Naringenin-functionalized multi-walled carbon nanotubes: a potential approach for site-specific remote-controlled anticancer delivery for the treatment of lung cancer cells

Multi-walled carbon nanotubes functionalized with naringenin have been developed as new drug carriers to improve the performance of lung cancer treatment. The nanocarrier was characterized by Transmission Electron Microscopy (TEM), Fourier-Transform Infrared Spectroscopy (FTIR), X-ray photoelectron spectroscopy, Raman Spectroscopy, and Differential Scanning Calorimetry (DSC). Drug release rates were determined in vitro by the dialysis method. The cytotoxic profile was evaluated using the MTT assay, against a human skin cell line (hFB) as a model for normal cells, and against an adenocarcinomic human alveolar basal epithelial (A569) cell line as a lung cancer in vitro model. The results demonstrated that the functionalization of carbon nanotubes with naringenin occurred by non-covalent interactions. The release profiles demonstrated a pH-responsive behavior, showing a prolonged release in the tumor pH environment. The naringenin-functionalized carbon nanotubes showed lower cytotoxicity on non-malignant cells (hFB) than free naringenin, with an improved anticancer effect on malignant lung cells (A549) as an in vitro model of lung cancer.This work was supported by the Banco do Nordeste (grant FUNDECI/2016.0015), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Fundação de Apoio à Pesquisa e à Inovação Tecnológica do Estado de Sergipe (Fapitec) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES). Eliana B. Souto would like to acknowledge the contributions from the Portuguese Science and Technology Foundation (FCT/MCT) and from European Funds (PRODER/COMPETE) for the projects M-ERA-NET/0004/2015-PAIRED and UIDB/04469/2020 (strategic fund), co-financed by FEDER, under the Partnership Agreement PT2020.info:eu-repo/semantics/publishedVersio

Cashew gum (Anacardium occidentale) as a potential source for the production of tocopherol-loaded nanoparticles: formulation, release profile and cytotoxicity

Every year, more than thirty thousand tons of Cashew gum (Anacardium occidentale, family: Anacardiaceae) are produced in Brazil; however, only a small amount is used for different applications in foodstuff and in pharmaceutical industries. As a raw material for the production of drug delivery systems, cashew gum is still regarded as an innovative compound worth to be exploited. In this work, cashew gum was extracted from the crude exudate of cashew tree employing four methodologies resulting in a light brown powder in different yields (40.61% to 58.40%). The total ashes (0.34% to 1.05%) and moisture (12.90% to 14.81%) were also dependent on the purification approach. FTIR spectra showed the typical bands of purified cashew gum samples, confirming their suitability for the development of a pharmaceutical product. Cashew gum nanoparticles were produced by nanoprecipitation resulting in particles of low polydispersity (<0.2) and an average size depending on the percentage of the oil. The zeta potential of nanoparticles was found to be below 20 mV, which promotes electrostatic stability. Encapsulation efficiencies were above 99.9%, while loading capacity increased with the increase of the percentage of the oil content of particles. The release of the oil from the nanoparticles followed the KorsmeyerPeppas kinetics model, while particles did not show any signs of toxicity when tested in three distinct cell lines (LLC-MK2, HepG2, and THP-1). Our study highlights the potential added value of using a protein-, lignans-, and nucleic acids-enriched resin obtained from crude extract as a new raw material for the production of drug delivery systems.This research received funding from the Coordenação Aperfeiçoamento de Pessoal de Nivel Superior (CAPES), Fundação de Ámparo à Pesquisa do Estado de Sergipe (FAPITEC) (PROCESSO: 88887.159533/2017-00 extração, encapsulação e caracterização de bioativos para o interesse biotecnologico) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq 301964/2019-0 Chamada 06/2019, and Chamada CNPq nº 01/2019), from Portuguese Foundation for Science and Technology (FCT/MEC) through national funds, and co-financed by FEDER, under the Partnership Agreement PT2020 for the project UIDB/04469/2020.info:eu-repo/semantics/publishedVersio

Funding for Chagas Disease: A 10-Year (2009–2018) Survey

Chagas disease was discovered in 1909 by the Brazilian scientist Carlos Chagas. After more than 110 years, many outcomes have been achieved in all research fields; however, Chagas disease remains a serious public health problem, mainly in Latin America, being one of the most neglected tropical diseases in the world. As a neglected disease, it receives very little financial support. Nevertheless, how much is actually spent? With this question in mind, the goal of the present work was to summarize all funding employed by multiple institutions in the Chagas disease field in a 10-year survey. From 2009 to 2018, Chagas disease received only USD 236.31 million, representing 0.67% of the total applied for all neglected diseases in this period. Mostly, the investments are concentrated in basic research (47%) and drug development (42.5%), with the public sector responsible for 74% of all funding, followed by the industry (19%) and philanthropy (7%). Relevantly, NIH (USA) alone accounted for more than half of the total investment. Taking into account that Chagas disease has a great socio-economic impact, it is clear that more investments are needed, especially from endemic countries. Furthermore, coordinated strategies to make better use of resources and incentives for the pharmaceutical industry must be adopted

Lopinavir and Nelfinavir Induce the Accumulation of Crystalloid Lipid Inclusions within the Reservosomes of Trypanosoma cruzi and Inhibit Both Aspartyl-Type Peptidase and Cruzipain Activities Detected in These Crucial Organelles

Several research groups have explored the repositioning of human immunodeficiency virus aspartyl peptidase inhibitors (HIV-PIs) on opportunistic infections caused by bacteria, fungi and protozoa. In Trypanosoma cruzi, HIV-PIs have a high impact on parasite viability, and one of the main alterations promoted by this treatment is the imbalance in the parasite’s lipid metabolism. However, the reasons behind this phenomenon are unknown. In the present work, we observed by transmission electron microscopy (TEM) that the treatment of T. cruzi epimastigotes with the HIV-PIs lopinavir and nelfinavir induced a huge accumulation of crystalloid-shaped lipids within the reservosomes, most of them deforming these key organelles. As previously reported, those structures are characteristic of lipid inclusions formed mostly of cholesterol and cholesterol-esters. The fractionation of nontreated epimastigotes generated two distinct fractions enriched in reservosomes: one mostly composed of lipid inclusion-containing reservosomes (Fraction B1) and one where lipid inclusions were much less abundant (Fraction B2). Interestingly, the extract of Fraction B2 presented enzymatic activity related to aspartyl-type peptidases 3.5 times higher than that found in the extract obtained from Fraction B1. The cleavage of cathepsin D substrate by this class of peptidases was strongly impaired by pepstatin A, a prototypical aspartyl PI, and the HIV-PIs lopinavir and nelfinavir. In addition, both HIV-PIs also inhibited (to a lesser extent) the cruzipain activity present in reservosomes. Finally, our work provides new evidence concerning the presence and supposed participation of aspartyl peptidases in T. cruzi, even as it adds new information about the mechanisms behind the alterations promoted by lopinavir and nelfinavir in the protozoan

Chagas Disease Control—Many Approaches to Prospect

Chagas disease is an emerging and neglected tropical disease caused by the protozoan parasite Trypanosoma cruzi, estimated to infect 8 to 10 million people worldwide, according to the World Health Organization [...

Decoding the Anti-Trypanosoma cruzi Action of HIV Peptidase Inhibitors Using Epimastigotes as a Model

Made available in DSpace on 2015-04-17T17:04:29Z (GMT). No. of bitstreams: 2 license.txt: 1914 bytes, checksum: 7d48279ffeed55da8dfe2f8e81f3b81f (MD5) rubem_mennabarretoetal2_IOC_2014.pdf: 2175139 bytes, checksum: 6a0196b654df473c1f1556cd07cf8120 (MD5) Previous issue date: 2014Universidade Federal do Rio de Janeiro (UFRJ). Instituto de Microbiologia Paulo de Góes (IMPG). Departamento de Microbiologia Geral. Laboratório de Investigação de Peptidases. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biologia Celular. Rio de Janeiro, RJ, BrasilFundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biologia Molecular e Doenças Endêmicas. Rio de Janeiro, RJ, Brasil.Universidade Federal do Rio de Janeiro (UFRJ). Instituto de Microbiologia Paulo de Góes (IMPG). Departamento de Microbiologia Geral. Laboratório de Investigação de Peptidases. Rio de Janeiro, RJ, Brasil / Universidade Federal do Rio de Janeiro. Instituto de Química. Programa de Pós-Graduação em Bioquímica. Rio de Janeiro, RJ, Brasil.Universidade Federal do Rio de Janeiro (UFRJ). Instituto de Microbiologia Paulo de Góes (IMPG). Departamento de Microbiologia Geral. Laboratório de Investigação de Peptidases. Rio de Janeiro, RJ, Brasil.Background: Aspartic peptidase inhibitors have shown antimicrobial action against distinct microorganisms. Due to an increase in the occurrence of Chagas’ disease/AIDS co-infection, we decided to explore the effects of HIV aspartic peptidase inhibitors (HIV-PIs) on Trypanosoma cruzi, the etiologic agent of Chagas’ disease. Methodology and Principal Findings: HIV-PIs presented an anti-proliferative action on epimastigotes of T. cruzi clone Dm28c, with IC50 values ranging from 0.6 to 14 mM. The most effective inhibitors, ritonavir, lopinavir and nelfinavir, also had an anti-proliferative effect against different phylogenetic T. cruzi strains. The HIVPIs induced some morphological alterations in clone Dm28c epimastigotes, as reduced cell size and swollen of the cellular body. Transmission electron microscopy revealed that the flagellar membrane, mitochondrion and reservosomes are the main targets of HIV-PIs in T. cruzi epimastigotes. Curiously, an increase in the epimastigote-into-trypomastigote differentiation process of clone Dm28c was observed, with many of these parasites presenting morphological alterations including the detachment of flagellum from the cell body. The pretreatment with the most effective HIV-PIs drastically reduced the interaction process between epimastigotes and the invertebrate vector Rhodnius prolixus. It was also noted that HIV-PIs induced an increase in the expression of gp63-like and calpain-related molecules, and decreased the cruzipain expression in epimastigotes as judged by flow cytometry and immunoblotting assays. The hydrolysis of a cathepsin D fluorogenic substrate was inhibited by all HIV-PIs in a dose-dependent manner, showing that the aspartic peptidase could be a possible target to these drugs. Additionally, we verified that ritonavir, lopinavir and nelfinavir reduced drastically the viability of clone Dm28c trypomastigotes, causing many morphological damages. Conclusions and Significance: The results contribute to understand the possible role of aspartic peptidases in T. cruzi physiology, adding new in vitro insights into the possibility of exploiting the use of HIV-PIs in the clinically relevant forms of the parasite

Effect of HIV PIs on the <i>T. cruzi</i> clone Dm28c epimastigote-into-trypomastigote differentiation.

<p>Epimastigotes were incubated for 72 h and 96 h in the absence or in the presence of HIV PIs at 1 and 10 µM. After this period, Giemsa-stained smears were prepared and the percentage of each morphological stage was calculated after counting at least 200 parasites for each system. The graphics show the percentage of differentiating forms (which was considered the intermediate morphological stage between typical epimastigotes and typical trypomastigotes) and trypomastigote cells. Data shown are the mean standard error (S.E.) of three independent experiments performed in triplicate. All values are statistically different from control except for: indinavir treatment for 72 h and after 96 h with 1 µM for trypomastigote forms; amprenavir treatment with 1 µM over 72 and 96 h (<i>P</i><0.05).</p

Effect of HIV PIs on the expression of gp63, calpain and cruzipain by <i>T. cruzi</i> clone Dm28c epimastigotes.

<p>At left, flow cytometric analysis of the anti-gp63, anti-Dm-calpain and anti-cruzipain antibodies binding to <i>T. cruzi</i> clone Dm28c epimastigotes after treatment with HIV PIs (IC₅₀ value) for 24 h. <b>C</b>, non-treated cells; <b>RTV</b>, ritonavir; <b>LPV</b>, lopinavir; <b>NFV</b>, nelfinavir; <b>SQV</b>, saquinavir; <b>IDV</b>, indinavir; <b>APV</b>, amprenavir. Data are expressed as the percentage of fluorescent cells. At right, Western blotting showing the proteins recognized by the anti-gp63, anti-Dm-calpain and anti-cruzipain antibodies in the whole cellular extract from <i>T. cruzi</i> clone Dm28c epimastigotes in non-treated cells and after treatment with nelfinavir (IC₅₀ value) for 24 h. Anti-α-tubulin monoclonal antibody was used as a control for sample loading in the blots. The apparent molecular masses of each band detected are shown, and the densitometric analysis of the reactive proteins is expressed as densitometric units (<b>D.U.</b>). The results represent means standard deviation of three independent experiments, and the symbol (♦) denotes statistic difference to control (<i>P</i><0.05).</p

Effect of HIV PIs on the interaction of <i>T. cruzi</i> clone Dm28c epimastigote forms with <i>R. prolixus</i> explanted gut.

<p>Epimastigotes were pre-treated for 1 h (A) or 24 h (B) with different concentrations of HIV PIs and subjected to interaction with the gut of the insect vector. After 1 h of parasite-vector interaction, the intestines were washed and the attached forms were counted. Results are expressed as the number of adhered parasites per gut and the symbol (♦) denotes significant differences compared to control (<i>P</i><0.05). All counts were performed in triplicate.</p

Antileishmanial Efficacy of the Calpain Inhibitor MDL28170 in Combination with Amphotericin B

The necessity of drug combinations to treat leishmaniasis came to the surface mainly because of the toxicity of current treatments and the emergence of resistant strains. The calpain inhibitor MDL28170 has previously shown anti-Leishmania activity, therefore its use in association with standard drugs could provide a new alternative for the treatment strategy against leishmaniasis. In this study, we analyzed the potential of the combination of MDL28170 and the antileishmanial drug amphotericin B against Leishmania amazonensis and Leishmania chagasi. The compounds were tested in the combination of the &frac12; &times; IC50 value of MDL28170 plus the &frac14; &times; IC50 value of amphotericin B, which led to an increment in the anti-promastigote activity when compared to the single drug treatments. This drug association revealed several and severe morphophysiological changes on parasite cells, such as loss of plasma membrane integrity, reduced size of flagellum, and depolarization of mitochondrial membrane potential besides increased reactive oxygen species production. In addition, the combination of both drugs had a deleterious effect on the Leishmania&ndash;macrophage interaction, reflecting in a significant anti-amastigote action, which achieved a reduction of 50% in the association index. These results indicate that the combination treatment proposed here may represent a new alternative for leishmaniasis chemotherapy