Dioclea violacea lectin inhibits tumorigenesis and tumor angiogenesis in vivo.

Dioclea violacea seed mannose-binding lectin (DvL) has attracted considerable attention because of its interesting biological activities, including antitumor, antioxidant, and anti-inflammatory activities. This study evaluated the cytotoxic effect of DvL on tumor and normal cells using the mitochondrial activity reduction (MTT) assay, the carcinogenic and anti-carcinogenic activity by the epithelial tumor test (ETT) in Drosophila melanogaster, and the anti-angiogenic effect by the chick embryo chorioallantoic membrane (CAM) assay. Data demonstrated that DvL promoted strong selective cytotoxicity against tumor cell lines, especially A549 and S180 cells, whereas normal cell lines were weakly affected. Furthermore, DvL did not promote carcinogenesis in D. melanogaster at any concentration tested, but modulated DXR-induced carcinogenesis at the highest concentrations tested. In the CAM and immunohistochemical assays, DvL inhibited sarcoma 180-induced angiogenesis and promoted the reduction of VEGF and TGF-ß levels at all concentrations tested. Therefore, our results demonstrated that DvL is a potent anticancer, anti-angiogenic, and selective cytotoxic agent for tumor cells, suggesting its potential application as a prototype molecule for the development of new drugs with chemoprotective and/or antitumor effects.

Carbon nanotubes and nanofibers seen as emerging threat to fish: Historical review and trends.

Since the discovery of the third allotropic carbon form, carbon-based one-dimensional nanomaterials (1D-CNMs) became an attractive and new technology with different applications that range from electronics to biomedical and environmental technologies. Despite their broad application, data on environmental risks remain limited. Fish are widely used in ecotoxicological studies and biomonitoring programs. Thus, the aim of the current study was to summarize and critically analyze the literature focused on investigating the bioaccumulation and ecotoxicological impacts of 1D-CNMs (carbon nanotubes and nanofibers) on different fish species. In total, 93 articles were summarized and analyzed by taking into consideration the following aspects: bioaccumulation, trophic transfer, genotoxicity, mutagenicity, organ-specific toxicity, oxidative stress, neurotoxicity and behavioral changes. Results have evidenced that the analyzed studies were mainly carried out with multi-walled carbon nanotubes, which were followed by single-walled nanotubes and nanofibers. Zebrafish (Danio rerio) was the main fish species used as model system. CNMs' ecotoxicity in fish depends on their physicochemical features, functionalization, experimental design (e.g. exposure time, concentration, exposure type), as well as on fish species and developmental stage. CNMs' action mechanism and toxicity in fish are associated with oxidative stress, genotoxicity, hepatotoxicity and cardiotoxicity. Overall, fish are a suitable model system to assess the ecotoxicity of, and the environmental risk posed by, CNMs.

Colorectal Cancer Cell Invasion and Functional Properties Depend on Peri-Tumoral Extracellular Matrix.

We investigated how the extracellular matrix (ECM) affects LoVo colorectal cancer cells behavior during a spatiotemporal invasion. Epithelial-to-mesenchymal transition (EMT) markers, matrix-degrading enzymes, and morphological phenotypes expressed by LoVo-S (doxorubicin-sensitive) and higher aggressive LoVo-R (doxorubicin-resistant) were evaluated in cells cultured for 3 and 24 h on Millipore filters covered by Matrigel, mimicking the basement membrane, or type I Collagen reproducing a desmoplastic lamina propria. EMT and invasiveness were investigated with RT-qPCR, Western blot, and scanning electron microscopy. As time went by, most gene expressions decreased, but in type I Collagen samples, a strong reduction and high increase in MMP-2 expression in LoVo-S and -R cells occurred, respectively. These data were confirmed by the development of an epithelial morphological phenotype in LoVo-S and invading phenotypes with invadopodia in LoVo-R cells as well as by protein-level analysis. We suggest that the duration of culturing and type of substrate influence the morphological phenotype and aggressiveness of both these cell types differently. In particular, the type I collagen meshwork, consisting of large fibrils confining inter fibrillar micropores, affects the two cell types differently. It attenuates drug-sensitive LoVo-S cell aggressiveness but improves a proteolytic invasion in drug-resistant LoVo-R cells as time goes by. Experimental studies on CRC cells should examine the peri-tumoral ECM components, as well as the dynamic physical conditions of TME, which affect the behavior and aggressiveness of both drug-sensitive and drug-resistant LoVo cells differently.

Substrate Type and Concentration Differently Affect Colon Cancer Cells Ultrastructural Morphology, EMT Markers, and Matrix Degrading Enzymes.

Aim of the study was to understand the behavior of colon cancer LoVo-R cells (doxorubicin-resistant) vs. LoVo-S (doxorubicin sensitive) in the initial steps of extracellular matrix (ECM) invasion. We investigated how the matrix substrates Matrigel and type I collagen-mimicking the basement membrane (BM) and the normal or desmoplastic lamina propria, respectively-could affect the expression of epithelial-to-mesenchymal transition (EMT) markers, matrix-degrading enzymes, and phenotypes. Gene expression with RT-qPCR, E-cadherin protein expression using Western blot, and phenotypes using scanning electron microscopy (SEM) were analyzed. The type and different concentrations of matrix substrates differently affected colon cancer cells. In LoVo-S cells, the higher concentrated collagen, mimicking the desmoplastic lamina propria, strongly induced EMT, as also confirmed by the expression of Snail, metalloproteases (MMPs)-2, -9, -14 and heparanase (HPSE), as well as mesenchymal phenotypes. Stimulation in E-cadherin expression in LoVo-S groups suggests that these cells develop a hybrid EMT phenotype. Differently, LoVo-R cells did not increase their aggressiveness: no changes in EMT markers, matrix effectors, and phenotypes were evident. The low influence of ECM components in LoVo-R cells might be related to their intrinsic aggressiveness related to chemoresistance. These results improve understanding of the critical role of tumor microenvironment in colon cancer cell invasion, driving the development of new therapeutic approaches.

Metallated phthalocyanines and their hydrophilic derivatives for multi-targeted oncological photodynamic therapy.

BACKGROUND AND AIM: A photosensitizer (PS) delivery and comprehensive tumor targeting platform was developed that is centered on the photosensitization of key pharmacological targets in solid tumors (cancer cells, tumor vascular endothelium, and cellular and non-cellular components of the tumor microenvironment) before photodynamic therapy (PDT). Interstitially targeted liposomes (ITLs) encapsulating zinc phthalocyanine (ZnPC) and aluminum phthalocyanine (AlPC) were formulated for passive targeting of the tumor microenvironment. In previous work it was established that the PEGylated ITLs were taken up by cultured cholangiocarcinoma cells. The aim of this study was to verify previous results in cancer cells and to determine whether the ITLs can also be used to photosensitize cells in the tumor microenvironment and vasculature. Following positive results, rudimentary in vitro and in vivo experiments were performed with ZnPC-ITLs and AlPC-ITLs as well as their water-soluble tetrasulfonated derivatives (ZnPCS4 and AlPCS4) to assemble a research dossier and bring this platform closer to clinical transition. METHODS: Flow cytometry and confocal microscopy were employed to determine ITL uptake and PS distribution in cholangiocarcinoma (SK-ChA-1) cells, endothelial cells (HUVECs), fibroblasts (NIH-3T3), and macrophages (RAW 264.7). Uptake of ITLs by endothelial cells was verified under flow conditions in a flow chamber. Dark toxicity and PDT efficacy were determined by cell viability assays, while the mode of cell death and cell cycle arrest were assayed by flow cytometry. In vivo systemic toxicity was assessed in zebrafish and chicken embryos, whereas skin phototoxicity was determined in BALB/c nude mice. A PDT efficacy pilot was conducted in BALB/c nude mice bearing human triple-negative breast cancer (MDA-MB-231) xenografts. RESULTS: The key findings were that (1) photodynamically active PSs (i.e., all except ZnPCS4) were able to effectively photosensitize cancer cells and non-cancerous cells; (2) following PDT, photodynamically active PSs were highly toxic-to-potent as per anti-cancer compound classification; (3) the photodynamically active PSs did not elicit notable systemic toxicity in zebrafish and chicken embryos; (4) ITL-delivered ZnPC and ZnPCS4 were associated with skin phototoxicity, while the aluminum-containing PSs did not exert detectable skin phototoxicity; and (5) ITL-delivered ZnPC and AlPC were equally effective in their tumor-killing capacity in human tumor breast cancer xenografts and superior to other non-phthalocyanine PSs when appraised on a per mole administered dose basis. CONCLUSIONS: AlPC(S4) are the safest and most effective PSs to integrate into the comprehensive tumor targeting and PS delivery platform. Pending further in vivo validation, these third-generation PSs may be used for multi-compartmental tumor photosensitization.

Strategies for Improving Photodynamic Therapy Through Pharmacological Modulation of the Immediate Early Stress Response.

Photodynamic therapy (PDT) is a minimally to noninvasive treatment modality that has emerged as a promising alternative to conventional cancer treatments. PDT induces hyperoxidative stress and disrupts cellular homeostasis in photosensitized cancer cells, resulting in cell death and ultimately removal of the tumor. However, various survival pathways can be activated in sublethally afflicted cancer cells following PDT. The acute stress response is one of the known survival pathways in PDT, which is activated by reactive oxygen species and signals via ASK-1 (directly) or via TNFR (indirectly). The acute stress response can activate various other survival pathways that may entail antioxidant, pro-inflammatory, angiogenic, and proteotoxic stress responses that culminate in the cancer cell's ability to cope with redox stress and oxidative damage. This review provides an overview of the immediate early stress response in the context of PDT, mechanisms of activation by PDT, and molecular intervention strategies aimed at inhibiting survival signaling and improving PDT outcome.

Inhibition of the HIF-1 Survival Pathway as a Strategy to Augment Photodynamic Therapy Efficacy.

Photodynamic therapy (PDT) is a non-to-minimally invasive treatment modality that utilizes photoactivatable drugs called photosensitizers to disrupt tumors with locally photoproduced reactive oxygen species (ROS). Photosensitizer activation by light results in hyperoxidative stress and subsequent tumor cell death, vascular shutdown and hypoxia, and an antitumor immune response. However, sublethally afflicted tumor cells initiate several survival mechanisms that account for decreased PDT efficacy. The hypoxia inducible factor 1 (HIF-1) pathway is one of the most effective cell survival pathways that contributes to cell recovery from PDT-induced damage. Several hundred target genes of the HIF-1 heterodimeric complex collectively mediate processes that are involved in tumor cell survival directly and indirectly (e.g., vascularization, glucose metabolism, proliferation, and metastasis). The broad spectrum of biological ramifications culminating from the activation of HIF-1 target genes reflects the importance of HIF-1 in the context of therapeutic recalcitrance. This chapter elaborates on the involvement of HIF-1 in cancer biology, the hypoxic response mechanisms, and the role of HIF-1 in PDT. An overview of inhibitors that either directly or indirectly impede HIF-1-mediated survival signaling is provided. The inhibitors may be used as pharmacological adjuvants in combination with PDT to augment therapeutic efficacy.

In Vitro Evaluation of Acellular Collagen Matrices Derived from Porcine Pericardium: Influence of the Sterilization Method on Its Biological Properties.

The aim of this study were characterize acellular collagen matrices derived from porcine pericardium (PP) and to evaluate their properties after sterilization by ethylene oxide and gamma ray. PP matrices were subjected to alkaline hydrolysis (AH), and samples were characterized for biological stability, membrane thickness measurements, differential scanning calorimetry (DSC) and scanning electron microscopy (SEM). Subsequently, the matrices were frozen, lyophilized and sterilized by ethylene oxide or gamma radiation. For in vitro assays, CHO-K1 cell culture was used and evaluated for cytotoxicity, clonogenic survival assay, genotoxicity and mutagenicity. Analysis of variance (ANOVA) was used, followed by Dunnett's post-test, with a significance level of 5%. After AH, there was no significant change in matrix thickness. The relative biodegradability of the material after implantation was observed. Morphology and dimensions had small changes after AH. As for cell viability, none of the tested matrices showed a statistically significant difference (p > 0.05; Dunnett) regardless of the sterilization method. Furthermore, it was found that PP matrices did not interfere with the proliferation capacity of CHO-K1 cells (p > 0.05; Dunnett). As for genotoxicity, when sterilized with ethylene oxide (NP, P12 and P24), it showed genotoxic potential, but it was not genotoxic when sterilized by gamma radiation. No mutagenic effects were observed in either group. PP-derived collagen matrices hydrolyzed at different times were not cytotoxic. It is concluded that the best method of sterilization is through gamma radiation, since no significant changes were observed in the properties of the PP matrices.

Cholesterol-rich nanoemulsion (LDE) as a novel drug delivery system to diagnose, delineate, and treat human glioblastoma.

We have prepared and characterized a cholesterol-rich nanoemulsion called LDE, a mimic of classic lipoprotein macromolecules, that can be applied as a new drug delivery system for aluminum phthalocyanine chloride (PcAlCl). The LDE containing PcAlCl system prepared herein had mean size and zeta potential of 127 nm and -29 mV, respectively, and encapsulation rate efficiency was 81%, and stability of 17 months. Compared to classical liposomes, LDE was more efficient, especially in brain diseases like glioblastoma (GBM), as revealed by tests on the U-87 MG cell line. The LDEPc formulation did not display dark cytotoxicity, as expected. The best light dose for LDEPc was 1.0 J·cm-2: its activity was 55% higher than PcAlCl in a compatible organic medium. In the U-87 MG cells, apoptosis was the preferential pathway activated by PDT. These results strongly support the use of LDE as a new theranostic system.

Attritional evaluation of lipophilic and hydrophilic metallated phthalocyanines for oncological photodynamic therapy.

BACKGROUND AND AIM: Oncological photodynamic therapy (PDT) relies on photosensitizers (PSs) to photo-oxidatively destroy tumor cells. Currently approved PSs yield satisfactory results in superficial and easy-to-access tumors but are less suited for solid cancers in internal organs such as the biliary system and the pancreas. For these malignancies, second-generation PSs such as metallated phthalocyanines are more appropriate. Presently it is not known which of the commonly employed metallated phtahlocyanines, namely aluminum phthalocyanine (AlPC) and zinc phthalocyanine (ZnPC) as well as their tetrasulfonated derivatives AlPCS4 and ZnPCS4, is most cytotoxic to tumor cells. This study therefore employed an attritional approach to ascertain the best metallated phthalocyanine for oncological PDT in a head-to-head comparative analysis and standardized experimental design. METHODS: ZnPC and AlPC were encapsulated in PEGylated liposomes. Analyses were performed in cultured A431 cells as a template for tumor cells with a dysfunctional P53 tumor suppressor gene and EGFR overexpression. First, dark toxicity was assessed as a function of PS concentration using the WST-1 and sulforhodamine B assay. Second, time-dependent uptake and intracellular distribution were determined by flow cytometry and confocal microscopy, respectively, using the intrinsic fluorescence of the PSs. Third, the LC50 values were established for each PS at 671 nm and a radiant exposure of 15 J/cm2 following 1-h PS exposure. Finally, the mode of cell death as a function of post-PDT time and cell cycle arrest at 24 h after PDT were analyzed. RESULTS: In the absence of illumination, AlPC and ZnPC were not toxic to cells up to a 1.5-µM PS concentration and exposure for up to 72 h. Dark toxicity was noted for AlPCS4 at 5 µM and ZnPCS4 at 2.5 µM. Uptake of all PSs was observed as early as 1 min after PS addition to cells and increased in amplitude during a 2-h incubation period. After 60 min, the entire non-nuclear space of the cell was photosensitized, with PS accumulation in multiple subcellular structures, especially in case of AlPC and AlPCS4. PDT of cells photosensitized with ZnPC, AlPC, and AlPCS4 yielded LC50 values of 0.13 µM, 0.04 µM, and 0.81 µM, respectively, 24 h post-PDT (based on sulforhodamine B assay). ZnPCS4 did not induce notable phototoxicity, which was echoed in the mode of cell death and cell cycle arrest data. At 4 h post-PDT, the mode of cell death comprised mainly apoptosis for ZnPC and AlPC, the extent of which was gradually exacerbated in AlPC-photosensitized cells during 8 h. ZnPC-treated cells seemed to recover at 8 h post-PDT compared to 4 h post-PDT, which had been observed before in another cell line. AlPCS4 induced considerable necrosis in addition to apoptosis, whereby most of the cell death had already manifested at 2 h after PDT. During the course of 8 h, necrotic cell death transitioned into mainly late apoptotic cell death. Cell death signaling coincided with a reduction in cells in the G0/G1 phase (ZnPC, AlPC, AlPCS4) and cell cycle arrest in the S-phase (ZnPC, AlPC, AlPCS4) and G2 phase (ZnPC and AlPC). Cell cycle arrest was most profound in cells that had been photosensitized with AlPC and subjected to PDT. CONCLUSIONS: Liposomal AlPC is the most potent PS for oncological PDT, whereas ZnPCS4 was photodynamically inert in A431 cells. AlPC did not induce dark toxicity at PS concentrations of up to 1.5 µM, i.e., > 37 times the LC50 value, which is favorable in terms of clinical phototoxicity issues. AlPC photosensitized multiple intracellular loci, which was associated with extensive, irreversible cell death signaling that is expected to benefit treatment efficacy and possibly immunological long-term tumor control, granted that sufficient AlPC will reach the tumor in vivo. Given the differential pharmacokinetics, intracellular distribution, and cell death dynamics, liposomal AlPC may be combined with AlPCS4 in a PS cocktail to further improve PDT efficacy.

Extracellular Matrix-Mediated Breast Cancer Cells Morphological Alterations, Invasiveness, and Microvesicles/Exosomes Release.

Breast cancer is a leading disease in women. Several studies are focused to evaluate the critical role of extracellular matrix (ECM) in various biochemical and molecular aspects but also in terms of its effect on cancer cell morphology and therefore on cancer cell invasion and metastatic potential. ECM fibrillar components, such as collagen and fibronectin, affect cell behavior and properties of mammary cancer cells. The aim of this study was to investigate using the scanning electron microscopy (SEM) how the highly invasive MDA-MB-231 breast cancer cells, interplaying with ECM substrates during cell migration/invasion, modify their morphological characteristics and cytoplasmic processes in relation to their invasive potential. In particular we reproduced and analyzed how natural structural barriers to cancer cell invasion, such as the basement membrane (Matrigel) and fibrillar components of dermis (fibronectin as well as the different concentrations/array of type I collagen), could induce morphological changes in 3D cultures. Interestingly, we demonstrate that, even with different effects, all collagen concentrations/arrays lead to morphological alterations of breast cancer cells. Intriguingly, the elongated mesenchymal shaped cells were more prominent in 3D cultures with a dense and thick substrate (thick Matrigel, high concentrated collagen network, and densely packed collagen fibers), even though cells with different shape produced and released microvesicles and exosomes as well. It is therefore evident that the peri-tumoral collagen network may act not only as a barrier but also as a dynamic scaffold which stimulates the morphological changes of cancer cells, and modulates tumor development and metastatic potential in breast cancer.

The redox function of apurinic/apyrimidinic endonuclease 1 as key modulator in photodynamic therapy.

Photodynamic therapy (PDT) is an anticancer modality depicting an induced oxidative stress as the mechanism of action that ultimately culminates in cell death. The apurinic/apyrimidinic endonuclease 1/redox factor-1 (APE1/Ref-1) is a key protein promoting bad prognostic in several cancer types. APE1/Ref-1 is able to regulate cell response to oxidative stress by two basic protein activities, including a reduction-oxidation-function and a DNA repair-function. Therefore, the efficacy of anticancer therapies is negatively affected by APE1-overexpression. Thus, here it was evaluated the potential of APE1-chemical inhibitors as sensitizers for PDT in two different cancer cell lines (A549 and HeLa cells). Both functions of APE1 were addressed using E3330 (redox-function) and CRT0044876 (DNA repair-function) molecules. A detailed cytotoxicity screening (cell viability, cell cycle kinetics, mitochondrial perturbation, and cell death) indicated HeLa cells as extremely sensitive (~ 3.5×) to the combination of PDT with E3330 when compared to A549 cells. The treatment using PDT with E3330 induced downregulation of APE1 as detected by Western Blot. The APE1's downregulation correlated to an increase of DNA fragmentation (17% and 66% in A549 and HeLa cells, respectively) and cell death rate (total: 24% and 74% in A549 and HeLa cells, respectively) characterized by annexin V and 7-AAD markers as well as a considerable difference in superoxide detected in mitochondria (29% and 78% in A549 and HeLa cells, respectively). This study definitively detected an increase in PDT efficacy when APE1's redox function is dysregulated by E3330.

Toxicity of therapeutic contact lenses based on bacterial cellulose with coatings to provide transparency.

Therapeutic contact lenses were developed from bacterial cellulose (BC) by the Institute of Chemistry at Brazil's São Paulo State University (UNESP). In a previous study, cyclodextrins (CD) and medications such as ciprofloxacin (CP) and diclofenac sodium (DS) were incorporated into the lenses to provide therapeutic properties and control drug release. However, significant opacity was seen in the material inherent to cellulose. In order to achieve full material transparency, the lenses were coated with an organic-inorganic hybrid compound containing aluminum alkoxide and glycidoxypropyltrimethoxysilane (GPTS)(H), or chitosan (Q) nanoparticles. This study evaluated the toxicity of these contact lenses to ensure the safety of these materials for future availability to the medical device industry. Lenses composed of BC and coated with either GPTS (H) or chitosan (Q), incorporating ciclodextrin (CD) to release diclofenac sodium (DS) or ciprofloxacin (CP), were submitted to cytotoxicity assays (XTT and Clonogenic Survival), genotoxicity (Comet Assay) and mutagenicity (Cytokinesis-blocked micronucleus assay) directly in cell culture. Statistical analyses were performed using the Tukey and Dunnett or Kruskal-Wallis and Dunn tests. All of the nanoparticles used in the lense coatings did not show cytotoxic effects by the XTT test (p > 0.05; Dunnett). Only materials associated with diclofenac sodium (BC-H-CD-DS and BC-Q-CD-DS) presented significantly different survival fractions compared to negative control (p < 0.001; Dunnett). Genotoxicity evaluation revealed a genotoxic effect in BC-H-CD-DS (p < 0.05; Dunn). All tested lenses did not present any mutagenic effect. These results indicate that improvements in DS incorporation are needed to eliminate toxicity. We demonstrated promising results in the safety of employing BC lenses functionalized with a drug delivery system permitting the bioavailability of ophthalmic drugs. Further studies utilizing other specific tests, such as corneal lineage are required before safe and efficient ophthalmologic use.

Silver nanoparticles: An integrated view of green synthesis methods, transformation in the environment, and toxicity.

Nowadays, silver nanoparticles (AgNPs) are the most widely used nanoparticles (NPs) in the industry due to their peculiar biocidal features. However, the use of these NPs still runs into limitations mainly because of the low efficiency of environmental friendly synthesis methods and lack of size standardization. When NPs are release in the environment, they can be transformed by oxidation, adsorption or aggregation. These modification shows a dual role in toxic response of AgNPs. The adsorption of natural organic matter from environment on AgNPs, for example, can decrease their toxicity. Otherwise oxidation occurred in the environment is also able to increase the release of toxic Ag+ from NPs. Thus, the current review proposes an integrated approach of AgNP synthetic methods using bacteria, fungi, and plants, AgNP cytotoxic and genotoxic effects as well as their potential therapeutic applications are also presented.

Combining Photodynamic Therapy and Chemotherapy: Improving Breast Cancer Treatment with Nanotechnology.

Nanomedical approaches are the major transforming factor in cancer therapies. Based on important previous works in the field of drug delivery nanomaterials, recent years have brought a broad array of new and improved intelligent nanoscale platforms that are suited to deliver drugs. In this context, the purpose of this study was to investigate the action of different nanoemulsions designed to encapsulate chloroaluminum phthalocyanine, a hydrophobic photosensitizer used in photodynamic therapy, and doxorubicin, a well-known chemotherapeutic agent used to treat aggressive breast cancer cells. The mean nanostructured system size ranged from 170.8 to 181.0 nm, and the nanoemulsions presented spherical morphology. All formulations exhibited negative zeta potential values (-68.7 to -75.0 mV) and suitable polydispersity values (0.20 to 0.28), explaining their colloidal stability up to three months. Murine breast cancer cells (4T1) were incubated with nanoemulsions for three hours at various concentrations and were subjected to cell viability tests to find the concentration dependence profile. Thereafter, the in vitro phototoxic effect was evaluated in the presence of the visible laser light irradiation. Less than 10% of 4T1 viable cells were observed when photodynamic therapy and chemotherapy were combined at a 1.0 J · cm-2 laser light dose with 1.0 µM phthalocyanine and 0.5 µM doxorubicin. The cell death assay and cell cycle arrest analysis confirmed the therapy efficiency demonstrating an increase in the apoptosis rate and in the cell cycle arrest on G2. Additionally, 15 genes related to apoptosis and 25 target genes of anti-cancer drugs were overexpressed. Four genes related to apoptosis and four target genes of anti-cancer drugs were downregulated in 4T1 cells after treatment with nanoemulsion with phthalocyanine and doxorubicin associated with photodynamic therapy. Thus, the nanoemulsions loaded with phthalocyanine and doxorubicin presented appropriate physical stability, improved photophysical properties, and remarkable activity in vitro to be considered as promising formulations for photodynamic therapy and chemotherapeutic use in breast cancer treatment.

Detection of DNA Damage Induced by Cerium Dioxide Nanoparticles: From Models to Molecular Mechanism Activated.

This chapter will present an original effort to summarize the relevant data about the cyto-genotoxicity induced by cerium dioxide nanoparticles (nanoceria) in physiologically (in vivo and in vitro) relevant models. In this way, this chapter should be extremely useful to everyone who wants to plan their research and publishing their results. Massive application of nanoceria at different fields is increasing year after year, and it is urgent to address and discuss their use and its safety-related issues. Specifically, the nanoceria are being designed for nanomedicine, cosmetics, polishing materials and additives for automotive fuels. Their unique properties include the ability to absorb UV radiation, antioxidant potential and the rapid exchange of valence between Ce4+ and Ce3+ ions associated to oxygen storage. In this chapter, the state of the art regarding the physicochemical properties of nanoceria, nanogenotoxicity detected by in vitro and in vivo systems and the general aspects in the cyto-genotoxic mechanism of nanoceria are summarized. The cyto-genotoxicity will be discussed in terms of evaluations by Comet assay, Micronucleus test, DNA damage response and oxidative stress detected in cell culture systems and in vivo test. We also described the dose dependent cyto-genotoxic effects of nanoceria based on their physical-chemical nature. Paradoxically, these particles have been characterized as either pro-oxidant or anti-oxidant in dependence of microenvironment and physiological conditions such as pH. Finally, this chapter will contribute to point out aspects of the development of new in vitro and in vivo methodologies to detect cyto-genotoxic effects of the nanoceria.

Toxic and Beneficial Potential of Silver Nanoparticles: The Two Sides of the Same Coin.

Nanotechnology has allowed great changes in chemical, biological and physical properties of metals when compared to their bulk counterparts. Within this context, silver nanoparticles (AgNPs) play a major role due to their unique properties, being widely used in daily products such as fabrics, washing machines, water filters, food and medicine. However, AgNPs can enter cells inducing a "Trojan-horse" type mechanism which potentially leads to cellular autophagy, apoptosis or necrosis. On the other hand, this cytotoxicity mechanism can be optimized to develop drug nanocarriers and anticancer therapies. The increasing use of these NPs entails their release into the environment, damaging ecosystems balance and representing a threat to human health. In this context, the possible deleterious effects that these NPs may represent for the biotic and abiotic ecosystems components represent an obstacle that must be overcome in order to guarantee the safety use of their unique properties.

DNA polymeric films as a support for cell growth as a new material for regenerative medicine: Compatibility and applicability.

DNA polymeric films (DNA-PFs) are a promising drug delivery system (DDS) in modern medicine. In this study, we evaluated the growth behavior of oral squamous cell carcinoma (OSCC) cells on DNA-PFs. The morphological, biochemical, and cytometric features of OSCC cell adhesion on DNA-PFs were also assessed. An initial, temporary alteration in cell morphology was observed at early time points owing to the inhibition of cell attachment to the film, which then returned to a normal morphological state at later time points. MTT and resazurin assays showed a moderate reduction in cell viability related to increased DNA concentration in the DNA-PFs. Flow cytometry studies showed low cytotoxicity of DNA-PFs, with cell viabilities higher than 90% in all the DNA-PFs tested. Flow cytometric cell cycle analysis also showed average cell cycle phase distributions at later time points, indicating that OSCC cell growth is maintained in the presence of DNA-PFs. These results show high biocompatibility of DNA-PFs and suggest their use in designing "dressing material," where the DNA film acts as a support for cell growth, or with incorporation of active or photoactive compounds, which can induce tissue regeneration and are useful to treat many diseases, especially oral cancer.

Light source is critical to induce glioblastoma cell death by photodynamic therapy using chloro-aluminiumphtalocyanine albumin-based nanoparticles.

Selection of an efficient light source is fundamental in the development of photodynamic therapy (PDT) protocols. However, few studies provide a comparison of different light sources with regard to phototoxic effects. Here, we compared the cell death induced by photoactivation of chloro-aluminiumphtalocyanine (AlClPc)-loaded human serum albumin nanoparticles under irradiation with different light sources: continuous laser (CL), pulsed laser (PL), and light-emitting diode (LED). Cells were exposed to three different AlClPc concentrations (1, 3, and 5µM) and three different light doses (200, 500, and 700mJ/cm2) for each light source. Cell death and differentiation of apoptosis and necrosis pathway were measured by flow cytometry. CL was the best light source for improving the photodynamic action of AlClPc-loaded albumin nanoparticles in glioblastoma cells and avoiding undesirable side effects, especially at low photosensitizer doses (200mJ/cm2). In addition, apoptosis was the main cell death pathway in all evaluated cases (70% for CL, and greater than 50% for PL and LED). In conclusion, the search for optimal light sources and light/photosensitizer doses is a crucial step in improving PDT outcomes and enhancing the clinical translation of PDT.

Nanocellulose-collagen-apatite composite associated with osteogenic growth peptide for bone regeneration.

Despite advances in the field of biomaterials for bone repair/regeneration, some challenges for developing an ideal bone substitute need to be overcome. Herein, this study synthesized and evaluated in vitro a nanocomposite based on bacterial cellulose (BC), collagen (COL), apatite (Ap) and osteogenic growth peptide (OGP) or its C-terminal pentapeptide [OGP(10-14)] for bone regeneration purposes. The BC-COL nanocomposites were successfully obtained by carbodiimide-mediated coupling as demonstrated by spectroscopy analysis. SEM, FTIR and 31P NMR analyses revealed that in situ synthesis to apatite was an effective route for obtaining of bone-like apatite. The OGP-containing (BC-COL)-Ap stimulated the early development of the osteoblastic phenotype. Additionally, the association among collagen, apatite, and OGP peptides enhanced cell growth compared with OGP-containing BC-Ap. Furthermore, none of the nanocomposites showed cytotoxic, genotoxic or mutagenic effects. These promising results suggest that the (BC-COL)-Ap associated with OGP peptides might be considered a potential candidate for bone tissue engineering applications.