Investigação do efeito de nanoemulsão à base de óleo de pequi e de sua associação com docetaxel e ácido anacárdico em células de câncer de mama, in vitro e in vivo

Tese (Doutorado) — Universidade de Brasília, Instituto de Ciências Biológicas, Programa de Pós-Graduação em Nanociência e Nanobiotecnologia, 2020.O câncer de mama triplo negativo (CMTN) representa a forma mais agressiva da doença e apresenta elevada taxa de mortalidade atribuída à metástase. O docetaxel (DTX) é um quimioterapeutico frequentemente usado no tratamento do CMTN. No entanto, efeitos adversos limitam sua aplicação como monoterapia. A biodiversidade brasileira representa fonte rica de moléculas bioativas que podem contribuir para o tratamento do CMTN. O óleo de pequi (Caryocar brasiliense Camb), planta originária do Cerrado brasileiro, é constituído principalmente por ácidos graxos e carotenoides que lhe conferem diversas propriedades biológicas, incluindo efeito antitumoral. Além disso, o ácido anacárdico (AA), isolado da casca da castanha de caju (Anacardium occidentale), também apresenta potencial anticâncer. Entretanto, a hidrofobicidade dessas moléculas representa limite à suas administrações. Portanto, nanoemulsão à base de óleo de pequi (PeNE) foi projetada como sistema de transporte e liberação do óleo sozinho ou em associação com DTX e AA baseando-se em terapia combinatória. Neste trabalho, as investigações foram conduzidas em 4 capítulos: (I) síntese e caracterização de PeNE e avaliação da citotoxicidade em células 4T1 e NIH3T3; (II) investigação dos efeitos antitumorais do PeNE em células 4T1, in vitro, na proliferação celular, morfologia, funcionamento de organelas (lisossomo, mitocôndria, núcleo), mecanismos celulares (produção de ROS e liberação de cálcio) e perfil de morte; (III) síntese, caracterização e avaliação do efeito antitumoral da associação da PeNE com DTX (PDTX) e AA (PAA) em células 4T1 na morfologia, proliferação celular, organelas, mecanismos celulares, perfil de morte, in vitro (IV) e avaliação da eficácia e toxicidade por meio de análises clínicas, bioquímicas, hematológicas e histopatológicas em modelo de camundongos fêmeas balb/c, in vivo. Os resultados demonstraram sucesso na obtenção de PeNE estável e com características adequadas à proposta de ser utilizada como plataforma para transporte de biomoléculas. Além disso, PeNE apresenta atividade citotóxica em células 4T1 dose e tempo dependente com diminuição da proliferação celular, indução de lesão da membrana plasmática, bloqueio do ciclo celular, fragmentação de DNA, permeabilidade da membrana lisossomal, despolarização da membrana mitocondrial, alteração da morfologia celular, da produção intracelular de ROS e no nível de cálcio. Adicionalmente, a associação de DTX e AA à PeNE aumentou a eficácia dos tratamentos, in vitro. A combinação PAA+PDTX apresentou efeito aditivo e foi considerada como tratamento mais promissor, in vitro. Contudo, os ensaios in vivo sugeriram que a combinação AA+DTX livres administrados por via intratumoral é o tratamento de maior eficácia com diminuição significativo do volume tumoral, regressão tumoral em 50% dos animais tratados e impedindo processo de metástase em 100% dos animais. Apesar dessa diferença observada entre modelos in vitro e in vivo, pode-se concluir que a associação de AA e DTX, livres (in vivo) ou nanoencapsulado (in vitro) apresentou melhoria quando comparado com os demais tratamentos. Dessa forma, essas combinações são promissoras e merecem futuros estudos para investigar melhor o potencial anticâncer.Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES); Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) e Fundação de Apoio à Pesquisa do Distrito Federal (FAP/DF).Triple negative breast cancer (CMTN) is the most aggressive form of breast cancer and presents high mortality rate attributed to metastasis process. Docetaxel (DTX) is a chemotherapeutic frequently used in the treatment of CMTN. However, adverse effects limit its application as monotherapy. Brazilian biodiversity represents a rich source of bioactive molecules that can contribute to the treatment of CMTN. Pequi oil (Caryocar brasiliense Camb), a plant native to the Brazilian Cerrado, is mainly constitute of fatty acids and carotenoids that give it several biological properties, including antitumor effect. In addition, anacardic acid (AA), isolated from the cashew nut shell (Anacardium occidentale), also demonstrates anticancer potential. However, the hydrophobicity of these molecules represents a limit to their administrations. Therefore, pequi oil-based nanoemulsion (PeNE) was designed as a transport and release system for oil alone or in association with DTX and AA based on combinatorial therapy. In this work, investigations were conducted in 4 chapters: (I) synthesis and characterization of PeNE and evaluation of cytotoxicity in 4T1 and NIH3T3 cells; (II) investigation of the antitumor effects of PeNE in 4T1 cells, in vitro, on cell proliferation, morphology, organelle functioning (lysosome, mitochondria, nucleus), cellular mechanisms (production of ROS and release of calcium) and death profile; (III) synthesis, characterization and evaluation of the antitumor effect of the association of PeNE with DTX (PDTX) and AA (PAA) in 4T1 cells in morphology, cell proliferation, organelles, cellular mechanisms, death profile, in vitro (IV) and evaluation of efficacy and toxicity by clinical, biochemical, hematological and histopathological analyzes in a model of female balb / c mice, in vivo. The results demonstrated success in obtained stable PeNE with characteristics appropriate to the proposal to be used as a platform for transporting biomolecules. In addition, PeNE shows dose and time dependent cytotoxic activity in 4T1 cells with decreased of cell proliferation, induction of plasma membrane damage, cell cycle block, DNA fragmentation, lysosomal membrane permeability, mitochondrial membrane depolarization and alteration of cells morphology, intracellular production of ROS and the level of calcium. Additionally, the association of DTX and AA with PeNE increased the effectiveness of treatments, in vitro. The PAA + PDTX combination presented an additive effect and this treatment group was considered the most promising, in vitro. However, in vivo trials suggested that the combination AA + DTX administered intratumorally is the most effective treatment with significant reduction in tumor volume, tumor regression in 50% of treated animals and preventing metastasis in 100% of animals. Despite this difference observed between in vitro and in vivo models, it can be concluded that the association of AA and DTX, free (in vivo) or nanoencapsulated (in vitro) showed improvement when compared with the other treatments. Thus, these combinations are promising and deserve future studies to better investigate their anticancer potential

In vivo efficacy and toxicity of curcumin nanoparticles in breast cancer treatment : a systematic review

Breast cancer is one of the most prevalent types of malignant tumors in the world, resulting in a high incidence of death. The development of new molecules and technologies aiming to apply more effective and safer therapy strategies has been intensively explored to overcome this situation. The association of nanoparticles with known antitumor compounds (including plant-derived molecules such as curcumin) has been considered an effective approach to enhance tumor growth suppression and reduce adverse effects. Therefore, the objective of this systematic review was to summarize published data regarding evaluations about efficacy and toxicity of curcumin nanoparticles (Cur-NPs) in in vivo models of breast cancer. The search was carried out in the databases: CINAHL, Cochrane, LILACS, Embase, FSTA, MEDLINE, ProQuest, BSV regional portal, PubMed, ScienceDirect, Scopus, and Web of Science. Studies that evaluated tumor growth in in vivo models of breast cancer and showed outcomes related to Cur-NP treatment (without association with other antitumor molecules) were included. Of the 528 initially gathered studies, 26 met the inclusion criteria. These studies showed that a wide variety of NP platforms have been used to deliver curcumin (e.g., micelles, polymeric, lipid-based, metallic). Attachment of poly(ethylene glycol) chains (PEG) and active targeting moieties were also evaluated. Cur-NPs significantly reduced tumor volume/weight, inhibited cancer cell proliferation, and increased tumor apoptosis and necrosis. Decreases in cancer stem cell population and angiogenesis were also reported. All the studies that evaluated toxicity considered Cur-NP treatment to be safe regarding hematological/biochemical markers, damage to major organs, and/or weight loss. These effects were observed in different in vivo models of breast cancer (e.g., estrogen receptor-positive, triple-negative, chemically induced) showing better outcomes when compared to treatments with free curcumin or negative controls. This systematic review supports the proposal that Cur-NP is an effective and safe therapeutic approach in in vivo models of breast cancer, reinforcing the currently available evidence that it should be further analyzed in clinical trials for breast cancer treatments

Síntese verde de nanopartículas de prata a partir de extrato aquoso do tubérculo de Curcuma longa associadas à quitosana e avaliação da atividade antitumoral in vitro em câncer de pele não melanoma (linhagem A431)

O câncer de pele não melanoma (CPNM) é o tipo de tumor de maior incidência no Brasil, e o subtipo maligno denominado de carcinoma epidermoide representa 70% das mortes por CPNM. Os tratamentos convencionais apresentam grande chance de cura, entretanto, são geralmente associados a vários efeitos colaterais adversos localizados e/ou sistêmicos. Portanto, o desenvolvimento de novas estratégias terapêuticas é de grande interesse. O uso de compostos naturais e de nanopartículas de prata (AgNPs) demonstram uma grande relevância para o tratamento de vários tipos de câncer. Adicionalmente, a síntese verde de AgNPs consiste em utilizar organismos biológicos e/ou biomoléculas isoladas como agentes redutores e estabilizantes. Portanto, o método de síntese se torna mais eco-amigável, de menor custo e biocompatível. Diante do exposto, o objetivo deste trabalho é de sintetizar AgNPs utilizando extrato aquoso (EA) do tubérculo de açafrão (Curcuma longa) e nitrato de prata (AgNO3), associá-las com polímeros de quitosana, caracterizá-las, avaliar a citotoxicidade em células tumorais de CPNM (A431) e não tumorais (queratinócitos - HaCAT) e investigar seus possíveis mecanismos de ação em linhagem de CPNM (A431), in vitro. O rendimento da síntese de AgNPs foi dependente da variação de temperatura de armazenamento da C. longa, do método de extração, da temperatura de síntese e da concentração de EA e de AgNO3. Após a otimização da síntese (C. longa armazenada a temperatura ambiente, extração por fervura, síntese a 75°C por 24 horas com 2 mg/mL de EA e 1 mM de AgNO3) a quitosana foi utilizada para recobrir as AgNPs devido às suas propriedades de biocompatibilidade, biodegradabilidade e bioadesividade. As AgNPs obtidas apresentaram uma homogeneidade e estabilidade coloidal moderadas (PdI de 0,319 ± 0,043 e potencial Zeta de -24,8 ± 2,2 mV) com diâmetro hidrodinâmico (DH) de 210,3 ± 57,1 nm. Em paralelo, as AgNPs recobertas por quitosana (CH-AgNPs) apresentaram DH de 328,0 ± 61,6 nm, moderada homogeneidade (PdI de 0,372 ± 0,059) e estabilidade coloidal (potencial Zeta de +53,9 ± 3,1 mV). A forma das nanopartículas foi avaliada por técnicas de microscopia. As amostras demonstraram ser estáveis por 30 dias a 4°C. Além disso, ambas demonstraram atividade citotóxica dose-dependente em células A431 e HaCAT. A investigação dos possíveis mecanismos de ação das AgNPs expostas às células A431 por 24 horas a 50μM demonstrou a indução de lesão de membrana plasmática (50%), diminuição da proliferação celular (50%), aumento da taxa de fragmentação de DNA (40%) e do potencial de membrana mitocondrial. Além disso, as CH-AgNPs bloquearam as células em fase G1 do ciclo celular e apresentaram maior marcação por Anexina-V, sugerindo morte celular por apoptose. Em suma, pode-se concluir que a síntese verde demonstrou ser uma boa alternativa para a obtenção de AgNPs com grande potencial citotóxico. A associação com a quitosana pode representar um potencial promissor para o tratamento de CPMN. _______________________________________________________________________________________________ ABSTRACTNonmelanoma skin cancers (NMSC) represents the most incident tumor in Brazil, and the malignant subtype squamous cell carcinoma accounts for 70% of deaths from NMSC. Conventional treatments have great chance of cure, however, they are usually associated with several localized and/or systemic adverse side effects. Therefore, the development of new therapeutic strategies is of great interest. The use of natural compounds and silver nanoparticles (AgNPs) showed a great importance for the treatment of various cancers. Additionally, the green synthesis of AgNPs consists of using biological organisms and/or biomolecules isolated as reducing agents and stabilizers. Therefore, this method of synthesis becomes more eco-friendly, less costly and biocompatible. Given the above, the objective of this study is to synthesize AgNPs using aqueous extract (EA) of turmeric tuber (Curcuma longa) and silver nitrate (AgNO3), associate them with chitosan polymers, characterize them, analyze the cytotoxicity NMSC tumor (A431) and non-tumor cells (keratinocytes - HaCaT) and investigate the possible mechanisms of action in NMSC cells line (A431) in vitro. The yield of synthesis was dependent on the variation of the storage temperature of C. longa, the method of extraction, the temperature of synthesis and the concentration of EA and AgNO3. After optimization of the synthesis parameters (C. longa stored at room temperature, extraction by boiling, and synthesis at 75°C for 24 hours with 2 mg/ml EA and 1 mM AgNO3) chitosan was used to coat AgNPs due to its properties of biocompatibility, biodegradability and bioadhesiveness. The obtained AgNPs showed a moderate homogeneity and colloidal stability (0.319 ± 0.043 PDI and Zeta potential of - 24.8 ± 2.2 mV) with hydrodynamic diameter (HD) of 210.3 ± 57.1 nm. In parallel, covered with chitosan (CH-AgNPs) exhibited HD of 328.0 ± 61.6 nm, moderate homogeneity (PDI 0.372 ± 0.059), and colloidal stability (Zeta potential of +53.9 ± 3.1 mV). The shape of the nanoparticles was analyzed by microscopy techniques. The samples proved to be stable for 30 days at 4°C. In addition, both showed dosedependent cytotoxic activity in HaCaT and A431 cells. The investigation of possible mechanisms of action of AgNPs exposed to A431 cells for 24 hours at 50 μM demonstrated the induction of plasma membrane injury (50%), reduction of cell proliferation (50%), increased of DNA fragmentation rate (40%) and mitochondrial membrane potential and induction of cell death by apoptosis. Furthermore, CH-AgNPs blocked cells in G1 phase of the cell cycle and presented anexin-V dialing suggesting cell death by apoptosis. In short, it can be concluded that the green synthesis proved to be a good alternative for obtaining AgNPs with high cytotoxic potential. The association with chitosan may represent a promising potential for the treatment of CPMN

Melittin sensitizes skin squamous carcinoma cells to 5-fluorouracil by affecting cell proliferation and survival

Combined 5-fluorouracil (5-FU) and melittin (MEL) is believed to enhance cytotoxic effects on skin squamous cell carcinoma (SCC). However, the rationale underlying cytotoxicity is fundamentally important for a proper design of combination chemotherapy, and to provide translational insights for future therapeutics in the dermatology field. The aim was to elucidate the effects of 5-FU/MEL combination on the viability, proliferation and key structures of human squamous cell carcinoma (A431). Morphology, plasma membrane, DNA, mitochondria, oxidative stress, cell viability, proliferation and cell death pathways were targeted for investigation by microscopy, MTT, trypan blue assay, flow cytometry and real-time cell analysis. 5-FU/MEL (0.25 µM/0.52 µM) enhanced the cytotoxic effect in A431 cells (74.46%, p < .001) after 72 h exposure, showing greater cytotoxic effect when compared to each isolated compound (45.55% 5-FU and 61.78% MEL). The results suggest that MEL induces plasma membrane alterations that culminate in a loss of integrity at subsequent times, sensitizing the cell to 5-FU action. DNA fragmentation, S and G2/M arrest, disruption of mitochondrial metabolism, and alterations in cell morphology culminated in proliferation blockage and apoptosis. 5-FU/MEL combination design optimizes the cytotoxic effects of each drug at lower concentrations, which may represent an innovative strategy for SCC therapy

Evaluation of biocompatibility, anti-inflammatory, and antinociceptive activities of pequi oil-based nanoemulsion in in vitro and in vivo models

Pequi oil (Caryocar brasiliense) contains bioactive compounds capable of modulating the inflammatory process; however, its hydrophobic characteristic limits its therapeutic use. The encapsulation of pequi oil in nanoemulsions can improve its biodistribution and promote its immunomodulatory effects. Thus, the objective of the present study was to formulate pequi oil-based nanoemulsions (PeNE) to evaluate their biocompatibility, anti-inflammatory, and antinociceptive effects in in vitro (macrophages—J774.16) and in vivo (Rattus novergicus) models. PeNE were biocompatible, showed no cytotoxic and genotoxic effects and no changes in body weight, biochemistry, or histology of treated animals at all concentrations tested (90–360 µg/mL for 24 h, in vitro; 100–400 mg/kg p.o. 15 days, in vivo). It was possible to observe antinociceptive effects in a dose-dependent manner in the animals treated with PeNE, with a reduction of 27 and 40% in the doses of 100 and 400 mg/kg of PeNE, respectively (p < 0.05); however, the treatment with PeNE did not induce edema reduction in animals with carrageenan-induced edema. Thus, the promising results of this study point to the use of free and nanostructured pequi oil as a possible future approach to a preventive/therapeutic complementary treatment alongside existing conventional therapies for analgesia.Instituto de Ciências Biológicas (IB)Departamento de Biologia Celular (IB CEL)Faculdade UnB Ceilândia (FCE)Curso de Farmácia (FCE-FAR)Faculdade de Medicina (FM)Faculdade UnB Gama (FGA)Faculdade UnB Planaltina (FUP)Programa de Pós-Graduação em Nanociência e NanobiotecnologiaPrograma de Pós-Graduação em Ciências e Tecnologias em Saúd

Nanocapsules for the co-delivery of selol and doxorubicin to breast adenocarcinoma 4T1 cells <i>in vitro</i>

<p>Nanocapsules (NCS-DOX) with an oily core of selol and a shell of poly(methyl vinyl ether-co-maleic anhydride) covalently conjugated to doxorubicin were developed. These nanocapsules are spherical, with an average hydrodynamic diameter of about 170 nm, and with negative zeta potential. NCS-DOX effectively co-delivered the selol and the doxorubicin into 4T1 cells and changed the intracellular distribution of DOX from the nuclei to the mitochondria. Moreover, a significantly increased cytotoxicity against 4T1 cells was observed, which is suggestive of additive or synergic effect of selol and doxorubicin. In conclusion, PVM/MA nanocapsules are suitable platforms to co-deliver drugs into cancer cells.</p

A novel N95 respirator with chitosan nanoparticles: mechanical, antiviral, microbiological and cytotoxicity evaluations

Abstract Background It is known that some sectors of hospitals have high bacteria and virus loads that can remain as aerosols in the air and represent a significant health threat for patients and mainly professionals that work in the place daily. Therefore, the need for a respirator able to improve the filtration barrier of N95 masks and even inactivating airborne virus and bacteria becomes apparent. Such a fact motivated the creation of a new N95 respirator which employs chitosan nanoparticles on its intermediate layer (SN95 + CNP). Results The average chitosan nanoparticle size obtained was 165.20 ± 35.00 nm, with a polydispersity index of 0.36 ± 0.03 and a zeta potential of 47.50 ± 1.70 mV. Mechanical tests demonstrate that the SN95 + CNP respirator is more resistant and meets the safety requisites of aerosol penetration, resistance to breath and flammability, presenting higher potential to filtrate microbial and viral particles when compared to conventional SN95 respirators. Furthermore, biological in vitro tests on bacteria, fungi and mammalian cell lines (HaCat, Vero E6 and CCL-81) corroborate the hypothesis that our SN95 + CNP respirator presents strong antimicrobial activity and is safe for human use. There was a reduction of 96.83% of the alphacoronavirus virus and 99% of H1N1 virus and MHV-3 betacoronavirus after 120 min of contact compared to the conventional respirator (SN95), demonstrating that SN95 + CNP have a relevant potential as personal protection equipment. Conclusions Due to chitosan nanotechnology, our novel N95 respirator presents improved mechanical, antimicrobial and antiviral characteristics