Selection of internalizing RNA aptamers into human breast cancer cells derived from primary sites.

Breast cancer is the most common cancer in women. Although chemotherapy is still broadly used in its treatment, adverse effects remain a challenge. In this scenario, aptamers emerge as a promising alternative for theranostic applications. Studies using breast cancer cell lines provide useful information in laboratory and preclinical investigations, most of which use cell lines established from metastatic sites. However, these cell lines correspond to cell populations of the late stage of tumor progression. On the other hand, studies using breast cancer cells established from primary sites make it possible to search for new theranostic approaches in the early stages of the disease. Therefore, this work aimed to select RNA aptamers internalized by MGSO-3 cells, a human breast cancer cell line, derived from a primary site previously established in our laboratory. Using the Cell-Internalization SELEX method, we have selected two candidate aptamers (ApBC1 and ApBC2). We evaluated their internalization efficiencies, specificities, cellular localization by Reverse Transcription-qPCR (RT-qPCR) and confocal microscopy assays. The results suggest that both aptamers were efficiently internalized by human breast cancer cells, MACL-1, MDA-MB-231, and especially by MGSO-3 cells. Furthermore, both aptamers could effectively distinguish human breast cancer cells derived from normal human mammary cell (MCF 10A) and prostate cancer cell (PC3) lines. Therefore, ApBC1 and ApBC2 could be promising candidate molecules for theranostic applications, even in the early stages of tumor progression.

Kisspeptin (Kp-10) inhibits in vitro osteogenic differentiation of multipotent mesenchymal stromal cells extracted from the bone marrow of adult rats.

Kisspeptin (Kp-10) is a neuropeptide that binds to GPR54 receptors, exerting several functions mainly in the nervous and reproductive systems of the body. However, its effects and mechanisms of action on the skeletal system remain poorly understood. This study evaluated the effects of different concentrations of Kp-10 on in vitro osteogenic differentiation of multipotent mesenchymal stromal cells (MSCs) extracted from the bone marrow (BM) of adult Wistar rats. Two-month-old female rats were euthanized to extract BM from long bones to obtain MSCs. Four experimental groups were established in vitro: a control and Kp-10 at concentrations of 0.01, 0.05 and, 0.1 µg/mL. After induction of osteogenic differentiation, cell viability was evaluated using the 3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyl tetrazolium bromide (MTT) assay, alkaline phosphatase activity, collagen synthesis, percentage of area covered by MSCs/field and mineralized nodules/field, and immunocytochemistry of the GPR54 receptor tests. Furthermore, evaluation of gene transcripts for type I collagen, Runx-2, Bmp-2, bone sialoprotein, osteocalcin and osteopontin was performed using real-time RT-qPCR. It was observed that MSCs expressed GPR54 receptor to which Kp-10 binds during osteogenic differentiation, promoting a negative effect on osteogenic differentiation. This effect was observed at all the Kp-10 concentrations in a concentration-dependent manner, characterized by a decrease in the activity of alkaline phosphatase, collagen synthesis, mineralized nodules, and decreased expression of gene transcripts for type I collagen, osteocalcin, osteopontin, and Runx-2. Thus, Kp-10 inhibits in vitro osteogenic differentiation of MSCs extracted from the BM of adult Wistar rats.

Leishmania (L.) infantum BH401 strain induces classic renal lesions in dogs: Histological and confocal microscopy study.

Extracellular matrix (ECM) alterations in visceral leishmaniasis are related mainly to collagen deposition (fibropoiesis). In canine visceral leishmaniasis (CVL), an intense fibrosis associated to chronic inflammation in organs such as kidneys is described. However, renal fibropoiesis has not been described in natural or experimental infections with L. (L.) infantum. We aimed to characterize renal nephropathies by histology and confocal microscopy comparing renal lesions in dogs naturally and experimentally infected with L. (L.) infantum. Sixty-two mixed-breed symptomatic dogs naturally infected with L. (L.) infantum, sixteen beagles experimentally infected with two strains of L. infantum (eleven dogs with the BH400 strain and five dogs with the BH401 strain), and five uninfected beagles (controls) were used. Samples were stained with hematoxylin & eosin for routine histology. Congo red was used to visualize amyloid protein deposits, periodic acid-Schiff to identify glomerular basal membrane anomalies, Masson's trichrome for collagen deposits, and Jones' methenamine silver to reveal membranous glomerulonephropathy. Immunohistochemistry was used to identify Leishmania amastigotes, and confocal microscopy was used for macrophage characterization (L1/calprotectin and CD163 antigen receptors). The most common lesions were chronic glomerular and interstitial nephritis, which was found in all naturally infected dogs and dogs experimentally infected with L. infantum strain BH401 but not with the BH400 strain. Glomeruloesclerosis was the main lesion presented in all BH401 group. Morphometric analysis revealed positive correlation of renal glomeruli tufts with cellular expression of L1/calprotectin and CD163 antigens. Leishmania infantum strain BH401 shows pathogenicity that may be sufficient to induce classic chronic visceral renal leishmaniasis.

Induction of Corneal Epithelial Differentiation of Induced Pluripotent and Orbital Fat-Derived Stem Cells Seeded on Decellularized Human Corneas.

Up to 40% of donor corneas are deemed unsuitable for transplantation, aggravating the shortage of graft tissue. In most cases, the corneal extracellular matrix is intact. Therefore, their decellularization followed by repopulation with autologous cells may constitute an efficient alternative to reduce the amount of discarded tissue and the risk of immune rejection after transplantation. Although induced pluripotent (hiPSCs) and orbital fat-derived stem cells (OFSCs) hold great promise for corneal epithelial (CE) reconstruction, no study to date has evaluated the capacity of decellularized corneas (DCs) to support the attachment and differentiation of these cells into CE-like cells. Here, we recellularize DCs with hiPSCs and OFSCs and evaluate their differentiation potential into CE-like cells using animal serum-free culture conditions. Cell viability and adhesion on DCs were assessed by calcein-AM staining and scanning electron microscopy. Cell differentiation was evaluated by RT-qPCR and immunofluorescence analyses. DCs successfully supported the adhesion and survival of hiPSCs and OFSCs. The OFSCs cultured under differentiation conditions could not express the CE markers, TP63, KRT3, PAX6, and KRT12, while the hiPSCs gave rise to cells expressing high levels of these markers. RT-qPCR data suggested that the DCs provided an inductive environment for CE differentiation of hiPSCs, supporting the expression of PAX6 and KRT12 without the need for any soluble induction factors. Our results open the avenue for future studies regarding the in vivo effects of DCs as carriers for autologous cell transplantation for ocular surface reconstruction.

Disruption of Active Trans-Sialidase Genes Impairs Egress from Mammalian Host Cells and Generates Highly Attenuated Trypanosoma cruzi Parasites.

Trans-sialidases (TS) are unusual enzymes present on the surface of Trypanosoma cruzi, the causative agent of Chagas disease. Encoded by the largest gene family in the T. cruzi genome, only few members of the TS family have catalytic activity. Active trans-sialidases (aTS) are responsible for transferring sialic acid from host glycoconjugates to mucins, also present on the parasite surface. The existence of several copies of TS genes has impaired the use of reverse genetics to study this highly polymorphic gene family. Using CRISPR-Cas9, we generated aTS knockout cell lines displaying undetectable levels of TS activity, as shown by sialylation assays and labeling with antibodies that recognize sialic acid-containing mucins. In vitro infection assays showed that disruption of aTS genes does not affect the parasite's capacity to invade cells or to escape from the parasitophorous vacuole but resulted in impaired differentiation of amastigotes into trypomastigotes and parasite egress from the cell. When inoculated into mice, aTS mutants were unable to establish infection even in the highly susceptible gamma interferon (IFN-γ) knockout mice. Mice immunized with aTS mutants were fully protected against a challenge infection with the virulent T. cruzi Y strain. Altogether, our results confirmed the role of aTS as a T. cruzi virulence factor and indicated that aTS play a major role during the late stages of intracellular development and parasite egress. Notably, mutants lacking TS activity are completely avirulent in animal models of infection and may be used as a live attenuated vaccine against Chagas disease. IMPORTANCE Trypanosoma cruzi is the causative agent of Chagas disease, a neglected tropical disease that affects approximately 6 to 8 million people and for which there is no effective treatment or vaccine. The parasite expresses a family of surface proteins, named trans-sialidases, responsible for transferring sialic acid from host glycoconjugates to parasite mucins. Although recognized as a main virulence factor, the multiple roles of these proteins during infection have not yet been fully characterized, mainly because the presence of several copies of aTS genes has impaired their study using reverse genetics. By applying CRISPR-Cas9, we generated aTS knockout parasites and showed that, although aTS parasite mutants were able to infect cells in vitro, they have an impaired capacity to egress from the infected cell. Importantly, aTS mutants lost the ability to cause infection in vivo but provided full protection against a challenge infection with a virulent strain.

Roles of mesenchymal stromal cells in the head and neck cancer microenvironment.

Head and neck cancer (HNC), a common malignancy worldwide, is associated with high morbidity and mortality rates. Squamous cell carcinoma is the most common HNC type, followed by salivary gland carcinomas, head and neck sarcomas, and lymphomas. The microenvironment of HNCs comprises various cells that regulate tumor development. Recent studies have reported that the tumor microenvironment, which modulates cancer progression, regulates cancer treatment response. However, the presence of different types of stromal cells in cancers is a major challenge to elucidate the role of individual cells in tumor progression. The role of mesenchymal stromal cells (MSCs), which are a component of the tumor microenvironment, in HNC is unclear. The major impediment for characterizing the role of MSCs in cancer progression is the lack of MSC-specific markers and their phenotypic similarity with stromal cells. This review aimed to summarize the latest findings on the role of MSCs in the progression of HNC to improve our understanding of HNC pathophysiology.

The basis of nuclear phospholipase C in cell proliferation.

Ca2+ is a highly versatile intracellular signal that regulates many biological processes such as cell death and proliferation. Broad Ca2+-signaling machinery is used to assemble signaling systems with a precise spatial and temporal resolution to achieve this versatility. Ca2+-signaling components can be organized in different regions of the cell and local increases in Ca2+ within the nucleus can regulate different cellular functions from the increases in cytosolic Ca2+. However, the mechanisms and pathways that promote localized increases in Ca2+ levels in the nucleus are still under investigation. This review presents evidence that the nucleus has its own Ca2+ stores and signaling machinery, which modulate processes such as cell proliferation and tumor growth. We focus on what is known about the functions of nuclear Phospholipase C (PLC) in the generation of nuclear Ca2+ transients that are involved in cell proliferation.

Synthesis and characterization of gold nanorods coated by mesoporous silica MCM-41 as a platform bioapplication in photohyperthermia.

Gold nanoparticles have been widely investigated for biomedical applications due to their optical properties. These particles present the interesting feature of absorbing light when stimulated with laser radiation to generate heating. Among the possible morphologies for synthetic gold nanoparticles, gold nanorods have properties of great interest for applications in the photohyperthermia processes. Due to their morphology, gold nanorods can absorb light at longer wavelengths comprising specific regions of the electromagnetic spectrum, such as the region of the biological window, in which laser radiation has less interaction with tissues. However, these nanoparticles present limitations in biomedical applications, such as low colloidal and thermal stabilities that can be overcome by coating the gold nanorods with silica MCM-41. The silicate covering can provide greater stability for gold nanorods and allow multifunctionality in treating different diseases through photohyperthermia. This work developed a specific chemical route through seed and growth solutions to synthesize gold nanorods with controlled particle size, rod morphology, and silica covering for photohyperthermia applications. The synthesized samples were characterized through a multi-technique approach that successfully demonstrated the presence of gold nanorods inside the silica coating, presenting high stability and desirable textural and morphological characteristics for bioapplications. Furthermore, silica-coated gold nanorods exhibit high biocompatibility and great performance in generating therapeutic heating by absorbing laser radiation in the biological window range, making the system developed in this work a promising agent in photohyperthermia.

Adipose-derived stem/stromal cell secretome modulates breast cancer cell proliferation and differentiation state towards aggressiveness.

It is becoming increasingly evident that mesenchymal stem/stromal cells are recruited by cancer cells from nearby endogenous host stroma and promote events such as tumor proliferation, angiogenesis, invasion, and metastasis, as well as mediate therapeutic resistance. Consequently, understanding the regulatory mechanisms of ASCs that influence the tumor microenvironment may provide an avenue for further treatment. To understand the role of the ASC secretome in breast cancer cell proliferation, death, and phenotype alteration, adipose-derived stem cell-conditioned medium (mASC) was used to cultivate MCF-7 and MDA-MB-231 cells. These breast cancer cells in mASC showed a shorter doubling time, higher frequency of EdU positivity, and higher levels of phosphorylated histone 3. In addition, increased expression of cyclin B1 was observed, suggesting that proliferation was induced. The mASC was also able to increase apoptosis in MCF-7 cells, which was confirmed by caspase-7 activation. The number of tumor-initiating cells (CD44+ CD24-/low) and migration capacity were increased in cells cultivated in mASC. These data collectively suggest that ASC-conditioned medium can induce selective pressure by increasing cell proliferation, giving rise to a more aggressive phenotype in MCF-7 and MDA-MB-231 cells. Our study provides a foundation for further elucidation of the precise mechanism underlying ASCs in breast cancer cells and the modulation of ASCs in potential therapeutic uses.

Selection of DNA Aptamers for Differentiation of Human Adipose-Derived Mesenchymal Stem Cells from Fibroblasts.

In recent years, stem cell therapy has shown promise in regenerative medicine. The lack of standardized protocols for cell isolation and differentiation generates conflicting results in this field. Mesenchymal stem cells derived from adipose tissue (ASC) and fibroblasts (FIB) share very similar cell membrane markers. In this context, the distinction of mesenchymal stem cells from fibroblasts has been crucial for safe clinical application of these cells. In the present study, we developed aptamers capable of specifically recognize ASC using the Cell-SELEX technique. We tested the affinity of ASC aptamers compared to dermal FIB. Quantitative PCR was advantageous for the in vitro validation of four candidate aptamers. The binding capabilities of Apta 2 and Apta 42 could not distinguish both cell types. At the same time, Apta 21 and Apta 99 showed a better binding capacity to ASC with dissociation constants (Kd) of 50.46 ± 2.28 nM and 72.71 ± 10.3 nM, respectively. However, Apta 21 showed a Kd of 86.78 ± 9.14 nM when incubated with FIB. Therefore, only Apta 99 showed specificity to detect ASC by total internal reflection microscopy (TIRF). This aptamer is a promising tool for the in vitro identification of ASC. These results will help understand the differences between these two cell types for more specific and precise cell therapies.

Phenotypic changes on central nervous system (CNS) tumor cell lines cultured in vitro 2D and 3D models and treated with cisplatin.

Despite aggressive therapy, most patients with brain tumors present disease relapse due to the cellular and molecular nature of these tumors. One of the models that best explains the heterogeneity observed in CNS tumors is the presence of cancer stem cells (CSCs). In this paper, we evaluated platinum-based response in brain tumor U-87 MG, LN-18, and KELLY cell lines cultured in monolayer (2D) and neurosphere (CSC enrichment- 3D) models. We evaluated mRNA expression of heat shock proteins (HSPA1A, HSPB1, HSPA1AL, TRAP1, and HSPD1), and DNA repair gene ERCC1. Changes in cell cycle and glycosylation profile were assessed by flow cytometry. After treatment with cisplatin, we found that the mRNA expression of HSPs markedly increased in the U-87 MG and LN-18 neurosphere cells. In KELLY monolayer cells, cisplatin induced upregulation of all genes. In KELLY neurosphere cells, only the HSPA1A, HSPB1, TRAP1, and HSPD1 genes were upregulated. The proportion of cells in the G0/G1 phase was significantly higher in U-87 MG neurosphere cisplatin-treated cells. A trend towards a greater proportion of cells in the S phase of U-87 MG monolayer cisplatin-treated cells was also observed. On the other hand, a significant decrease in the number of cells in the S phase and an increase in G2/M was observed in LN-18 monolayer cisplatin-treated cells. Glycosylation analysis using lectins showed a higher surface binding for PNA in the U-87 MG treated monolayer and a lower binding for Concanavalin A in the treated neurospheres. The binding of Isolectin GS-IB4, GSII, and SBA in KELLY monolayer cisplatin-treated cells was lower whereas the proportion of cells labeled with Concanavalin A was higher. In the KELLY neurosphere cisplatin-treated cells, the binding of Concanavalin A was lower than nontreated cells. Thus, our findings strongly supported the idea that definitions of phenotypic characteristics may help to establish better therapeutic strategies for brain tumors.

Dual anticancer and antibacterial activities of bismuth compounds based on asymmetric [NN'O] ligands.

Two new bismuth(III) complexes, [BiL1Cl2] (1) and [BiL2Cl2] (2), in which L1 is (2-hydroxy-4-6-di-tert-butylbenzyl-2-pyridylmethyl)amine and L2 is 2,4-diiodo-6-((pyridine-2-ylmethylamino)methyl)phenol, were synthesized and characterized by elemental and conductivity analyses, atomic absorption spectrometry, infrared and 1H NMR spectroscopies. The molecular structure of 1 reveals that the NN'O ligand forms a 1:1 complex with bismuth through coordination via the nitrogen of the aliphatic amine, the nitrogen of the pyridine ring and the oxygen of the phenolate. The coordination sphere is completed with two chloride anions in a distorted square pyramidal geometry. Bismuth exhibits the same coordination mode in compound 2. The cytotoxic activity of 1 and 2 was investigated in a chronic myelogenous leukemia cell line. The complexes are approximately three times more potent than the corresponding free ligands, with the IC50 values 0.30 and 0.38 µM for complex 1 and 2, respectively. To address the cellular mechanisms underlying cell demise, apoptosis was quantified by flow cytometry analysis. From 0.1 µM, both complexes induce apoptosis and there is a remarkable concentration-dependent increase in the population of cells in apoptosis. The complexes were also evaluated against Gram-positive and Gram-negative bacteria. Both inhibited the bacterial growth in a concentration-dependent way, with remarkable activity in some of the tested strains, for example, complex 2 was more active than its free ligand against all bacterial strains and approximately fourteen times more potent against S. dysenteriae and S. typhimurium.

Neuroprotective Effect of siRNA Entrapped in Hyaluronic Acid-Coated Lipoplexes by Intravitreal Administration.

Since the possibility of silencing specific genes linked to retinal degeneration has become a reality with the use of small interfering RNAs (siRNAs), this technology has been widely studied to promote the treatment of several ocular diseases. Despite recent advances, the clinical success of gene silencing in the retina is significantly reduced by inherent anatomical and physiological ocular barriers, and new strategies are required to achieve intraocular therapeutic effectiveness. In this study, we developed lipoplexes, prepared with sodium alginate as an adjuvant and strategically coated with hyaluronic acid (HA-LIP), and investigated the potential neuroprotective effect of these systems in a retinal light damage model. Successful functionalization of the lipoplexes with hyaluronic acid was indicated in the dynamic light scattering and transmission electron microscopy results. Moreover, these HA-LIP nanoparticles were able to protect and deliver siRNA molecules targeting caspase-3 into the retina. After retinal degeneration induced by high light exposure, in vitro and in vivo quantitative reverse transcription-PCR (RT-qPCR) assays demonstrated significant inhibition of caspase-3 expression by HA-LIP. Furthermore, these systems were shown to be safe, as no evidence of retinal toxicity was observed by electroretinography, clinical evaluation or histology.

The synthetic peptide PnPP-19 potentiates erectile function via nNOS and iNOS.

PnPP-19 peptide has a primary sequence design based on molecular modeling studies of PnTx2-6 toxin. It comprises the amino acid residues that are potentially significant for the pharmacological action of PnTx2-6. Ex vivo and in vivo experiments in normotensive, hypertensive, or diabetic murine models have shown a significant improvement in penile erection after administration of PnPP-19. Given the potential use of PnPP-19 in pharmaceutical formulations to treat erectile dysfunction and the lack of information concerning its mode of action, the present work investigates its activities on the nitrergic system. PnPP-19 induced a significant increase in nitric oxide (NO) and cGMP levels in corpus cavernosum (cc). These effects were inhibited by l-NAME, a non-selective inhibitor of nitric oxide synthase (NOS); were partially inhibited by 7- Nitroindazole, a selective inhibitor of neuronal NOS (nNOS); and were abolished by L-NIL, a selective inhibitor of inducible NOS (iNOS). This potentiating effect was not affected by atropine. PnPP-19 also led to changes in mRNA levels, protein expression and phosphorylation at specific sites of NOS, in cc. Assays using cavernous tissue from knockout mice to endothelial NOS (eNOS), nNOS or iNOS showed that PnPP-19 potentiates relaxation only in eNOS-knockout mice, which suggests an essential role for nNOS. Surprisingly, iNOS enhanced the potentiation of erectile function evoked by PnPP-19. Our results demonstrate that this new synthetic peptide potentiates erectile function via nitric oxide activation and reinforce its role as a new pharmacological tool for the treatment of erectile dysfunction.

Human adipose-derived stromal/stem cells are distinct from dermal fibroblasts as evaluated by biological characterization and RNA sequencing.

Human adipose-derived stromal/stem cells (ASC) have immunomodulatory properties and the potential to differentiate into several cell lines, important for application in regenerative medicine. However, the contamination with dermal fibroblasts (FIB) can impair the beneficial effects of ASC in cell therapy. It is then essential to develop new strategies that contribute to the distinction between these two cell types. In this study, we performed functional assays, high-throughput RNA sequencing (RNA-Seq) and quantitative PCR (qPCR) to find new markers that can distinguish ASC and FIB. We showed that ASC have adipogenic and osteogenic differentiation capacity and alkaline phosphatase activity, not observed in FIB. Gene expression variation analysis identified more than 2000 differentially expressed genes (DEG) between these two cell types. We validated 16 genes present in the list of DEG, including the alkaline phosphatase gene (ALPL). In conclusion, we showed that ASC and FIB have distinct biological properties as demonstrated by alkaline phosphatase activity and differentiation capacity, besides having different gene expression profiles. SIGNIFICANCE OF THE STUDY: Although many differences between stromal stem cells derived from human adipose tissue (ASC) and human dermal fibroblasts (FIB) are described, it is still difficult to find specific markers to differentiate them. This problem can interfere with the therapeutic use of ASC. This work aimed to find new markers to differentiate these two cell populations. Our findings suggest that these cells can be distinguished by biological and molecular characteristics, such as adipogenic and osteogenic differentiation, alkaline phosphatase activity and differential gene expression profiles. The DEG were related to the regulation of the cell cycle, development process, structural organization of the cell and synthesis of the extracellular matrix. This study helps to find new cellular markers to distinguish the two populations and to better understand the properties of these cells, which can improve cell therapy.

Long-term dexamethasone treatment increases the engraftment efficiency of human breast cancer cells in adult zebrafish.

The host immune system tends to reject xenogenic-implanted cells making tumor development in adult host animal models difficult. Immune system suppression is used for successful xenotransplantation of human cancer cells in many animal models. The studies of cancer development processes in vivo offer opportunities to understand cancer biology and discover new therapeutic strategies. In this context, zebrafish is a model that has been widely applied in the study of human diseases, such as cancer. However, the long-term immunosuppression of these adult zebrafish is still under study as a xenograft animal model for human cancer. This work aimed to evaluate the effects of 21 days of (long-term) exposure of dexamethasone in zebrafish-transplanted with MGSO-3 cells, human breast tumor cell line. Our results show that the animals, while kept on dexamethasone treatment, remained with a 50% reduction in the number of peripheral lymphocytes. In vitro data demonstrated that up to 7 days of dexamethasone treatment did not alter the morphology, proliferation, or viability of MGSO-3 cells. The animals that received a prolonged dexamethasone treatment allowed the engraftment of tumor cells in 100% of the zebrafish tested. These animals also showed tumor progression over 21 days. The experimental group that received only previous exposure to dexamethasone had their tumors regressed after 14 days. In conclusion, the prolonged use of dexamethasone in zebrafish showed a potential strategy for in vivo monitoring of xenograft tumor growth for development studies, as well as in anticancer drug discovery.

Cobalt-containing bioactive glass mimics vascular endothelial growth factor A and hypoxia inducible factor 1 function.

Bioactive glasses (BGs) have shown great potential for tissue regeneration and their composition flexibility allows the incorporation of different ions with physiological activities and therapeutic properties in the glass network. Among the many ions that could be incorporated, cobalt (Co) is a significant one, as it mimics hypoxia, triggering the formation of new blood vessels by the vascular endothelial growth factor A (VEGFA), due to the stabilizing effect on the hypoxia inducible factor 1 subunit alpha (HIF1A), an activator of angiogenesis-related genes, and is therefore of great interest for tissue engineering applications. However, despite its promising properties, the effects of glasses incorporated with Co on angiogenesis, through human umbilical cord vein endothelial cells (HUVECs) studies, need to be further investigated. Therefore, this work aimed to evaluate the biocompatibility and angiogenic potential of a new sol-gel BG, derived from the SiO2 -CaO-P2 O5 -CoO system. The structural evaluation showed the predominance of an amorphous glass structure, and the homogeneous presence of cobalt in the samples was confirmed. in vitro experiments showed that Co-containing glasses did not affect the viability of HUVECs, stimulated the formation of tubes and the gene expression of HIF1A and VEGFA. in vivo experiments showed that Co-containing glasses stimulated VEGFA and HIF1A expression in blood vessels and cell nuclei, respectively, in the deep dermis layer of the dorsal region of rats, featuring considerable local stimulation of the angiogenesis process due to Co-release. Co-containing glasses showed therapeutic effect, and Co incorporation is a promising strategy for obtaining materials with superior angiogenesis properties for tissue engineering applications.

Human Stem Cell-Derived Retinal Pigment Epithelial Cells as a Model for Drug Screening and Pre-Clinical Assays Compared to ARPE-19 Cell Line.

BACKGROUND AND OBJECTIVES: Eye diseases have a high socioeconomic impact on society and may be one of the fields in which most stem cell-related scientific accomplishments have been achieved recently. In this context, human Pluripotent Stem Cell (hPSC) technology arises as an important tool to produce and study human Embryonic Stem cell derived-Retinal Pigmented Epithelial Cells (hES-RPE) for several applications, such as cell therapy, disease modeling, and drug screening. The use of this technology in pre-clinical phases attends to the overall population desire for animal-free product development. Here, we aimed to compare hES-RPE cells with ARPE-19, one of the most commonly used retinal pigmented epithelial immortalized cell lines. METHODS AND RESULTS: Functional, cellular and molecular data obtained suggest that hES-RPE cells more closely resembles native RPEs compared to ARPE-19. Furthermore, hES-RPE revealed an interesting robustness when cultured on human Bruch's membrane explants and after exposure to Cyclosporine (CSA), Sirolimus (SRL), Tacrolimus (TAC), Leflunomide (LEF) and Teriflunomide (TER). On these conditions, hES-RPE cells were able to survive at higher drug concentrations, while ARPE-19 cell line was more susceptible to cell death. CONCLUSIONS: Therefore, hES-RPEs seem to have the ability to incur a broader range of RPE functions than ARPE-19 and should be more thoroughly explored for drug screening.

Nanostructured lipid carriers as a novel tool to deliver sclareol: physicochemical characterisation and evaluation in human cancer cell lines

Sclareol (SC) is arousing great interest due to its cytostatic and cytotoxic activities in several cancer cell lines. However, its hydrophobicity is a limiting factor for its in vivo administration. One way to solve this problem is through nanoencapsulation. Therefore, solid lipid nanoparticles (SLN-SC) and nanostructured lipid carriers (NLC-SC) loaded with SC were produced and compared regarding their physicochemical properties. NLC-SC showed better SC encapsulation than SLN-SC and was chosen to be compared with free SC in human cancer cell lines (MDA-MB-231 and HCT-116). Free SC had slightly higher cytotoxicity than NLC-SC and produced subdiploid DNA content in both cell lines. On the other hand, NLC-SC led to subdiploid content in MDA-MB-231 cells and G2/M checkpoint arrest in HCT-116 cells. These findings suggest that SC encapsulation in NLC is a way to allow the in vivo administration of SC and might alter its biological properties