Hydrophobic quercetin encapsulated hemoglobin nanoparticles: formulation and spectroscopic characterization.

Various natural proteins are finding application in drug delivery for their high biodegradability and biocompatibility. Albumins are well explored and now focus is shifting to other proteins like hemoglobin (Hb) with unique structural properties. In the present study Hb is allowed to denature at pH 5.0 and model hydrophobic drug quercetin (Q) is encapsulated via self-assembly and hydrophobic interactions. Fluorimetric titrations record highest binding between Hb and Q at pH 5.0, rendering significant structural changes in Hb as captured in CD spectra. A decrease in fluorescence life time of tryptophan residues from 3.31 ns in Hb to 2.89 ns in presence of Q at pH 5.0; surmises efficient binding of Q at the hydrophobic core housing tryptophan. Peak shifts in Fourier transform infrared spectroscopy spectra of Hb-Q compared to Hb evidence significant interactions between them at pH 5.0. Significant spectral changes in soret band region of Hb on addition of Q at pH 5.0 envisages unfolding of porphyrin ring and binding influence of Q. Efficient formation of Hb-Q nanoparticles (NPs) at pH 5.0 is established by DLS, SEM and TEM.Communicated by Ramaswamy H. Sarma.

An evaluation of DNA double strand break formation and excreted guanine species post whole body PET/CT procedure.

Ionizing radiation-induced oxidation and formation of deoxyribonucleic acid (DNA) double strand breaks (DSBs) are considered the exemplar of genetic lesions. Guanine bases are most prone to be oxidized when DNA and Ribonucleic acid (RNA) are damaged. The repair processes that are initiated to correct this damage release multiple oxidized guanine species into the urine. Hence, the excretion of guanine species can be related with the total repair process. Our study quantified the total DSBs formation and the amount of guanine species in urine to understand the DNA break and repair process after whole body (WB) exposure to 18F-FDG positron emission tomography/computed tomography (PET/CT). A total of 37 human participants were included with control and test groups and the average radiation dose was 27.50 ± 2.91 mSv. γ-H2AX foci assay in the collected blood samples was performed to assess the DSBs, and excreted guanine species in urine were analyzed by a competitive ELISA method. We observed a significant increase of DNA damage that correlated well with the increasing dose (p-value 0.009) and body weight (p-value 0.05). In the test group, excreted guanine species in urine sample significantly increased (from 24.29 ± 5.82 to 33.66 ± 7.20 mg/mmol creatinine). A minimum (r2 = 0.0488) correlation was observed between DSBs formation and excreted guanine species. A significant difference of DNA damage and 8-OHdG formation was seen in the test group compared to controls. Larger population studies are needed to confirm these observations, describe the fine-scale timing of changes in the biomarker levels after exposure, and further clarify any potential risks to patients from PET/CT procedures.

Physicochemical characterization of dual action liposomal formulations: anticancer and antimicrobial.

BACKGROUND: Cancer till date remains one of the world's most life threatening disease accompanied by risk of secondary infections. Therefore formulations carrying anticancer drugs which can also decrease the risk of secondary infection are inevitable. Chemotherapeutic drug doxorubicin along with flavonoids quercetin and epigallocatechin gallate (EGCG) is simultaneously loaded on liposomal formulation exploiting the amphiphilic property of the liposomes. RESULTS: Atomic force microscope imaging reveal the size of liposomal formulation loaded with doxorubicin, quercetin and EGCG to be greater than void liposome confirming the presence of drugs. Liposomal stability is improved by PEGylation; adding to the drug release time in vitro. The charge of phosphatidylcholine is rendered positive by coating the formulation with histone. The average size of the formulation is 342 nm. The encapsulation efficiency of doxorubicin, quercetin and EGCG is found to be 65.8%, 96.8% and 98% respectively. The above formulation demonstrated both anticancer and antimicrobial activity. CONCLUSION: The formulation will provide dual anticancer and antimicrobial therapy thereby evading secondary infection in cancer patients along with chemotherapy.

Interaction of flavonols with human serum albumin: a biophysical study showing structure-activity relationship and enhancement when coated on silver nanoparticles.

Binding affinities of flavonols namely quercetin, myricetin, and kaempferol to human serum albumin (HSA) were determined fluorimetrically and the order was observed to be myricetin > quercetin > kaempferol demonstrating structure-activity relationship. Quercetin-coated silver nanoparticles (AgNPs) show higher binding affinity to HSA compared to free quercetin with binding constants 6.04 × 107 M-1 and 4.2 × 106 M-1, respectively. Using site-specific markers it is concluded that free quercetin and that coated on AgNPs bind at different sites. Significant structural changes in circular dichroism (CD) spectra of HSA were recorded with quercetin-coated AgNPs compared to free quercetin. These results were further substantiated by time-resolved fluorescence spectroscopy where fluorescence life time of the tryptophan residue in HSA-quercetin-coated AgNPs complex decreased to 3.63 ns from 4.22 ns in HSA-quercetin complex. Isothermal calorimetric studies reveal two binding modes for quercetin-coated AgNPs and also higher binding constants compared to free quercetin. These higher binding affinities are attributed to altered properties of quercetin when coated on AgNPs enabling it to reach the binding sites other than site II where free quercetin mainly binds.

Catalase inhibition an anti cancer property of flavonoids: A kinetic and structural evaluation.

Flavonoids are dietary polyphenols that present abundantly in fruits and vegetables. Flavonoids have inhibitory effects on enzymes and catalase is one among them. Catalase is a common enzyme ubiquitously found in all living organisms exposed to oxygen. It catalyzes the decomposition of hydrogen peroxide to water and oxygen (2H2O2→2H2O+O2). Inhibition of pure and cellular catalase from K562 cells by flavonoids was similar and exhibited the following efficacy; Myrecetin>Quercetin>Kaempferol and Quercetin>Luteolin>Apigenin demonstrating structure activity relationship. Circular Dichroism (CD) spectra have shown distinct loss in α-helical structure of the catalase on interaction with the flavonoids. All flavonoids inhibited the catalase activity by uncompetitive mechanism. The Km and Vmax values of pure catalase were observed to be 294mM-1 and 0.222mM-1s-1 respectively and on inhibition with myrecetin the values decreased to a minimum of 23mM-1 and 0.014mM-1s-1 respectively. Inhibition of catalase will directly results in increased production of Reactive Oxygen Species (ROS) and pro-oxidant property of flavonoids. This inhibition was reversed in presence of Cu2+ ions because of the chelating affect of flavonoids.

Solvent effect in the synthesis of hydrophobic drug-loaded polymer nanoparticles.

Quercetin is an abundant flavonoid in fruits, vegetables such as onion, tea leaves, cranberry, radish leaves etc. with numerous biological activities and widely used as an effective antioxidant. Its low solubility in water and chemical decomposition in intestinal environment are predicaments in delivery through dietary or oral intake. Noble polymeric nanoparticles are of particular interest today because of their applications in many areas. Polymer nanoparticles have attracted the interest of many research groups and have been utilised in an increasing number of fields such as site targeted drug delivery in cancer research during the last decades. Various techniques can be used to produce polymer nanoparticles, such as solvent evaporation, salting-out, dialysis, supercritical fluid technology etc. The choice of method depends on a number of factors, such as, particle size, particle size distribution, area of application, etc. In the present study, single emulsion-solvent evaporation technique has been utilised with two different organic solvents: acetone and chloroform/methanol to prepare quercetin loaded poly(D,L-lactide-co-glycolide) nanoparticles. According to the authors' observations acetone is a better solvent for encapsulating quercetin in polymer nanoparticles owing to its physical and chemical properties.

Correlation of binding efficacies of DNA to flavonoids and their induced cellular damage.

Flavonoids are dietary intakes which are bestowed with several health benefits. The most studied property of flavonoids is their antioxidant efficacy. Among the chosen flavonoids Quercetin, Kaempferol and Myricetin is catagorized as flavonols whereas Apigenin and Luteolin belong to the flavone group. In the present study anti-cancer properties of flavonoids are investigated on the basis of their binding efficacy to ct-DNA and their ability to induce cytotoxicity in K562 leukaemic cells. The binding affinities of the flavonoids with calf thymus DNA (ct-DNA) are in the order Quercetin>Myricetin>Luteolin>Kaempferol>Apigenin. Quercetin with fewer OH than myricetin has higher affinity towards DNA suggesting that the number and position of OH influence the binding efficacies of flavonoids to ct-DNA. CD spectra and EtBr displacement studies evidence myricetin and apigenin to be stronger intercalators of DNA compared to quercetin. From comet assay results it is observed that quercetin and myricetin when used in combination induce higher DNA damage in K562 leukemic cells than when tested individually. Higher binding efficacy has been recorded for quercetin to DNA at lower pH, which is the micro environment of cancerous cells, and hence quercetin can act as a potential anti-cancer agent. Presence of Cu also increases cellular damage as recorded by comet assay.

Understanding nucleosomal histone and DNA interactions: a biophysical study.

Histones are associated with DNA to form nucleosome essential for chromatin structure and major nuclear processes like gene regulation and expression. Histones consist of H1, H2A, H2B and H3, H4 type proteins. In the present study, combined histones from calf thymus were complexed with ct DNA and their binding affinities were measured fluorimetrically. All the five histones were resolved on SDS page and their binding with DNA was visualized. The values of biding affinities varied with pH and salt concentration. Highest affinity (4.0 × 105 M-1) was recorded at pH 6.5 in 50 mM phosphate buffer and 1.5 × 104 M-1 in 2 M NaCl at pH 7.0. The CD spectra support the highest binding affinity with maximum conformational changes at pH 7.0. The time-resolved fluorescence data recorded two life times for histone tyrosine residues at 300 nm emission in phosphate buffer pH 6.5. These life times did not show much change upon binding with DNA in buffer as well as in 2 M NaCl. The isothermal calorimetric studies yielded thermodynamic parameters ΔG, ΔH and ΔS as -1.6 × 105 cal/mol, -1.13 × 103 cal/mol and -3.80 cal/mol/deg, respectively, evidencing a spontaneous exothermic reaction. The dominant binding forces in building the nucleosome are electrostatic interactions.

Anthracycline Drugs on Modified Surface of Quercetin-Loaded Polymer Nanoparticles: A Dual Drug Delivery Model for Cancer Treatment.

Polymer nanoparticles are vehicles used for delivery of hydrophobic anti-cancer drugs, like doxorubicin, paclitaxel or chemopreventors like quercetin (Q). The present study deals with the synthesis and characterisation of nano formulations (NFs) from Q loaded PLGA (poly lactic-co-glycolic acid) nano particles (NPs) by surface modification. The surface of Q-loaded (NPs) is modified by coating with biopolymers like bovine serum albumin (BSA) or histones (His). Conventional chemotherapeutic drugs adriamycin (ADR) and mitoxantrone (MTX) are bound to BSA and His respectively before being coated on Q-loaded NPs to nano formulate NF1 and NF2 respectively. The sizes of these NFs are in the range 400-500 nm as ascertained by SEM and DLS measurements. Encapsulation of Q in polymer NPs is confirmed from shifts in FT-IR, TGA and DSC traces of Q-loaded NPs compared to native PLGA and Q. Surface modification in NFs is evidenced by three distinct regions in their TEM images; the core, polymer capsule and the coated surface. Negative zeta potential of Q-loaded NPs shifted to positive potential on surface modification in NF1 and NF2. In vitro release of Q from the NFs lasted up to twenty days with an early burst release. NF2 is better formulation than NF1 as loading of MTX is 85% compared to 23% loading of ADR. Such NFs are expected to overcome multi-drug resistance (MDR) by reaching and treating the target cancerous cells by virtue of size, charge and retention.

Antagonistic effects of black tea against gamma radiation-induced oxidative damage to normal lymphocytes in comparison with cancerous K562 cells.

The potential of naturally occurring antioxidants to reduce the cellular oxidative damage induced by ionizing radiation has been studied for more than a decade for their pharmacological application during cancer treatment. It is already known that radioprotective efficacy of phytochemicals might influence various end points of radiation damage. Flavonoids are well-known natural radioprotectors, and their biological effects depend upon their chemical structure. In the present study, radioprotective effect of black tea rich in flavonoids was evaluated against gamma radiation-induced oxidative damage on normal lymphocytes and compared with erythroleukemic K562 cells. Pre-treatment with black tea extract (BTE) significantly reduced radiation-induced loss of cell viability, generation of reactive oxygen species, mitochondrial dysfunction, activation of caspase-3 and apoptosis in normal lymphocytes compared to K562 cells. BTE also regulates the activity of endogenous antioxidant enzymes. The changes in the mRNA expression of bax, bcl2, p53 and Nrf2 were also followed to evaluate regulation of radiation-induced apoptosis by BTE. These findings suggest that black tea may have the potential of a natural radioprotective agent which can be used as adjunct with radiation during cancer treatment.

Inhibition of catalase by tea catechins in free and cellular state: a biophysical approach.

Tea flavonoids bind to variety of enzymes and inhibit their activities. In the present study, binding and inhibition of catalase activity by catechins with respect to their structure-affinity relationship has been elucidated. Fluorimetrically determined binding constants for (-)-epigallocatechin gallate (EGCG) and (-)-epicatechin gallate (ECG) with catalase were observed to be 2.27×106 M(-1) and 1.66×106 M(-1), respectively. Thermodynamic parameters evidence exothermic and spontaneous interaction between catechins and catalase. Major forces of interaction are suggested to be through hydrogen bonding along with electrostatic contributions and conformational changes. Distinct loss of α-helical structure of catalase by interaction with EGCG was captured in circular dichroism (CD) spectra. Gallated catechins demonstrated higher binding constants and inhibition efficacy than non-gallated catechins. EGCG exhibited maximum inhibition of pure catalase. It also inhibited cellular catalase in K562 cancer cells with significant increase in cellular ROS and suppression of cell viability (IC50 54.5 µM). These results decipher the molecular mechanism by which tea catechins interact with catalase and highlight the potential of gallated catechin like EGCG as an anticancer drug. EGCG may have other non-specific targets in the cell, but its anticancer property is mainly defined by ROS accumulation due to catalase inhibition.

Mutation induced conformational changes in genomic DNA from cancerous K562 cells influence drug-DNA binding modes.

Normal human genomic DNA (N-DNA) and mutated DNA (M-DNA) from K562 leukemic cells show different thermodynamic properties and binding affinities on interaction with anticancer drugs; adriamycin (ADR) and daunomycin (DNM). Isothermal calorimetric thermograms representing titration of ADR/DNM with N-DNA and M-DNA on analysis best fitted with sequential model of four and three events respectively. From Raman spectroscopy it has been identified that M-DNA is partially transformed to A form owing to mutations and N-DNA on binding of drugs too undergoes transition to A form of DNA. A correlation of thermodynamic contribution and structural data reveal the presence of different binding events in drug and DNA interactions. These events are assumed to be representative of minor groove complexation, reorientation of the drug in the complex, DNA deformation to accommodate the drugs and finally intercalation. Dynamic light scattering and zeta potential data also support differences in structure and mode of binding of N and M DNA. This study highlights that mutations can manifest structural changes in DNA, which may influence the binding efficacy of the drugs. New generation of drugs can be designed which recognize the difference in DNA structure in the cancerous cells instead of their biochemical manifestation.

A review on structure-affinity relationship of dietary flavonoids with serum albumins.

Flavonoids are a class of plant secondary metabolites and among thousands of flavonoids few are considered as dietary flavonoids. Serum albumin (SA), the most abundant protein in plasma, functions as the most important carrier of vital drugs, including dietary flavonoids. The binding affinity of dietary flavonoids to SA is demonstrated to be governed by structure-affinity relationship (SAR) and its bioavailability. The present review summarizes the interactions of flavonoids categorized as flavanol, flavonol, flavone, isoflavone, flavanones, and anthocyanidins with SAs (bovine serum albumin and human serum albumin) in light of SAR. The key findings are: (1) the position and degree of hydroxylation highly influence the affinity of flavonoids to SAs, (2) glycosylation decreases and substitution of methoxy group increases the affinity of flavonoids for SAs, (3) catechin gallates have higher binding affinity to SAs than catechins and gallocatechins, (4) inorganic metal ions modulate the binding affinity of flavonoids to SAs, and (5) hydrophobic interaction plays a major role in the interactions of all flavonoids with SAs.

Studies on black tea (Camellia sinensis) extract as a potential antioxidant and a probable radioprotector.

Positive health effects of tea (Camellia sinensis) on a wide range of physiological problems and diseases are well known and are in part due to its copious antioxidant content. The effect of black tea extract (BTE), which is rich in polyphenolic antioxidants, against the consequences of radiation exposure has not been properly identified. The functional properties of BTE were analyzed and its radioprotective effect on V79 cells was explored in the present study. BTE scavenged free radicals and inhibited Fenton reaction-mediated 2-deoxyribose degradation and lipid peroxidation in a dose-dependent fashion, establishing its antioxidant properties. The radioprotective effects of BTE on strand break induction in pBR322 plasmid DNA were 100 % at 80 µg/ml and higher. In V79 cells, BTE was effective in decreasing the frequency of radiation-induced micronucleated cells and the yields of reactive oxygen species (ROS) and also in restoring the integrity of cellular mitochondrial membrane potential significantly. BTE exerted maximum protection against radiation-induced damage in V79 at a dose of 5 µg/ml. Due to the functional properties of BTE-flavonoids, which have been identified by HPLC, it is envisaged that the key player in radioprotection is elimination of ROS.

Biophysical studies of mutated K562 DNA (erythroleukemic cells) binding to adriamycin and daunomycin reveal that mutations induce structural changes influencing binding behavior.

K562 cells are erythroleukemic cells derived from a chronic myeloid leukemia patient in blast crisis. Comparison of the genome from K562 cells and normal human genome has been very useful strategy, in uncovering eight genes, implicated in acute myeloid leukemia (AML). These genes carry mutations in K562 genome and the role of these mutations in the progression and treatment of AML is still not known. Consequences of these mutations on drug DNA binding are also not known exactly. In the present study, mutation induced structural changes in K562 genome, compared to normal genome, are identified by Fourier transform infra red (FTIR) and circular dichroism (CD) spectroscopy. These structural changes in native K562 DNA favor stronger binding with binding constants 2.0 × 108 and 1.9 × 109 M⁻¹ with antileukemic drugs adriamycin and daunomycin (DNM), respectively, compared to normal DNA. On binding, these drugs disrupt the native B form structure of normal DNA to a greater extent, compared to A-like structure of K562 DNA. Fluorescence and absorption studies reveal higher intercalation as well as mixed groove binding of these drugs with K562 DNA compared to normal DNA. Among the drugs, DNM has higher affinity for K562 DNA.

Black tea extract: a supplementary antioxidant in radiation-induced damage to DNA and normal lymphocytes.

Myriad research has contributed significantly toward the understanding and identification of health benefits stemming from tea polyphenols and many other naturally occurring flavonoids present in fruits and vegetables. These flavonoids are known to mitigate reactive oxygen species-induced damage by scavenging them. In this study, hot-water black tea extract rich in flavonoids is evaluated as a supplementary antioxidant. The antioxidant efficacy of black tea extract was investigated by evaluating radioprotection conferred to pBR322 DNA, calf thymus DNA, and normal lymphocytes during gamma irradiation. The protection was measured by gel electrophoresis, fluorimetric study, cell viability assay, cytokinesis-blocked micronuclei assay, and comet assay. The 2,2-diphenyl-1-picrylhydrazyl scavenging ability of the tea extract used increased in a dose-dependent manner (IC50: 182.45 µg/mL). Positive correlation of radioprotection with antioxidant activity of black tea extract was observed in all systems. Maximum protection against radiation-induced damage was observed in pBR322 DNA and calf thymus DNA at ≥200 µg/mL of black tea extract. At a dose of black tea extract as low as 5 µg/mL, efficient radioprotection was observed in normal lymphocytes, which is encouraging and can be tested in the future as a natural antioxidant supplement during radiotherapy.

Influence of galloyl moiety in interaction of epicatechin with bovine serum albumin: a spectroscopic and thermodynamic characterization.

The health benefits stemming from green tea are well known, but the exact mechanism of its biological activity is not elucidated. Epicatechin (EC) and epicatechin gallate (ECG) are two dietary catechins ubiquitously present in green tea. Serum albumins functionally carry these catechins through the circulatory system and eliminate reactive oxygen species (ROS) induced injury. In the present study ECG is observed to have higher antioxidant activity; which is attributed to the presence of galloyl moiety. The binding affinity of these catechins to bovine serum albumin (BSA) will govern the efficacy of their biological activity. EC and ECG bind with BSA with binding constants 1.0 × 10(6) M(-1) and 6.6 × 10(7) M(-1), respectively. Changes in secondary structure of BSA on interaction with EC and ECG have been identified by circular dichroism (CD) and Fourier transform infrared (FT-IR) spectroscopy. Thermodynamic characterization reveals the binding process to be exothermic, spontaneous and entropy driven. Mixed binding forces (hydrophobic, electrostatic and hydrogen bonding) exist between ECG and BSA. Binding site for EC is primarily site-II in sub-domain IIIA of BSA and for ECG; it is site-I in sub-domain IIA. ECG with its high antioxidant activity accompanied by high affinity for BSA could be a model in drug designing.

Intercalation and induction of strand breaks by adriamycin and daunomycin: a study with human genomic DNA.

The anticancer drugs Adriamycin (ADR) and Daunomycin (DNM) of the anthracycline family are effective in treating a variety of cancers. Although their interactions with other cellular targets may play a role in the selective cytotoxicity of these drugs, it is generally believed that intercalation with DNA is essential for their activity. However, a relationship has not yet been established between intercalation and cellular processes leading to cytotoxicity. The present study was designed to investigate the relationship, if any, between intercalation and DNA strand breaks. ADR and DNM were observed to be strong intercalators of human genomic DNA by absorption and fluorimetric methods that were further substantiated by rise in thermal melting temperature. DNM is the better intercalator of the two, which is also evident from circular dichroic spectral changes. DNA strand breaks, considered to be an index of genotoxicity, was assayed by single cell gel electrophoresis (SCGE; comet assay). ADR and DNM induced equivalent genotoxicity in normal human lymphocytes at a clinically used dose, which was observed to be independent of intercalation efficiency though positively correlated to yield of reactive oxygen species.

The lifetimes of Pharaonis phoborhodopsin signaling states depend on the rates of proton transfers--effects of hydrostatic pressure and stopped flow experiments.

Pharaonis phoborhodopsin (ppR), a negative phototaxis receptor of Natronomonas pharaonis, undergoes photocycle similar to the light-driven proton pump bacteriorhodopsin (BR), but the turnover rate is much slower due to much longer lifetimes of the M and O intermediates. The M decay was shown to become as fast as it is in BR in the L40T/F86D mutant. We examined the effects of hydrostatic pressure on the decay of these intermediates. For BR, pressure decelerated M decay but slightly affected O decay. In contrast, with ppR and with its L40T/F86D mutant, pressure slightly affected M decay but accelerated O decay. Clearly, the pressure-dependent factors for M and O decay are different in BR and ppR. In order to examine the deprotonation of Asp75 in unphotolyzed ppR we performed stopped flow experiments. The pH jump-induced deprotonation of Asp75 occurred with 60 ms, which is at least 20 times slower than deprotonation of the equivalent Asp85 in BR and about 10-fold faster than the O decay of ppR. These data suggest that proton transfer is slowed not only in the cytoplasmic channel but also in the extracellular channel of ppR and that the light-induced structural changes in the O intermediate of ppR additionally decrease this rate.