay. Affinity resin was prepared by coupling the purified HbsAg with commercially availableyanogens bromide activated sepharose 4-B. Fifty clinically HBV positive human blood samples with medical history were collected from different hospitals of Lahore whereas five normal serum samples were collected as a negative control. The HBV in the sera samples was inactivated and used for immunochemical tests. An ELISA of all the serum samples was performed which revealed the presence of PreS2 epitope of HBV in positive sera. Immunodotblot and western blot analysis also showed the reactivity of PreS2 epitope towards the antibodies by giving purple blue color reaction product. So, in all these immunoassay...
Background: Herbs and spices are very important and useful as therapeutic agent against many path... more Background: Herbs and spices are very important and useful as therapeutic agent against many pathological infections. Increasing multidrug resistance of pathogens forces to find alternative compounds for treatment of infectious diseases. Methods: In the present study the antimicrobial potency of garlic and ginger has been investigated against eight local clinical bacterial isolates. Three types of extracts of each garlic and ginger including aqueous extract, methanol extract and ethanol extract had been assayed separately against drug resistant Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis, Staphylococcus aureus, Klebsiella pneumoniae, Shigella sonnei, Staphylococcus epidermidis and Salmonella typhi. The antibacterial activity was determined by disc diffusion method. Results: All tested bacterial strains were most susceptible to the garlic aqueous extract and showed poor susceptibility to the ginger aqueous extract. The (minimum inhibitory concentration) MIC of different bacterial species varied from 0.05 mg/ml to 1.0 mg/ml. Conclusion: In the light of several socioeconomic factors of Pakistan mainly poverty and poor hygienic condition, present study encourages the use of spices as alternative or supplementary medicine to reduce the burden of high cost, side effects and progressively increasing drug resistance of pathogens.
Background and Objectives: Molecular epidemiology of hepatitis C virus (HCV) is very important fo... more Background and Objectives: Molecular epidemiology of hepatitis C virus (HCV) is very important for the treatment of hepatitis C infection. The aim of this study was to determine the distribution of HCV genotypes in Bushehr province (South West of Iran). Materials and Methods: A total of 100 patients who were detected as positive for HCV antibody (by using ELISA method and RIBA test) referred to Arya Virology Laboratory between 2007-2009 in order to molecular diagnosis and furthermore virus genotyping. After detection of HCV, RNA genotyping of virus was done by using genotype specific primers. Results: Genotype 1a was found in 49% of the patients and genotype 3a was found in 40% of the patients and 1b in 5% of patients, while the genotype of the virus could not be identified in 5% of the patients. Finally, in 1% of patients coinfection due to 1a-3a genotypes was identified. Conclusion: The dominant genotype of HCV in Bushehr province, Iran, was determined as 1a.
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