Materials Science and Engineering C 36 (2014) 180–186

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Photodynamic therapy in the cattle protozoan Tritrichomonas foetus cultivated on superhydrophilic carbon nanotube Susane Moreira Machado a, Cristina Pacheco-Soares a,b, Fernanda Roberta Marciano c, Anderson Oliveira Lobo c, Newton Soares da Silva a,b,⁎ a Laboratory of Tissue and Cell Biology, Development and Research Institute (IP&D), Universidade do Vale do Paraíba (UNIVAP), Av. Shishima Hifumi 2911, Urbanova, 12244-000 São José dos Campos, SP, Brazil b Laboratory of Dynamics of Cellular Compartments, Development and Research Institute (IP&D), Universidade do Vale do Paraíba (UNIVAP), Av. Shishima Hifumi 2911, Urbanova, 12244-000 São José dos Campos, SP, Brazil c Laboratory of Biomedical Nanotechnology, Development and Research Institute (IP&D), Universidade do Vale do Paraíba (UNIVAP), Av. Shishima Hifumi 2911, Urbanova, 12244-000 São José dos Campos, SP, Brazil

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Article history: Received 20 December 2012 Received in revised form 21 November 2013 Accepted 6 December 2013 Available online 14 December 2013 Keywords: Multiwalled carbon nanotubes Vertically aligned Suerhydrophilic Tritrichomonas foetus Photodynamic therapy Tetrasulfonated aluminum phthalocyanine

a b s t r a c t Superhydrophilic vertically aligned carbon nanotubes (VACNT-O2) were used for the first time as scaffolds for photodynamic therapy (PDT) to induce inhibition of cell division in eukaryotic cells. VACNT-O2 scaffolds were produced on Ti substrates using plasma enhanced chemical vapor deposition technique and functionalized by oxygen plasma. Scanning electron microscopy (SEM) analysis was performed to characterize the surface changes of the protozoan and interaction with VACNT-O2. Characterization of lipid and total protein expression was performed with protozoa that were or not treated with PDT. Quantification of protein was conducted using Qubit fluorometer and separated on a polyacrylamide gel. SEM analysis showed the release of lipid vesicles by protozoa after the PDT. These vesicles were characterized by the PKH26 fluorescent probe. The results demonstrated a greater amount of protein released after PDT than in the control. When analyzing the protein material in polyacrylamide gel, a significant protein expression of approximately 65 kDa was found. A model identified the programmed death of Tritrichomonas foetus after the PDT was also proposed. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Tritrichomonas foetus is a flagellate protozoan and the etiological agent of bovine genital trichomonosia [1,2], which is an infectious venereal disease. This protozoan is found on the urogenital mucosal surface of males and females, causing bovine trichomonosis. This infectious disease with venereal transmission causes infertility and abortion in cattle, thereby increasing herd management expenses [3,4]. In the bull, the parasite is found on the penis, preputial cavity, and in some cases, the urethral opening. Penile mucosa and adjacent areas of the preputial mucosa have a large concentration of protozoa, which are not invasive, situated in the surface mucosa, secretions, and glandular light [5,6]. In cows, the parasite inhabits the vagina and uterus, which can inhibit the attachment of embryo or rupture its membranes after attachment, leading to abortion [5–7]. Infection often leads to a moderate vaginitis with purulent discharge, with or without mild endometriosis and transitory infertility, leading to pyometra, salpingitis, and cervicitis [5,7–9]. Because T. foetus is an amitochondrial and aerotolerant organism, energy production under low O2 stress in the protozoan is done via

⁎ Corresponding author. Tel.: +55 1239471143; fax: +55 1239471149. E-mail address: [email protected] (N.S. da Silva). 0928-4931/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.msec.2013.12.004

hydrogenosome, which, as the name suggests, is the organelle where H2 is generated [10–12]. Photodynamic therapy (PDT) involves the activation of photosensitive substances with a light source (laser), which generates cytotoxic oxygen species and free radicals. This promotes the selective destruction of the target tissue to selectively induce death in neoplastic cells [12,13]. This technique uses visible light to activate photosensitizer compounds, leading to a photo-oxidation process of biological tissue, which can induce apoptosis or necrosis both in vivo and in vitro. PDT can be used to treat tumors [13]. Carbon Nanotube (CNT) can absorb bacteria [3–5,14] and shows strong antimicrobial activity toward Escherichia coli [6]. Hydrophilic surfaces are favorable to the adhesion, spread, and growth of various cell types [7,15,16]. However, some studies have shown that raw vertically aligned CNT (VACNT) are superhydrophobic [17–19], which may limit their application as nanostructures for cellular and bacterial attachment [20,21]. Lobo et al. [22] found that simple functionalization using oxygen plasma effectively made VACNT superhydrophilic (VACNT-O2). Machado et al. [23] also showed, for the first time, that T. foetus can adhere on VACNT-O2 films. Therefore, it seems possible to apply VACNT-O2 films to understand spreading mechanisms of protozoan and the specifically recognize adhesion proteins.

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T. foetus is usually associated with the mucosal surface of the urogenital tract in bovines. Studies that focus on trichomonas-host cell interaction have shown that the cytotoxicity of T. foetus can be triggered by physical contact been the surfaces of the parasite and host cell, resulting in the secretion of extracellular proteases and glycosidases [21–23]. Such enzymes seem not only to induce cytolysis or disrupt epithelial junctions, but also to remodel the surrounding extracellular matrix (ECM) [24]. Because these factors are involved in the recognition of ECM proteins and VACNTs present physical dimensions similar to ECM components [7,15], the objective in this study was to investigate the behavior of the protozoan parasite T. foetus before and after PDT, when placed to adhere on a surface coated with VACNT-O2.

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used the pulsed direct current plasma functioned with an oxygen flow rate of 1 sccm, at a pressure of 85 mTorr, −700 V, and a frequency of 20 kHz. After this simple treatment convert them to superhydrophilic (VACNT-O2). Details about this process were described elsewhere [27]. A Krüss Easy Drop system in sessile drop method was used to measure the contact angle at room temperature to evaluate the wettability of as-grown and VACNT-O2 films. SEM images (Jeol JSM-5310 and JSM-6330 F) examined morphological arrangement of VACNT-O2 surfaces and high-resolution SEM images evaluated modification details of surface morphology. 2.4. Scanning electron microscopy (SEM)

T. foetus, K strain, was kindly provided by Dr. Fernando Costa e Silva Filho, from the Institute of Biophysics Carlos Chagas Filho, Brazil (UFRJ-RJ) and Dr. Marlene Benchimol, from the Santa Ursula University. The parasite was kept in TYM Diamond's medium [25] pH 6.8 supplemented with 10% fetal calf serum for 48 h at 37 °C in a humidified atmosphere containing 5% CO2. The number of parasites was standardized at 1 × 106 cell mL− 1.

VACNT-O2 scaffolds were sterilized for 24 h under UV irradiation. After this, they were placed in individual wells of 24-well culture plates. The suspension containing T. foetus (1 × 10 6 cells mL − 1 ) after PDT treatment or not, was put into each well. After incubation period (1 h at 37 °C in a 5% CO2 atmosphere), T. foetus, attached to the VACNT-O2 films, was fixed with a 3% glutaraldehyde 4% paraformaldehyde in PBS for 1 h and dehydrated in a graded acetone solution series (50%, 70%, 90%, 100%), for 10 min each. The drying stage used a 1:1 solution of acetone with hexamethyldisilazane (HMDS) and the samples were dried with pure HMDS at room temperature. After deposition of a thin gold layer, the specimens were examined with a SEM Zeiss EVOMA10.

2.2. Photosensitizer

2.5. Fluorescence microscopy (lipids—PKH26)

The drug chloroaluminum phthalocyanine tetrasulfonate (AlPcS4) (Porphyrin Products, Inc.) was dissolved in PBS to a stock concentration of 1 mM and stored in the dark at 4 °C until use. For the experiments, the AlPcS4 was diluted to 10 μM.

We used the same methodology to sterilization and suspension for interaction assay described in details at 2.4 section. After incubation period (1 h at 37 °C in a 5% CO2 atmosphere), T. foetus attached to the VACNT-O2 films were incubated (5 min, RT) with 10 μL fluorescent dye PKH26, lipids specific membrane. After this, 100 μL of fetal bovine serum (FBS) was added and incubated again for 1 min. Observations were performed on Leica DMLB fluorescence microscope and images were capture via digital video camera Leica DFC 300FX.

2. Materials and methods 2.1. T. foetus

2.2.1. Treatment of T. foetus with AlPcS4 Parasites were distributed at 1 × 106 cells mL−1. Six vials were left without treatment (control and for light treatment only), and the other six vials were treated with AlPcS4 (10 μM) and incubated for 60 min in the dark at 37 °C in a humidified atmosphere containing 5% CO2. After this period, they were washed with PBS twice to remove the photosensitizer that had not been taken up by cells. In addition, 500 μL of fresh TYM Diamond's medium without serum was added before irradiation. 2.2.2. Irradiation Three vials with parasites that had undergone treatments with AlPcS4 and three others vials that had not been treated were subjected to irradiation in the dark with a semiconductor laser InGaAlP (Thera Lase-DMC), (λ = 685 nm; P = 26 mW; D.E. = 4.5 J/cm2; t = 3,52 s). After irradiation, the parasite was kept in TYM Diamond's medium [25] supplemented with 10% fetal calf serum at 37 °C in a humidified atmosphere containing 5% CO2, according to each assay described below. 2.3. VACNT synthesis and functionalization VACNT films were produced using a microwave plasma chamber at 2.45 GHz [24,26]. VACNTs were grown on Ti substrates (10 mm × 10 mm × 1 mm) covered by 10 nm Ni layers deposited by e-beam evaporation. The Ni layers were pre-treated to promote nanocluster formation, which served as the catalyst for VACNT growth. The 5 min pretreatment in N2/H2 (10/90 sccm) plasma, at a substrate temperature of nearly 760 °C, promoted nano-catalyst formation. VACNT growth started by introducing CH4 (14 sccm) into the gas mixture, with a substrate temperature at 800 °C. The reactor pressure was 30 Torr during the whole 2 min process. Oxygen plasma functionalized the VACNT tips by incorporating oxygen-containing groups. Briefly, we

2.6. Total protein evaluation In order to evaluate changes in expression of proteins in T. foetus before and after PDT, the following protocols were established: Solubilization of proteins. T. foetus (1 × 106 cells mL−1) was resuspended in phosphate-buffered sulfate (PBS, pH 7.2), sonicated in an Ultrasonic Processor (Z412619—Aldrich) for 3 × 30 s/60 mHz, and subjected to low-speed centrifugation (500 g for 15 min, 4 °C). The resulting supernatant and pellet were separated for further analysis. Protein determination. Total protein content was determined in the supernatant and pellet by Qubit® 2.0 Fluorometer using a kit provided by the manufacturer (Qubit® Protein Assay Kit). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE). SDS-PAGE was carried out on a 10% gel, to which 4.5 μg of protein from the supernatant or pellet was applied. The material was resuspended in sample buffer containing 2-mercaptoethanol and was then boiled and applied to the gel. SeeBlue® Plus2 PreStained Standard Markers (Novex® LC5925 Life Technologies) were used to evaluate the molecular mass of the protein bands. The standard proteins were Myosin (250 kDa), Phosphorylase B (148 kDa), BSA (68 kDa), Glutamic Dehydrogenase (64 kDa), Alcohol Dehydrogenase (50 kDa), Carbonic Anhydrase (36 kDa), Myoglobin-Red (22 kDa), Lysozyme (16 kDa), Aprotinin (6 kDa), and Insulin B chain (4 kDa). Gels were stained with Coomassie Brilliant Blue R in 50% trichloroacetic acid and destained in 10% acetic acid.

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2.7. Statistical analyses Data were collected from five different experiments and expressed as the average ± the standard deviation (SD). The statistical differences were analyzed by 2-way Anova (Graph PadPrism 5®). The populations from the analyses were obtained with normal distribution and independent for each experiment. P-values less than 0.05 were considered to indicate the statistical difference. 3. Results Fig. 1 shows the efficiency of the oxygen plasma treatment on VACNT surfaces in promoting the transition from superhydrophobic to superhydrophilic behavior. FEG-SEM was used to analyze the possible morphological changes. Fig. 1A shows the morphological and structural VACNT analyses after exposure to the oxygen plasma. The VACNTs had a length of 8–15 μm (Fig. 1A). The comparison of wettability of as-grown VACNTs before (Fig. 1a.1) and after the plasma treatment was studied using contact angle (Fig. 1a.2). Notice that the as-grown VACNT films exhibited superhydrophobic character, with a contact angle of ~ 154° (Fig. 1a.1). The superhydrophilic character was obtained after only 2 min of the oxygen plasma etching

(Fig. 1a.2). Clearly, the nanotube walls were attacked by the oxygen plasma. Fig. 1b.1 shows that the surface of as-grown samples has entangled arrangements, without any defect. After 2 min of treatment in oxygen plasma, the surface morphology, shown in Fig. 1b.2, kept its severely damaged appearance. Hence, the VACNT surface switched from superhydrophobic to superhydrophilic, showing the high efficiency of this treatment. Fig. 2 shows that the protozoan parasite T. foetus strongly adhered to the VACNT-O2. The arrows in Fig. 2A point to projections (lamelipodia) in the anterior region of the parasite. In Fig. 2B, the adherence follows the recurrent flagellum (undulating membrane) of the protozoa. Fig. 2C and D evidence changes in the surface of the protozoa (2C) and release of vesicles (2B—arrowhead) that changed the arrangement of the VACNT-O2. Fig. 3 illustrates the arrangement of lipids by using the fluorescent marker PKH26 to characterize these vesicles. The control group (T. foetus without application of PDT) had a higher concentration of vesicles in the interior of the protozoa (Fig. 3a). After PDT (Fig. 3b), these vesicles migrated to the periphery. The analysis of total protein expression of the protozoa before and after PDT demonstrated a greater release of total protein after PDT (Fig. 4 PDTs), compared to the control. Upon analyzing the protein

Fig. 1. (A) SEM images of the superhydrophilic VACNT shows the alignment. Effect of oxygen plasma functionalization on the VACNTs. Optical microscopy images of the contact angle between deionized water (magnification 200×) (a.1) before and (a.2) after the oxygen plasma treatment. FEG-SEM images show the morphological structures of VACNT before (b.1) and after (b.2) oxygen plasma treatment.

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Fig. 2. SEM photomicrograph of the interaction of the protozoan parasite T. foetus with a titanium surface coated with VACNT-O2. Figs. 1A and B are SEM-images of non-PDT treated protozoa. Figs. 1C and D show images of protozoa after PDT. Arrow—filopodia adhered to the VACNT-O2. Arrowhead—vesicles released by T. foetus after PDT (Fig. A, B, and C 5000 ×; FIG. D 15,000 ×).

material in polyacrylamide gel (SDS-PAGE), significant expression of a protein of approximately 65 kDa was founded.

4. Discussion The cellular mechanism by which T. foetus colonizes mucosal surfaces has not been not well defined; therefore, further study is necessity to understand better the pathogenic effects of this parasite and the parasite–host relationship. Lobo et al. [22] analyzed a hydrophilic surface of a VACNT and with functionality. Using an oxygen plasma producing VACNT-O2, they obtained a structure with properties that mimic the structural components of extracellular matrix (MEC). With the same VACNT-O2 surface film, this study examined the adhesion of the protozoa and the mechanism of specific protein recognition, according to the viability of the parasite. Previous studies [23] demonstrated that the

Fig. 3. Fluorescence Optical Microscopy using marker PKH26. (A) T. foetus without treatment, (B) T. foetus after treatment with PDT (Fig. a and b 1000 ×).

mobility of VACNT-O2 films could have a pattern of exceptional adherence to T. foetus with lamelipodia type projections, showing adherence of the protozoa to VACNT-O2. Our results confirm those obtained previously (Fig. 2A and B) when the protozoa adhered without any treatment. After PDT, the protozoa showed a change in the disposition of VACNT-O2s through the release of vesicles (Fig. 2C and D). These vesicles were identified with fluorescent marker PKH26, which is specific for lipid. These vesicles were concentrated within the parasite prior to the PDT (Fig. 3a). After PDT, these vesicles migrated to the periphery of the protozoa, confirming the observations by SEM. We believe that after PDT, these vesicles are released to the outside, acting in the external environment. For many years, lysosomes have been thought to be solely involved in necrotic and autophagic cell death, with their role in apoptosis being limited to the digestion of engulfed apoptotic bodies [28,29]. These concepts now seem outdated by a growing body of evidence that strongly points to a role of lysosomes in apoptosis beyond that of simple “garbage disposals.” The key-factor in determining the type of cell death (necrosis vs. apoptosis) mediated by lysosomal enzymes seems to be the magnitude of lysosomal permeabilization, and consequently, the amount of proteolytic enzymes released into the cytosol [30]. However, the precise mechanisms by which lysosomes are involved in apoptosis are still largely unknown and currently under intense investigation. Photodynamic therapy, a process employing UV-light and photosensitizers to kill cancer cells, as well as accumulation of lysosomotropic agents within the organelle, also triggers the lysosomal pathway of cell death [30–32]. The application of PDT in T. foetus [33] caused intense vacuolization of the cytoplasm after 24 h. Da Silva et al. [33] also observed the presence of dense electron material inside after 48 h. In addition, they [33] found giant vacuoles near the plasma membrane. Many lysosomal enzymes, including cathepsins, are overexpressed in cancer [34]. Many authors have suggested that enhanced secretion of these proteases by exocytosis promotes cancer growth, invasion, and metastasis by promoting degradation of extracellular connective matrices and angiogenesis [34]. Our observations suggest that the vesicles characterized by fluorescent marker PKH26 can be lysosomal vacuoles. The observation of changes in the arrangement of VACNT-O2

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Fig. 4. Analysis of total protein expression of protozoa before and after PDT. CTRp—precipitated control; CTRs—supernatant control; PDTp—precipitated after PDT; PDTs—precipitated after PDT. Column A—standard molecular weight; Column B—supernatant after PDT; Column C—control supernatant.

confirms the properties similar to MEC and the contents of these vesicles are released at this point of contact (Fig. 2c and d). The release of lysosomal contents in the exterior to destabilize the MEC was also observed by Zdolsek et al. [35] after cell damage by oxidative stress. In order to understand the mechanisms of adhesion, death, and release of vesicles by protozoa after PDT, we performed expression analysis of total proteins of the protozoa before and after PDT. In the quantification of the proteins, a higher production in the supernatant fraction was found (Fig. 4 PDTs). For the separation on SDS-PAGE, the expression of a protein with molecular weight of approximately 65 kDa was significant (Fig. 4—Column B). Adhesion of Trichomonas vaginalis to host cells, a preparatory step to infection, is complex and involves at least four surface adhesin proteins. The proteins exhibit functional diversity based on cellular location. AP65 is a prominent T. vaginalis adhesin and is targeted to both the plasma membrane surface and hydrogenosomes. This compartmentalization is in part modulated by iron. Furthermore, the surface placement of AP65 is increased upon contact with host cells [36,37]. Solano-González et al. [8] identified and characterized a protease of 65 kDa (CP65) that causes damage to the host cell. CP65 participates in host parasite interaction and cellular destruction throughout a cellbinding domain that specifically recognizes its putative receptor on the surface of the host cell. According to a study by Singh et al. [9], cysteine proteinases and adhesins on the surface of T. foetus are directly involved in parasite interactions with host cells. Cysteine proteases, soluble or associated with the surface of T. foetus, are more involved in cytotoxicity. These proteins induce the apoptosis in bovine vaginal epithelial cells (BVECs), suggesting that this mechanism of cell death may be involved in the pathogenesis of these protozoa. Apoptosis or autophagic cell death after PDT in T. foetus facilitates the release of lysosomal contents, shown in Fig. 2 of our experiments, further increasing its pathogenesis [27]. In the last decade, apoptosis has attracted great scientific interest. In many biological systems, cell suicide indicates the involvement of an autophagic lysosomal compartment [2]. Recently, Silverman et al. [38] shows evidence supporting a role for Leishmania exosomes during early infection. They suggest a model in

which Leishmania secreted microvesicles released into the extracellular milieu deliver effector cargo to host target cells. This cargo mediates immunosuppression and functionally primes host cells for Leishmania invasion. Leishmania ssp. release microvesicles and the amount of vesicle release and the specific protein cargo of the vesicles is sensitive to changes in environmental conditions that mimic infection. Torrecilhas et al. [39] show that different types of shed vesicles, for example, exosomes, plasma-membrane-derived vesicles, or microparticles, are the focus of intense research in view of their potential role in cell-cell communication and under the perspective that they might be good tools for immunotherapy, vaccination, or diagnostic purposes. This review discusses ways employed by pathogenic trypanosomatids to interact with the host by shedding vesicles that contain molecules important for the establishment of infection, as opposed to previous beliefs considering them as a waste of cellular metabolism [39]. Extracellular microvesicles (eMVs) are a class of membrane bound organelles secreted by various cell types [40]. eMVs include (i) exosomes: 40–100 nm diameter membranous vesicles of endocytic origin; (ii) ectosomes (also referred to as shedding microvesicles, SMVs): large membranous vesicles (50–1000 nm diameter) that are shed directly from the plasma membrane (PM); and (iii) apoptotic blebs (50–5000 nm diameter): released by dying cells. Over the last five years, the field of eMVs has witnessed tremendous growth (more than 3500 research articles published) mainly due to their purported role in intercellular signaling and possible source of disease biomarkers [41]. In our results, the release of vesicles of T. foetus after PDT (Fig. 2D) were observed, and this vesicle possibly acted in the external environment as observed in the modification of the disposition of the VACNTO2. Cellular interactions are pivotal for the progression, angiogenesis, and invasiveness of tumors [42]. Such interactions are presumed to be regulated by membrane surface molecules (e.g., EGFR) and soluble secreted proteins (e.g., IL-12) that activate the target cells by interacting with the target cell surface receptors. Another mode of intercellular communication that has recently gained immense scientific interest is mediated by exosomes. Additionally, such fusion might change some of the membrane features of the target cell including varied lipid

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Fig. 5. Mechanism of cell death in T. foetus after PDT. The photosensitizer after activation with LASER produces reactive oxygen species (ROS) that interact with the membrane of the lysosome leading to permeabilization with release of lysosomal proteases. These proteases activate procaspases inducing the protozoa to the apoptosis. Lysosomes also fuse with the plasma membrane releasing their contents into the extracellular environment. These proteases act on the basal membrane of the host cells.

concentrations and the transfer of exosomal membrane proteins on the target cell surface [43]. In contrast to the fate of proteins trafficked for degradation to the lysosomal system, secreted exosomes are biologically active entities that are important for a variety of pathways. Although extensive information on the biosynthesis of exosomes has been demonstrated using in vitro cell lines, many questions regarding the biological role of exosomes in complex cellular systems remain to be addressed. It is clear that exosome-like microvesicles are present in body fluids such as blood, urine, amnionic fluid ascites, and pleural effusions under healthy and disease conditions. However, the origin of these exosomes and their destination for stimulation of distal cells remains unclear. It has been demonstrated that exosomes can be taken up by other cells. However, whether this establishes a novel mechanism of cell–cell communication is an intriguing yet unanswered question. Although functional roles of exosomes are only recently becoming clear, future investigations are likely to indicate the importance of these mediators in biological processes [44]. We believe that the vesicles release by the parasitic protozoan T. foetus have proteic contents with an activity similar to observed by Zdolsek et al. [35], where the content of the lysosomal vesicle destabilized the MEC. This research evidenced the microvesicles release mechanisms by parasitic protozoan after death (apoptosis and/or necrosis) induced by PDT. According to our results and brief review about the role of exosomes (microvesicles) and for a better comprehension of the release in the environment (host cell), the authors first propose a model of programmed cell death in T. foetus after PDT (Fig. 5). In this model, we show the action of PDT in the parasitic protozoan, in which the reactive oxygen species (ROS) induce the permeabilization of lysosomes in cytoplasm and activate the processing of procaspases to form active caspase. The active caspase induces the cell death process (apoptosis or necrosis). The same mechanism (lysosomal activation by

ROS) makes the lysosome plasma membrane fusion occur, releasing the content into the extracellular microenvironment. Thus, this process helps degrade basal lamina, as shown in Fig. 1, with the change in the distribution of VACNT-O2 that mimics the dimensions of the structural components of the extracellular matrix, promoting protein interactions responsible for cell adhesion. This proposed mechanism is the beginning of a study of T. foetus VACNT-O2 interaction with emphasis on adhesion and cell death. 5. Conclusion This study showed for the first time that the protozoan parasite T. foetus adheres strongly to VACNT-O2 after PDT. A significant release of a protein of approximately 65 kDa was also observed, analyzed on polyacrylamide gel (SDS-PAGE). This same mechanism induces the release of this protease that leads to destruction of the basal lamina, which favors the process of pathogenesis and operates as a signaling process showing the mechanism of this altruistic protozoan. Future works will examine the adhesion mechanism of the protozoan to VACNT-O2 scaffolds associated to PDT. Acknowledgments This work has been supported by FAPESP (2008/06654-4), (2011/ 17877-7), and (2011/20345-7) and CNPq (309699/2009-6). Special thanks to Priscila Leite for scanning electron microscopy images and Alene Alder-Rangel for English revisions. References [1] A.O. Pellegrin, R.C. Leite, Atualização sobre Tricomonose genital bovina, Embrapa Pantanal, Corumbá, 2003.

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Photodynamic therapy in the cattle protozoan Tritrichomonas foetus cultivated on superhydrophilic carbon nanotube.

Superhydrophilic vertically aligned carbon nanotubes (VACNT-O2) were used for the first time as scaffolds for photodynamic therapy (PDT) to induce inh...
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