Nanoparticle-Mediated Drug Delivery of Doxorubicin Induces a Differentiated Clonogenic Inactivation in 3D Tumor Spheroids In Vitro.

Involvement of 3D tumor cell models in the in vitro biological testing of novel nanotechnology-based strategies for cancer management can provide in-depth information on the real behavior of tumor cells in complex biomimetic architectures. Here, we used polyethylene glycol-encapsulated iron oxide nanoparticles for the controlled delivery of a doxorubicin chemotherapeutic substance (IONPDOX), and to enhance cytotoxicity of photon radiation therapy. The biological effects of nanoparticles and 150 kV X-rays were evaluated on both 2D and 3D cell models of normal human keratinocytes (HaCaT) and tumor cells-human cervical adenocarcinoma (HeLa) and human squamous carcinoma (FaDu)-through cell survival. In all 2D cell models, nanoparticles were similarly internalized in a peri-nuclear pattern, but resulted in different survival capabilities following radiation treatment. IONP on normal keratinocytes showed a protective effect, but a cytotoxic effect for cancer cells. In 3D tumor cell models, IONPDOX were able to penetrate the cell spheroids towards the hypoxic areas. However, IONPDOX and 150 kV X-rays led to a dose-modifying factor DMFSF=0.1 = 1.09 ± 0.1 (200 µg/mL IONPDOX) in HeLa spheroids, but to a radioprotective effect in FaDu spheroids. Results show that the proposed treatment is promising in the management of cervical adenocarcinoma.

Impaired Integrated Stress Response and Mitochondrial Integrity Modulate Genotoxic Stress Impact and Lower the Threshold for Immune Signalling.

Mitochondria-nucleus communication during stress dictates cellular fate with consequences on the etiopathology of multiple age-related diseases. Impaired mitochondrial quality control through loss of function of the mitochondrial protease HtrA2 associates with accumulation of damaged mitochondria and triggers the integrated stress response, implicating the transcription factor CHOP. Here we have employed a combined model of impaired mitochondria quality control, namely HtrA2 loss of function, and/or integrated stress response, namely CHOP loss of function, and genotoxicity to address the distinctive roles of these cellular components in modulating intracellular and intercellular responses. The genotoxic agents employed were cancer therapeutic agents such as irradiation with X-ray and protons or treatment with the radiomimetic bleomycin. The irradiation had an enhanced effect in inducing DNA damage in cells with CHOP loss of function, while the bleomycin treatment induced more DNA damage in all the transgenic cells as compared to the control. The genetic modifications impaired the transmission of DNA damage signalling intercellularly. Furthermore, we have dissected the signalling pathways modulated by irradiation in selected genotypes with RNA sequencing analysis. We identified that loss of HtrA2 and CHOP function, respectively, lowers the threshold where irradiation may induce the activation of innate immune responses via cGAS-STING; this may have a significant impact on decisions for combined therapeutic approaches for various diseases.

Novel Antitumor Agents Based on Fluorescent Benzofurazan Derivatives and Mesoporous Silica.

Two novel fluorescent mesoporous silica-based hybrid materials were obtained through the covalent grafting of [4-hydrazinyl-7-nitrobenz-[2,1,3-d]-oxadiazole (NBDH) and N1-(7-nitrobenzo[c][1,2,5]-oxadiazol-4-yl) benzene-1,2-diamine (NBD-PD), respectively, inside the channels of mesoporous silica SBA-15. The presence of fluorescent organic compounds (nitrobenzofurazan derivatives) was confirmed by infrared spectroscopy (IR), X-ray photoelectron spectroscopy (XPS), thermal analysis (TG), and fluorescence spectroscopy. The nitrogen physisorption analysis showed that the nitrobenzofurazan derivatives were distributed uniformly on the internal surface of SBA-15, the immobilization process having a negligible effect on the structure of the support. Their antioxidant activity was studied by measuring the ability to reduce free radicals DPPH (free radical scavenging activity), in order to formulate potential applications of the materials obtained. Cytotoxicity of the newly synthesized materials, SBA-NBDH and SBA-NBD-PD, was evaluated on human B16 melanoma cells. The morphology of these cells, internalization and localization of the investigated materials in melanoma and fibroblast cells were examined through fluorescence imaging. The viability of B16 (3D) spheroids after treatment with SBA-NBDH and SBA-NBD-PD was evaluated using MTS assay. The results showed that both materials induced a selective antiproliferative effect, reducing to various degrees the viability of melanoma cells. The observed effect was enhanced with increasing concentration. SBA-NBD-PD exhibited a higher antitumor effect compared to SBA-NBDH starting with a concentration of 125 µg/mL. In both cases, a significantly more pronounced antiproliferative effect on tumor cells compared to normal cells was observed. The viability of B16 spheroids dropped by 40% after treatment with SBA-NBDH and SBA-NBD-PD at 500 µg/mL concentration, indicating a clear cytotoxic effect of the tested compounds. These results suggest that both newly synthesized biomaterials could be promising antitumor agents for applications in cancer therapy.

Intracellular Delivery of Doxorubicin by Iron Oxide-Based Nano-Constructs Increases Clonogenic Inactivation of Ionizing Radiation in HeLa Cells.

In this study, we determined the potential of polyethylene glycol-encapsulated iron oxide nanoparticles (IONPCO) for the intracellular delivery of the chemotherapeutic doxorubicin (IONPDOX) to enhance the cytotoxic effects of ionizing radiation. The biological effects of IONP and X-ray irradiation (50 kV and 6 MV) were determined in HeLa cells using the colony formation assay (CFA) and detection of γH2AX foci. Data are presented as mean ± SEM. IONP were efficiently internalized by HeLa cells. IONPCO radiomodulating effect was dependent on nanoparticle concentration and photon energy. IONPCO did not radiosensitize HeLa cells with 6 MV X-rays, yet moderately enhanced cellular radiosensitivity to 50 kV X-rays (DMFSF0.1 = 1.13 ± 0.05 (p = 0.01)). IONPDOX did enhance the cytotoxicity of 6 MV X-rays (DMFSF0.1 = 1.3 ± 0.1; p = 0.0005). IONP treatment significantly increased γH2AX foci induction without irradiation. Treatment of HeLa cells with IONPCO resulted in a radiosensitizing effect for low-energy X-rays, while exposure to IONPDOX induced radiosensitization compared to IONPCO in cells irradiated with 6 MV X-rays. The effect did not correlate with the induction of γH2AX foci. Given these results, IONP are promising candidates for the controlled delivery of DOX to enhance the cytotoxic effects of ionizing radiation.

Development of New Collagen/Clay Composite Biomaterials.

The fabrication of collagen-based biomaterials for skin regeneration offers various challenges for tissue engineers. The purpose of this study was to obtain a novel series of composite biomaterials based on collagen and several types of clays. In order to investigate the influence of clay type on drug release behavior, the obtained collagen-based composite materials were further loaded with gentamicin. Physiochemical and biological analyses were performed to analyze the obtained nanocomposite materials after nanoclay embedding. Infrared spectra confirmed the inclusion of clay in the collagen polymeric matrix without any denaturation of triple helical conformation. All the composite samples revealed a slight change in the 2-theta values pointing toward a homogenous distribution of clay layers inside the collagen matrix with the obtaining of mainly intercalated collagen-clay structures, according X-ray diffraction analyses. The porosity of collagen/clay composite biomaterials varied depending on clay nanoparticles sort. Thermo-mechanical analyses indicated enhanced thermal and mechanical features for collagen composites as compared with neat type II collagen matrix. Biodegradation findings were supported by swelling studies, which indicated a more crosslinked structure due additional H bonding brought on by nanoclays. The biology tests demonstrated the influence of clay type on cellular viability but also on the antimicrobial behavior of composite scaffolds. All nanocomposite samples presented a delayed gentamicin release when compared with the collagen-gentamicin sample. The obtained results highlighted the importance of clay type selection as this affects the performances of the collagen-based composites as promising biomaterials for future applications in the biomedical field.

Enhanced Internalization of Nanoparticles Following Ionizing Radiation Leads to Mitotic Catastrophe in MG-63 Human Osteosarcoma Cells.

This study aims to investigate whether ionizing radiation combined with doxorubicin-conjugated iron oxide nanoparticles (NP-DOX) improves the internalization and cytotoxic effects of the nano-carrier-mediated drug delivery in MG-63 human osteosarcoma cells. NP-DOX was designed and synthesized using the co-precipitation method. Highly stable and crystalline nanoparticles conjugated with DOX were internalized in MG-63 cells through macropinocytosis and located in the perinuclear area. Higher nanoparticles internalization in MG-63 cells previously exposed to 1 Gy X-rays was correlated with an early accumulation of cells in G2/M, starting at 12 h after treatment. After 48 h, the application of the combined treatment led to higher cytotoxic effects compared to the individual treatment, with a reduction in the metabolic capacity and unrepaired DNA breaks, whilst a low percent of arrested cells, contributing to the commitment of mitotic catastrophe. NP-DOX showed hemocompatibility and no systemic cytotoxicity, nor histopathological alteration of the main organs.

Unveiling Nanoparticles: Recent Approaches in Studying the Internalization Pattern of Iron Oxide Nanoparticles in Mono- and Multicellular Biological Structures.

Nanoparticle (NP)-based solutions for oncotherapy promise an improved efficiency of the anticancer response, as well as higher comfort for the patient. The current advancements in cancer treatment based on nanotechnology exploit the ability of these systems to pass biological barriers to target the tumor cell, as well as tumor cell organelles. In particular, iron oxide NPs are being clinically employed in oncological management due to this ability. When designing an efficient anti-cancer therapy based on NPs, it is important to know and to modulate the phenomena which take place during the interaction of the NPs with the tumor cells, as well as the normal tissues. In this regard, our review is focused on highlighting different approaches to studying the internalization patterns of iron oxide NPs in simple and complex 2D and 3D in vitro cell models, as well as in living tissues, in order to investigate the functionality of an NP-based treatment.

Chondrosarcoma Resistance to Radiation Therapy: Origins and Potential Therapeutic Solutions.

Chondrosarcoma is a malignant cartilaginous tumor that is particularly chemoresistant and radioresistant to X-rays. The first line of treatment is surgery, though this is almost impossible in some specific locations. Such resistances can be explained by the particular composition of the tumor, which develops within a dense cartilaginous matrix, producing a resistant area where the oxygen tension is very low. This microenvironment forces the cells to adapt and dedifferentiate into cancer stem cells, which are described to be more resistant to conventional treatments. One of the main avenues considered to treat this type of tumor is hadrontherapy, in particular for its ballistic properties but also its greater biological effectiveness against tumor cells. In this review, we describe the different forms of chondrosarcoma resistance and how hadrontherapy, combined with other treatments involving targeted inhibitors, could help to better treat high-grade chondrosarcoma.

In vitro hyperspectral biomarkers of human chondrosarcoma cells in nanoparticle-mediated radiosensitization using carbon ions.

New therapeutic approaches are needed for the management of the highly chemo- and radioresistant chondrosarcoma (CHS). In this work, we used polyethylene glycol-encapsulated iron oxide nanoparticles for the intracellular delivery of the chemotherapeutic doxorubicin (IONPDOX) to augment the cytotoxic effects of carbon ions in comparison to photon radiation therapy. The in vitro biological effects were investigated in SW1353 chondrosarcoma cells focusing on the following parameters: cell survival using clonogenic test, detection of micronuclei (MN) by cytokinesis blocked micronucleus assay and morphology together with spectral fingerprints of nuclei using enhanced dark-field microscopy (EDFM) assembled with a hyperspectral imaging (HI) module. The combination of IONPDOX with ion carbon or photon irradiation increased the lethal effects of irradiation alone in correlation with the induction of MN. Alterations in the hyperspectral images and spectral profiles of nuclei reflected the CHS cell biological modifications following the treatments, highlighting possible new spectroscopic markers of cancer therapy effects. These outcomes showed that the proposed combined treatment is promising in improving CHS radiotherapy.

Mitochondria - Nucleus communication in neurodegenerative disease. Who talks first, who talks louder?

Mitochondria - nuclear coadaptation has been central to eukaryotic evolution. The dynamic dialogue between the two compartments within the context of multiorganellar interactions is critical for maintaining cellular homeostasis and directing the balance survival-death in case of cellular stress. The conceptualisation of mitochondria - nucleus communication has so far been focused on the communication from the mitochondria under stress to the nucleus and the consequent signalling responses, as well as from the nucleus to mitochondria in the context of DNA damage and repair. During ageing processes this dialogue may be better viewed as an integrated bidirectional 'talk' with feedback loops that expand beyond these two organelles depending on physiological cues. Here we explore the current views on mitochondria - nucleus dialogue and its role in maintaining cellular health with a focus on brain cells and neurodegenerative disease. Thus, we detail the transcriptional responses initiated by mitochondrial dysfunction in order to protect itself and the general cellular homeostasis. Additionally, we are reviewing the knowledge of the stress pathways initiated by DNA damage which affect mitochondria homeostasis and we add the information provided by the study of combined mitochondrial and genotoxic damage. Finally, we reflect on how each organelle may take the lead in this dialogue in an ageing context where both compartments undergo accumulation of stress and damage and where, perhaps, even the communications' mechanisms may suffer interruptions.

Influence of Polymer Shell Molecular Weight on Functionalized Iron Oxide Nanoparticles Morphology and In Vivo Biodistribution.

Iron oxide nanoparticles (IONPs) have been extensively used in different biomedical applications due to their biocompatibility and magnetic properties. However, different functionalization approaches have been developed to improve their time-life in the systemic circulation. Here, we have synthesized IONPs using a modified Massart method and functionalized them in situ with polyethylene glycol with different molecular weights (20 K and 35 K). The resulting nanoparticles were characterized in terms of morphology, structure, and composition using transmission electron microscopy (TEM) and selected area electron diffraction (SAED). In vivo biodistribution was evaluated in Balb/c mice, the presence of IONP being evidenced through histopathological investigations. IONP morphological characterization showed a change in shape (from spherical to rhombic) and size with molecular weight, while structural characterization proved the obtaining of highly crystalline samples of spinel structured cubic face-centered magnetite. In vivo biodistribution in a mice model proved the biocompatibility of all of the IONP samples. All NPs were cleared through the liver, spleen, and lungs, while bare IONPs were also evidenced in kidneys.

Benzofurazan derivatives modified graphene oxide nanocomposite: Physico-chemical characterization and interaction with bacterial and tumoral cells.

Two novel graphene oxide-benzofuran derivatives composites were obtained through the covalent immobilization of [4-hydrazinyl-7nitrobenz-[2,1,3-d]-oxadiazole (NBDH) and respectively, N1-(7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)benzene-1,2-diamine (NBD-PD), on graphene oxide. This covalent functionalization was achieved by activating the carboxylic groups on the surface of graphene oxide by the reaction with thionyl chloride followed by coupling with the amino group of benzofurazane derivatives to obtain the NBD derivatives grafted on graphene oxide. The formation of new materials was check by Raman spectroscopy, fluorescence, infrared spectroscopy and X-ray photoelectron spectroscopy, thermal analysis, scanning electron microscopy, and elemental mapping. The antimicrobial effect of the new composites was evaluated on Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa, both on planktonic and adherent biofilm populations. The cytotoxic effects of the materials on human colon cancer HCT-116 cell line and the normal human fibroblast BJ cell line were evaluated by investigating cell viability and membrane integrity. Apoptosis and colony forming ability of tumor cells were also investigated following exposure to new materials. The biological results of this study have shown that the new materials have potential in combating biofilm formation and also, the tested materials induced cytotoxicity in human colon cancer HCT-116 cell line with limited effects on normal BJ fibroblasts, suggesting their antitumor potential.

Intracellular and Intercellular Signalling Mechanisms following DNA Damage Are Modulated By PINK1.

Impaired mitochondrial function and accumulation of DNA damage have been recognized as hallmarks of age-related diseases. Mitochondrial dysfunction initiates protective signalling mechanisms coordinated at nuclear level particularly by modulating transcription of stress signalling factors. In turn, cellular response to DNA lesions comprises a series of interconnected complex protective pathways, which require the energetic and metabolic support of the mitochondria. These are involved in intracellular as well as in extracellular signalling of damage. Here, we have initiated a study that addresses how mitochondria-nucleus communication may occur in conditions of combined mitochondrial dysfunction and genotoxic stress and what are the consequences of this interaction on the cell system. In this work, we used cells deficient for PINK1, a mitochondrial kinase involved in mitochondrial quality control whose loss of function leads to the accumulation of dysfunctional mitochondria, challenged with inducers of DNA damage, namely, ionizing radiation and the radiomimetic bleomycin. Combined stress at the level of mitochondria and the nucleus impairs both mitochondrial and nuclear functions. Our findings revealed exacerbated sensibility to genotoxic stress in PINK1-deficient cells. The same cells showed an impaired induction of bystander phenomena following stress insults. However, these cells responded adaptively when a challenge dose was applied subsequently to a low-dose treatment to the cells. The data demonstrates that PINK1 modulates intracellular and intercellular signalling pathways, particularly adaptive responses and transmission of bystander signalling, two facets of the cell-protective mechanisms against detrimental agents.