Cytotoxic Effects Caused by Functionalized Carbon Nanotube in Murine Macrophages

Background/Aims: The development of new nanomaterials has been growing in recent decades to bring benefits in several areas, especially carbon-based nanoparticles, which have unique physical-chemical properties and allow to take on several applications. Consequently, the use of new nanomaterials without previous toxicological studies raises concern about possible harmful health effects. The aim of this study was to investigate the cytotoxic profile of a new multi-walled carbon nanotube (MWCNT) functionalized with tetraethylenepentamine called OCNT-TEPA using in vitro assays in murine macrophage cells linage J774 A.1. Methods: OCNT-TEPA was characterized by transmission electron microscopy (TEM) and high resolution TEM (HR-TEM), scanning electron microscopy (SEM), zeta potential and dynamic light scattering (DLS), and its cytotoxic effects were evaluated at 24 and 48 hours by cell viability assays (MTT and NR), morphology and cell recovery (optic microscopy and clonogenic assay), formation of reactive oxygen (ROS) and nitric oxide (NO) species, inflammatory profile (IL-6 and TNF cytokines), mitochondrial membrane potential analysis (MMP), activation of the caspase 3 pathway and cell death (flow cytometry). Results: The data showed a significant decrease in cell viability, increased production of ROS and NO, alteration of mitochondrial membrane potential, increased levels of inflammatory cytokines, alteration of cell morphology, activation of the Caspase 3 pathway and consequently cell death, in the highest concentrations of OCNT-TEPA tested in the periods of 24 and 48 hours. Conclusion: The analyses showed that OCNT-TEPA has a dose-dependent cytotoxic profile, which may be harmful to murine macrophages (J774 A.1) and may represent a health risk

Bacterial cellulose-based biomaterials on third-degree burns in rats

Burns are cutaneous lesions that present  high rate of morbidity and mortality worldwide. In order to innovate the treatment strategies currently applied new biomaterials are being investigated. The aim of the present study was to evaluate the action of bacterial cellulose in both membrane and gel form, in the treatment of third degree burns in rats. For this, 24 Wistar rats were used, divided into three distinct groups. The lesion was performed with the aid of a soldering iron heated at 150 °C pressed on the back of the animal for 10 seconds. Treatment was performed immediately after wound induction, and skin samples were collected on the tenth day post-injury. Statistical analysis was performed using a significance level of 5% (p?0.05). The histological results show differences in the healing process presented by each group. The group that received bacterial cellulose in the membrane format presented the best results, such as discrete inflammatory infiltrate and better morphological quality of the tissue, characterizing an advanced stage of the healing process, also proven in the collagen quantitative analysis. On the other hand, the group that received the cellulose gel showed characteristics of an inflammatory phase with the presence of evident ulcerations, which corresponds to a delay in the healing process even when compared to CG alone. Thus, it was concluded that before the biomaterials tested cellulose membrane in the format presented more favorable results both in terms of environmental protection as a contribution to an adequate tissue recovery.

Efeitos da terapia laser de baixa intensidade e de membranas de celulose bacteriana no tratamento de queimaduras de terceiro grau em ratos

Burn injuries represent a high risk of morbidity and mortality worldwide. In severe and deep injuries, the wound healing process is complex and requires the participation of different types of cells. Among the existing treatments, biomaterials and LLLT are highlighted for having properties that favor and stimulate the healing process. Thus, three studies were conducted to evaluate the effects of bacterial cellulose membranes in its pure state or enriched with lidocaine and LLLT (660 nm) in two different fluences (12.5J/cm2 and 25J/cm2) used independently or associated, on third-degree burns in rats. The burn was induced with an aluminum plate at 150°C, pressed onto the animal's back for 10 seconds. In the first study the action of bacterial cellulose membrane in its pure state and enriched with lidocaine, as biological dressings was evaluated. Therefore, the rats were divided in three experimental groups, CG (control group), MG (group treated with the pure bacterial cellulose membrane), and MLG (group treated with the bacterial cellulose membrane with lidocaine). The treated groups showed an advanced wound healing when compared to the control group. In the immunohystochemical analysis of COX-2, the treated groups showed a light immunoexpression, with the characteristics of repaired tissue. Thus, bacterial cellulose-based biological dressings were effective and provided a favorable environment for the development of the healing process. In the second study, the effects of LLLT with two different fluences (12.5J/cm2 and 25J/cm2) in three experimental groups, divided into CG (control group), LG12.5 (burning treated group 12.5 J/cm2) and LG25 (burn group treated with 25 J/cm2) were evaluated. The animals received laser application immediately after the induction of the lesion and the subsequent doses 2, 4, 6 and 8 days after the induction, at five different points, four on the edges of the wound and one in the central region. The LG25 had better results, with higher number of blood vessels, increased immunoexpression of VEGF (Vascular Endothelial Growth Factor) and decreased immunoexpression of COX-2 (Cyclooxygenase-2) when compared to CG and GL12.5 groups. The LG12.5 showed the longest delay in the progression of the healing process, due to its intense inflammation and tissue fibrosis when compared to CG and LG25. In the third study the association of pure bacterial cellulose membrane and LLLT (660 nm, 25 J/cm2) was investigated. Four groups were evaluated, CG (control group), MG (burn group treated with pure bacterial cellulose membrane), LG (burn group treated with laser 25 J/cm2) and MG + L (burn group treated with bacterial cellulose membrane + LLLT). Histological findings demonstrated that the treated group showed better results in the healing process. The (GM + L) showed results similar to those found in the GL, evidencing the stimulatory effects of angiogenesis provided by the laser light. GM showed improvement in the healing process, indicating the proliferative phase. However, although LLLT presented the expected proinflam matory effects, which modulate the inflammatory phase and favor tissue regeneration, the isolated action of the bacterial cellulose membrane proved to be advantageous by presenting tissue characteristics, which are compatible with a more advanced phase of the healing process.Não recebi financiamentoQueimaduras representam mundialmente alto risco de morbidade e mortalidade. Nas consideradas severas ou profundas, o processo de cicatrização é complexo e necessita da participação de diversas linhagens celulares. Dentre os tratamentos existentes, os biomateriais e a Terapia laser de baixa intensidade (LLLT) vêm se destacando por apresentar propriedades que favorecem e estimulam o processo de cicatrização. Assim, foram realizados três estudos com o objetivo de avaliar os efeitos das membranas de celulose bacteriana pura e com lidocaína e da LLLT (660 nm) em duas fluências diferentes (12,5J/cm2 e 25J/cm2), utilizados independentemente ou associados, em queimaduras de terceiro grau em ratos. A queimadura foi realizada através de uma placa de alumínio acoplada a um ferro de solda aquecido a 150°C, pressionado no dorso do animal por 10 segundos. No primeiro estudo a ação das membranas de celulose bacteriana pura e acrescida de lidocaína foram avaliados. Para isso, foram estabelecidos três grupos experimentais divididos em GC (grupo controle), GM (grupo queimadura tratado com a membrana de celulose bacteriana pura) e GML (grupo queimadura tratado com a membrana de celulose acrescida de lidocaína). Os grupos tratados com as membranas demonstraram um processo de cicatrização avançado quando comparado ao grupo controle. Na análise da imunoexpressão da COX-2, os grupos tratados apresentaram a imunoexpressão de forma leve, o que evidencia características de tecido reparado. Assim, concluímos que os curativos biológicos a base de celulose bacteriana, foram efetivos, proporcionando um ambiente favorável para a evolução do processo de cicatrização. No segundo estudo, foram avaliados os efeitos da LLLT com duas diferentes fluências (12,5J/cm2 e 25J/cm2) em três grupos experimentais, divididos em GC (grupo controle), GL12,5 (grupo queimadura tratado com 12,5J/cm2) e GL25 (grupo queimadura tratado com 25J/cm2). Os animais receberam a aplicação da LLLT imediatamente após a indução da lesão e nos dias 2, 4, 6 e 8 subsequentes, em cinco pontos distintos, sendo quatro localizados nas bordas da ferida e um na região central. O GL25 demonstrou os melhores resultados, com maior imunoexpressão do VEGF (Fator de crescimento endotelial vascular), maior quantidade de vasos sanguíneos e menor imunoexpressão da COX-2 Ciclooxigenase-2) quando comparado aos grupos GC e GL12,5. Com isso, foi possível concluir que a maior fluência, bem como a maior energia depositada no tecido foi mais eficaz em estimular o processo de cicatrização em queimaduras de terceiro grau em ratos. No terceiro estudo foi abordada a associação da membrana de celulose bacteriana pura com a LLLT (660 nm, 25J/cm2). Quatro grupos foram avaliados, GC (grupo controle), GM (grupo queimadura tratado com membrana de celulose bacteriana pura), GL (grupo queimadura tratado com laser 25J/cm2) e GM+L (grupo queimadura tratado com membrana de celulose bacteriana + LLLT). Os achados histológicos demonstraram que os grupos que receberam os tratamentos apresentaram melhores resultados no processo de cicatrização quando comparados ao grupo controle. O (GM+L) apresentou resultados similares aos achados no GL, evidenciando os efeitos estimuladores da angiogênese fornecidos pela luz laser. O GM apresentou avanço no processo de cicatrização, evidenciando a fase proliferativa. Assim, apesar da LLLT apresentar os efeitos pró-inflamatórios esperados, que modulam a fase inflamatória e favorecem a regeneração tecidual, a ação isolada da membrana de celulose bacteriana demonstrou vantagens no tratamento por apresentar características morfológicas teciduais compatíveis a um processo de cicatrização mais avançado

Carbon Black CB-EDA Nanoparticles in Macrophages: Changes in the Oxidative Stress Pathway and in Apoptosis Signaling

The influence of black carbon nanoparticles on J774.A1 murine cells was investigated with the objective of exploring the cytotoxicity of black carbon functionalized with ethylenediamine CB-EDA. The results showed that CB-EDA has a cytotoxic profile for J774.A1 macrophages in a time- and dose-dependent manner. When phagocytosed by the macrophage, CB-EDA triggers a mechanism that leads to apoptosis. In this process, there is an increase in oxidative stress pathways due to the activation of nitric oxide and then ROS. This causes an imbalance in redox function and a disruption of membrane integrity that occurs due to high levels of LDH, in addition to favoring the release of the pro-inflammatory cytokines IL-6, IL-12, and tumor necrosis factor (TNF) in an attempt to modulate the cell. However, these stimuli are not sufficient to repair the cell and the level of mitochondrial integrity is affected, causing a decrease in cell viability. This mechanism may be correlated with the activation of the caspasse-3 pathway, which, when compromised, cleaves and induces cells death via apoptosis, either through early or late apoptosis. In view of this, the potential for cell damage was investigated by analyzing the oxidative and inflammatory profile in the macrophage lineage J774.A1 and identifying potential mechanisms and metabolic pathways connected to these processes when cells were exposed to NP CB-EDA for both 24 h and 48 h.The authors would like to thank Márcia Regina Cominetti (Departamento de Gerontologia, Universidade Federal de São Carlos, São Carlos, SP, Brazil) for availability of equipment. M.A. was supported by the Margarita Salas postdoctoral contract MGS/2021/21 (UP2021-021) financed by the European Union—Next Generation EU. The authors would also like to express their gratitude for the financial aid from the Fundação de Amparo à Pesquisa do Estado de São Paulo—FAPESP (2013/07296-2), the Financiadora de Estudos e Projetos—FINEP, the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior—CAPES (Financial Code 001) and the Conselho Nacional de Desenvolvimento Científico e Tecnológico—CNPq