Development of Magnetic Nanosystems with Potential for the Treatment of Inner Ear Pathologies.

Hearing loss (HL) affects more than 5% of the global population, with projections indicating an impact of up to 50% on young individuals in the next years. HL treatments remain limited due to the inner ear's hermeticism. HL often involves inflammatory processes, underscoring the need for enhanced delivery of antiinflammatory agents to the inner ear. Our research focuses on the development of a directed therapy based on magnetic nanoparticles (MNPs). We previously synthesized biocompatible folic acid-coated iron oxide-core nanoparticles (MNPs@FA) as potential carriers for the anti-inflammatory Diclofenac (Dfc). This study aims to incorporate Dfc onto MNPs@FA to facilitate targeted drug delivery to the inner ear. Through optimizing the loading procedure, we achieved optimal loading capacity. Dfc release was studied in the simulated target fluid and the administration vehicle. Complete characterization is also shown. In vitro biocompatibility testing ensured the biosafety of the resulting formulation. Subsequent ex vivo targeting assays on murine cochleae validated the nanosystems' ability to penetrate the round window membrane, one of the main HL therapy barriers. These findings serve as validation before continuing to more complex in vivo studies. Together, the data here presented represent an advancement in addressing unmet medical needs in HL therapy.

Morin loaded mesoporous molecular sieves as novel devices to the potential treatment of tumor pathologies.

Morin is an antioxidant and anticancer flavonoid, extracted from natural sources, that may exert beneficial effects for several pathologies. Despite this, the administration of morin represents a challenge due to its low aqueous solubility. Mesoporous silica materials have emerged as biocompatible tools for drug delivery, as their pore size can be modulated for maximum surface area to volume ratio. In this contribution, we evaluate the ability of iron-modified mesoporous materials, for morin loading and controlled delivery. The SBA-15 and MCM-41 sieves were synthesized and modified with iron (metal content 4.02 and 6.27 % wt, respectivily). Characterization by transmission electron microscopy, XRD and UV-Vis revealed adequate pore size and agglomerates of very small metallic nanospecies (nanoclusters), without larger iron oxide nanoparticles. FT-IR spectra confirmed the presence of silanol groups in the solid hosts, which can interact with different groups present in morin molecules. SBA-15 materials were more efficient in terms of morin loading capacity (LC) due to their larger pore diameter. LC was more than 35% for SBA-15 materials when adsorptions studies were carried out with 9 mg of drug. Antioxidant activity were assayed by using DPPH test. Free iron materials presented a significate improvement as antioxidants after morin incorporation, reaching a scavenging activity of almost a 90%. On the other hand, in iron modified mesoporous materials, the presence of morin did not affect the scavenging activity. The results could be related with the formation of a complex between the flavonoid and the iron. Finally, biosafety studies using normal epithelial cells revealed that neither the loaded nor the unloaded materials exerted toxicity, even at doses of 1 mg/ml. These findings expand knowledge about mesoporous materials as suitable carriers of flavonoids with the aim of improving therapies for a wide range of pathologies.

Tumor microenvironment involvement in colorectal cancer progression via Wnt/ß-catenin pathway: Providing understanding of the complex mechanisms of chemoresistance.

Colorectal cancer (CRC) continues to be one of the main causes of death from cancer because patients progress unfavorably due to resistance to current therapies. Dysregulation of the Wnt/ß-catenin pathway plays a fundamental role in the genesis and progression of several types of cancer, including CRC. In many subtypes of CRC, hyperactivation of the ß-catenin pathway is associated with mutations of the adenomatous polyposis coli gene. However, it can also be associated with other causes. In recent years, studies of the tumor microenvironment (TME) have demonstrated its importance in the development and progression of CRC. In this tumor nest, several cell types, structures, and biomolecules interact with neoplastic cells to pave the way for the spread of the disease. Cross-communications between tumor cells and the TME are then established primarily through paracrine factors, which trigger the activation of numerous signaling pathways. Crucial advances in the field of oncology have been made in the last decade. This Minireview aims to actualize what is known about the central role of the Wnt/ß-catenin pathway in CRC chemoresistance and aggressiveness, focusing on cross-communication between CRC cells and the TME. Through this analysis, our main objective was to increase the understanding of this complex disease considering a more global context. Since many treatments for advanced CRC fail due to mechanisms involving chemoresistance, the data here exposed and analyzed are of great interest for the development of novel and effective therapies.

Biomedical Applications of Iron Oxide Nanoparticles: Current Insights Progress and Perspectives.

The enormous development of nanomaterials technology and the immediate response of many areas of science, research, and practice to their possible application has led to the publication of thousands of scientific papers, books, and reports. This vast amount of information requires careful classification and order, especially for specifically targeted practical needs. Therefore, the present review aims to summarize to some extent the role of iron oxide nanoparticles in biomedical research. Summarizing the fundamental properties of the magnetic iron oxide nanoparticles, the review's next focus was to classify research studies related to applying these particles for cancer diagnostics and therapy (similar to photothermal therapy, hyperthermia), in nano theranostics, multimodal therapy. Special attention is paid to research studies dealing with the opportunities of combining different nanomaterials to achieve optimal systems for biomedical application. In this regard, original data about the synthesis and characterization of nanolipidic magnetic hybrid systems are included as an example. The last section of the review is dedicated to the capacities of magnetite-based magnetic nanoparticles for the management of oncological diseases.

Novel Insights and Perspectives for the Diagnosis and Treatment of Hearing Loss through the Implementation of Magnetic Nanotheranostics.

Hearing loss (HL) is a sensory disability that affects 5 % of the world's population. HL predominantly involves damage and death to the cochlear cells. Currently, there is no cure or specific medications for HL. Furthermore, the arrival of therapeutic molecules to the inner ear represents a challenge due to the limited blood supply to the sensory cells and the poor penetration of the blood-cochlear barrier. Superparamagnetic iron oxide nanoparticles (SPIONs) perfectly coordinate with the requirements for controlled drug delivery along with magnetic resonance imaging (MRI) diagnostic and monitoring capabilities. Besides, they are suitable tools to be applied to HL, expecting to be more effective and non-invasive. So far, the published literature only refers to some preclinical studies of SPIONs for HL management. This contribution aims to provide an integrated view of the best options and strategies that can be considered for future research punctually in the field of magnetic nanotechnology applied to HL.

Involvement of parathyroid hormone-related peptide in the aggressive phenotype of colorectal cancer cells.

Colorectal cancer (CRC) remains one of the leading causes of mortality from malignant diseases worldwide. In general terms, CRC presents high heterogeneity due to the influence of different genetic and environmental factors; also, the neoplastic cells are strongly influenced by the extracellular matrix and several surrounding cells, known together as the tumor microenvironment (TME). Bidirectional communication takes place between the tumor and the TME through the release of autocrine and paracrine factors. Parathyroid hormone-related peptide (PTHrP) is a cytokine secreted by a wide variety of tissues and is able to regulate several cellular functions both in physiological as well as in pathological processes. It exerts its effects as a paracrine/autocrine factor, although its mode of action is mainly paracrine. It has been shown that this peptide is expressed by several tumors and that the tumor secretion of PTHrP is responsible for the malignant humoral hypercalcemia. Eight years ago, when our research group started studying PTHrP effects in the experimental models derived from intestinal tumors, the literature available at the time addressing the effects of PTHrP on colorectal tumors was limited, and no articles had been published regarding to the paracrine action of PTHrP in CRC cells. Based on this and on our previous findings regarding the role of PTH in CRC cells, our purpose in recent years has been to explore the role of PTHrP in CRC. We analyzed the behavior of CRC cells treated with exogenous PTHrP, focalizing in the study of the following events: Survival, cell cycle progression and proliferation, migration, chemoresistance, tumor-associated angiogenesis, epithelial to mesenchymal transition program and other events also associated with invasion, such us the induction of cancer stem cells features. This work summarizes the major findings obtained by our investigation group using in vitro and in vivo CRC models that evidence the participation of PTHrP in the acquisition of an aggressive phenotype of CRC cells and the molecular mechanisms involved in these processes. Recently, we found that this cytokine induces this malignant behavior not only by its direct action on these intestinal cells but also through its influence on cells derived from TME, promoting a communication between CRC cells and surrounding cells that contributes to the molecular and morphological changes observed in CRC cells. These investigations establish the basis for our next studies in order to address the clinical applicability of our findings. Recognizing the factors and mechanisms that promote invasion in CRC cells, evasion to the cytotoxic effects of current CRC therapies and thus metastasis is decisive for the identification of new markers with the potential to improve early diagnosis and/or to predict prognosis, to predetermine drug resistance and to provide treatment guidelines that include targeted therapies for this disease.

Doxorubicin delivery by magnetic nanotheranostics enhances the cell death in chemoresistant colorectal cancer-derived cells.

Colorectal cancer (CRC) is a major cause of cancer death with a high probability of treatment failure. Doxorubicin (DOXO) is an efficient antitumor drug; however, most CRC cells show resistance to its effects. Magnetic nanoparticles (MNPs) are potential cancer management tools that can serve as diagnostic agents and also can optimize and personalize treatments. This work aims to evaluate the aptitude of magnetic nanotheranostics composed of magnetite (Fe3O4) nanoparticles coated with folic acid intended to the sustained release of DOXO. The administration of DOXO by means of these MNPs resulted in the enhancement of cell death respect to the free drug administration. Chromatin compaction and cytoplasmic protrusions were observed. Mitochondrial transmembrane potential disruption and increased PARP protein cleavage confirmed apoptosis. The nanosystem was also tested as a vectoring tool by exposing it to the stimuli of a static magnetic field in vitro. CRC-related magnetic nanotechnology still remains in pre-clinical trials. In this context, this contribution expands the knowledge of the behavior of MNPs in contact with in vitro models and proposes the nanodevices studied here as potential theranostic agents for the monitoring of the progress of CRC and the evolution of its treatment.

In vitro Biological Tests as the First Tools To Validate Magnetic Nanotheranostics for Colorectal Cancer Models.

Colorectal cancer (CRC) remains a leading cause of cancer death. Nanotechnology has focused on reaching more effective treatments. In this concern, magnetic nanoparticles (MNPs) have been studied for a wide range of biomedical applications related to CRC, such as diagnostic imaging, drug delivery and thermal therapy. However, limited research is currently found in the open literature that refers to nanosystems combining all these mentioned areas (theranostics). When developing nanosystems intended as theranostics applied to CRC, possible variations between patients must be considered. Therefore, multiple in vitro assays are required as guidance for future preclinical and clinical trials. The objective of this contribution is to evaluate the available and recent literature regarding the interactions of MNP and CRC models, aiming to critically analyze the information given by the commonly used assays and evaluate the data provided by each one with a view to implementing this novel technology in CRC diagnostics and therapy.

Selective contrast agents with potential to the earlier detection of tumors: Insights on synthetic pathways, physicochemical properties and performance in MRI assays.

Magnetic iron oxide nanoparticles (MNPs) have been prepared and stabilized with three organic acids (tartaric, malic and ascorbic) in order to obtain biocompatible and water dispersible MNPs with potential to bind specifically to tumoral cancer cells. An in deep characterization was performed aiming to verify the presence and effect of the coating and stabilizer on MNPs surface. Besides the mechanisms followed by the different acids to bind MNPs were elucidated and used to justify the differences in the physicochemical properties of each formulation. Data related to characterization revealed that MNPs coated with ascorbic acid (MNPs-AA) resulted the most suitable in terms of their size, surface charge and stability along the time. Besides, ascorbic acid may be recognized by GLUTs receptors that are overexpressed in several kinds of tumoral cells. Therefore, MNPs-AA was selected to explore its performance in both MRI and in vitro assays using human colon cancer cells HCT 116. MRI experiments were performed in clinical equipment using a series of aqueous dispersions of MNPs-AA that were evaluated as T2 contrast agent. The T2- weighted images obtained as well as the calculated r2, indicated that MNPs-AA could act as efficient T2 contrast agent for MRI. Regarding in vitro assays, MNPs-AA did not alter the cellular function neither exert cytotoxicity using the three explored doses. The internalization of the nanoparticles on the cellular structure was confirmed quanti and qualitatively using atomic absorption spectroscopy and Prussian blue techniques respectively. From these results, it emerges that ascorbic acid coated-magnetite nanoparticles may be used as alternative contrast agent to avoid or minimize some toxicological issues related to the widely used gadolinium.

Case Report: Penile Squamous Cell Carcinoma Associated With Severe Hypercalcemia and High Levels of Parathyroid Hormone-related Peptide Expressed in Metastatic Skin Tissue.

It is known that high levels of parathyroid hormone-related protein (PTHrP) correlate with a bad prognostic in malignancies. Here we present a patient with advanced penile cancer (PC) without antecedents of human papillomavirus infections and bone metastases but with severe hypercalcemia. By quantitative polymerase chain reaction, we observed high levels of PTHrP messenger RNA in metastatic cutaneous tissue. This is the first reported case in Argentina of hypercalcemia induced by PTHrP in human PC. Furthermore, the association of PTHrP and this disease through quantitative polymerase chain reaction allows us to consider this molecular technique as a novel tool for diagnosis in patients with PC.

Potential therapeutic targets for growth arrest of colorectal cancer cells exposed to PTHrP.

Although PTHrP is implicated in several cancers, its role in chemoresistance is not fully elucidated. We found that in CRC cells, PTHrP exerts proliferative and protective effects and induces cell migration. The aim of this work was to further study the effects of PTHrP in CRC cells. Herein we evidenced, for the first time, that PTHrP induces resistance to CPT-11 in Caco-2 and HCT116 cells; although both cell lines responded to the drug through different molecular mechanisms, the chemoresistance by PTHrP in these models is mediated through ERK, which in turn is activated by PCK, Src and Akt. Moreover, continue administration of PTHrP in nude mice xenografts increased the protein levels of this MAPK and of other markers related to tumorigenic events. The understanding of the molecular mechanisms leading to ERK 1/2 activation and the study of ERK targets may facilitate the development of new therapeutic strategies for CRC treatment.

RSK activation via ERK modulates human colon cancer cells response to PTHrP.

Parathyroid hormone-related peptide (PTHrP) is associated with several human cancers such as colon carcinoma. This disease is a complex multistep process that involves enhanced cell cycle progression and migration. Recently we obtained evidence that in the human colorectal adenocarcinoma Caco2 cells, exogenous PTHrP increases the proliferation and positively modulates cell cycle progression via ERK1/2, p38 MAPK and PI3K. The purpose of this study was to explore if the serine/threonine kinase RSK, which is involved in the progress of many cancers and it is emerging as a potential therapeutic target, mediates PTHrP effects on cancer colon cells. Western blot analysis revealed that PTHrP increases RSK phosphorylation via ERK1/2 signaling pathway but not through p38 MAPK. By performing subcellular fractionation, we found that the peptide also induces the nuclear localization of activated RSK, where many of its substrates are located. RSK participates in cell proliferation, in the upregulation of cyclin D1 and CDK6 and in the downregulation of p53 induced by PTHrP. Wound healing and transwell filter assays revealed that cell migration increased after PTHrP treatment. In addition, the hormone increases the protein expression of the focal adhesion kinase FAK, a regulator of cell motility. We observed that PTHrP induces cell migration and modulates FAK protein expression through ERK/RSK signaling pathway but not via p38 MAPK pathway. Finally, in vivo studies revealed that the hormone activates RSK in xenografts tumor. Taken together, our findings provide new insights into the deregulated cell cycle and migration that is characteristic of tumor intestinal cells.

Characterization of the key antigenic components of pertussis vaccine based on outer membrane vesicles.

Pertussis has resurged during the last two decades in different countries. In particular in the 2010-2013 period large outbreaks were detected in US, Australia, UK and The Netherlands with significant mortality in infants. The epidemiological situation of pertussis points out the need to develop new vaccines and in this regard we previously developed a new vaccine based on outer membrane vesicles (OMVs) which have been shown to be safe and to induce protection in mice. Here we have further investigated the properties of OMVs vaccines; in particular we studied the contribution of pertussis toxin (PTx) and pertactin (Prn) in OMVs-mediated protection against pertussis. PTx-deficient OMVs and Prn-deficient OMVs were obtained from defective Bordetella pertussis mutants. The absence of PTx or Prn did compromise the protective capacity of the OMVs formulated as Tdap vaccine. Whereas the protective efficacy of the PTx-deficient OMVs in mice was comparable to Prn-deficient OMVs, the protective capacity of both of them was significantly impaired when it was compared with the wild type OMVs. Interestingly, using OMVs obtained from a B. pertussis strain which does not express any of the virulence factors but expresses the avirulent phenotype; we observed that the protective ability of such OMVs was lower than that of OMVs obtained from virulent B. pertussis phase. However, it was surprising that although the protective capacity of avirulent OMVs was lower, they were still protective in the used mice model. These results allow us to hypothesize that OMVs from avirulent phase shares protective components with all OMVs assayed. Using an immune proteomic strategy we identified some common components that could play an important role in protection against pertussis.

Molecular mechanisms associated with PTHrP-induced proliferation of colon cancer cells.

Parathyroid Hormone-related Protein (PTHrP) is normally produced in many tissues and is recognized for its endocrine, paracrine, autocrine and intracrine modes of action. PTHrP is also implicated in different types of cancer and its expression correlates with the severity of colon carcinoma. Using the human colon cell line Caco-2 we recently obtained evidence that PTHrP, through a paracrine pathway, exerts a protective effect under apoptotic conditions. However, if exogenous PTHrP is able or not to induce the proliferation of these intestinal tumor cells is not known. We found that PTHrP treatment increases the number of live Caco-2 cells. The hormone induces the phosphorylation and nuclear translocation of ERK 1/2, α p38 MAPK, and Akt, without affecting JNK phosphorylation. In addition, PTHrP-dependent ERK phosphorylation is reverted when PI3K activity was inhibited. Following MAPKs nuclear translocation, the transcription factors ATF-1 and CREB were activated in a biphasic manner. In addition PTHrP induces the translocation into the nucleus of ß-catenin, protein that plays key role in maintaining the growth and proliferation of colorectal cancer, and increases the amount of both positive cell cycle regulators c-Myc and Cyclin D. Studies with ERK1/2, α p38 MAPK, and PI3K specific inhibitors showed that PTHrP regulates Caco-2 cell proliferation via these signaling pathways. In conclusion, the results obtained in this work expand our knowledge on the role of exogenous PTHrP in intestinal tumor cells and identify the signaling pathways that are involved in the mitogenic effect of the hormone on Caco-2 cells.

Involvement of ERK1/2, p38 MAPK, and PI3K/Akt signaling pathways in the regulation of cell cycle progression by PTHrP in colon adenocarcinoma cells.

Parathyroid hormone-related peptide (PTHrP) is distributed in most fetal and adult tissues, and its expression correlates with the severity of colon carcinoma. Recently we obtained evidence that in Caco-2 cells, a cell line from human colorectal adenocarcinoma, exogenous PTHrP increases the number of live cells, via ERK1/2, p38 MAPK, and PI3-kinase and induces the expression of cyclin D1, a cell cycle regulatory protein. In this study, we further investigated the role of PTHrP in the regulation of the cell cycle progression in these intestinal cells. Flow cytometry analysis revealed that PTHrP treatment diminishes the number of cells in the G0/G1 phase and increases the number in both S and G2/M phases. The hormone increases the expression of CDK6 and diminishes the amount of negative cell cycle regulators p27Kip1, p15INK4B, and p53. However, PTHrP does not modify the expression of cyclin D3, CDK4, and p16INK4A. In addition, inhibitors of ERK1/2 (PD98059), p38 MAPK (SB203580), and PI3Kinase (LY294002) reversed PTHrP response in Caco-2 cells. Taken together, our results suggest that PTHrP positively modulates cell cycle progression and changes the expression of proteins involved in cell cycle regulation via ERK1/2, p38 MAPK, and PI3K signaling pathways in Caco-2 cells.