Engineered nanomicelles targeting proliferation and angiogenesis inhibit tumour progression by impairing the synthesis of ceramide-1-phosphate.

Tumour cells secrete various proangiogenic factors like VEGF, PDGF, and EGF that result in the formation of highly vascularized tumours with an immunosuppressive tumour microenvironment. As tumour growth and metastasis are highly dependent on angiogenesis, targeting tumour vasculature along with rapidly dividing tumour cells is a potential approach for cancer treatment. Here, we specifically engineered sub-100 sized nanomicelles (DTX-CA4 NMs) targeting proliferation and angiogenesis using an esterase-sensitive phosphocholine-tethered docetaxel conjugate of lithocholic acid (LCA) (PC-LCA-DTX) and a poly(ethylene glycol) (PEG) derivative of an LCA-combretastatin A4 conjugate (PEG-LCA-CA4). DTX-CA4 NMs effectively inhibit the tumour growth in syngeneic (CT26) and xenograft (HCT116) colorectal cancer models, inhibit tumour recurrence, and enhance the percentage survival in comparison with individual drug-loaded NMs. DTX-CA4 NMs enhance the T cell-mediated anti-tumour immune response and DTX-CA4 NMs in combination with an immune checkpoint inhibitor, anti-PDL1 antibody, enhance the anti-tumour response. We additionally showed that DTX-CA4 NMs effectively attenuate the production of ceramide-1-phosphate, a key metabolite of the sphingolipid pathway, by downregulating the expression of ceramide kinase at both transcriptional and translational levels. Therefore, this study presents the engineering of effective DTX-CA4 NMs for targeting the tumour microenvironment that can be explored further for clinical applications.

Engineered nanomicelles inhibit the tumour progression via abrogating the prostaglandin-mediated immunosuppression.

Cancer treatment is challenged due to immunosuppressive inflammatory tumour microenvironment (TME) caused by infiltration of tumour-promoting and inhibition of tumour-inhibiting immune cells. Here, we report the engineering of chimeric nanomicelles (NMs) targeting the cell proliferation using docetaxel (DTX) and inflammation using dexamethasone (DEX) that alters the immunosuppressive TME. We show that a combination of phospholipid-DTX conjugate and PEGylated-lipid-DEX conjugate can self-assemble to form sub-100 nm chimeric NMs (DTX-DEX NMs). Anti-cancer activities against syngeneic and xenograft mouse models showed that the DTX-DEX NMs are more effective in tumour regression, enhance the survival of mice over other treatment modes, and alter the tumour stroma. DTX-DEX NMs cause a significant reduction in myeloid-derived suppressor cells, alter the polarization of macrophages, and enhance the accumulation of cytotoxic CD4+ and CD8+ T cells in tumour tissues, along with alterations in cytokine expression. We further demonstrated that these DTX-DEX NMs inhibit the synthesis of prostaglandins, especially PGE2, by targeting the cyclooxygenase 2 that is partly responsible for immunosuppressive TME. Therefore, this study presents, for the first time, the engineering of lithocholic acid-derived chimeric NMs that affect the prostaglandin pathway, alter the TME, and mitigate tumour progression with enhanced mice survival.

Socio-economic inequalities in burden of communicable and non-communicable diseases among older adults in India: Evidence from Longitudinal Ageing Study in India, 2017-18.

Developing countries like India grapple with significant challenges due to the double burden of communicable and non-communicable disease in older adults. Examining the distribution of the burden of different communicable and non-communicable diseases among older adults can present proper evidence to policymakers to deal with health inequality. The present study aimed to determine socioeconomic inequality in the burden of communicable and noncommunicable diseases among older adults in India. This study used Longitudinal Ageing study in India (LASI), Wave 1, conducted during 2017-2018. Descriptive statistics along with bivariate analysis was used in the present study to reveal the initial results. Binary logistic regression analysis was used to estimate the association between the outcome variables (communicable and non-communicable disease) and the chosen set of separate explanatory variables. For measurement of socioeconomic inequality, concentration curve and concentration index along with state wise poor-rich ratio was calculated. Additionally, Wagstaff's decomposition of the concentration index approach was used to reveal the contribution of each explanatory variable to the measured health inequality (Communicable and non- communicable disease). The study finds the prevalence of communicable and non-communicable disease among older adults were 24.9% and 45.5% respectively. The prevalence of communicable disease was concentrated among the poor whereas the prevalence of NCDs was concentrated among the rich older adults, but the degree of inequality is greater in case of NCD. The CI for NCD is 0.094 whereas the CI for communicable disease is -0.043. Economic status, rural residence are common factors contributing inequality in both diseases; whereas BMI and living environment (house type, drinking water source and toilet facilities) have unique contribution in explaining inequality in NCD and communicable diseases respectively. This study significantly contributes in identifying the dichotomous concentration of disease prevalence and contributing socio- economic factors in the inequalities.

Gender differences in premature mortality for cardiovascular disease in India, 2017-18.

BACKGROUND: The present study tries to provide a comprehensive estimate of gender differences in the years of life lost due to CVD across the major states of India during 2017-18. METHODS: The information on the CVD related data were collected from medical certification of causes of death (MCCD reports, 2018). Apart from this, information from census of India (2001, 2011), SRS (2018) were also used to estimate YLL. To understand the variation in YLL due to CVD at the state level, nine sets of covariates were chosen: share of elderly population, percentage of urban population, literacy rate, health expenditure, social sector expenditure, labour force participation, HDI Score and co-existence of other NCDs such as diabetes, & obesity. The absolute number of YLL and YLL rates were calculated. Further, Pearson's correlation had been calculated and to understand the effect of explanatory variables on YLL due to CVD, multiple linear regression analysis had been applied. RESULTS: Men have a higher burden of premature mortality in terms of Years of life lost (YLL) due to CVD than women in India, with pronounced differences at adult ages of 50-54 years and over. The age pattern of YLL rate suggests that the age group 85 + makes the highest contribution to the overall YLL rate due to CVD. YLL rate showed a J-shaped relationship with age, starting high at ages below 1 years, dropping to their lowest among children aged 1-4 years, and rising again to highest levels at 85 + years among both men and women. In all the states except Bihar men had higher estimated YLL due to CVD for all ages than women. Among men the YLL due to CVD was higher in Tamil Nadu followed by Madhya Pradesh and Chhattisgarh. On the other hand, the YLL due to CVD among men was lowest in Jharkhand followed by Assam. Similarly, among women the YLL due to CVD was highest in Tamil Nadu followed by Madhya Pradesh and Chhattisgarh. While, the YLL due to CVD among women was lowest in Jharkhand. Irrespective of gender, all factors except state health expenditure were positively linked with YLL due to CVD, i.e., as state health expenditure increases, the years of life lost (YLL) due to CVDs falls. Among all the covariates, the proportion of a state's elderly population emerges as the most significant predictor variable for YLL for CVDs (r = 0.42 for men and r = 0.50 for women). CONCLUSION: YLL due to cardiovascular disease varies among men and women across the states of India. The state-specific findings of gender differences in years of life lost due to CVD may be used to improve policies and programmes in India.

Strategies To Modify the Surface and Bulk Properties of 3D-Printed Solid Scaffolds for Tissue Engineering Applications.

3D printing is one of the effective scaffold fabrication techniques that emerged in the 21st century that has the potential to revolutionize the field of tissue engineering. The solid scaffolds developed by 3D printing are still one of the most sought-after approaches for developing hard-tissue regeneration and repair. However, applications of these solid scaffolds get limited due to their poor surface and bulk properties, which play a significant role in tissue integration, loadbearing, antimicrobial/antifouling properties, and others. As a result, several efforts have been directed to modify the surface and bulk of these solid scaffolds. These modifications have significantly improved the adoption of 3D-printed solid scaffolds and devices in the healthcare industry. Nevertheless, the in vivo implant applications of these 3D-printed solid scaffolds/devices are still under development. They require attention in terms of their surface/bulk properties, which dictate their functionality. Therefore, in the current review, we have discussed different 3D-printing parameters that facilitate the fabrication of solid scaffolds/devices with different properties. Further, changes in the bulk properties through material and microstructure modification are also being discussed. After that, we deliberated on the techniques that modify the surfaces through chemical and material modifications. The computational approaches for the bulk modification of these 3D-printed materials are also mentioned, focusing on tissue engineering. We have also briefly discussed the application of these solid scaffolds/devices in tissue engineering. Eventually, the review is concluded with an analysis of the choice of surface/bulk modification based on the intended application in tissue engineering.

Surface Engineering of a Bioartificial Membrane for Its Application in Bioengineering Devices.

Membrane technology is playing a crucial role in cutting-edge innovations in the biomedical field. One such innovation is the surface engineering of a membrane for enhanced longevity, efficient separation, and better throughput. Hence, surface engineering is widely used while developing membranes for its use in bioartificial organ development, separation processes, extracorporeal devices, etc. Chemical-based surface modifications are usually performed by functional group/biomolecule grafting, surface moiety modification, and altercation of hydrophilic and hydrophobic properties. Further, creation of micro/nanogrooves, pillars, channel networks, and other topologies is achieved to modify physio-mechanical processes. These surface modifications facilitate improved cellular attachment, directional migration, and communication among the neighboring cells and enhanced diffusional transport of nutrients, gases, and waste across the membrane. These modifications, apart from improving functional efficiency, also help in overcoming fouling issues, biofilm formation, and infection incidences. Multiple strategies are adopted, like lysozyme enzymatic action, topographical modifications, nanomaterial coating, and antibiotic/antibacterial agent doping in the membrane to counter the challenges of biofilm formation, fouling challenges, and microbial invasion. Therefore, in the current review, we have comprehensibly discussed different types of membranes, their fabrication and surface modifications, antifouling/antibacterial strategies, and their applications in bioengineering. Thus, this review would benefit bioengineers and membrane scientists who aim to improve membranes for applications in tissue engineering, bioseparation, extra corporeal membrane devices, wound healing, and others.

Caregiving: a risk factor of poor health and depression among informal caregivers in India- A comparative analysis.

BACKGROUND: In an ageing society, the provision of long-term care is the prime need. In Indian cultural setting, family members are the informal, albeit primary caregivers to the elderly. Caregiving demands intense emotional and financial involvement. While taking care of elderly persons' health and wellbeing, these family members, acting as informal caregivers, may themselves become vulnerable to poor health due to additional stress and burden. Using a nationally representative survey, the study tried to identify how health condition varies within caregivers and a comparative analysis of how in similar socio-economic background health condition varies between caregivers and non-caregivers. METHOD: The data, used for the analysis, is taken from Longitudinal Ageing Study in India (LASI), Wave I. Both descriptive and multivariable regression analysis are done in different models along with interaction effect of caregiving to understand the difference in health status between caregiver and non-caregivers. RESULTS: Nearly 29% and 11% of the informal caregivers, reported to have depressive symptoms and poor self-rated health (SRH), respectively. Almost half of the caregivers, who provide care for more than 40 h a week, are diagnosed to have depressive symptoms. They are also at higher risk of having depressive symptoms (AOR 1.59 CI 1.16-2.18) and poor SRH (AOR 1.73 CI 1.11-2.69) than those who invest less than 40 h in a week. In almost every socio-economic condition, caregivers are at a higher risk of having depression and poor health than non-caregivers. Caregivers, who are widowed, live in rural areas or are not satisfied with current living arrangement are more vulnerable to have depressive symptoms. On the other hand, caregivers of age 45-59 years, widowed, male and who live only with their children with spouse absent, have almost 2 times higher odds of poor SRH than non-caregivers. CONCLUSION: Caregivers are more susceptible to depression and poor self-rated health compared to non-caregivers irrespective of their socio-economic characteristics, only the magnitude of vulnerability varies.

Diffusion Reflection Method for Early Detection of Oral Squamous Cell Carcinoma Specifically Targeted by Circulating Gold-Nanorods Bio-Conjugated to Anti-Epidermal Growth Factor Receptor.

BACKGROUND: Translation of nanomedical developments into clinical application is receiving an increasing interest. However, its use for oral squamous cell carcinoma (OSCC) diagnosis remains limited. We present an advanced nanophotonic method for oral cancer detection, based on diffusion reflection (DR) measurements of gold-nanorods bio-conjugated to anti-epidermal growth factor receptor (C-GNRs) specifically attached to OSCC cells. OBJECTIVE: To investigate in a rat model of oral carcinogenesis the targeting potential of C-GNRs to OSCC by using the DR optical method. MATERIALS AND METHODS: OSCC was induced by the carcinogen 4-nitroquinoline-N-oxide (4NQO). C-GNRs were introduced locally and systemically and DR measurements were recorded from the surface of the rat tongue following illumination with red laser beam. Rats were divided into experimental and control groups. The results were compared with the histologic diagnosis. RESULTS: A total of 75 Wistar-derived rats were enrolled in the study. Local application did not reveal any statistical results. DR measurements following intravenous injection of C-GNRs revealed a significant increase in light absorption in rats with OSCC compare with rats without cancer (p<0.02, sensitivity 100%, specificity 89%). In addition, absorption of light increased significantly in cases of severe dysplasia and cancer (high risk) compared to rats without cancer and rats with mild dysplasia (low risk) (86% sensitivity and 89% specificity, AUC=0.79). CONCLUSION: Combining nanotechnology and nanophotonics for in vivo diagnosis of OSCC serves as additional tier in the translation of advanced nanomedical developments into clinical applications. The presented method shows a promising potential of nanophotonics for oral cancer identification, and provides support for the use of C-GNRs as a selective drug delivery.

The Scattering of Gold Nanorods Combined with Differential Uptake, Paving a New Detection Method for Macrophage Subtypes Using Flow Cytometery.

The strategy of identification for M1 and M2 macrophages both in vivo and in vitro would help to predict the health condition of the individual. Here, we introduced a solution to this problem with the advantage of both the phagocytic nature of macrophages and the scattering effect of gold nanorods (GNRs). The internalized GNRs, relating to their extent of intake, caused a conspicuous scattering profile at the red channel in flow cytometry, overruling the contribution of the cellular side scatters. This internalization is solely governed by the surface chemistry of GNRs. The PAH-GNRs showed maximum intake potency followed by Cit-, PSS-, and PEG-GNRs. On a substantial note, PAH-GNRs lead to differential uptake between M1 and M2 cells, with three times higher intake in M2 cells over M1. This is the first report of employing the scattering of unlabeled GNRs to discriminate M1 and M2 cell types using a flow cytometer.

Surface Modification by Media Organics Reduces the Bacterio-toxicity of Cupric Oxide Nanoparticle against Escherichia coli.

Prevalence of antibiotic-resistant bacteria demands alternatives to antibiotics. Copper-based nanoparticles with a high antibacterial property may be a solution to the problem. It is, therefore, important to understand the mode of antibacterial action of the nanoparticles (NPs). Despite reports on induction of reactive oxygen species (ROS) in bacteria by copper and copper-oxide nanoparticles and involvement of such ROS in cell killing, it is still unclear (a) if surface modification of the nanoparticles by media organics has any role on their antibacterial potency and (b) whether the bactericidal effects of these NPs are 'particle-specific' or 'ion-specific' in nature. We address these issues for cupric oxide nanoparticle (CuO-NP) in this study. Instead of nutrient medium, when E. coli bacterial cells were suspended in saline (0.9% NaCl), CuO-NP had a more anti-bacterial effect, with MBC (minimum bactericidal concentration) value of 6 µg/mL, than in nutrient medium with MBC value of 160 µg/mL. Moreover, the lysine-modified CuO-NP in saline had MBC at 130 µg/mL. Thus, unmodified CuO-NP was more efficient killer than modified one. Our finding further revealed that in saline;CuO-NP had 'particle-specific' antibacterial effect through generation of ROS and consequent oxidative damage by lipid peroxidation, protein oxidation and DNA degradation in cells.

Algorithm for in vivo detection of tissue type from multiple scattering light phase images.

In vivo physiological assessments are typically done by either imaging techniques or by sensing changes in the attenuation coefficient. Using visible or near-infrared (NIR), imaging is mainly possible for thin tissues. On the other hand, clinical information can also be detected by examining changes in tissue optical properties. The most challenging aspect in sensing techniques is the spectral dependent scattering, which varies with the physiological state and tissue type. We have previously published our novel noninvasive nanophotonics technique for detecting tissue scattering based on reflectance measurements: the iterative multi-plane optical property extraction (IMOPE). The IMOPE reconstructs the reemitted light phase using an iterative algorithm and extracts the scattering properties based on a theoretical model. This paper presents the in vivo application of distinguishing between different mouse tissue areas. The reconstructed phase images reveal different areas in the inner thigh of a mouse, which are related to the muscle, bone, and skin. The IMOPE uses the reconstructed phases for sensing and detecting unseen components beneath the skin surface. This technique could be further applied to the diagnosis of various physiological states.

Hyperlipidemic mice as a model for a real-time in vivo detection of atherosclerosis by gold nanorods-based diffusion reflection technique.

Atherosclerosis (AS), the leading cause of morbidity and mortality in cardiovascular disease, needs an early detection for treatment and prevention of fatal events. Here, for the first time, we applied gold nanorods (GNRs)-assisted diffusion reflection (DR), a noninvasive technique for in vivo detection of AS in a high-fat-diet-induced c57bl mouse model, which resembles the manifestation of AS in humans. DR simply detects the change in light reflection profile of tissue due to the accumulation of GNRs in the AS plaques and enables clear detection of AS lesions in carotid and femoral arteries of these hyperlipidemic mice. After 24 hours post-GNRs injection, DR showed the highest efficiency of AS detection. Moreover, the sensitivity of the DR method is much higher than computed tomography (CT) and is comparable to ex vivo high-resolution CT. Our results strongly suggest that the DR method can detect early atherosclerotic lesions in a sensitive and specific manner.

Self-Calibration Phenomenon for Near-Infrared Clinical Measurements: Theory, Simulation, and Experiments.

An irradiated turbid medium scatters the light in accordance to its optical properties. Near-infrared (NIR) clinical methods, which are based on spectral-dependent absorption, suffer from an inherent error due to spectral-dependent scattering. We present here a unique spatial point, that is, iso-pathlength (IPL) point, on the surface of a tissue at which the intensity of re-emitted light remains constant. This scattering-indifferent point depends solely on the medium geometry. On the basis of this natural phenomenon, we suggest a novel optical method for self-calibrated clinical measurements. We found that the IPL point exists in both cylindrical and semi-infinite tissue geometries (Supporting Information, Video file). Finally, in vivo human finger and mice measurements are used to validate the crossing point between the intensity profiles of two wavelengths. Hence, measurements at the IPL point yield an accurate absorption assessment while eliminating the scattering dependence. This finding can be useful for oxygen saturation determination, NIR spectroscopy, photoplethysmography measurements, and a wide range of optical sensing methods for physiological aims.

Three-Dimensional Highly Sensitive Diffusion Reflection-Based Imaging Method for the in Vivo Localization of Atherosclerosis Plaques Following Gold Nanorods Accumulation.

In this work, we present a novel, simple, and highly accurate three-dimensional (3D) diffusion reflection (DR) imaging system and method for the detection of accumulation sites of gold nanorods (GNRs) within the tissue. GNRs are intensively used for diagnosis purposes of varied diseases, mainly because of their ability to well absorb visible light, which introduces them as terrific contrast agents in various imaging and theranostics methods. Lately, these GNRs unique absorption properties have served in DR intensity-based measurements, suggesting a novel diagnostic tool, DR-GNRs. In this paper, we show a new measurement system and method for DR, based on its radial collection from the tissue. These radial measurements enabled a unique 3D presentation of the DR-GNR, introducing the dimensions ρ for the radius, θ for the angle, and Γ for the reflected intensity. On the basis of the diffusion model, which enables to correlate between the sample's optical properties and its reflectance, a unique, radial map is presented. This map introduces the slopes of the DR curves in each measured angle, which are linearly correlated with the tissue's optical properties and with the GNRs concentrations within the tissue, thus enables the exact radial localization of the GNRs in the sample. We show the detection of macrophage accumulation in tissue-like phantoms, as well as the localization of unstable plaques in hyperlipidemic mice, in vivo. This highly accurate, powerful technology paves the way toward a real-time detection method that can be successfully integrated in the rapid increasing field of personalized medicine.

Haemocompatibility of Modified Nanodiamonds.

This study reports the interactions of modified nanodiamond particles in vitro with human blood. Modifications performed on the nanodiamond particles include oxygenation with a chemical method and hydrogenation upon chemical vapor deposition (CVD) plasma treatment. Such nanodiamonds were later incubated in whole human blood for different time intervals, ranging from 5 min to 5 h. The morphology of red blood cells was assessed along with spectral measurements and determination of haemolysis. The results showed that no more than 3% of cells were affected by the nanodiamonds. Specific modifications of the nanodiamonds give us the possibility to obtain nanoparticles which are biocompatible with human blood. They can form a basis for the development of nanoscale biomarkers and parts of sensing systems and devices useful in biomedical environments.

Metallic copper nanoparticles induce apoptosis in a human skin melanoma A-375 cell line.

In two earlier communications (Chatterjee et al 2012 Nanotechnology 23 085103, Chatterjee et al 2014 Nanotechnology 25 135101), we reported the development of a simple and unique method of synthesizing highly stable metallic copper nanoparticles (Cu NPs) with high antibacterial activity. Here we report on the cytotoxic potency of the NPs against cancer cells. The value of the IC50 dose of the Cu NPs against human skin cancer cell A-375 was found to be 1.71 µg ml-1 only, which was much less than values reported so far, and this concentration had no cytotoxic effect on normal white blood cells. The NPs caused (i) lowering of cell membrane rigidity, (ii) DNA degradation, (iii) chromosomal condensation, (iv) cell cycle arrest in the G2/M phase, (v) depolarization of the mitochondrial membrane and (vi) apoptosis of cells. Cellular apoptosis occurred in the caspase-9-mediated intrinsic pathway. This study revealed that our Cu NPs had high anticancer properties by killing tumor cells through the apoptotic pathway. Since this particle has high antibacterial activity, our Cu NPs might be developed in future as a dual action drug-anticancer as well as antibacterial.

A simple, fast and cost-effective method of synthesis of cupric oxide nanoparticle with promising antibacterial potency: Unraveling the biological and chemical modes of action.

BACKGROUND: Gradual attainment of bacterial resistance to antibiotics led us to develop a robust method of synthesis of stable, colloidal cupric oxide nanoparticle of physiological pH with potential antibacterial action. METHODS: Cu(II) oxide NP was synthesized by reduction-oxidation of CuCl2, using polyvinyl alcohol as stabilizer. Characteristics and antibacterial activity of the particles were investigated by techniques like UV-Vis spectrophotometry, DLS, AFM, TEM, EDS, FTIR, AAS, agar plating, FACS, gel electrophoresis and XPS. RESULTS: The NPs were about 50 nm in size and cubic in shape with two surface plasmon peaks at 266 and 370 nm and had semi-conducting behavior with a band gap of 3.40 and 3.96 eV. About 80% of precursor CuCl2 was converted to NP. The minimum inhibitory and the minimum bactericidal concentrations of CuO-NP were respectively 120 and 160 µg/mL for Escherichia coli and 180 and 195 µg/mL for Staphylococcus aureus in Luria-Bertani medium. In growth media, the NPs got modified by media organics with displacement of the stabilizer PVA molecules. This modified NP (around 240 nm) killed cells by generating ROS, which finally caused membrane lipid per-oxidation and chromosomal DNA degradation in NP-treated cells. CONCLUSION: Reports indicate that we are among the few who had prepared CuO-NP in colloidal form. The antibacterial potency of our particle in growth media was much promising than other reports. Our findings demonstrated that 'particle-specific' effect, not 'ion-specific' one, was responsible for the NP action. GENERAL SIGNIFICANCE: The NP may be used as a sterilizing agent in various bioprocesses and as substituent of antibiotics, after thorough toxicological study.

Mechanism of antibacterial activity of copper nanoparticles.

In a previous communication, we reported a new method of synthesis of stable metallic copper nanoparticles (Cu-NPs), which had high potency for bacterial cell filamentation and cell killing. The present study deals with the mechanism of filament formation and antibacterial roles of Cu-NPs in E. coli cells. Our results demonstrate that NP-mediated dissipation of cell membrane potential was the probable reason for the formation of cell filaments. On the other hand, Cu-NPs were found to cause multiple toxic effects such as generation of reactive oxygen species, lipid peroxidation, protein oxidation and DNA degradation in E. coli cells. In vitro interaction between plasmid pUC19 DNA and Cu-NPs showed that the degradation of DNA was highly inhibited in the presence of the divalent metal ion chelator EDTA, which indicated a positive role of Cu(2+) ions in the degradation process. Moreover, the fast destabilization, i.e. the reduction in size, of NPs in the presence of EDTA led us to propose that the nascent Cu ions liberated from the NP surface were responsible for higher reactivity of the Cu-NPs than the equivalent amount of its precursor CuCl2; the nascent ions were generated from the oxidation of metallic NPs when they were in the vicinity of agents, namely cells, biomolecules or medium components, to be reduced simultaneously.

A simple robust method for synthesis of metallic copper nanoparticles of high antibacterial potency against E. coli.

A method for preparation of copper nanoparticles (Cu-NPs) was developed by simple reduction of CuCl2 in the presence of gelatin as a stabilizer and without applying stringent conditions like purging with nitrogen. The NPs were characterized by spectrophotometry, dynamic light scattering, x-ray diffraction, transmission electron microscopy, atomic force microscopy and x-ray photoelectron spectroscopy. The particles were about 50-60 nm in size and highly stable. The antibacterial activity of this Cu-NP on Gram-negative Escherichia coli was demonstrated by the methods of agar plating, flow cytometry and phase contrast microscopy. The minimum inhibitory concentration (3.0 µg ml(-1)), minimum bactericidal concentration (7.5 µg ml(-1)) and susceptibility constant (0.92) showed that this Cu-NP is highly effective against E. coli at a much lower concentration than that reported previously. Treatment with Cu-NPs made E. coli cells filamentous. The higher the concentration of Cu-NPs, the greater the population of filamentous cells; average filament size varied from 7 to 20 µm compared to the normal cell size of ∼2.5 µm. Both filamentation and killing of cells by Cu-NPs (7.5 µg ml(-1)) also occurred in an E. coli strain resistant to multiple antibiotics. Moreover, an antibacterial effect of Cu-NPs was also observed in Gram-positive Bacillus subtilis and Staphylococcus aureus, for which the values of minimum inhibitory concentration and minimum bactericidal concentration were close to that for E. coli.