Prachi Joshi

Contrasting Effect of Gold Nanoparticles and Nanorods with Different Surface Modifications on the Structure and Activity of Bovine Serum Albumin

Nanoparticles exposed to biofluids become coated with proteins, thus making protein-nanoparticle interactions of particular interest. The consequence on protein conformation and activity depends upon the extent of protein adsorption on the nanoparticle surface. We report the interaction of bovine serum albumin (BSA) with gold nanostructures, particularly gold nanoparticles (GNP) and gold nanorods (GNR). The difference in the geometry and surface properties of nanoparticles is manifested during complexation in terms of different binding modes, structural changes, thermodynamic parameters, and the activity of proteins. BSA is found to retain native-like structure and properties upon enthalpy-driven BSA-GNP complexation. On the contrary, the entropically favored BSA-GNR complexation leads to substantial loss in protein secondary and tertiary structures with the release of a large amount of bound water, as indicated by isothermal calorimetry (ITC), circular dichroism (CD), and Fourier transform infrared (FTIR) and fluorescence spectroscopies. The esterase activity assay demonstrated a greater loss in BSA activity after complexation with GNR, whereas the original activity is retained in the presence of GNP. The formation of large assemblies (aggregates) and reduced average lifetime, as evidenced from dynamic light scattering and fluorescence decay measurements, respectively, suggest that GNR induces protein unfolding at its surface. The effect of temperature on the CD spectra of BSA-GNP was found to be similar to that of pristine BSA, whereas BSA-GNR shows distortion in CD spectra at lower wavelengths, strengthening the perception of protein unfolding. High binding constant and entropy change for BSA-GNR complexation determined by ITC are consistent with large surfacial interaction that may lead to protein unfolding. The present work highlights the differential response of a protein depending on the nature of the nanostructure and its surface chemistry, which need to be modulated for controlling the biological responses of nanostructures for their potential biomedical applications.

Structure and Activity of Lysozyme on Binding to ZnO Nanoparticles

The interaction between ZnO nanoparticles (NPs) and lysozyme has been studied using calorimetric as well as spectrophotometric techniques, and interpreted in terms of the three-dimensional structure. The circular dichroism spectroscopic data show an increase in alpha-helical content on interaction with ZnO NPs. Glutaraldehyde cross-linking studies indicate that the monomeric form occurs to a greater extent than the dimer when lysozyme is conjugated with ZnO NPs. The enthalpy-driven binding between lysozyme and ZnO possibly involves the region encompassing the active site in the molecule, which is also the site for the dimer formation in a homologous structure. The enzyme retains high fraction of its native structure with negligible effect on its activity upon attachment to NPs. Compared to the free protein, lysozyme-ZnO conjugates are more stable in the presence of chaotropic agents (guanidine hydrochloride and urea) and also at elevated temperatures. The possible site of binding of NP to lysozyme has been proposed to explain these observations. The stability and the retention of a higher level of activity in the presence of the denaturing agent of the NP-conjugated protein may find useful applications in biotechnology ranging from diagnostic to drug delivery.

Interaction of Polyethyleneimine-Functionalized ZnO Nanoparticles with Bovine Serum Albumin

In biological fluids, nanoparticles are always surrounded by proteins. As the protein is adsorbed on the surface, the extent of adsorption and the effect on the protein conformation and stability are dependent on the chemical nature, shape, and size of the nanoparticle (NP). We have carried out a detailed investigation on the interaction of bovine serum albumin (BSA) with polyethyleneimine-functionalized ZnO nanoparticles (ZnO-PEI). ZnO-PEI was synthesized using a wet chemical method with a core size of ~3-7 nm (from transmission electron microscopy). The interaction of BSA with ZnO-PEI was examined using a combination of calorimetric, spectroscopic, and computational techniques. The binding was studied by ITC (isothermal titration calorimetry), and the result revealed that the complexation is enthalpy-driven, indicating the possible involvement of electrostatic interaction. To investigate the nature of the interaction and the location of the binding site, a detailed domain-wise surface electrostatic potential calculation was performed using adaptive Poisson-Boltzmann software (APBS). The result shows that the protein surface can bind the nanoparticle. On binding ZnO-PEI, the protein gets destabilized to some extent, as displayed by CD (circular dichroism) and FTIR (Fourier transform infrared) spectroscopy. Chemical and thermal denaturation of BSA, when carried out in the presence of ZnO-PEI, also indicated a small perturbation in the protein structure. A comparison of the enthalpy and entropy components of binding with those derived for the interaction of BSA with ZnO nanoparticles explains the effect of hydrophilic cationic species attached on the NP surface. The effect of the NP surface modification on the structure and stability of BSA would find useful applications in nanobiotechnology.

Binding of chloroquine–conjugated gold nanoparticles with bovine serum albumin

We have conjugated chloroquine, an anti-malarial, antiviral and anti-tumor drug, with thiol-functionalized gold nanoparticles and studied their binding interaction with bovine serum albumin (BSA) protein. Gold nanoparticles have been synthesized using sodium borohydride as reducing agent and 11-mercaptoundecanoic acid as thiol functionalizing ligand in aqueous medium. The formation of gold nanoparticles was confirmed from the characteristic surface plasmon absorption band at 522 nm and transmission electron microscopy revealed the average particle size to be ~7 nm. Chloroquine was conjugated to thiolated gold nanoparticles by using EDC/NHS chemistry and the binding was analyzed using optical density measurement and Fourier transform infrared spectroscopy. The chloroquine-conjugated gold nanoparticles (GNP-Chl) were found to interact efficiently with BSA. Thermodynamic parameters suggest that the binding is driven by both enthalpy and entropy, accompanied with only a minor alteration in proteins structure. Competitive drug binding assay revealed that the GNP-Chl bind at warfarin binding site I in subdomain IIA of BSA and was further supported by Trp212 fluorescence quenching measurements. Unraveling the nature of interactions of GNP-Chl with BSA would pave the way for the design of nanotherapeutic agents with improved functionality, enriching the field of nanomedicine.

The anticancer activity of chloroquine-gold nanoparticles against MCF-7 breast cancer cells.

In the present study, 11-mercaptoundecanoic acid-modified gold nanoparticles (∼7 nm) were conjugated with chloroquine to explore their potential application in cancer therapeutics. The anticancer activity of chloroquine-gold nanoparticle conjugates (GNP-Chl) was demonstrated in MCF-7 breast cancer cells. The MCF-7 cells were treated with different concentrations of GNP-Chl conjugates, and the cell viability was assayed using trypan blue, resulting in an IC(50) value of 30 ± 5 μg/mL. Flow cytometry analysis revealed that the major pathway of cell death was necrosis, which was mediated by autophagy. The drug release kinetics of GNP-Chl conjugates revealed the release of chloroquine at an acidic pH, which was quantitatively estimated using optical absorbance spectroscopy. The nature of stimuli-responsive drug release and the inhibition of cancer cell growth by GNP-Chl conjugates could pave the way for the design of combinatorial therapeutic agents, particularly nanomedicine, for the treatment of cancer.

The effect of zinc oxide nanoparticles on the structure of the periplasmic domain of the Vibrio cholerae ToxR protein.

Proteins adsorbed on nanoparticles (NPs) are being used as biosensors and in drug delivery. However, our understanding of the effect of NPs on the structure of proteins is still in a nascent state. In this work we report the unfolding behavior of the periplasmic domain of the ToxR protein (ToxRp) of Vibrio cholerae on zinc oxide (ZnO) nanoparticles with a diameter of 2.5 nm. This protein plays a crucial role in regulating the expression of several virulence factors in the pathogenesis of cholera. Thermodynamic analysis of the equilibrium of unfolding, induced both by urea and by guanidine hydrochloride (GdnHCl), and measured by fluorescence spectroscopy, revealed a two‐state process. NPs increased the susceptibility of the protein to denaturation. The midpoints of transitions for the free and the NP‐bound ToxRp in the presence of GdnHCl were 1.5 and 0.5 m respectively, whereas for urea denaturation, the values were 3.3 and 2.4 m, respectively. Far‐UV CD spectra showed a significant change in the protein conformation upon binding to ZnO NPs, which was characterized by a substantial decrease in the α‐helical content of the free protein. Isothermal titration calorimetry, used to quantify the thermodynamics of binding of ToxRp with ZnO NPs, showed an exothermic binding isotherm (ΔH = −9.8 kcal·mol−1 and ΔS = −5.17 cal·mol−1·K−1).

Tuning of emission colors in zinc oxide quantum dots

High brightness zinc oxide quantum dots were made with intentional alkali metal doping using quantum-confined atom model. The tuning of emission spectrum in the range of 480–562nm was achieved by dispersing them in solvents with varying index of refraction. The observed emission bands are quite distinct from the nonstructured green emission of zinc oxide at 2.4eV (515nm) but are attributed to donor-acceptor recombination involving the zinc vacancy and Li+∕Na+, or the modifications assigned to the surface states by the surrounding medium. The photoluminescence shifts are found to be sensitive to refractive index term n2−1∕2n2+1 useful for practical applications.

A multiple antibiotic and serum resistant oligotrophic strain, Klebsiella pneumoniae MB45 having novel dfrA30, is sensitive to ZnO QDs

BackgroundThe aim of this study was to describe a novel trimethoprim resistance gene cassette, designated dfrA30, within a class 1 integron in a facultatively oligotrophic, multiple antibiotic and human serum resistant test strain, MB45, in a population of oligotrophic bacteria isolated from the river Mahananda; and to test the efficiency of surface bound acetate on zinc oxide quantum dots (ZnO QDs) as bactericidal agent on MB45.MethodsDiluted Luria broth/Agar (10-3) media was used to cultivate the oligotrophic bacteria from water sample. Multiple antibiotic resistant bacteria were selected by employing replica plate method. A rapid assay was performed to determine the sensitivity/resistance of the test strain to human serum. Variable region of class 1 integron was cloned, sequenced and the expression of gene coding for antibiotic resistance was done in Escherichia coli JM 109. Identity of culture was determined by biochemical phenotyping and 16S rRNA gene sequence analyses. A phylogenetic tree was constructed based on representative trimethoprim resistance-mediating DfrA proteins retrieved from GenBank. Growth kinetic studies for the strain MB45 were performed in presence of varied concentrations of ZnO QDs.Results and conclusionsThe facultatively oligotrophic strain, MB45, resistant to human serum and ten antibiotics trimethoprim, cotrimoxazole, ampicillin, gentamycin, netilmicin, tobramycin, chloramphenicol, cefotaxime, kanamycin and streptomycin, has been identified as a new strain of Klebsiella pneumoniae. A novel dfr gene, designated as dfrA30, found integrated in class 1 integron was responsible for resistance to trimethoprim in Klebsiella pneumoniae strain MB45. The growth of wild strain MB45 was 100% arrested at 500 mg/L concentration of ZnO QDs. To our knowledge this is the first report on application of ZnO quantum dots to kill multiple antibiotics and serum resistant K. pneumoniae strain.

Graphene oxide–chloroquine nanoconjugate induce necroptotic death in A549 cancer cells through autophagy modulation

AIM Chloroquine (Chl) has shown its potential in cancer therapy and graphene oxide (GO) exhibited excellent tumor-targeting ability, biocompatibility and low toxicity. We have endeavored to conjugate Chl to GO sheets and investigated the nonproliferation action on A549 cell lines along with cell signaling pathways. MATERIALS & METHODS Cellular toxicity, autophagic flux modulation and cell death mechanism induced by GO-Chl have been investigated on A549 cell lines. RESULTS & CONCLUSION GO-Chl induces accumulation of autophagosomes (monodansylcadaverine staining, green fluorescence protein-tagged LC3 plasmid and transmission electron microscopy observations) in A549 cells through the blockade of autophagic flux that serves as scaffold for necrosome assembling and activates necroptotic cell death. GO-Chl nanoconjugate could be used as an effective cancer therapeutic agent, by targeting the autophagy necroptosis axis.

Cytotoxicity of Graphene Oxide (GO) and Graphene Oxide Conjugated Losartan Potassium (GO-LP) on Neuroblastoma (NB41A3) Cells

Despite several advancements in the biomedical sciences, an efficient cancer therapy still remains a challenge. Nanomedicines have shown potential to overcome certain roadblocks faced in the existing treatment modalities. Losartan potassium (LP) which is a known vasodilator also exhibits anti fibrolytic and anti-metastatic properties altogether. Further, also being a potential angiotensin II type 1 receptor antagonist, it has been well explored for down regulating tumourogenic biomarkers like VEGF-A (Vascular endothelial growth factor A) and suppression of neovascularization, making it a suitable drug to target for cancer treatment. Besides this, it too reflected the stimulation of pro apoptotic signaling pathways. But due to its lower bioavailability and extensive hepatic metabolism its therapeutic index reduces down. Thus, the present study is focused on designing a nano-delivery system using graphene oxide (GO) as a nano-vehicle and conjugated the LP with it. Then, the successful synthesis of GO and GO-LP nano conjugates were characterized by high-resolution transmission electron microscopy, X-ray diffraction, FTIR and UV visible spectroscopy, confirming the formation of nanosheets. The qualitative morphological evaluation of NB41A3 neuroblastoma cell line treated with bare GO, LP and GO-LP using microscopy and DAPI staining revealed the inhibitory action of GO-LP nano conjugate on cell proliferation. Additionally, the cytotoxicity was also estimated using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), Nitric oxide (NO) and Lactate dehydrogenase (LDH) assays. The results show that GO-LP significantly suppresses the cell viability in comparison to control and bare GO suggesting that the designed system may express its potential to be used with existing chemo drugs for the treatment of neural cancers.