Carbon nanotubes conjugated with cisplatin activate different apoptosis signaling pathways in 2D and 3D-spheroid triple-negative breast cancer cell cultures: a comparative study.

The type of experimental model for the in vitro testing of drug formulations efficiency represents an important tool in cancer biology, with great attention being granted to three-dimensional (3D) cultures as these offer a closer approximation of the clinical sensitivity of drugs. In this study, the effects induced by carboxyl-functionalized single-walled carbon nanotubes complexed with cisplatin (SWCNT-COOH-CDDP) and free components (SWCNT-COOH and CDDP) were compared between conventional 2D- and 3D-spheroid cultures of human breast cancer cells. The 2D and 3D breast cancer cultures were exposed to various doses of SWCNT-COOH (0.25-2 µg/mL), CDDP (0.158-1.26 µg/mL) and the same doses of SWNCT-COOH-CDDP complex for 24 and 48 h. The anti-tumor activity, including modulation of cell viability, oxidative stress, proliferation, apoptosis, and invasion potential, was explored by spectrophotometric and fluorometric methods, immunoblotting, optical and fluorescence microscopy. The SWCNT-COOH-CDDP complex proved to have high anti-cancer efficiency on 2D and 3D cultures by inhibiting cell proliferation and activating cell death. A dose of 0.632 µg/mL complex triggered different pathways of apoptosis in 2D and 3D cultures, by intrinsic, extrinsic, and reticulum endoplasmic pathways. Overall, the 2D cultures showed higher susceptibility to the action of complex compared to 3D cultures and SWCNT-COOH-CDDP proved enhanced anti-tumoral activity compared to free CDDP.

Biomedical Promise of Sustainable Microwave-Engineered Symmetric Curcumin Derivatives.

Curcumin is a polyphenol of the Curcuma longa plant, which can be used for various medicinal purposes, such as inflammation and cancer treatment. In this context, two symmetric curcumin derivatives (D1-(1E,6E)-1,7-bis(4-acetamidophenyl)hepta-1,6-diene-3,5-dione and D2-p,p-dihydroxy di-cinnamoyl methane) were obtained by the microwave-based method and evaluated for their antitumoral effect on human cervix cancer in comparison with toxicity on non-tumoral cells, taking into account that they were predicted to act as apoptosis agonists or anti-inflammatory agents. The HeLa cell line was incubated for 24 and 72 h with a concentration of 50 µg/mL of derivatives that killed almost half of the cells compared to the control. In contrast, these compounds did not alter the viability of MRC-5 non-tumoral lung fibroblasts until 72 h of incubation. The nitric oxide level released by HeLa cells was higher compared to MRC-5 fibroblasts after the incubation with 100 µg/mL. Both derivatives induced the decrease of catalase activity and glutathione levels in cancer cells without targeting the same effect in non-tumoral cells. Furthermore, the Western blot showed an increased protein expression of HSP70 and a decreased expression of HSP60 and MCM2 in cells incubated with D2 compared to control cells. We noticed differences regarding the intensity of cell death between the tested derivatives, suggesting that the modified structure after synthesis can modulate their function, the most prominent effect being observed for sample D2. In conclusion, the outcomes of our in vitro study revealed that these microwave-engineered curcumin derivatives targeted tumor cells, much more specifically, inducing their death.

TRP Channels in Tumoral Processes Mediated by Oxidative Stress and Inflammation.

The channels from the superfamily of transient receptor potential (TRP) activated by reactive oxygen species (ROS) can be defined as redox channels. Those with the best exposure of the cysteine residues and, hence, the most sensitive to oxidative stress are TRPC4, TRPC5, TRPV1, TRPV4, and TRPA1, while others, such as TRPC3, TRPM2, and TRPM7, are indirectly activated by ROS. Furthermore, activation by ROS has different effects on the tumorigenic process: some TRP channels may, upon activation, stimulate proliferation, apoptosis, or migration of cancer cells, while others inhibit these processes, depending on the cancer type, tumoral microenvironment, and, finally, on the methods used for evaluation. Therefore, using these polymodal proteins as therapeutic targets is still an unmet need, despite their draggability and modulation by simple and mostly unharmful compounds. This review intended to create some cellular models of the interaction between oxidative stress, TRP channels, and inflammation. Although somewhat crosstalk between the three actors was rather theoretical, we intended to gather the recently published data and proposed pathways of cancer inhibition using modulators of TRP proteins, hoping that the experimental data corroborated clinical information may finally bring the results from the bench to the bedside.

Biological Response of Human Gingival Fibroblasts to Zinc-Doped Hydroxyapatite Designed for Dental Applications-An In Vitro Study.

This study aimed to investigate the biological response induced by hydroxyapatite (HAp) and zinc-doped HAp (ZnHAp) in human gingival fibroblasts and to explore their antimicrobial activity. The ZnHAp (with xZn = 0.00 and 0.07) powders, synthesized by the sol-gel method, retained the crystallographic structure of pure HA without any modification. Elemental mapping confirmed the uniform dispersion of zinc ions in the HAp lattice. The size of crystallites was 18.67 ± 2 nm for ZnHAp and 21.54 ± 1 nm for HAp. The average particle size was 19.38 ± 1 nm for ZnHAp and 22.47 ± 1 nm for HAp. Antimicrobial studies indicated an inhibition of bacterial adherence to the inert substrate. In vitro biocompatibility was tested on various doses of HAp and ZnHAp after 24 and 72 h of exposure and revealed that cell viability decreased after 72 h starting with a dose of 31.25 µg/mL. However, cells retained membrane integrity and no inflammatory response was induced. High doses (such as 125 µg/mL) affected cell adhesion and the architecture of F-actin filaments, while in the presence of lower doses (such as 15.625 µg/mL), no modifications were observed. Cell proliferation was inhibited after treatment with HAp and ZnHAp, except the dose of 15.625 µg/mL ZnHAp at 72 h of exposure, when a slight increase was observed, proving an improvement in ZnHAp activity due to Zn doping.

Dextran-Coated Iron Oxide Nanoparticles Loaded with 5-Fluorouracil for Drug-Delivery Applications.

This study aims to design and test different formulations composed of dextran-coated iron oxide nanoparticles (IONPs) loaded with 5-Fluorouracil (5-FU) with varying nanoparticle:drug ratios on colorectal cancer cells. The stable suspension of IONPs s was synthesized by the adapted co-precipitation method. The stable suspension of IONPs was mixed with a solution of dextran and 5-FU solubilized in a saline solution. The final suspensions with optimized ratios of IONP:5-FU in the final suspension were 0.5:1, 1:1, and 1.5:1. The information on the morphology and size distribution of the IONPs suspension and IONP loads with 5-FU was obtained using scanning electron microscopy (SEM). The presence of 5-FU and dextran on the surface of the IONPs was highlighted by energy-dispersive X-ray spectroscopy (EDS) studies. The determination of the surface charge of the nanoparticles in the final suspensions of IONP:5-FU was achieved by measuring the zeta potential (ζ). The hydrodynamic diameter of the resulting suspensions of IONP:5-FU was determined by dynamic light scattering (DLS). A cytocompatibility analysis was performed using Caco-2 (human epithelial colorectal adenocarcinoma) cells. In this research, our goal was to find a relationship between the formulation ratio of nanoparticles and drug, and the cellular response after exposure, as a strategy to increase the efficacy of this drug-delivery system. The nanoparticle uptake and antitumor activity, including modulation of oxidative stress, apoptosis, and proliferation biomarkers, were analyzed. The present study showed that the nanoformulation with the ratio IONP:5-FU 1.5:1 had the highest anti-tumor efficiency. Moreover, decreased MCM-2 expression in Caco-2 cells exposed to dextran-coated iron oxide nanoparticles loaded with 5-FU was demonstrated for the first time.

Photodynamic Activity of TMPyP4/TiO2 Complex under Blue Light in Human Melanoma Cells: Potential for Cancer-Selective Therapy.

The combination of TiO2 nanoparticles (NPs) and photosensitizers (PS) may offer significant advantages in photodynamic therapy (PDT) of melanoma, such as improved cell penetration, enhanced ROS production, and cancer selectivity. In this study, we aimed to investigate the photodynamic effect of 5,10,15,20-(Tetra-N-methyl-4-pyridyl)porphyrin tetratosylate (TMPyP4) complexes with TiO2 NPs on human cutaneous melanoma cells by irradiation with 1 mW/cm2 blue light. The porphyrin conjugation with the NPs was analyzed by absorption and FTIR spectroscopy. The morphological characterization of the complexes was performed by Scanning Electron Microscopy and Dynamic Light Scattering. The singlet oxygen generation was analyzed by phosphorescence at 1270 nm. Our predictions indicated that the non-irradiated investigated porphyrin has a low degree of toxicity. The photodynamic activity of the TMPyP4/TiO2 complex was assessed on the human melanoma Mel-Juso cell line and non-tumor skin CCD-1070Sk cell line treated with various concentrations of the PS and subjected to dark conditions and visible light-irradiation. The tested complexes of TiO2 NPs with TMPyP4 presented cytotoxicity only after activation by blue light (405 nm) mediated by the intracellular production of ROS in a dose-dependent manner. The photodynamic effect observed in this evaluation was higher in melanoma cells than the effect observed in the non-tumor cell line, demonstrating a promising potential for cancer-selectivity in PDT of melanoma.

Iron-Oxide-Nanoparticles-Doped Polyaniline Composite Thin Films.

Iron-oxide-doped polyaniline (PANI-IO) thin films were obtained by the polymerization of aniline monomers and iron oxide solutions in direct current glow discharge plasma in the absence of a buffer gas for the first time. The PANI-IO thin films were deposited on optical polished Si wafers in order to study surface morphology and evaluate their in vitro biocompatibility. The characterization of the coatings was accomplished using scanning electron microscopy (SEM), Fourier-transform infrared spectroscopy (FTIR), atomic force microscopy (AFM), metallographic microscopy (MM), and X-ray photoelectron spectroscopy (XPS). In vitro biocompatibility assessments were also conducted on the PANI-IO thin films. It was observed that a uniform distribution of iron oxide particles inside the PANI layers was obtained. The constituent elements of the coatings were uniformly distributed. The Fe-O bonds were associated with magnetite in the XPS studies. The surface morphology of the PANI-IO thin films was assessed by atomic force microscopy (AFM). The AFM topographies revealed that PANI-IO exhibited the morphology of a uniformly distributed and continuous layer. The viability of Caco-2 cells cultured on the Si substrate and PANI-IO coating was not significantly modified compared to control cells. Moreover, after 24 h of incubation, we observed no increase in LDH activity in media in comparison to the control. In addition, our results revealed that the NO levels for the Si substrate and PANI-IO coating were similar to those found in the control sample.

Carboxyl-Functionalized Carbon Nanotubes Loaded with Cisplatin Promote the Inhibition of PI3K/Akt Pathway and Suppress the Migration of Breast Cancer Cells.

PI3K/Akt signaling is one of the most frequently dysregulated pathways in cancer, including triple-negative breast cancer. With considerable roles in tumor growth and proliferation, this pathway is studied as one of the main targets in controlling the therapies' efficiency. Nowadays, the development of nanoparticle-drug conjugates attracts a great deal of attention due to the advantages they provide in cancer treatment. Hence, the main purpose of this study was to design a nanoconjugate based on single-walled carbon nanotubes functionalized with carboxyl groups (SWCNT-COOH) and cisplatin (CDDP) and to explore the potential of inhibiting the PI3K/Akt signaling pathway. MDA-MB-231 cells were exposed to various doses (0.01-2 µg/mL SWCNT-COOH and 0.00632-1.26 µg/mL CDDP) of SWCNT-COOH-CDDP and free components for 24 and 48 h. In vitro biological tests revealed that SWCNT-COOH-CDDP had a high cytotoxic effect, as shown by a time-dependent decrease in cell viability and the presence of a significant number of dead cells in MDA-MB-231 cultures at higher doses. Moreover, the nanoconjugates induced the downregulation of PI3K/Akt signaling, as revealed by the decreased expression of PI3K and p-Akt in parallel with PTEN activation, the promotion of Akt protein degradation, and inhibition of tumor cell migration.

Biological properties and development of hypoxia in a breast cancer 3D model generated by hanging drop technique.

Hanging drop represents a simple approach designed for the generation of 3D models that have potential to be used for the study of solid tumors characteristics. The aim of the study was to develop and characterize the breast cancer 3D cellular models obtained through hanging drop technique using MDA-MB-231 cells. The biological characteristics such as: morphology, cellular viability, proliferation capacity and hypoxia, were monitored for a six-day time period. The morphological evaluation indicated that the 3D models presented the aspect of compact (seeding density of 2500 and 5000 cells/drop) and loose (seeding density of 8000 cells/drop) aggregates, with a decrease in diameter and an increase of their circularity. The cellular viability and proliferation capacity decreased in time and the level of lactate dehydrogenase (LDH) increased in a time-dependent manner, suggesting the presence of necrotic cells that were dispersed in the cellular aggregates. The occurrence of hypoxia process was suggested by the up-regulation of Hsp70 protein expression and increased level of nitric oxide (NO). Moreover, the up-regulation of HIF-1α and poli-ubiquitinated Nrf2 protein expressions and decreased level of reduced glutathione (GSH) indicated the presence of an acute hypoxic environment in MDA-MB-231 3D aggregates. In conclusion, the MDA-MB-231 3D models generated through hanging drop are compact and loose aggregates characterized by an acute hypoxic condition.

Low Blue Dose Photodynamic Therapy with Porphyrin-Iron Oxide Nanoparticles Complexes: In Vitro Study on Human Melanoma Cells.

The purpose of this study was to investigate the effectiveness in photodynamic therapy of iron oxide nanoparticles (γ-Fe2O3 NPs), synthesized by laser pyrolysis technique, functionalized with 5,10,15,20-(Tetra-4-sulfonatophenyl) porphyrin tetraammonium (TPPS) on human cutaneous melanoma cells, after only 1 min blue light exposure. The efficiency of porphyrin loading on the iron oxide nanocarriers was estimated by using absorption and FTIR spectroscopy. The singlet oxygen yield was determined via transient characteristics of singlet oxygen phosphorescence at 1270 nm both for porphyrin functionalized nanoparticles and rose bengal used as standard. The irradiation was performed with a LED (405 nm, 1 mW/cm2) for 1 min after melanoma cells were treated with TPPS functionalized iron oxide nanoparticles (γ-Fe2O3 NPs_TPPS) and incubated for 24 h. Biological tests revealed a high anticancer effect of γ-Fe2O3 NPs_TPPS complexes indi-cated by the inhibition of tumor cell proliferation, reduction of cell adhesion, and induction of cell death through ROS generated by TPPS under light exposure. The biological assays were combined with the pharmacokinetic prediction of the porphyrin.

Doxorubicin-Conjugated Iron Oxide Nanoparticles Synthesized by Laser Pyrolysis: In Vitro Study on Human Breast Cancer Cells.

Even today, breast cancer remains a global public problem, with a high mortality rate among women. Nanoparticle (NP) based systems are developed to enhance drug delivery, reducing the toxic effect of medicine molecules. By using iron oxide nanoparticles for cancer treatment, several advantages were highlighted: the ability to target specific locations derived from their magnetic properties and reduced side effects. The aim of this study was to examine on breast cancer cell line the anticancer potential of γ-Fe2O3 NPs loaded with doxorubicin (DOX) and stabilized with carboxymethylcellulose sodium (CMCNa). The γ-Fe2O3 NPs were synthesized by laser pyrolysis technique and their nanometric size and crystallinity were confirmed by X-ray diffraction and transmission electron microscopy. The loading efficiency was estimated by using absorption and fluorescence spectroscopy. The DOX conjugated//CMCNa coated γ-Fe2O3 NPs proved through the biological studies to have a good anticancer effect through the inhibition of tumoral cell proliferation, disruption of the cellular membrane, induction of cell death and reduced effects on normal breast cells. Our data showed that DOX cytotoxicity increases significantly when conjugated with É£-Fe2O3 and É£-Fe2O3_CMCNa, a 50% reduction of cancer cell viability was obtained with a concentration around 0.1 µg/mL.

Influence of Matrigel on Single- and Multiple-Spheroid Cultures in Breast Cancer Research.

This study aimed to develop and compare single and multiple 3D models such as multicellular tumor spheroids and to investigate the influence of Matrigel on their morphological and functional behavior. MDA-MB-231 3D models were generated in the presence and absence of Matrigel and their key biological properties within 6 days of culture were monitored. Our results revealed the formation of well-defined 3D models in the presence of Matrigel, with a uniform morphology, increased diameter, good circularity, and increased expression of a proliferation marker (PCNA). In comparison, 3D models generated without Matrigel were characterized by an irregular border, reduced dimensions and circularity, and a decrease of PCNA expression. Similarities between the single and multiple 3D cultures were found in their viability, Nrf2 expression, and glutathione (GSH) content. The influence of Matrigel on MDA-MB-231 spheroids metabolism under hypoxic conditions was highlighted by released lactate dehydrogenase and nitric oxide, GSH levels and expression of Nrf2 and Hsp70 proteins. Based on the increased expression of PCNA and the development of the hypoxia process in the presence of extracellular matrix, our study showed that the addition of Matrigel improves the growing environment of tumor spheroids, making it closer to that of in vivo tumor conditions.

Designing cotton fibers impregnated with photocatalytic graphene oxide/Fe, N-doped TiO2 particles as prospective industrial self-cleaning and biocompatible textiles.

Our study reports the fabrication and characterization (surface morphology, hydrophobicity/hydrophilicity, photocatalytic efficiency) of cotton fibers treated by various methods with graphene oxide decorated with Fe, N-doped TiO2 nanoparticles. Designed as prospective industrial self-cleaning, antimicrobial and biocompatible textiles, microbiological and cytotoxicity tests were performed on these particles-treated fibers to validate their qualities. The photocatalytic effect was dependent on chemicals used to disperse the nanoparticles, the parameters of the treatment, the fiber structure and composition of the material. The double and triple treatment of the textiles with the same particle dispersion resulted in a relatively uniform coverage of cotton fibers with relatively large amounts of particles. A larger amount of doped TiO2 particles demonstrated a better photocatalytic effect under visible light. The material's hydrophobicity increased with the number of treatments due to the deposition of successive layers of reduced graphene, ensuring self-cleaning properties. The photocatalyst-treated cotton fabrics exhibited an increased resistance to Enterococcus faecalis and Escherichia coli colonization, and also high biocompatibility, as they did not affect the cell viability, membrane integrity and morphology, nor induce inflammation. All these data confirm the improved properties of cotton fibers treated with graphene oxide decorated with Fe, N-doped TiO2 particles in order to be used as industrial self-cleaning and biocompatible textiles.

Cisplatin Loaded Multiwalled Carbon Nanotubes Induce Resistance in Triple Negative Breast Cancer Cells.

In this paper we developed a method for multiwalled carbon nanotubes (MWCNTs) use as carriers for a drug based on platinum in breast cancer therapy. The method of functionalization involves the carboxyl functionalization of nanotubes and encapsulation of cisplatin (CDDP) into MWCNTs. The biological properties of MWCNTs loaded with CDDP (MWCNT-COOH-CDDP) and of individual components MWCNT-COOH and free CDDP were evaluated on MDA-MB-231 cells. Various concentrations of CDDP (0.316⁻2.52 µg/mL) and MWCNTs (0.5⁻4 µg/mL) were applied on cells for 24 and 48 h. Only at high doses of CDDP (1.26 and 2.52 µg/mL) and MWCNT-COOH-CDDP (2 and 4 µg/mL) cell morphological changes were observed. The cellular viability decreased only with approx. 40% after 48 h of exposure to 2.52 µg/mL CDDP and 4 µg/mL MWCNT-COOH-CDDP despite the high reactive oxygen species (ROS) production induced by MWCNTs starting with 24 h. After 48 h, ROS level dropped as a result of the antioxidant defence activation. We also found a significant decrease of caspase-3 and p53 expression after 48 h, accompanied by a down-regulation of NF-κB in cells exposed to MWCNT-COOH-CDDP system which promotes apoptosis escape and thus failing to overcome the triple negative breast cancer (TNBC) cells resistance.

Coating Dependent In Vitro Biocompatibility of New Fe-Si Nanoparticles.

Magnetic nanoparticles offer multiple utilization possibilities in biomedicine. In this context, the interaction with cellular structures and their biological effects need to be understood and controlled for clinical safety. New magnetic nanoparticles containing metallic/carbidic iron and elemental silicon phases were synthesized by laser pyrolysis using Fe(CO)5 vapors and SiH4 gas as Fe and Si precursors, then passivated and coated with biocompatible agents, such as l-3,4-dihydroxyphenylalanine (l-DOPA) and sodium carboxymethyl cellulose (CMC-Na). The resulting magnetic nanoparticles were characterized by XRD, EDS, and TEM techniques. To evaluate their biocompatibility, doses ranging from 0⁻200 µg/mL hybrid Fe-Si nanoparticles were exposed to Caco2 cells for 24 and 72 h. Doses below 50 μg/mL of both l-DOPA and CMC-Na-coated Fe-Si nanoparticles induced no significant changes of cellular viability or membrane integrity. The cellular internalization of nanoparticles was dependent on their dispersion in culture medium and caused some changes of F-actin filaments organization after 72 h. However, reactive oxygen species were generated after exposure to 25 and 50 μg/mL of both Fe-Si nanoparticles types, inducing the increase of intracellular glutathione level and activation of transcription factor Nrf2. At nanoparticles doses below 50 μg/mL, Caco2 cells were able to counteract the oxidative stress by activating the cellular protection mechanisms. We concluded that in vitro biological responses to coated hybrid Fe-Si nanoparticles depended on particle synthesis conditions, surface coating, doses and incubation time.