Engineering alginate/carboxymethylcellulose scaffolds to establish liver cancer spheroids: Evaluation of molecular variances between 2D and 3D models.

Natural polymeric hydrogels represent an optimal framework for 3D culture development. This study demonstrates a freeze-thaw-based ionic crosslinking technique for fabricating alginate/carboxymethylcellulose scaffold for culturing human hepatocellular carcinoma, Huh-7 cells to generate 3D spheroids. Consolidating morphological and biomechanical characterization of Alg/CMC scaffolds shows the formation of uniform hydrogels with significant crosslinking (ATR-FTIR), multiscale pores (FE-SEM), swelling/water absorbance, softer texture, viscoelasticity (rheology), spreading nature (contact angle), and degradation rate optimal for 3D culture establishment. The influence of cell seeding density and time with spheroid formation reveals a maximal size of 250-300 µm on day 7. Calcein AM and Propidium iodide staining confirm that a culmination of viable and dead cells generates spheroidal heterogeneity. RT-qPCR in 3D culture against RPL-13 and 2D culture controls indicate an upregulation of E-cadherin, N-cadherin, fibronectin, and integrin α9/ß6. Further, western blotting and immunofluorescence confirm the collective display of cellular interactions in 3D spheroids. Thus, the expression profile signifies the role of key genes during the assembly and formation of 3D spheroids in 1%Alg/1%CMC scaffolds with a profound epithelial characteristic. In the future, this study will bring a 3D spheroid model in a platter for elucidating epithelial to mesenchymal transition of cells during in vitro disease modeling.

Investigation of the hydration process and biological activity of a novel nanosilver incorporated dicalcium silicate based retrograde filling material.

Background: Although several materials have been used for retrograde filling following apical surgeries, there is no consensus on a single best material. Tricalcium silicate-based types of cement have been developed as root-end filling materials mainly due to tricalcium silicate's hydraulic properties. However, its unfavorable setting characteristics and minimal antimicrobial properties have necessitated the introduction of new additives into the existing commercially available materials. To design an affordable product based on a dicalcium silicate with a shorter set time, minimal cytotoxic complications, and enhanced antibacterial activity, we developed a new endodontic cement from pure raw materials, intending to satisfy the prerequisites of ideal retrograde material. Methods: The composition of the experimental calcium silicate-based cement included the addition of calcium chloride and silver nanoparticles in varying concentrations. Structural characterization was carried out using energy dispersive analysis by X-rays using scanning electron microscope (EDAX SEM) and hydration characteristics were performed using an X-ray diffractometer (XRD). The experimental material was further evaluated for biocompatibility using MTT ([3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide)assay and antibacterial activity was evaluated using an agar diffusion test against Enterococcus faecalis. Results: The structural characterization and hydration characteristics revealed that the experimental cement was dicalcium silicate based with favorable biocompatibility and enhanced antibacterial activity. Tricalcium silicate based mineral trioxide aggregate (MTA) also had favourable biocompatibility, however, its antibacterial activity was significantly decreased when compared to the novel cement. Conclusion: All hydraulic cements that are available in the dental market are predominantly tricalcium silicate-based materials. There has been no evidence in the literature to date wherein it has been explored whether a dicalcium silicate-based hydraulic cement can solely be used in root-end cavities. The findings of the study revealed a dicalcium silicate based retrograde filling material with favourable biocompatibility exhibited immediately as well as in the set samples. Incorporation of silver nanoparticles boosted the antibacterial activity when compared to that of ProRoot MTA. This material could potentially reinstate the usual hype created with tricalcium silicate types of cement since dicalcium silicate cements also exhibit similar properties.

Effect of shape of titanium dioxide nanofillers on the properties of dental composites.

The main objective of the present study was to evaluate the effect of the morphology of titanium dioxide nanofillers on the flexural strength and shear bond strength of the dental composite. Spherical and rhombic-shaped nano titanium dioxide fillers were synthesized via solvothermal method and were characterized. Subsequently, these fillers were incorporated into a flowable composite (Filtek™ Z350 XT Flowable Restorative) at 0.5 wt.% and 1.5 wt.% and the prepared specimens were stored in water for 24 h. The specimens were then evaluated for flexural strength using a universal testing machine. Similarly, the shear bond strength of modified composites to the tooth was evaluated and bond failures were analyzed using stereomicroscope magnification. Incorporation of nanofillers significantly enhanced the flexural strength of flowable composite (p = 0.009) with a significant increase at 0.5wt.% of spherical (p = 0.015) and rhomboidal-shaped fillers (p = 0.010). However, no statistically significant difference in flexural strength was observed among the different shapes of nanofillers. The results of our study did not show a significant effect on the shear bond strength of the composites. Thus the reinforcing ability of titanium dioxide nanofillers on dental composite was confirmed in this study, although the effect of using nanofillers with different morphology was not significant.

Systematic toxicity assessment of CdTe quantum dots in Drosophila melanogaster.

The risk assessment of cadmium (Cd)-based quantum dots (QDs) used for biomedical nanotechnology applications has stern toxicity concerns. Despite cytotoxicity studies of cadmium telluride (CdTe) QDs, the systematic in vivo study focusing on its organismal effects are more relevant to public health. Therefore, the present study aims to investigate the effect of chemically synthesized 3-mercapto propionic acid-functionalized CdTe QDs on organisms' survival, development, reproduction, and behaviour using Drosophila melanogaster as a model. The sub-cellular impact on the larval gut was also evaluated. First/third instar larvae or the adult Drosophila were exposed orally to green fluorescence emitting CdTe QDs (0.2-100 µM), and organisms' longevity, emergence, reproductive performance, locomotion, and reactive oxygen species (ROS), and cell death were assessed. Uptake of semiconductor CdTe QDs was observed as green fluorescence in the gut. A significant decline in percentage survivability up to 80% was evident at high CdTe QDs concentrations (25 and 100 µM). The developmental toxicity was marked by delayed and reduced fly emergence after CdTe exposure. The teratogenic effect was evident with significant wing deformities at 25 and 100 µM concentrations. However, at the reproductive level, adult flies' fecundity, fertility, and hatchability were highly affected even at low concentrations (1 µM). Surprisingly, the climbing ability of Drosophila was unaffected at any of the used CdTe QDs concentrations. In addition to organismal toxicity, the ROS level and cell death were elevated in gut cells, confirming the sub-cellular toxicity of CdTe QDs. Furthermore, we observed a significant rescue in CdTe QDs-associated developmental, reproductive, and survival adversities when organisms were co-exposed with N-acetyl-cysteine (NAC, an antioxidant) and CdTe QDs. Overall, our findings indicate that the environmental release of aqueously dispersible CdTe QDs raises a long-lasting health concern on the development, reproduction, and survivability of an organism.

Identification and evaluation of an appropriate housekeeping gene for real time gene profiling of hepatocellular carcinoma cells cultured in three dimensional scaffold.

BACKGROUND: Assessing an optimal reference gene as an internal control for target gene normalization is important during quantitative real time polymerase chain reaction (RT-qPCR) of three dimensional (3D) cell culture. Especially, gene profiling of cancer cells under a complex 3D microenvironment in a polymer scaffold provides a deeper understanding of tumor functioning in vivo. METHODS AND RESULTS: Expression of six housekeeping genes (HKG's): Glyceraldehyde-3-phosphodehydrogenase (GAPDH), ß-actin (ACTB), beta-2-microglobulin (B2M), 18S ribosomal RNA (18S rRNA), peptidyl-propyl-isomerase A (PPIA), and ribosomal protein L13 (RPL-13) during two dimensional (2D) culture, and alginate-carboxymethylcellulose scaffold based 3D culture conditioned up to 21 days was analysed for hepatocellular carcinoma (Huh-7) cells. The gene expression studies were performed by determining primer efficiency, melting curve and threshold cycle analysis. Further, RT-qPCR data was validated statistically using geNorm and NormFinder softwares. The study indicated RPL-13, 18S rRNA and B2M to be stable among selected referral HKG candidates. CONCLUSION: An exploration of a reliable HKG is necessary for normalization of gene expression in RT-qPCR during varying cell culture conditions.

Fabrication techniques of biomimetic scaffolds in three-dimensional cell culture: A review.

In the last four decades, several researchers worldwide have routinely and meticulously exercised cell culture experiments in two-dimensional (2D) platforms. Using traditionally existing 2D models, the therapeutic efficacy of drugs has been inappropriately validated due to the failure in generating the precise therapeutic response. Fortunately, a 3D model addresses the foregoing limitations by recapitulating the in vivo environment. In this context, one has to contemplate the design of an appropriate scaffold for favoring the organization of cell microenvironment. Instituting pertinent model on the platter will pave way for a precise mimicking of in vivo conditions. It is because animal cells in scaffolds oblige spontaneous formation of 3D colonies that molecularly, phenotypically, and histologically resemble the native environment. The 3D culture provides insight into the biochemical aspects of cell-cell communication, plasticity, cell division, cytoskeletal reorganization, signaling mechanisms, differentiation, and cell death. Focusing on these criteria, this paper discusses in detail, the diversification of polymeric scaffolds based on their available resources. The paper also reviews the well-founded and latest techniques of scaffold fabrication, and their applications pertaining to tissue engineering, drug screening, and tumor model development.

Antihepatoma activity of multifunctional polymeric nanoparticles via inhibition of microtubules and tyrosine kinases.

Aim: Synthesis of poly-L-lactic acid nanoparticles comprising of microtubule-inhibitor docetaxel and tyrosine kinase inhibitor sorafenib (PLDS NPs) for hepatoma treatment. Materials & methods: PLDS NPs were prepared by the emulsion solvent evaporation method and the anticancer activity was evaluated in Huh7 hepatoma cells. Results: Real-time imaging of quantum dots incorporating poly-L-lactic acid nanoparticles showed a rapid internalization of the nanoparticles in Huh7 cells. PLDS NPs exerted stronger antiproliferative, apoptotic and antiangiogenic effects than free single drug counterparts. They strongly promoted microtubule bundling, multinucleation and increased mitotic index in Huh7 cells. They also inhibited the expression of pERK1/2, pAKT and cyclin D1. Conclusion: We developed a single-nanoscale platform for dual drug delivery and high-sensitivity quantum dots imaging for hepatoma treatment. [Formula: see text].

Mechanism of apoptosis induction in human breast cancer MCF-7 cell by Ruviprase, a small peptide from Daboia russelii russelii venom.

Ruviprase, a 4.4 kDa peptide isolated from Daboia russelii russelii venom demonstrated antiproliferative activity against EMT6/AR1, U-87MG, HeLa and MCF-7 cancer cells with an IC50 value of 23.0, 8.8, 5.8 and 4.0 µg ml(-1), respectively. However, it was nontoxic to non-cancerous human embryonic kidney cell and human peripheral blood lymphocytes. Flow-cytometric analysis confirmed the apoptosis induction in MCF-7 cells by Ruviprase where it induced DNA condensation but did not cause mitotic blockage or chromosomal aberration in treated-cells. Immunofluorescence microscopic analysis indicated Ruviprase induced apoptosis in MCF-7 cells through p53 and p21-mediated pathways. Ruviprase generated reactive oxygen species (ROS), altered the mitochondrial transmembrane potential, and significantly decreased the cellular glutathione (GSH) content of MCF-7 cells. Immunoblotting and quantitative real-time PCR (qRT-PCR) analyses suggested that Ruviprase down-regulated the expression of anti-apoptotic protein Bcl-2, increased cleavage of poly (ADP-ribose) polymerase (PARP) protein, and up-regulated the expression of pro-apoptotic protein Bax, as well as executer protein caspase-7 to induced apoptosis in MCF-7 cells via intrinsic pathway. This is the first report on the characterization of the anticancer potential of a small, non-toxic and anticoagulant peptide purified from Russell's viper venom.

Intracellular interactions of electrostatically mediated layer-by-layer assembled polyelectrolytes based sorafenib nanoparticles in oral cancer cells.

In this paper, we report the preparation of LbL-nanoSraf (100-300nm) comprising of layer-by-layer (LbL) assembled polyelectrolytes dextran-sulfate/poly-l-arginine, with a multikinase inhibitor sorafenib (Sraf) encapsulated calcium carbonate (CaCO3) nanoparticles for oral cancer therapy in vitro. The zeta potential of LbL-nanoSraf exhibited a negative charge of the polyanionic dextran sulfate, which alternated with a positive charge of polycationic poly-l-arginine indicating a successful LbL assembly of the two polyelectrolyte bilayers on the CaCO3 nanoparticles. The LbL-nanoSraf exhibited an encapsulation efficiency of 61±4%. The LbL-nanoSraf was characterized using field-emission gun scanning electron microscopy, X-ray powder diffraction, atomic force microscopy and confocal laser scanning microscopy. Confocal laser scanning microscopy, flow cytometry and transmission electron microscopic investigations showed the internalization of LbL-nanoSraf in human oral cancer (KB) cells. The LbL-nanoSraf exhibited more potent antiproliferative, apoptotic and antimigratory activities in KB cells than the free drug Sraf. The findings could promote the application of nano-sized LbL assembled polyelectrolytes for the delivery of Raf-kinase inhibitors and provide mechanistic insights for oral cancer therapy.

A Chimeric Cetuximab-Functionalized Corona as a Potent Delivery System for Microtubule-Destabilizing Nanocomplexes to Hepatocellular Carcinoma Cells: A Focus on EGFR and Tubulin Intracellular Dynamics.

In this study, we have developed microtubule destabilizing agents combretastatin A4 (CA4) or 2-methoxyestradiol (2ME) encapsulated poly(d,l-lactide-co-glycolide)-b-poly(ethylene glycol) (PLGA-b-PEG) nanocomplexes for targeted delivery to human hepatocellular carcinoma (HCC) cells. An epidermal growth factor receptor (EGFR) is known to be overexpressed in HCC cells. Therefore, the targeting moiety cetuximab (Cet), an anti-EGFR chimeric monoclonal antibody, is functionalized on the surface of these diblock copolymeric coronas. Cetuximab is associated with the extracellular domain of the EGFR; therefore, the uptake of the cetuximab conjugated nanocomplexes occurred efficiently in EGFR overexpressing HCC cells indicating potent internalization of the complex. The cetuximab targeted-PLGA-b-PEG nanocomplexes encapsulating CA4 or 2ME strongly inhibited phospho-EGFR expression, depolymerized microtubules, produced spindle abnormalities, stalled mitosis, and induced apoptosis in Huh7 cells compared to the free drugs, CA4 or 2ME. Further, the combinatorial strategy of targeted nanocomplexes, Cet-PLGA-b-PEG-CA4 NP and Cet-PLGA-b-PEG-2ME NP, significantly reduced the migration of Huh7 cells, and markedly enhanced the anticancer effects of the microtubule-targeted drugs in Huh7 cells compared to the free drugs, CA4 or 2ME. The results indicated that EGFR receptor-mediated internalization via cetuximab facilitated enhanced uptake of the nanocomplexes leading to potent anticancer efficacy in Huh7 cells. Cetuximab-functionalized PLGA-b-PEG nanocomplexes possess a strong potential for the targeted delivery of CA4 or 2ME in EGFR overexpressed HCC cells, and the strategy may be useful for selectively targeting microtubules in these cells.

Mechanism of Anti-Cancer Activity of Benomyl Loaded Nanoparticles in Multidrug Resistant Cancer Cells.

Polymeric chitosan-poly(D,L-lactide-co-glycolide) nanoparticles loaded with benomyl as anticancer drug formulation against multidrug-resistant EMT6/AR1 cells were synthesized by amine-carboxylate reaction. Using transmission electron microscopy, the average size of chitosan-poly(D,L-lactide-co-glycolide) nanoparticles and benomyl-encapsulated polymeric chitosan-poly(D,L-lactide-co-glycolide) nanoparticles was estimated to be 155 ± 20 nm and 160 ± 25 nm, respectively. Fourier transform infrared spectroscopy revealed that poly(D,L-lactide-co-glycolide) and chitosan are linked by covalent bonds. Zeta potentials of benomyl-encapsulated polymeric chitosan-poly(D,L-lactide-co-glycolide) nanoparticles at pH 4, 7.2, and 10 were 30 ± 1.8, 19 ± 0.65, and -22 ± 0.15 mV, respectively, indicating the formation of stable, hydrophilic nanoparticles. The release of benomyl from benomyl-encapsulated polymeric chitosan-poly(D,L-lactide-co-glycolide) nanoparticles followed pH-dependent kinetics. The uptake of fluorescein isothiocyanate-labeled chitosan-poly(D,L-lactide-co-glycolide) nanoparticles was concentration-dependent in both MCF-7 and multidrug-resistant EMT6/AR1 cells. EMT6/AR1 cells showed 10-fold higher resistance to benomyl compared to MCF-7 cells; in contrast, benomyl-encapsulated polymeric chitosan-poly(D,L-lactide-co-glycolide) nanoparticles effectively inhibited proliferation of MCF-7 and EMT6/AR1 cells with a half-maximal inhibitory concentration of 4 ± 0.5 and 9 ± 0.5 pM, respectively. In the presence of a P-glycoprotein inhibitor, the activity of benomyl was increased, suggesting that benomyl is a substrate for P-glycoprotein. Further, benomyl-encapsulated polymeric chitosan-poly(D,L-lactide-co-glycolide) nanoparticles depoly-merized microtubules both in interphase and mitosis. It blocked cell cycle progression at G2/M and induced apoptosis in EMT6/AR1 cells, suggesting that benomyl-encapsulated polymeric chitosan-poly(D,L-lactide-co-glycolide) nanoparticles have chemotherapeutic activity against multidrug-resistant cancer cells.

CXI-benzo-84 reversibly binds to tubulin at colchicine site and induces apoptosis in cancer cells.

Here, we have discovered CXI-benzo-84 as a potential anticancer agent from a library of benzimidazole derivatives using cell based screening strategy. CXI-benzo-84 inhibited cell cycle progression in metaphase stage of mitosis and accumulated spindle assembly checkpoint proteins Mad2 and BubR1 on kinetochores, which subsequently activated apoptotic cell death in cancer cells. CXI-benzo-84 depolymerized both interphase and mitotic microtubules, perturbed EB1 binding to microtubules and inhibited the assembly and GTPase activity of tubulin in vitro. CXI-benzo-84 bound to tubulin at a single binding site with a dissociation constant of 1.2±0.2µM. Competition experiments and molecular docking suggested that CXI-benzo-84 binds to tubulin at the colchicine-site. Further, computational analysis provided a significant insight on the binding site of CXI-benzo-84 on tubulin. In addition to its potential use in cancer chemotherapy, CXI-benzo-84 may also be useful to screen colchicine-site agents and to understand the colchicine binding site on tubulin.