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Background. Development of pre-clinical models of long-lasting inflammation is needed for the study of inflammatory diseases. Osteoarthritis (OA), the most common inflammatory joint pathology, is characterized by an abundance of M1-like macrophages producing several pro-inflammatory cytokines and proteolytic enzymes damaging the local tissues. Here we developed a novel in vitro model of long-lasting inflammation using primary human monocyte-derived macrophages continuously cultured for 15 days. Results. We first screened synovial fluids from 11 OA patients using multiplex Ella® and we found measurable levels of IL-6, CCL2, CCL5, CXCL8, CXCL10, TNFa, IL-1b, using them as read-outs of the following experiments. We then found an increased survival of macrophages differentiated with both M-CSF and GM-CSF (M-/GM-Mf) compared either with macrophages differentiated with M-CSF alone (M-Mf) or macrophages differentiated with GM-CSF alone (GM-Mf) and, consequently, decided to use them to set up the in vitro model of long-lasting inflammation. Consistently with the increased survival, the repeated stimulation of M-/GM-Mfs either with lipopolysaccharide + interferon-g (LPS+IFN-g) or Pam3CysSerLys4 (Pam3CSK4) led to the sustained production of IL-6, CCL2 and CXCL8 over time. Finally, to investigate the usefulness of our model for OA research, we repeatedly exposed stimulated macrophages to dexamethasone (DEX) and/or celecoxib (CEL), two anti-inflammatory drugs commonly used for OA therapy. We found an excellent anti-inflammatory activity but significant toxicity of DEX for concentrations over 10 nM, while CEL was not toxic and efficiently inhibited PGE2 secretion. Of interest, the inflammatory profile of macrophages differentiated with M-CSF or GM-CSF and activated with the two pro-inflammatory stimuli (LPS+IFN-g vs Pam3CSK4) was not identical, overall showing production of more inflammatory mediators with GM-CSF-macrophages stimulated with Pam3CSK4. Conclusions. We introduce here a novel easy-to-use in vitro culture model of long-lasting inflammation using primary human macrophages, that could be useful for the screening of new compounds and drug delivery systems, to improve the therapy of inflammatory disorders.

Tumor-associated macrophages (TAMs) play a key immunosuppressive role that limits the ability of the immune system to fight cancer and hinder the anti-tumoral efficacy of most treatments currently applied in the clinic. However, a key feature of macrophages is their phenotypical and functional plasticity, which called their attention as promising targets for therapeutic intervention based on their elimination or reprogramming toward M1-like cytotoxic effector cells, with anti-tumor functions. This polarization status of macrophages can be studied in terms of molecular markers and functional activities, using an appropriate combination of experimental methodologies, both in vitro and in vivo. Here we focus on describing in vitro protocols to isolate primary monocytes from buffy coats and to study macrophage phenotype and function, after exposure to new therapies, by a combination of flow cytometry, RT-PCR, and ELISA analysis. We also provide the methodology to evaluate in vitro the cytotoxic activity of treated macrophages toward cancer cells.

In most solid cancers, tumor-associated macrophages (TAMs) infiltrating the tumor microenvironment (TME) represent a major population of immunosuppressive cells. This correlates with poor prognosis and resistance to antitumoral therapies, including immune checkpoint inhibitors. Although initial preclinical studies were primarily meant to deplete macrophages in the TME or prevent their recruitment at tumor sites, recent evidence has indicated that the reprogramming of macrophages into cytotoxic effectors might be more beneficial in eliciting an effective antitumor immune response. Taking this into consideration, the comprehensive analysis of the phenotype and function of macrophages in the TME, and their interaction with cancer cells or other immune cells, has become of paramount importance in oncological research. Accordingly, here we explain the experimental procedures for the in vivo evaluation of tumor progression and response to therapy, with a particular focus on the detailed analysis of TAMs and related immune cells in the TME by flow cytometry, RNA analysis, and multiplex immunophenotyping. The output generated through these experiments allow researchers to test the efficacy of new therapeutic strategies on targeting.

The stroma of most solid tumors is populated by myeloid cells, which mostly represent macrophages. Tumor-associated macrophages (TAMs), strongly influenced by cancer cell–derived factors, are key drivers of immunosuppression and support tumor growth and spread to distant sites. Their accurate quantification and characterization in the tumor microenvironment are gaining prognostic value: increasing evidence demonstrates their ability to hamper cancer patients' response to chemotherapy, as well as to immunotherapies based on checkpoint inhibition. Therefore, strategies to counteract their negative effects are nowadays gaining momentum at preclinical, translational, and clinical levels. Our knowledge of the biology of TAMs has greatly advanced in the last years; several strategies to target and reprogram their functions to become antitumor effectors have proven successful in experimental preclinical tumor models; on the other hand, few approaches have so far been effectively translated into clinic practice. A growing interest in the therapeutic manipulation of TAMs is evidenced by numerous early-phase clinical trials, which are continuously fueled by new discoveries from basic research. This gives us hope that the targeting and sustained reprogramming of TAMs will be more specific to synergize with current therapies and maximize antitumor responses in patients.

Pancreatic ductal adenocarcinoma (PDAC) represents one of the deadliest malignancies worldwide. The lack of clinical symptoms in the early stages of the disease and the poor immunogenicity of PDAC are two major causes of late diagnosis and inefficient therapies. The tumor microenvironment of PDAC is rich in stromal cells inducing desmoplasia, complicating the arrival of medical compounds, as well as in immune cells, that are suppressed by several mediators produced by cancer cells. In this setting, it has been shown that some medications can restore the antitumoral activity of innate immune cells that, in turn, re-activate also adaptive immune cells. In this study, we present the combination of the TLR agonists poly(I:C) (pIC - TLR3) and resiquimod (R848 - TLR7/8) as an immunotherapeutic tool in PDAC. In a subcutaneous model of PDAC (K8484 cells, isolated from KPC mice that spontaneously develop PDAC and recapitulate the features of human pancreatic cancer), the intratumoral administration of pIC+R848 for 5 times every 2-3 days led to the complete regression of the tumor in 100% of the treated mice. Moreover, all mice were protected from a second and a third rechallenge with the same cell line, performed months after the first treatments. Mechanistic studies performed on the tumors with flow cytometry demonstrated an increase of MHC-II positive cells in the monocytic (Ly6C+) compartment, as well as an increase in the number of mature CD8+ T cells and NK cells. Similar findings were demonstrated also in another heterotopic model obtained injecting Panc02 cells, another cell line with a sarcomatous phenotype. Consistently with the flow cytometry data, the depletion of CD4+ and CD8+ T cells abolished the antitumoral efficacy of pIC+R848, while the depletion of NK cells and CSF-1R+ cells was not sufficient to abolish the antitumoral response, suggesting a strong involvement of the adaptive immunity. In line with these findings, the intratumoral administration of pIC+R848 in mice unable to produce IFN-γ failed to reduce the tumor growth. In conclusion, our work clearly demonstrates that pIC+R848 are an effective treatment for PDAC when injected intratumorally and that this activity strongly relies on IFN-γ and adaptive immunity. Further investigations are ongoing to assess their potential in orthotopic models using different routes of administration, in a setting closer to the real clinical situation. Citation Format: Aldo Ummarino, Clément Anfray, Andrea Mariancini, Domenico Supino, Cecilia Garlanda, Fernando Torres Andòn, Alberto Mantovani, Paola Allavena. Combined administration of poly(I:C) and resiquimod triggers effective antitumoral response in mouse models of pancreatic adenocarcinoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5140.

CERVOXY COLL; International audience; The O2 and CO2 sorption properties of nanosized zeolite X with faujasite type structure through a partial ionic exchange of sodium (Na +) by trivalent cations (Gd 3+ and Ce 3+) were evaluated. Three faujasite samples were studied, the as-synthesized Na-X possessing Na + solely, and the modified samples Na-Gd-X and Na-Ce-X containing Gd 3+ (1.8 wt%) and Ce 3+ (0.82 wt%), respectively. Incorporating scarce amounts of trivalent cations modified the adsorption affinity of zeolites towards O2 and CO2 as demonstrated by in situ Fourier-transform infrared spectroscopy (FTIR). While Na-Ce-X encounters the highest O2 physisorption capacity, the Na-Gd-X is adsorbing the highest quantities of molecular CO2. All three samples exhibit the chemisorbed CO2 in the form of carbonates, while the Na-X stores carbonates in monodentate and polydentate forms, the Na-Gd-X and Na-Ce-X allow the formation of polydentate carbonates only. Density functional theory (DFT) calculations revealed that trivalent cations tend to adsorb gases through two cations simultaneously which explains the presence of polydentate carbonates exclusively in the corresponding modified zeolites. The DFT results confirmed the higher affinity of Na-Gd-X and Na-Ce-X nanocrystals towards O2 in the presence of CO2. The affinity of Na-Gd-X and Na-Ce-X nanocrystals towards O2 opens the door of their use as oxygen transporters for medical applications where CO2 is constantly present. The toxicity of the nanosized zeolites and their performance in O2 release are reported too.

Tumor-associated macrophages (TAMs), a class of immune cells that play a key role in tumor immunosuppression, are recognized as important targets to improve cancer prognosis and treatment. Consequently, the engineering of drug delivery nanocarriers that can reach TAMs has acquired special relevance. This work describes the development and biological evaluation of a panel of hyaluronic acid (HA) nanocapsules (NCs), with different compositions and prepared by different techniques, designed to target macrophages. The results showed that plain HA NCs did not significantly influence the polarization of M0 and M2-like macrophages towards an M1-like pro-inflammatory phenotype; however, the chemical functionalization of HA with mannose (HA-Man) led to a significant increase of NCs uptake by M2 macrophages in vitro and to an improved biodistribution in a MN/MNCA1 fibrosarcoma mouse model with high infiltration of TAMs. These functionalized HA-Man NCs showed a higher accumulation in the tumor compared to non-modified HA NCs. Finally, the pre-administration of the liposomal liver occupying agent Nanoprimer™ further increased the accumulation of the HA-Man NCs in the tumor. This work highlights the promise shown by the HA-Man NCs to target TAMs and thus provides new options for the development of nanomedicine and immunotherapy-based cancer treatments Open Access funding provided thanks to the CRUE-CSIC agreement with Springer Nature. This work was supported by the 2^2-INTRATARGET project (A20/00028) funded by the ISCIII under the umbrella of the ERA NET EuroNanoMed GA N 723770 of the EU Horizon 2020 Research and Innovation Programme. This work was also supported by the Xunta de Galicia (ED431C 2018/30, and “Centro singular de investigación de Galicia” accreditation 2019 − 2022, ED431G2019/03), and the European Union (European Regional Development Fund-ERDF) SI

BackgroundTumor-associated macrophages (TAMs) play a key immunosuppressive role that limits the ability of the immune system to fight cancer and hinder the antitumoral efficacy of most treatments currently applied in the clinic. Previous studies have evaluated the antitumoral immune response triggered by (TLR) agonists, such as poly(I:C), imiquimod (R837) or resiquimod (R848) as monotherapies; however, their combination for the treatment of cancer has not been explored. This study investigates the antitumoral efficacy and the macrophage reprogramming triggered by poly(I:C) combined with R848 or with R837, versus single treatments.MethodsTLR agonist treatments were evaluated in vitro for toxicity and immunostimulatory activity by Alamar Blue, ELISA and flow cytometry using primary human and murine M-CSF-differentiated macrophages. Cytotoxic activity of TLR-treated macrophages toward cancer cells was evaluated with an in vitro functional assay by flow cytometry. For in vivo experiments, the CMT167 lung cancer model and the MN/MCA1 fibrosarcoma model metastasizing to lungs were used; tumor-infiltrating leukocytes were evaluated by flow cytometry, RT-qPCR, multispectral immunophenotyping, quantitative proteomic experiments, and protein–protein interaction analysis.ResultsResults demonstrated the higher efficacy of poly(I:C) combined with R848 versus single treatments or combined with R837 to polarize macrophages toward M1-like antitumor effectors in vitro. In vivo, the intratumoral synergistic combination of poly(I:C)+R848 significantly prevented tumor growth and metastasis in lung cancer and fibrosarcoma immunocompetent murine models. Regressing tumors showed increased infiltration of macrophages with a higher M1:M2 ratio, recruitment of CD4+ and CD8+ T cells, accompanied by a reduction of immunosuppressive CD206+ TAMs and FOXP3+/CD4+ T cells. The depletion of both CD4+ and CD8+ T cells resulted in complete loss of treatment efficacy. Treated mice acquired systemic antitumoral response and resistance to tumor rechallenge mediated by boosted macrophage cytotoxic activity and T-cell proliferation. Proteomic experiments validate the superior activation of innate immunity by poly(I:C)+R848 combination versus single treatments or poly(I:C)+R837, and protein–protein-interaction network analysis reveal the key activation of the STAT1 pathway.DiscussionThese findings demonstrate the antitumor immune responses mediated by macrophage activation on local administration of poly(I:C)+R848 combination and support the intratumoral application of this therapy to patients with solid tumors in the clinic.

CERVOXY COLL; International audience; A growing interest is currently directed to find an efficient NO and CO2 gas carriers for biomedical applications. The gas adsorption properties of sodium- (Na-X) and copper- (Cu-X) containing FAU nanozeolites toward nitric oxide and carbon dioxide were studied. The materials were fully characterised by XRD, TEM, ICP, DLS and in-situ FTIR. The as-prepared Na-X showed higher gas adsorption ability towards carbon dioxide, whereas the ion-exchanged Cu-X was more efficient adsorbent with regards to nitric oxide. In addition, the cytotoxicity tests disclosed that both nanozeolites have no toxicity making them suitable for further tests in biomedical field as gas transporters.

Simple Summary Glioblastoma (GBM) is one of the greatest challenges facing neuro-oncology today. Current treatments are far from satisfactory and, given the poor prognosis of the disease, therapeutic efforts are focused on palliative management rather than curative intervention. Here, we review the cellular heterogeneity of GBM, including tumor cells and microglia/macrophages among others, as well as the genetic, epigenetic and metabolic alterations controlling its initiation and progression. Then, we describe the genetic and xenotransplantation zebrafish models established in the last few years for the study of GBM physiopathology and for testing new drugs to improve the treatment of the disease. Taking this information into account, forthcoming studies using zebrafish models of GBM are expected to shed light on better diagnosis and treatments, thus providing hope for GBM patients. Abstract Glioblastoma (GBM) is the most common of all brain malignant tumors; it displays a median survival of 14.6 months with current complete standard treatment. High heterogeneity, aggressive and invasive behavior, the impossibility of completing tumor resection, limitations for drug administration and therapeutic resistance to current treatments are the main problems presented by this pathology. In recent years, our knowledge of GBM physiopathology has advanced significantly, generating relevant information on the cellular heterogeneity of GBM tumors, including cancer and immune cells such as macrophages/microglia, genetic, epigenetic and metabolic alterations, comprising changes in miRNA expression. In this scenario, the zebrafish has arisen as a promising animal model to progress further due to its unique characteristics, such as transparency, ease of genetic manipulation, ethical and economic advantages and also conservation of the major brain regions and blood–brain–barrier (BBB) which are similar to a human structure. A few papers described in this review, using genetic and xenotransplantation zebrafish models have been used to study GBM as well as to test the anti-tumoral efficacy of new drugs, their ability to interact with target cells, modulate the tumor microenvironment, cross the BBB and/or their toxicity. Prospective studies following these lines of research may lead to a better diagnosis, prognosis and treatment of patients with GBM.

In the last decade, it has been well-established that tumor-infiltrating myeloid cells fuel not only the process of carcinogenesis through cancer-related inflammation mechanisms, but also tumor progression, invasion, and metastasis. In particular, tumor-associated macrophages (TAMs) are the most abundant leucocyte subset in many cancers and play a major role in the creation of a protective niche for tumor cells. Their ability to generate an immune-suppressive environment is crucial to escape the immune system and to allow the tumor to proliferate and metastasize to distant sites. Conventional therapies, including chemotherapy and radiotherapy, are often not able to limit cancer growth due to the presence of pro-tumoral TAMs; these are also responsible for the failure of novel immunotherapies based on immune-checkpoint inhibition. Several novel therapeutic strategies have been implemented to deplete TAMs; however, more recent approaches aim to use TAMs themselves as weapons to fight cancer. Exploiting their functional plasticity, the reprogramming of TAMs aims to convert immunosuppressive and pro-tumoral macrophages into immunostimulatory and anti-tumor cytotoxic effector cells. This shift eventually leads to the reconstitution of a reactive immune landscape able to destroy the tumor. In this review, we summarize the current knowledge on strategies able to reprogram TAMs with single as well as combination therapies F.T.A. was supported by the AECC (“Asociación Española Contra el Cáncer, Spain). E.D. was supported by AIRC (Associazione Italiana per la Ricerca contro il Cancro) SI

Tumor hypoxia is known to limit the efficacy of ionizing radiations, a concept called oxygen enhancement ratio (OER). OER depends on physical factors such as pO2 and linear energy transfer (LET). Biological pathways, such as the hypoxia-inducible transcription factors (HIF), might also modulate the influence of LET on OER. Glioblastoma (GB) is resistant to low-LET radiation (X-rays), due in part to the hypoxic environment in this brain tumor. Here, we aim to evaluate in vitro whether high-LET particles, especially carbon ion radiotherapy (CIRT), can overcome the contribution of hypoxia to radioresistance, and whether HIF-dependent genes, such as erythropoietin (EPO), influence GB sensitivity to CIRT. Hypoxia-induced radioresistance was studied in two human GB cells (U251, GL15) exposed to X-rays or to carbon ion beams with various LET (28, 50, 100&thinsp, keV/&micro, m), and in genetically-modified GB cells with downregulated EPO signaling. Cell survival, radiobiological parameters, cell cycle, and ERK activation were assessed under those conditions. The results demonstrate that, although CIRT is more efficient than X-rays in GB cells, hypoxia can limit CIRT efficacy in a cell-type manner that may involve differences in ERK activation. Using high-LET carbon beams, or targeting hypoxia-dependent genes such as EPO might reduce the effects of hypoxia.

The therapeutic manipulation of the immune system is the most promising strategy for treating endemic chronic diseases of our time, such as cancer or autoimmune diseases. However, the key challenge for the clinical implementation of immunotherapies remains the controlled modulation of the immune system, to achieve therapeutic activity without off-target effects. A successful response to immunotherapy requires administration of the right immunomodulator dose during the right time at the right tissular, cellular, and even intracellular location. Biomedical nanotechnology, intended as engineering of advanced biomaterials and drug delivery systems, is especially well-suited to target the immune system in a controlled manner. This chapter reviews a series of colloidal nanoparticles (NPs) to improve the ability of immunomodulatory molecules to reach disease tissues, immune cells, or their intracellular compartments, in the context of chronic immune disorders. The applications of nano-immuno-oncological therapies, aimed at improving the ability of the patient's own immune system to recognize and kill cancer cells, are herein presented in several categories of NPs to enhance the efficacy of: (i) cancer vaccines including peptides, adjuvants, or even nucleic acids such as mRNA; (ii) immune checkpoint inhibitors or other monoclonal antibodies; and (iii) small drugs or other nano-immunostrategies to reprogram the tumor microenvironment. In the context of autoimmune diseases, we describe nano-immunotherapies for the treatment of Alzheimer disease, diabetes, inflammatory bowel disease, systemic lupus erythematosus, multiple sclerosis, and rheumatoid arthritis. Taking into consideration the biological basis of each disease, we discuss the opportunities and challenges of nano-immunotherapies for their application in the clinic, as an emerging area with enormous potential to the benefit of patients.

CERVOXY; International audience; Approaches able to counteract, at least temporarily, hypoxia, a well-known factor of resistance to treatment in solid tumors are highly desirable. Herein, we report the use of nanosized zeolite crystals as hyperoxic/hypercapnic gas carriers for glioblastoma. First, the non-toxic profile of nanosized zeolite crystals in living animals (mice, rats and non-human primates) and in various cell types is presented. Second, the ability of the nanosized zeolites to act as a vasoactive agent for a targeted re-oxygenation of the tumor after intravenous injection is shown. As attested by an MRI protocol, the zeolites were able to increase oxygenation and blood volume specifically within the brain tumor whilst no changes in the healthy-non tumoral brain-were observed. The first proof of concept for the use of metal-containing nanosized zeolites as a tool for vectorization of hyperoxic/hypercapnic gases in glioblastoma is revealed.

The potential toxicity of encapsulated silver in EMT-type nanosized zeolites on prokaryotic cells, human tumor cell lines from various origins, and primary cultures of neurons and astrocytes was investigated. Silver in cationic form (Ag+) was encapsulated in EMT-type nanosized zeolites via an ion exchange process (Ag+-EMT) and compared with the reduced silver (Ag0) in the zeolite (Ag0-EMT). As reference samples for the toxicity measurements, pure EMT-type zeolite and silver perchlorate were used. Cells were exposed to silver-containing zeolites (50, 100, and 400 μg/mL) for 24 and 48 h. After exposure to Ag+-EMT (50 μg/mL) for 24 h, a loss in cell viability independent of the cell type was observed, ranging from −34.37 ± 23.90% for astrocytes to −99.5 ± 0.24% for U87-MG cells. These results were comparable with the toxicity for silver perchlorate. The Ag0-EMT sample showed lower toxicity on human cell lines in comparison to that of Ag+-EMT. A decrease in cell viability, i.e., −73.46 ± 20.78% and −62.3 ± 17...

Fluorescein was entrapped inside FAU type zeolite (Y) by generating it from resorcinol and phthalic anhydride. Prior to the formation of fluorescein, zeolite Y was modified with various cations (H, Zn, Ce, Na, K). The modified zeolite acted as catalysts for the fluorescein Friedel–Crafts reaction synthesis, while at the same time it played a role of matrix for the stabilization and homogeneous dispersion of the guest molecules. The nature of the different cations influenced the content of fluorescein generated in the zeolite, and favoured the formation of some dye isomers (i.e. anion, dianion, cation or transition metal complex). The contribution of different fluorescein derivatives in the products was reflected in their absorption and fluorescence properties. The H-, Ce-, and Zn- containing Y zeolites revealed the highest content of fluorescein due to their distinct acid active sites. The composites with H- and ZnY zeolites indicated much higher fluorescence than that of the pristine fluorescein. Moreover, fluorescein in zeolite Y matrices did not show any photobleaching under continuous illumination (λ = 730 nm, in a two-photon experiment for 180 s), which suggests that these materials could be used for biomedical applications.

Established evidence demonstrates that tumor-infiltrating myeloid cells promote rather than stop-cancer progression. Tumor-associated macrophages (TAMs) are abundantly present at tumor sites, and here they support cancer proliferation and distant spreading, as well as contribute to an immune-suppressive milieu. Their pro-tumor activities hamper the response of cancer patients to conventional therapies, such as chemotherapy or radiotherapy, and also to immunotherapies based on checkpoint inhibition. Active research frontlines of the last years have investigated novel therapeutic strategies aimed at depleting TAMs and/or at reprogramming their tumor-promoting effects, with the goal of re-establishing a favorable immunological anti-tumor response within the tumor tissue. In recent years, numerous clinical trials have included pharmacological strategies to target TAMs alone or in combination with other therapies. This review summarizes the past and current knowledge available on experimental tumor models and human clinical studies targeting TAMs for cancer treatment.

Fluorescein (F) species were encapsulated inside nanosized zeolites L and Y by their synthesis from adsorbed precursors (phthalic anhydride (Ph) and resorcinol (R)). The applied nanozeolites were modified either with H+ or Zn2+ cations and they played roles of the catalysts for synthesis of F and of the matrices for resulting dye molecules. The F loading, its tautomeric forms and optical properties of obtained composites depended on zeolite structure, morphology and on introduced cations. The composites usually showed higher fluorescence intensity than the pristine dye. The highest fluorescence intensity was noticed for the sample with nano-ZnL matrix. This intensity was much higher than that observed for composite with conventional micro-sized Zn L zeolite. Regarding an efficient fluorescence and nanosize of the obtained composites they seem promising fluorescent tracer for medical (theranostic) applications.

CERVOXY COLL; International audience; The preparation of iron-loaded nanosized zeolite with EMT-type structure (Fe-EMT) is reported. The sorption capacity of the Fe-EMT zeolite crystals for NO and CO2 is evaluated. The application of Fe-EMT zeolite as a gas delivery system with high capacity for CO2 and NO in biomedicine is considered. Therefore the toxicity of the pure EMT and Fe-EMT zeolite nanocrystals is studied. Primary culture of astrocytes was exposed to zeolite nanocrystals. In addition, two human cell lines, U87-MG glioblastoma and HEK-293T were used. Cells were exposed to zeolites nanocrystals with concentrations of 50, 100 and 400 μg/ml for 24 h and 48 h. Cytotoxicity was assessed by a cell viability assay and no alteration of cell viability exposed to zeolite nanoparticles was observed.