Placental Drug Delivery to Treat Pre-Eclampsia and Fetal Growth Restriction.

Pre-eclampsia and fetal growth restriction (FGR) continue to cause unacceptably high levels of morbidity and mortality, despite significant pharmaceutical and technological advances in other disease areas. The recent pandemic has also impacted obstetric care, as COVID-19 infection increases the risk of poor pregnancy outcomes. This review explores the reasons why it lacks effective drug treatments for the placental dysfunction that underlies many common obstetric conditions and describes how nanomedicines and targeted drug delivery approaches may provide the solution to the current drug drought. The ever-increasing range of biocompatible nanoparticle formulations available is now making it possible to selectively deliver drugs to uterine and placental tissues and dramatically limit fetal drug transfer. Formulations that are refractory to placental uptake offer the possibility of retaining drugs within the maternal circulation, allowing pregnant individuals to take medicines previously considered too harmful to the developing baby. Liposomes, ionizable lipid nanoparticles, polymeric nanoparticles, and adenoviral vectors have all been used to create efficacious drug delivery systems for use in pregnancy, although each approach offers distinct advantages and limitations. It is imperative that recent advances continue to be built upon and that there is an overdue investment of intellectual and financial capital in this field.

Glucose-conjugated glutenin nanoparticles for selective targeting and delivery of camptothecin into breast cancer cells.

Receptor-mediated drug delivery systems are a promising tool for targeting malignant cells to suppress/inhibit the malignancy without disturbing healthy cells. Protein-based nanocarrier systems possess numerous advantages for the delivery of variety of chemotherapeutics, including therapeutic peptides and genes. In the present work, glucose-conjugated camptothecin-loaded glutenin nanoparticles (Glu-CPT-glutenin NPs) were fabricated to deliver camptothecin to MCF-7 cells via GLUT-1 transporter protein. Initially, Glu-conjugated glutenin polymer was successfully synthesized through reductive amination reaction, and this was confirmed by FTIR and 13C-NMR. Then, camptothecin (CPT) was loaded into Glu-conjugated glutenin polymer forming Glu-CPT-glutenin NPs. The nanoparticles were studied for their drug releasing capacity, morphological shape, size, physical nature, and zeta potential. The fabricated Glu-CPT-glutenin NPs were found to be spherical in shape and amorphous in nature with 200-nm size range and a zeta potential of - 30 mV. Furthermore, MTT assay using Glu-CPT-glutenin NPs confirmed concentration-dependent cytotoxicity against MCF-7 cells after 24-h treatment, and IC50 was found to be 18.23 µg mL-1. In vitro cellular uptake study demonstrated that the Glu-CPT-glutenin NPs had enhanced endocytosis and delivered CPT in MCF-7 cells. A typical apoptotic morphological change of condensed nuclei and distorted membrane bodies was found after treatment with IC50 concentration of NPs. The released CPT from NPs also targeted mitochondria of MCF-7 cells, significantly increasing the level of reactive oxygen species and causing the damage of mitochondrial membrane integrity. These outcomes confirmed that the wheat glutenin can positively serve as a significant delivery vehicle and enhance the anticancer potential of this drug.

Cytotoxic Potential of Bioactive Compounds from Aspergillus flavus, an Endophytic Fungus Isolated from Cynodon dactylon, against Breast Cancer: Experimental and Computational Approach.

Endophytic fungi are a diverse group of microorganisms that colonize the inter- or intracellular spaces of plants and exhibit mutual benefits. Their interactions with the host plant and other microbiomes are multidimensional and play a crucial role in the production of secondary metabolites. We screened bioactive compounds present in the extracts of Aspergillus flavus, an endophytic fungus isolated from the roots of the medicinal grass Cynodon dactylon, for its anticancer potential. An in vitro analysis of the Ethyl acetate extract from A. flavus showed significant cytostatic effects (IC50: 16.25 µg/mL) against breast cancer cells (MCF-7). A morphological analysis of the cells and a flow cytometry of the cells with annexin V/Propidium Iodide suggested that the extract induced apoptosis in the MCF-7 cells. The extract of A. flavus increased reactive oxygen species (ROS) generation and caused a loss of mitochondrial membrane potential in MCF-7 cells. To identify the metabolites that might be responsible for the anticancer effect, the extract was subjected to a gas chromatography-mass spectrometry (GC-MS) analysis. Interestingly, nine phytochemicals that induced cytotoxicity in the breast cancer cell line were found in the extract. The in silico molecular docking and molecular dynamics simulation studies revealed that two compounds, 2,4,7-trinitrofluorenone and 3α, 5 α-cyclo-ergosta-7,9(11), 22t-triene-6beta-ol exhibited significant binding affinities (-9.20, and -9.50 Kcal/mol, respectively) against Bcl-2, along with binding stability and intermolecular interactions of its ligand-Bcl-2 complexes. Overall, the study found that the endophytic A. flavus from C. dactylon contains plant-like bioactive compounds that have a promising effect in breast cancer.

Doxorubicin resistant choriocarcinoma cell line derived spheroidal cells exhibit stem cell markers but reduced invasion.

Cell cycle-specific cancer chemotherapy is based on the ability of a drug to halt, minimise or destroy rapidly dividing cells. However, their efficacy is limited by the emergence of a self-renewing cell pool called "cancer stem cells" (CSC). Choriocarcinoma is a tumour of trophoblastic tissue. We, in this study, analysed whether spheroids generated from doxorubicin-treated and non-treated choriocarcinoma cell lines exhibit markers of stem cells. Two choriocarcinoma cell lines, namely JEG-3 and BeWo, were used in this study. Spheroids were generated from doxorubicin-treated cells and the non-treated cells under non-adherent condition, followed by analysis of stem-cell markers' expression, namely NANOG, OCT4 and SOX2. Immunofluorescence analysis suggested a general increase in the markers' concentration in spheroids relative to the parental cells. RT-qPCR and immunoblots showed an increase in the stem-cell marker expression in spheroids generated from doxorubicin-treated when compared to non-treated cells. In spheroids, Sox2 was significantly upregulated in doxorubicin-treated spheroids, whereas Nanog and Oct4 were generally downregulated when compared to non-treated spheroids. Both 2D and 3D invasion assays showed that the spheroids treated with doxorubicin exhibited reduced invasion. Our data suggest that choriocarcinoma cell lines may have the potential to produce spheroidal cells, yet the drug-treatment affected the invasion potential of spheroids. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-022-03243-x.

Analyses of selected tumour-associated factors expression in normotensive and preeclamptic placenta.

INTRODUCTION: Human placenta is often considered a controlled-tumour because of shared properties such as invasion and angiogenesis. We assessed the status of a few selected tumour-associated factors (TAFs) in late onset pre-eclamptic (PE) and normotensive (NT) placentae, to understand their involvement in trophoblast invasion. These molecules include aldehyde dehydrogenase (ALDH3A1), aurora kinases (AURK-A/C), platelet derived growth factor receptor-α (PDGFRα), jagged-1 (JAG1) and twist related protein-1 (TWIST1). METHODS: The expression of TAF was compared in 13 NT and 11 PE (late onset) placentae using immunoblotting/immunohistochemistry. We then used a novel spheroidal cell model developed from transformed human first trimester trophoblast cell lines HTR8/SVneo and TEV-1 to determine the expression and localization of these six factors during invasion. We also compared the expression of these TAFs during migration and invasion. RESULTS: Our results suggest that expressions of ALDH3A1, AURK-A, PDGFRα, and TWIST1 are significantly upregulated in PE placentae (p < 0.05) when compared to NT placentae, whereas AURK-C and JAG1 are down-regulated (p < 0.05). The protein expression pattern of all the six factors were found to be similar in spheroids in comparison to their parental counterparts. The invasive potential of the spheroids was also enhanced when compared with the parental cells. DISCUSSION: Collectively, data from our present study suggests that these TAFs are involved in placental invasion and their altered expressions may be regarded as a compensatory mechanism against reduced invasion.

Exopolysaccharides from Lactobacillus acidophilus modulates the antioxidant status of 1,2-dimethyl hydrazine-induced colon cancer rat model.

The aim of the current study is to ascertain the anticancer activity of exopolysaccharides (EPS) from probiotic Lactobacillus acidophilus in the 1, 2-dimethyl hydrazine (DMH)-induced colon cancer rat model and to determine the antioxidant status. Rats were divided into five groups of six animals each. Group I served as control, group II served as cancer control (DMH alone administered), group III as standard drug control (5-FU along with DMH) and group IV and V received EPS in two doses (200 mg/kg body weight and 400 mg/kg body weight along with DMH). EPS administration was found to reduce the number of polyps formed (Group IV-8.25 ± 1.258 and Group V-8.50 ± 1.732 vs Group II-14.50 ± 2.380) and to increase the levels of antioxidant enzymes viz. Superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) and antioxidants like vitamin C (Vit. C), reduced glutathione (GSH) which was found to be reduced in colon cancer control rats. The status of lipid peroxidation (LPO) was also evaluated. All the values which were affected by the supplementation of DMH were brought to near normal levels by the treatment with EPS. The well-preserved histology of colon and the biochemical evaluation also show that EPS could be a potential agent for the prevention and treatment of colon cancer.

Targeting complement cascade: an alternative strategy for COVID-19.

The complement system is a stakeholder of the innate and adaptive immune system and has evolved as a crucial player of defense with multifaceted biological effects. Activation of three complement pathways leads to consecutive enzyme reactions resulting in complement components (C3 and C5), activation of mast cells and neutrophils by anaphylatoxins (C3a and C5a), the formation of membrane attack complex (MAC) and end up with opsonization. However, the dysregulation of complement cascade leads to unsolicited cytokine storm, inflammation, deterioration of alveolar lining cells, culminating in acquired respiratory destructive syndrome (ARDS). Similar pathogenesis is observed with the middle east respiratory syndrome (MERS), severe acquired respiratory syndrome (SARS), and SARS-CoV-2. Activation of the lectin pathway via mannose-binding lectin associated serine protease 2 (MASP2) is witnessed under discrete viral infections including COVID-19. Consequently, the spontaneous activation and deposits of complement components were traced in animal models and autopsy of COVID-19 patients. Pre-clinical and clinical studies evidence that the inhibition of complement components results in reduced complement deposits on target and non-target tissues, and aid in recovery from the pathological conditions of ARDS. Complement inhibitors (monoclonal antibody, protein, peptide, small molecules, etc.) exhibit great promise in blocking the activity of complement components and its downstream effects under various pathological conditions including SARS-CoV. Therefore, we hypothesize that targeting the potential complement inhibitors and complement cascade to counteract lung inflammation would be a better strategy to treat COVID-19.

Transcription factor AP2A affects sFLT1 expression and decidualization in decidual stromal cells: Implications to preeclampsia pathology.

Preeclampsia (PE) yields a spectrum of phenotypic expression, leading to varying degrees of hypertension, maternal renal dysfunction and placental insufficiency with resultant maternal and neonatal morbidity. Increased sFLT1 expression contributing to angiogenic factor imbalance, placental hypoxia, failed immune adaptation to the fetus and defective decidualization are among the commonly proposed theories of PE pathogenesis. Recently researchers have focused their attention on the events that occur at the maternal fetal interface as potential contributors to PE pathogenesis. Decidual stromal cells (DSC) isolated from preeclamptic women show diminished ability to decidualize upon stimulation and reduced capacity to downregulate sFlt-1 levels. In this study, we sought to gain insight into the molecular mechanism(s) involved in the aberrant decidualization capacity of PE DSC. Our findings using qRT-PCR show that PE DSCs have 6-fold higher basal levels of transcription factor AP2A (TFAP2A) RNA compared to women without PE and that expression of TFAP2A increases during decidualization but only in DSCs of normotensive (NT) women. Silencing of TFAP2A using Trilencer siRNA upregulated sFLT1 expression only in NT-DSCs but suppressed the expression of decidualization markers PRL, IGFBP1 and their regulator FOXO1 in cells from both groups. Collectively, our observations suggest that TFAP2A acts as a repressor of sFLT1 and plays a necessary role in decidualization possibly through interacting with another factor that is aberrantly expressed in PE DSCs.

Transcription factor ID1 is involved in decidualization of stromal cells: Implications in preeclampsia.

Decidual stromal cells (DSC) from women with preeclampsia (PE) show defective decidualization upon in vitro treatment with cAMP. Decidualization is associated with a multitude of gene expression changes and is a prerequisite for embryo implantation. We reason that the process of decidualization involves a cascade of changes in transcriptional regulators. Our prior studies have found defective decidualization of PE-DSCs as reflected by low prolactin (PRL) levels and other decidualization markers. Transcription factor array analysis identified inhibitor of DNA binding (ID1) and FOXO1 as top differentially expressed genes during decidualization. Unlike ID1, FOXO1 involvement in decidualization has been established. We hypothesized that ID1 plays a major role in regulating stromal cell decidualization. Our data shows basal ID1 mRNA expression is significantly higher in PE DSCs. Cyclic AMP-mediated decidualization significantly upregulates ID1 mRNA expression in DSCs and siRNA-mediated knockdown of ID1 significantly interferes with decidualization as shown by a reduction in PRL and FOXO1 expression, and morphologic criteria. Thus ID1 may serve as a master regulator of stromal cell differentiation and defects in ID1 expression may affect decidualization as seen in PE-DSCs.

Effects of Supraphysiologic Levels of Estradiol on Endometrial Decidualization, sFlt1, and HOXA10 Expression.

OBJECTIVE: Supraphysiologic estradiol (E2) levels associated with controlled ovarian hyperstimulation in high in vitro fertilization (IVF) responders may alter implantation and placentation and increase the risk of preeclampsia. Our hypothesis is that elevated E2 levels in vitro significantly alter endometrial decidualization, sFlt1, and HOXA10 expression. METHODS: Human endometrial stromal cells were treated with a decidualization cocktail of medroxyprogesterone, cyclic adenosine monophosphate, and 3 concentrations of E2 10 nM (standard), 100 nM (intermediate), or 1000 nM E2 (high). Effects on sFlt1, prolactin (PRL), insulin-like growth factor binding protein 1 (IGFBP-1), vascular endothelial growth factor (VEGF), and HOXA10 were studied. RESULTS: Prolactin, IGFBP-1, and VEGF significantly increased at all 3 E2 concentrations. While IGFBP-1 and VEGF did not change with increasing E2, PRL was less with high E2 (6.0 ng/mL ± 1.4 standard error of the mean) compared to standard (21.4 ± 3.2) and intermediate (19.8 ± 3.8). sFlt1 decrease was similar at all E2 concentrations. HOXA10 was lower at standard (10%) and intermediate (30%) as expected, but did not change with high E2. CONCLUSIONS: Supraphysiologic E2 levels associated with high IVF responders that exceed in vivo levels may impair in vitro endometrial decidualization. Although PRL did increase with high E2, the levels were, however, attenuated and 3.4-fold lower than standard and intermediate E2. sFlt1 was decreased under all 3 conditions with no differences between concentrations. Reduced HOXA10 was not observed with high E2. These findings suggest that elevated E2 levels in vitro may alter endometrial decidualization and subsequently affect implantation and placentation.

Retinoic Acid Is a Negative Regulator of sFLT1 Expression in Decidual Stromal Cells, and Its Levels Are Reduced in Preeclamptic Decidua.

sFLT1 (soluble VEGF [vascular endothelial growth factor] receptor-1) levels are increased in preeclampsia-a pathological condition of pregnancy. The mechanism of sFLT1 overexpression by gestational tissues, particularly the decidua, remains unknown. Mass spectrometry measurement of the active retinoid metabolite, all-trans retinoic acid (RA), showed significantly lower levels of RA in preeclamptic versus normotensive decidua. In this study, we investigated the involvement of RA in regulating decidual sFLT1 expression. When decidual stromal cells (DSCs) isolated from the decidua basalis of normotensive and preeclampsia placentas were treated with BMS493-a pan-RAR (RA nuclear receptor) antagonist-upregulation of sFLT1 expression was observed. Conversely, treatment with RA resulted in downregulation of sFLT1 in normotensive DSCs and preeclampsia DSCs. Unlike treatment with cAMP, which induces decidualization while downregulating sFLT1, RA treatment did not alter DSC expression of prolactin-a marker of decidualization-or FOXO1 (forkhead box protein 01)-a transcription factor required for prolactin upregulation. TFAP2A (transcription factor AP-2-alpha [activating enhancer-binding protein 2 alpha]), a different transcription factor was upregulated in normotensive DSCs but not in preeclampsia DSCs after RA treatment. Collectively, our data show that RA suppresses sFLT1 expression in DSCs independently of cellular decidualization. These findings suggest that reduced decidual RA levels may contribute to preeclampsia pathogenesis by allowing sFLT1 accumulation at the maternal-fetal interface.

Decidual cells from women with preeclampsia exhibit inadequate decidualization and reduced sFlt1 suppression.

Uterine stromal cell decidualization of maternal tissue is essential for implantation of and local adaptation to the fetal allograft, as well as growth and maintenance of the placenta in healthy pregnancies. Maternal defects in decidualization have been suggested as a possible driver of preeclampsia. Preeclampsia (PE) pregnancies demonstrate shallow implantation, inadequate spiral artery remodeling, and elevated levels of the anti-angiogenic protein, sFlt1. To test whether stromal cells (DSCs) isolated from PE placentas exhibit inadequate re-decidualization and increased expression of sFlt1, DSCs from normotensive (NT-DSCs) and PE (PE-DSCs) placentas were treated for 8 days (D8) with cAMP to induce decidualization and levels of decidualization markers (PRL, IGFBP1, VEGF) and sFlt1 were measured at day 0 (D0), D8, and after reversal of treatment. NT-DSCs achieved statistically significant elevations in PRL and IFGBP1 expression (25.72 [5.78-50.04], p = 0.0008 and 92.09 [1.79-543.10], p = 0.005). PE-DSCs increased PRL and IFGBP1 expression to 6.15 [2.30-10.73] (p = 0.18) and 8.67 [1.64-376.10] (p = 0.04). NT-DSCs reduced sFlt1 expression at D8 to 0.25 [0.17-0.49] (p = 0.0021) compared to 0.31 [0.25-0.82] (p = 0.087) in PE-DSCs. These results show that, when induced to decidualize, PE-DSCs fail to increase expression of decidualization markers to levels achieved by NT-DSCs. sFlt1 expression is higher in PE-DSCs during decidualization, suggesting inadequate suppression during the crucial implantation period. These defects at the maternal fetal interface may lead to the failed spiral artery modification, decreased placental invasion of the uterus, and elevated circulating sFlt1 levels seen in PE pathology.

Villous explants from preeclamptic placentas induce sFlt1 in PBMCs: An ex vivo co-culture study.

OBJECTIVES: Soluble Flt1 (sFlt1) is an anti-angiogenic protein linked to the pathology of preeclampsia (PE). While the placenta serves as the major organ producing sFlt1 during normal pregnancy, peripheral blood mononuclear cells (PBMCs), endothelial cells, and stromal cells also produce sFlt1. The key question is 'what drives the overexpression of sFlt1 observed during PE?' In the present work we show evidence for sFlt1 over-expression in PBMCs due to interaction with placental villi from PE patients. STUDY DESIGN: sFlt1 production by PBMCs is estimated by using two blood collection methods with different coagulation chemistry. PBMCs were then cultured with homologous villous explants and heterologous villous explants to determine the effects of the interaction between the two tissues. MAIN OUTCOME MEASURES: sFlt1 levels were estimated using real time PCR, ELISA, and gel electrophoresis. RESULTS: Plasma samples obtained using CTAD as anti-coagulant showed 16-23% less sFlt1 compared to plasma collected in EDTA. Preeclamptic PBMCs showed higher basal level of sFlt1 mRNA. In addition, we show evidence of placental interaction as a cause of sFlt1 overexpression in PBMCs using homologous and heterologous co-culture system. However, during co-culture, we observed that while the sFlt1 expression in PE PBMCs is increased, PE villous explants show reduced sFlt1 RNA expression. CONCLUSION: sFlt1 was produced in significant amounts by preeclamptic PBMCs, and ex vivo studies show that the placenta induces this over-expression. In contrast, exposure to PBMCs appears to decrease sFlt1 production by preeclamptic placenta.

In vitro evaluation of anticancer properties of exopolysaccharides from Lactobacillus acidophilus in colon cancer cell lines.

The present work aims at studying the effect of exopolysaccharides (EPS) from Lactobacillus acidophilus on the colon cancer cell lines in vitro. Initial analysis showed that EPS has antioxidative properties. EPS was also found to induce cytotoxicity in two colon cancer cell lines, viz. HCT15 and CaCo2 under normoxia and hypoxia. The membrane integrity was also found to be affected in EPS-treated cells. Once the toxic concentration was determined (5 mg/ml), the effect of EPS on the messenger RNA (mRNA) expression of various genes was studied by quantitative real-time (RT)-PCR under both normoxic and hypoxic conditions. The results suggest that EPS downregulated the expression of vascular endothelial growth factor (VEGF) and hypoxia-inducible factor-1α (HIF-1α) and upregulated the expression of tissue inhibitor of metalloproteinases-3 (TIMP-3), hypoxia-inducible factor-2α (HIF-2α), and hemeoxygenase-1 (HO-1). An increase in plasminogen activator inhibitor-1 (PAI-1) was also observed. These results show that EPS may inhibit the expressions of genes involved in tumor angiogenesis and survival. Increase in the expression of HO-1 also shows that EPS have antioxidative properties.

Optimization of anticancer exopolysaccharide production from probiotic Lactobacillus acidophilus by response surface methodology.

Colorectal cancer (CRC) is one of the leading causes of cancer-related deaths in the Western world. Recently, much attention has been focused on decreasing the risk of CRC by consuming probiotics. In the present study, exopolysaccharide (EPS) extracted from Lactobacillus acidophilus was found to inhibit the growth of CaCo2 colon cancer cell line in a dose-dependent manner. The experiment was performed in both normoxic and hypoxic conditions, and EPS was found to reduce the survival of CaCo2 cell line in both the conditions. Quantitative polymerase chain reaction (qPCR) studies demonstrated that EPS treatment upregulated the expression of peroxisome proliferator activator receptor-γ (PPAR-γ) in both normoxia and hypoxia conditions, whereas it upregulated the expression of erythropoietin (EPO) in the normoxic condition, but there was no significant expression under hypoxic conditions. Hence, the EPS production was optimized by Plackett-Burman design followed by central composite rotatory design. The optimized production of EPS at 24 hr was found to be 400 mg/L. During batch cultivation the production peaked at 21 hr, resulting in an EPS concentration of 597 mg/L.

PEG-PHB-glutaminase nanoparticle inhibits cancer cell proliferation in vitro through glutamine deprivation.

In this study, we demonstrate that L-glutaminase, a marine bacterial enzyme with a molecular weight of 37 kDa, inhibits cancer cell proliferation in vitro through glutamine deprivation. The concentration of the enzyme reducing the viability of HeLa cells to 50% was determined to be 12.5 µg/mL; the function of L-glutaminase in controlling cell proliferation was further analysed by BrdU assays. To increase its stability and bioavailability, the enzyme was immobilized on polyethyleneglycol (PEG)-polyhydroxybutyrate (PHB) nanoparticles. A dented anatomy of the HeLa cells was observed under fluorescence and confocal microscopy when they were incubated with L-glutaminase and in glutamine-free medium, as also a 3-fold increase in caspase-3 activity was observed under the same conditions. Blebbed cytoplasm and shrunken nuclei were observed in treated cells under transmission electron microscopy (TEM). Finally, the influence of the enzyme on cell cycle and DNA damage was evaluated using flow cytometry and DNA fragmentation assays. The results confirmed significant damage to the DNA of HeLa cells incubated with L-glutaminase and in glutamine-free medium. These studies attest to the significant role played by L-glutaminase against proliferation in cancer cells through glutamine deprivation.

Biologically synthesized fluorescent CdS NPs encapsulated by PHB.

Here an attempt was made to biologically synthesize fluorescent cadmium sulfide nanoparticles and to immobilize the synthesized nanoparticles in PHB nanoparticles. The present study uses Brevibacterium casei SRKP2 as a potential producer for the green synthesis of CdS nanoparticles. Biologically synthesized nanoparticles were characterized and confirmed using electron microscopy and XRD. The size distribution of the nanoparticles was found to be 10-30 nm followed by which the consequence of time, growth of the organism, pH, concentration of CdCl(2) and Na(2)S on the synthesis of nanoparticles were checked. Enhanced synthesis and fluorescence emission of CdS nanoparticles were achieved at pH 9. The synthesized CdS NPs were immobilized with PHB and were characterized. The fluorescent intensity of the CdS nanoparticles remained unaffected even after immobilization within PHB nanoparticles.

Synthesis of gold and silver nanoparticles using purified URAK.

This study aims at developing a new eco-friendly process for the synthesis of silver nanoparticles (AgNPs) and gold nanoparticles (AuNPs) using purified URAK. URAK is a fibrinolytic enzyme produced by Bacillus cereus NK1. The enzyme was purified and used for the synthesis of AuNPs and AgNPs. The enzyme produced AgNPs when incubated with 1 mM AgNO3 for 24 h and AuNPs when incubated with 1 mM HAuCl4 for 60 h. But when NaOH was added, the synthesis was rapid and occurred within 5 min for AgNPs and 12 h for AuNPs. The synthesized nanoparticles were characterized by a peak at 440 nm and 550 nm in the UV-visible spectrum. TEM analysis showed that AgNPs of the size 60 nm and AuNPs of size 20 nm were synthesized. XRD confirmed the crystalline nature of the nanoparticles and AFM showed the morphology of the nanoparticle to be spherical. FT-IR showed that protein was responsible for the synthesis of the nanoparticles. This process is highly simple, versatile and produces AgNPs and AuNPs in environmental friendly manner. Moreover, the synthesized nanoparticles were found to contain immobilized enzyme. Also, URAK was tested on RAW 264.7 macrophage cell line and was found to be non-cytotoxic until 100 µg/ml.

Biofilm inhibition and antimicrobial action of lipopeptide biosurfactant produced by heavy metal tolerant strain Bacillus cereus NK1.

Biosurfactants are worthful microbial amphiphilic molecules with efficient surface-active and biological properties applicable to several industries and processes. Among them lipopeptides represent a class of microbial surfactants with increasing scientific, therapeutic and biotechnological interests. A heavy metal tolerant Bacillus strain has been isolated and the biofilm inhibition and antimicrobial activity of biosurfactant produced by the strain have been studied. Biosurfactant production was confirmed by the conventional screening methods including hemolytic activity, drop collapsing test, oil displacement test, emulsification and lipase production assays. The biosurfactant produced by this strain was a lipopeptide and exhibited strong surface activity. The biosurfactant has been characterized using FTIR, TLC and HPLC. The minimum active dose of this biosurfactant when compared with the other chemical surfactants was found as 0.150±0.06 µg. The critical micelle concentration was found to be 45 mg/l. The biosurfactant was found to be stable and active over a wide range of pH, temperature and NaCl concentration. It was also able to emulsify a wide range of hydrocarbons and oils thereby extending its application for the bioremediation of oil contaminated sites. The biosurfactant exhibited significant reduction in biofilm formation by pathogens and showed potent antimicrobial activity against various gram positive, gram negative bacteria and fungi. Agar diffusion assay for heavy metal resistance showed that the isolate was resistant to ferrous, lead and zinc. Considering the biofilm inhibition and antimicrobial property of biosurfactant, it can be utilized as a potential therapeutic molecule for numerous microbial infections. The heavy metal resistance of the strain can also be harnessed as an invaluable biological tool for in situ bioremediation.

Mechanism of bactericidal activity of Silver Nitrate: a concentration dependent bi-functional molecule

Silver nitrate imparts different functions on bacteria depending upon its concentration. At lower concentration it induced synthesis of nanoparticles, whereas at higher concentrations it induced cell death. Bacillus licheniformis was used as model system. The MIC was 5 mM, and it induced catalase production, apoptotic body formation and DNA fragmentation.