Sumit Arora

Cellular responses induced by silver nanoparticles: In vitro studies.

A systematic study on the in vitro interactions of 7-20 nm spherical silver nanoparticles (SNP) with HT-1080 and A431 cells was undertaken as a part of an on-going program in our laboratory to develop a topical antimicrobial agent for the treatment of burn wound infections. Upon exposure to SNP (up to 6.25 microg/mL), morphology of both the cell types remained unaltered. However, at higher concentrations (6.25-50 microg/mL) cells became less polyhedral, more fusiform, shrunken and rounded. IC(50) values for HT-1080 and A431 as revealed by XTT assay were 10.6 and 11.6 microg/mL, respectively. When the cells were challenged with approximately 1/2 IC(50) concentration of SNP (6.25 microg/mL), clear signs of oxidative stress, i.e. decreased GSH ( approximately 2.5-folds in HT-1080, approximately 2-folds in A431) and SOD ( approximately 1.6-folds in HT-1080, 3-folds in A431) as well as increased lipid peroxidation ( approximately 2.5-folds in HT-1080, approximately 2-folds in A431) were seen. Changes in the levels of catalase and GPx in A431 cells were statistically insignificant in both cell types. DNA fragmentation in SNP-exposed cells suggested apoptosis. When the apoptotic thresholds of SNP were monitored with caspase-3 assay the concentrations required for the onset of apoptosis were found to be much lower (0.78 microg/mL in HT-1080, 1.56 microg/mL in A431) than the necrotic concentration (12.5 microg/mL in both cell types). These results can be used to define a safe range of SNP for the intended application as a topical antimicrobial agent after appropriate in vivo studies.

Superparamagnetic iron oxide nanoparticles: magnetic nanoplatforms as drug carriers.

A targeted drug delivery system is the need of the hour. Guiding magnetic iron oxide nanoparticles with the help of an external magnetic field to its target is the principle behind the development of superparamagnetic iron oxide nanoparticles (SPIONs) as novel drug delivery vehicles. SPIONs are small synthetic γ-Fe2O3 (maghemite) or Fe3O4 (magnetite) particles with a core ranging between 10 nm and 100 nm in diameter. These magnetic particles are coated with certain biocompatible polymers, such as dextran or polyethylene glycol, which provide chemical handles for the conjugation of therapeutic agents and also improve their blood distribution profile. The current research on SPIONs is opening up wide horizons for their use as diagnostic agents in magnetic resonance imaging as well as for drug delivery vehicles. Delivery of anticancer drugs by coupling with functionalized SPIONs to their targeted site is one of the most pursued areas of research in the development of cancer treatment strategies. SPIONs have also demonstrated their efficiency as nonviral gene vectors that facilitate the introduction of plasmids into the nucleus at rates multifold those of routinely available standard technologies. SPION-induced hyperthermia has also been utilized for localized killing of cancerous cells. Despite their potential biomedical application, alteration in gene expression profiles, disturbance in iron homeostasis, oxidative stress, and altered cellular responses are some SPION-related toxicological aspects which require due consideration. This review provides a comprehensive understanding of SPIONs with regard to their method of preparation, their utility as drug delivery vehicles, and some concerns which need to be resolved before they can be moved from bench top to bedside.

Silver nanoparticles in therapeutics: development of an antimicrobial gel formulation for topical use.

Silver is an effective antimicrobial agent with low toxicity, which is important especially in the treatment of burn wounds where transient bacteremia is prevalent and its fast control is essential. Drugs releasing silver in ionic forms are known to get neutralized in biological fluids and upon long-term use may cause cosmetic abnormality, e.g., argyria and delayed wound healing. Given its broad spectrum activity, efficacy and lower costs, the search for newer and superior silver based antimicrobial agents is necessary. Among the various options available, silver nanoparticles have been the focus of increasing interest and are being heralded as an excellent candidate for therapeutic purposes. This report gives an account of our work on development of an antimicrobial gel formulation containing silver nanoparticles (SNP) in the size range of 7-20 nm synthesized by a proprietary biostabilization process. The typical minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) against standard reference cultures as well as multidrug-resistant organisms were 0.78-6.25 microg/mL and 12.5 microg/mL, respectively. Gram-negative bacteria were killed more effectively (3 log(10) decrease in 5-9 h) than Gram-positive bacteria (3 log(10) decrease in 12 h). SNP also exhibited good antifungal activity (50% inhibition at 75 microg/mL with antifungal index 55.5% against Aspergillus niger and MIC of 25 microg/mL against Candida albicans). When the interaction of SNP with commonly used antibiotics was investigated, the observed effects were synergistic (ceftazidime), additive (streptomycin, kanamycin, ampiclox, polymyxin B) and antagonistic (chloramphenicol). Interestingly, SNP exhibited good anti-inflammatory properties as indicated by concentration-dependent inhibition of marker enzymes (matrix metalloproteinase 2 and 9). The post agent effect (a parameter measuring the length of time for which bacterial growth remains suppressed following brief exposure to the antimicrobial agent) varied with the type of organism (e.g., 10.5 h for P. aeruginosa, 1.3 h for Staphylococcus sp. and 1.6 h for Candida albicans) indicating that dose regimen of the SNP formulation should ensure sustained release of the drug. To meet this requirement, a gel formulation containing SNP (S-gel) was prepared. The antibacterial spectrum of S-gel was found to be comparable to that of a commercial formulation of silver sulfadiazine, albeit at a 30-fold less silver concentration. As part of toxicity studies, localization of SNP in Hep G2 cell line, cell viability, biochemical effects and apoptotic/necrotic potential were assessed. It was found that SNP get localized in the mitochondria and have an IC(50) value of 251 microg/mL. Even though they elicit an oxidative stress, cellular antioxidant systems (reduced glutathione content, superoxide dismutase, catalase) get triggered and prevent oxidative damage. Further, SNP induce apoptosis at concentrations up to 250 microg/mL, which could favor scarless wound healing. Acute dermal toxicity studies on SNP gel formulation (S-gel) in Sprague-Dawley rats showed complete safety for topical application. These results clearly indicate that silver nanoparticles could provide a safer alternative to conventional antimicrobial agents in the form of a topical antimicrobial formulation.

Nanotoxicology and in vitro studies: The need of the hour

Nanotechnology is considered as one of the key technologies of the 21st century and promises revolution in our world. Objects at nano scale, take on novel properties and functions that differ markedly from those seen in the corresponding bulk counterpart primarily because of their small size and large surface area. Studies have revealed that the same properties that make nanoparticles so unique could also be responsible for their potential toxicity. Nanotechnology is rapidly advancing, with more than 1000 nanoproducts already on the market. Considering the fact that intended as well as unintended exposure to nanomaterials is increasing and presently no clear regulatory guideline(s) on the testing/evaluation of nanoparticulate materials are available, the in vitro toxicological studies become extremely relevant and important. This review presents a summary of nanotoxicology and a concise account of the in vitro toxicity data on nanomaterials. For nanomaterials to move into the applications arena, it is important that nanotoxicology research uncovers and understands how these multiple factors influence their toxicity so that the ensuing undesirable effects can be avoided.

Interactions of silver nanoparticles with primary mouse fibroblasts and liver cells

Primary cells are ideal for in vitro toxicity studies since they closely resemble tissue environment. Here, we report a detailed study on the in vitro interactions of 7-20 nm spherical silver nanoparticles (SNP) with primary fibroblasts and primary liver cells isolated from Swiss albino mice. The intended use of silver nanoparticles is in the form of a topical antimicrobial gel formulation for the treatment of burns and wounds. Upon exposure to SNP for 24 h, morphology of primary fibroblasts and primary liver cells remained unaltered up to 25 microg/mL and 100 microg/mL SNP, respectively, although with minor decrease in confluence. IC(50) values for primary fibroblasts and primary liver cells as revealed by XTT assay were 61 microg/mL and 449 microg/mL, respectively. Ultra-thin sections of primary cells exposed to 1/2 IC(50) SNP for 24 h, visualized under Transmission electron microscope showed the presence of dark, electron dense, spherical aggregates inside the mitochondria, and cytoplasm, probably representing the intracellular SNP. When the cells were challenged with approximately 1/2 IC(50) concentration of SNP (i.e. 30 microg/mL and 225 microg/mL for primary fibroblasts and primary liver cells, respectively), enhancement of GSH (approximately 1.2 fold) and depletion of lipid peroxidation (approximately 1.4 fold) were seen in primary fibroblasts which probably protect the cells from functional damage. In case of primary liver cells; increased levels of SOD ( approximately 1.4 fold) and GSH ( approximately 1.1 fold) as compared to unexposed cells were observed. Caspase-3 activity assay indicated that the SNP concentrations required for the onset of apoptosis were found to be much lower (3.12 microg/mL in primary fibroblasts, 12.5 microg/mL in primary liver cells) than the necrotic concentration (100 microg/mL in primary fibroblasts, 500 microg/mL in primary liver cells). These observations were confirmed by CLSM studies by exposure of cells to 1/2 IC(50) SNP (resulting in apoptosis) and 2 x IC(50)) cells (resulting in necrosis). These results clearly suggest that although silver nanoparticles seem to enter the eukaryotic cells, cellular antioxidant mechanisms protect the cells from possible oxidative damage. This property, in conjunction with the finding that primary cells possess much higher SNP tolerance than the concentration in the gel (approximately 20 microg/g), indicates preliminary safety of the formulation and warrants further study for possible human application.

Honokiol Arrests Cell Cycle, Induces Apoptosis, and Potentiates the Cytotoxic Effect of Gemcitabine in Human Pancreatic Cancer Cells

Survival rates for patients with pancreatic cancer are extremely poor due to its asymptomatic progression to advanced and metastatic stage for which current therapies remain largely ineffective. Therefore, novel therapeutic agents and treatment approaches are desired to improve the clinical outcome. In this study, we determined the effects of honokiol, a biologically active constituent of oriental medicinal herb Magnolia officinalis/grandiflora, on two pancreatic cancer cell lines, MiaPaCa and Panc1, alone and in combination with the standard chemotherapeutic drug, gemcitabine. Honokiol exerted growth inhibitory effects on both the pancreatic cancer cell lines by causing cell cycle arrest at G1 phase and induction of apoptosis. At the molecular level, honokiol markedly decreased the expression of cyclins (D1 and E) and cyclin-dependent kinases (Cdk2 and Cdk4), and caused an increase in Cdk inhibitors, p21 and p27. Furthermore, honokiol treatment led to augmentation of Bax/Bcl-2 and Bax/Bcl-xL ratios to favor apoptosis in pancreatic cancer cells. These changes were accompanied by enhanced cytoplasmic accumulation of NF-κB with a concomitant decrease in nuclear fraction and reduced transcriptional activity of NF-κB responsive promoter. This was associated with decreased phosphorylation of inhibitor of kappa B alpha (IκB-α) causing its stabilization and thus increased cellular levels. Importantly, honokiol also potentiated the cytotoxic effects of gemcitabine, in part, by restricting the gemcitabine-induced nuclear accumulation of NF-κB in the treated pancreatic cancer cell lines. Altogether, these findings demonstrate, for the first time, the growth inhibitory effects of honokiol in pancreatic cancer and indicate its potential usefulness as a novel natural agent in prevention and therapy.

miRNA-708 Control of CD44 + Prostate Cancer–Initiating Cells

Tumor recurrence in prostate cancer has been attributed to the presence of CD44-expressing tumor-initiating cells. In this study, we report that miR-708 is a key negative regulator of this CD44(+) subpopulation of prostate cancer cells, with important implications for diagnosis and prognosis of this disease. miR-708 was underexpressed in CD44(+) cells from prostate cancer xenografts. Reconstitution of miR-708 in prostate cancer cell lines or CD44(+) prostate cancer cells led to decreased tumorigenicity in vitro. Intratumoral delivery of synthetic miR-708 oligonucleotides triggered regression of established tumors in a murine xenograft model of human prostate cancer. Conversely, miR-708 silencing in a purified CD44(-) population of prostate cancer cells promoted tumor growth. Functional studies validated CD44 to be a direct target of miR-708 and also identified the serine/threonine kinase AKT2 as an additional target. Clinically, low miR-708 expression was associated significantly with poor survival outcome, tumor progression, and recurrence in patients with prostate cancer. Together, our findings suggest that reduced miR-708 expression leads to prostate cancer initiation, progression, and development by regulating the expression of CD44 as well as AKT2. miR-708 therefore may represent a novel therapeutic target or diagnostic and prognostic biomarker in prostate cancer.

CXCL12/CXCR4 Protein Signaling Axis Induces Sonic Hedgehog Expression in Pancreatic Cancer Cells via Extracellular Regulated Kinase- and Akt Kinase-mediated Activation of Nuclear Factor κB IMPLICATIONS FOR BIDIRECTIONAL TUMOR-STROMAL INTERACTIONS

Background: CXCL12/CXCR4 and hedgehog pathways, predominantly acting in paracrine fashion, play important roles in pancreatic cancer pathobiology. Results: CXCL12/CXCR4 signaling regulates the expression of hedgehog ligand, the sonic hedgehog, in pancreatic cancer cells. Conclusion: Our findings indicate a novel molecular link between CXCL12/CXCR4 and hedgehog pathways. Significance: Our data provide a molecular basis for an active bidirectional tumor-stromal interaction in pancreatic cancer. Recent evidence suggests a major role of tumor-stromal interactions in pancreatic cancer pathobiology. The chemokine CXCL12 (stromal cell-derived factor 1 (SDF-1)), abundantly produced by stromal cells, promotes progression, metastasis, and chemoresistance of pancreatic cancer cells. On the other hand, pancreatic tumor cell-derived sonic hedgehog (SHH) acts predominantly on stromal cells to induce desmoplasia and, thus, has a paracrine effect on tumorigenesis and therapeutic outcome. In this study, we examined the association between these two proteins of pathological significance in pancreatic cancer. Our data demonstrate that CXCL12 leads to a dose- and time-dependent up-regulation of SHH in pancreatic cancer cells. CXCL12-induced SHH up-regulation is specifically mediated through the receptor CXCR4 and is dependent on the activation of downstream Akt and ERK signaling pathways. Both Akt and ERK cooperatively promote nuclear accumulation of NF-κB by inducing the phosphorylation and destabilization of its inhibitory protein, IκB-α. Using dominant negative IκB-α, a SHH promoter (deletion mutant) reporter, and chromatin immunoprecipitation assays, we demonstrate that CXCL12 exposure enhances direct binding of NF-κB to the SHH promoter and that suppression of NF-κB activation abrogates CXCL12-induced SHH expression. Finally, our data demonstrate a strong correlative expression of CXCR4 and SHH in human pancreatic cancer tissues, whereas their expression is not observed in the normal pancreas. Altogether, our data reveal a novel mechanism underlying aberrant SHH expression in pancreatic cancer and identify a molecular link facilitating bidirectional tumor-stromal interactions.

An Undesired Effect of Chemotherapy GEMCITABINE PROMOTES PANCREATIC CANCER CELL INVASIVENESS THROUGH REACTIVE OXYGEN SPECIES-DEPENDENT, NUCLEAR FACTOR κB- AND HYPOXIA-INDUCIBLE FACTOR 1α-MEDIATED UP-REGULATION OF CXCR4

Background: CXCR4 signaling protects pancreatic cancer cells from gemcitabine toxicity. However, the effect of gemcitabine on this resistance mechanism is unclear. Results: Gemcitabine up-regulates CXCR4 expression in pancreatic cancer cells and promotes their invasiveness. Conclusion: CXCR4 signaling serves as a counterdefense mechanism against gemcitabine. Significance: These findings are significant for the formulation of effective therapeutic strategies against pancreatic cancer. Recently, we have shown that CXCL12/CXCR4 signaling plays an important role in gemcitabine resistance of pancreatic cancer (PC) cells. Here, we explored the effect of gemcitabine on this resistance mechanism. Our data demonstrate that gemcitabine induces CXCR4 expression in two PC cell lines (MiaPaCa and Colo357) in a dose- and time-dependent manner. Gemcitabine-induced CXCR4 expression is dependent on reactive oxygen species (ROS) generation because it is abrogated by pretreatment of PC cells with the free radical scavenger N-acetyl-L-cysteine. CXCR4 up-regulation by gemcitabine correlates with time-dependent accumulation of NF-κB and HIF-1α in the nucleus. Enhanced binding of NF-κB and HIF-1α to the CXCR4 promoter is observed in gemcitabine-treated PC cells, whereas their silencing by RNA interference causes suppression of gemcitabine-induced CXCR4 expression. ROS induction upon gemcitabine treatment precedes the nuclear accumulation of NF-κB and HIF-1α, and suppression of ROS diminishes these effects. The effect of ROS on NF-κB and HIF-1α is mediated through activation of ERK1/2 and Akt, and their pharmacological inhibition also suppresses gemcitabine-induced CXCR4 up-regulation. Interestingly, our data demonstrate that nuclear accumulation of NF-κB results from phosphorylation-induced degradation of IκBα, whereas HIF-1α up-regulation is NF-κB-dependent. Lastly, our data demonstrate that gemcitabine-treated PC cells are more motile and exhibit significantly greater invasiveness against a CXCL12 gradient. Together, these findings reinforce the role of CXCL12/CXCR4 signaling in gemcitabine resistance and point toward an unintended and undesired effect of chemotherapy.

Protein and polymer immobilized La0.7Sr0.3MnO3 nanoparticles for possible biomedical applications

La0.7Sr0.3MnO3 (LSMO) is a mixed-valent room temperature ferromagnet with properties that are attractive for their applicability in biomedicine. We report, for the first time, immobilization of commonly used biocompatible molecules on LSMO nanoparticles, namely bovine serum albumin and dextran. The former was conjugated to LSMO using 1-ethyl-3-(3-dimethyl aminopropyl)-carbodiimide (CDI) as a coupling agent while the latter was used without any coupler. These bioconjugated nanoparticles exhibit several properties that suggest their applicability in the field of biomedicine, namely (a) no changes in the Curie temperature at ~360 K after conjugation with biomolecules, (b) rapid attainment of the desired temperature (48 °C) at low concentration (e.g. fluidized dextran-coated system at 80 µg ml−1) upon exposure to 20 MHz radio-frequency, (c) extremely low cytotoxicity in skin carcinoma, human fibrosarcoma and neuroblastoma cell lines and (d) high stability of the LSMO system with negligible leaching of ionic manganese into the delivery medium, indicating their safety in possible human applications.