Vaterite microparticle-loaded methylene blue for photodynamic activity in macrophages infected with Leishmania braziliensis.

Calcium carbonate (CaCO3) exhibits a variety of crystalline phases, including the anhydrous crystalline polymorphs calcite, aragonite, and vaterite. Developing porous calcium carbonate microparticles in the vaterite phase for the encapsulation of methylene blue (MB) as a photosensitizer (PS) for use in photodynamic therapy (PDT) was the goal of this investigation. Using an adsorption approach, the PS was integrated into the CaCO3 microparticles. The vaterite microparticles were characterized by scanning electron microscopy (SEM) and steady-state techniques. The trypan blue exclusion method was used to measure the biological activity of macrophages infected with Leishmania braziliensis in vitro. The vaterite microparticles produced are highly porous, non-aggregated, and uniform in size. After encapsulation, the MB-loaded microparticles kept their photophysical characteristics. The carriers that were captured allowed for dye localization inside the cells. The results obtained in this study indicated that the MB-loaded vaterite microparticles show promising photodynamic activity in macrophages infected with Leishmania braziliensis.

Synthesis, characterization, and evaluation of chloroaluminium phthalocyanine incorporated in poly(ε-caprolactone) nanoparticles for photodynamic therapy.

BACKGROUND: The use of nanotechnology has been widely used in biomedical science, including orthopedic implants, tissue engineering, cancer therapy and drug elution from nanoparticle systems, such as poly-caprolactone (PCL) nanoparticles, which stand out mainly for their biocompatibility, being considered as effective carriers for photosensitizing drugs (PS) in photodynamic therapy (PDT) protocols. METHODS: This manuscript describes the synthesis and characterization of PCL nanoparticles for controlled release of the drug chloro-aluminum phthalocyanine (ClAlPc) as a photosensitizer for application in PDT. The PCL-ClAlPc nanoparticles were developed by the nanoprecipitation process. The structure and morphology of the nanoparticles were studied with scanning electron microscopy (SEM) and with Fourier transform infrared (FTIR). The size of nanomaterials was studied using the Dynamic Light Scattering (DLS) method. Photophysical and photochemical characterizations were performed. Subsequently, photobiological studies were also used to characterize the system. RESULTS: The nanoparticles had an average diameter of 384.7 ± 138.6 nm and a polydispersity index of 0.153. SEM analysis revealed that the system formed a spherical shape typical of these delivery systems. Charging efficiency was 82.1% ± 1.2%. The phthalocyanine-loaded PCL nanoparticles maintained their photophysical behavior after encapsulation. Cell viability was determined after the dark toxicity test, and it was possible to observe that there was no evidence of toxicity in the dark, for all concentrations tested. The assay also revealed that adenocarcinoma cells treated with free ClAlPc and in the nanoformulation showed 100% cell death when subjected to PDT protocols. The intracellular location of the photosensitizer indicated a high potential for accumulation in the cytoplasm and nucleus. CONCLUSIONS: From the photophysical, photochemical and photobiological analyzes obtained, it was possible to observe that the development of PCL nanoparticles encapsulated with ClAlPc, by the nanoprecipitation method was adequate and that the in vivo release study is efficient to reduce the release rate and attenuate the burst of PS loaded on PCL nanoparticles. The results reinforce that the use of this system as drug delivery systems is useful in PDT protocols.

Synthesis and characterization of photosensitive gelatin-based hydrogels for photodynamic therapy in HeLa-CCL2 cell line.

BACKGROUND: Hydrogel systems are increasingly gaining visibility involving biomedicine, tissue engineering, environmental treatments, and drug delivery systems. These systems have a three-dimensional network composition and high-water absorption capacity, are biocompatible, allowing them to become an option as photosensitizer carriers (PS) for applications in Photodynamic Therapy (PDT) protocols. METHODS: A nanohydrogel system (NAHI), encapsulated with chloroaluminium phthalocyanine (ClAlPc) was synthesized for drug delivery.. NAHI was synthesized using gelatin as based polymer by the chemical cross-linking technique. The drug was encapsulated by immersing the hydrogel in a 1.0 mg.mL-1 ClAlPc solution. The external morphology of NAHI was examined by scanning electron microscopy (SEM). The degree of swelling of the synthesized system was evaluated to determine the water absorption potential. The produced nanohydrogel system was characterized by photochemical, photophysical and photobiologial studies. RESULTS: The images from the SEM analysis showed the presence of three-dimensional networks in the formulation. The swelling test demonstrated that the nanohydrogel freeze-drying process increases its water holding capacity. All spectroscopic results showed excellent photophysical parameters of the drug studied when served in the NAHI system. The incorporation efficiency was 70%. The results of trypan blue exclusion test have shown significant reduction (p < 0.05) in the cell viability for all groups treated with PDT, in all concentrations tested. In HeLa cells, PDT mediated by 0,5 mg.mL-1 ClAlPc encapsulated in NAHI showed a decrease in survival close to 95%. In the internalization cell study was possible to observe the internalization of phthalocyanine after one hour of incubation, at 37 °C, with the the accumulation of PS in the cytoplasm and inside the nucleus at both concentrations tested. CONCLUSIONS: Given the peculiar performance of the selected system, the resulting nanohydrogel is a versatile platform and display potential applications as controlled delivery systems of photosensitizer for photodynamic therapy application.

Finding reduced Raman spectroscopy fingerprint of skin samples for melanoma diagnosis through machine learning.

Early-stage detection of cutaneous melanoma can vastly increase the chances of cure. Excision biopsy followed by histological examination is considered the gold standard for diagnosing the disease, but requires long high-cost processing time, and may be biased, as it involves qualitative assessment by a professional. In this paper, we present a new machine learning approach using raw data for skin Raman spectra as input. The approach is highly efficient for classifying benign versus malignant skin lesions (AUC 0.98, 95% CI 0.97-0.99). Furthermore, we present a high-performance model (AUC 0.97, 95% CI 0.95-0.98) using a miniaturized spectral range (896-1039 cm-1), thus demonstrating that only a single fragment of the biological fingerprint Raman region is needed for producing an accurate diagnosis. These findings could favor the future development of a cheaper and dedicated Raman spectrometer for fast and accurate cancer diagnosis.

Molecular detection of HPV and FT-IR spectroscopy analysis in women with normal cervical cytology.

BACKGROUND: The increase in the incidence of Cervical Cancer in the female population worldwide has been an issue that deserves further attention from the scientific community. Several studies have already proven the relationship of its development with the molecular mechanisms that Human Papillomavirus (HPV) induces in cervical cells. The gene amplification provided by molecular biology techniques has been used as the gold standard diagnostic method of this virus because of its high specificity and sensitivity.However, the high investments associated with the acquisition of reagents, equipment and labor demonstrate the need for the development of more accessible techniques that present the same accuracy. FT-IR spectroscopy has been studied as an inexpensive and easily accessible technology that can provide the differentiation of malignant and benign cells. This study aimed to demonstrate the effectiveness and sensitivity of molecular analysis by PCR in relation to cytological analysis and to evaluate the sensitivity of FT-IR spectroscopy in the diagnosis of HPV for cervical cancer prevention. METHODS: Cervical fluid samples obtained from 50 patients with absence of cellular lesion by cytological analysis were analyzed by molecular and spectroscopic analyzes. Oncotic colpocitology analysis was performed by the Papanicolaou staining, amplification of the L1 viral gene by PCR was performed using primers MY09 and MY11 and biochemical analysis of the fluids by FT-IR was performed using the Spectrum 400 system equipped with a microscope. RESULTS: Of the 50 patients without evident morphological alteration of the cells, seven were diagnosed by molecular analysis as positive for presence of HPV. Principal component analysis of spectroscopy was not able to separate the negative samples from the HPV positive samples and, therefore, did not present as an effective diagnostic technique. CONCLUSIONS: We highlight the efficacy, sensitivity and specificity of molecular biology by PCR in the identification of the virus and we emphasize that more studies should be used for the application of FT-IR spectroscopy in the diagnosis of this infection and its application in the prevention of cervical cancer.

Biofilm formation by Candida albicans is inhibited by photodynamic antimicrobial chemotherapy (PACT), using chlorin e6: increase in both ROS production and membrane permeability.

Candida albicans is an opportunistic fungal producing both superficial and systemic infections in immunocompromised patients. Furthermore, it has been described an increase in the frequency of infections which have become refractory to standard antifungal therapy. Photodynamic antimicrobial chemotherapy (PACT) is a potential antimicrobial therapy that combines visible light and a nontoxic dye, known as a photosensitizer, producing reactive oxygen species (ROS) that can kill the treated cells. The objective of this study was to investigate the effects of PACT, using chlorin e6, as a photosensitizer on C. albicans. In this work, we studied the effect of PACT on both cell growth and biofilm formation by C. albicans. In addition, both ROS production and cell permeability were determined after PACT. PACT inhibited both growth and biofilm formation by C. albicans. We have also observed that PACT increased both ROS production (six times) and cell membrane permeability (five times) in C. albicans. PACT decreased both cell growth and biofilm development. The effect of PACT using chlorin e6 on C. albicans could be associated with an increase in ROS production, which could increase cell permeability, producing permanent damage to the cell membranes, leading to the cell death.

Evaluation of methylene blue as photosensitizer in promastigotes of Leishmania major and Leishmania braziliensis.

The cutaneous leishmaniasis is caused by the protozoan of the genus Leishmania. It is considered by WHO as a public health issue and a neglected disease, which affects rural workers and it is also a risk to travelers in endemic areas. The conventional treatment is toxic and leads to severe side effects. The photodynamic therapy has been studied as an alternative treatment to cutaneous leishmaniasis. This study aimed to evaluate the methylene blue internalization and the impact of the PDT in the viability and morphology of Leishmania major and Leishmania braziliensis promastigote in culture medium. The fluorescence microscopy was used to determine the MB localization. To evaluate the mitochondrial activity (MTT), viability (Trypan blue test) and the morphological alterations both species were incubated with the MB in concentrations starting in 500µg/ml, in serial dilution, until 7,8µg/ml. The fluorescence microscopy demonstrated that the MB is internalized by both species after one hour of incubation. The MB presented low toxicity at the dark and the PDT was capable of decreasing the viability in more than 70% in the higher concentrations tested. The PDT also triggered significant morphological alterations in the Leishmania promastigotes. The results presented in this study are an indicative that the MB is a photosensitizer with promising potential to clinical application, besides its low cost.

Amniotic membrane as an option for treatment of acute Achilles tendon injury in rats.

PURPOSE: To evaluate the effect of human amniotic membrane (hAM) fragment on inflammatory response, proliferation of fibroblast and organization of collagen fibers in injured tendon. METHODS: Sixty rats were divided into 3 groups: C - surgical procedures without tendon lesion and with simulation of hAM application; I - surgical procedures, tendon injury and simulation of hAM application; T - surgical procedures, tendon injury and hAM application. These groups were subdivided into four experimental times (3, 7, 14 and 28 days). The samples underwent histological analysis and ATR-FTIR spectroscopy. RESULTS: Histological analysis at 14 days, the T group showed collagen fibers with better alignment. At 28 days, the I group presented the characteristics described for the T group at 14 days, while this group presented aspects of a mature connective tissue. FT-IR analysis showed a clear distinction among the three groups at all experimental times and groups T and I presented more similarities to each other than to group C. CONCLUSION: Acute injury of tendon treated with human amniotic membrane fragment showed a faster healing process, reduction in inflammatory response, intense proliferation of fibroblasts and organization of collagen fibers.

Amniotic membrane as an option for treatment of acute Achilles tendon injury in rats

Abstract Purpose: To evaluate the effect of human amniotic membrane (hAM) fragment on inflammatory response, proliferation of fibroblast and organization of collagen fibers in injured tendon. Methods: Sixty rats were divided into 3 groups: C - surgical procedures without tendon lesion and with simulation of hAM application; I - surgical procedures, tendon injury and simulation of hAM application; T - surgical procedures, tendon injury and hAM application. These groups were subdivided into four experimental times (3, 7, 14 and 28 days). The samples underwent histological analysis and ATR-FTIR spectroscopy. Results: Histological analysis at 14 days, the T group showed collagen fibers with better alignment. At 28 days, the I group presented the characteristics described for the T group at 14 days, while this group presented aspects of a mature connective tissue. FT-IR analysis showed a clear distinction among the three groups at all experimental times and groups T and I presented more similarities to each other than to group C. Conclusion: Acute injury of tendon treated with human amniotic membrane fragment showed a faster healing process, reduction in inflammatory response, intense proliferation of fibroblasts and organization of collagen fibers.

FTIR and SEM analysis applied in tissue engineering for root recovering surgery.

Gingival recession is defined by the displacement of the gingival margin in the apical direction, which overcomes the cementum enamel junction. The etiology of gingival retraction is related to tissue inflammation caused by the accumulation of biofilm, by trauma from brushing action. Aesthetic periodontal surgery aims to return the root coverage to aesthetic harmony, and reduce the risk of periodontal disease and caries. To assist in the root coverage process, the porcine collagen matrix (PCM) has been widely studied. The objectives of this study are to identify the types of collagen that make up the PCM and analyze their morphology. For this, five PCM fragments, 2 mm (thickness) × 2.6 mm (width), were analyzed with the aid of scanning electron microscopy (SEM) and Fourier transform infrared spectroscopy (FTIR). The analysis by SEM showed that the PCM consists of two layers; the surface layer is compact, low porosity, and smooth surface, and a foamed underlying layer has high porosity. Through FTIR we identified that the surface and underlying layers are composed of collagen types I and III, respectively. This biomaterial is conducive to root coverage; it allows adsorption and cell proliferation following the matrix resorption and periodontal tissue neoformation. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 1326-1329, 2017.

Chlorin E6 phototoxicity in L. major and L. braziliensis promastigotes-In vitro study.

BACKGROUND: Cutaneous leishmaniasis is a zoonosis caused by protozoa of the genus Leishmania. Conventional treatments are long and aggressive, and they trigger a diversity of side effects. Photodynamic Therapy was originally proposed as a treatment for cancer, and it now appears to be a promising therapy for local treatment with fewer side effects of infectious diseases. METHODS: This study aimed to evaluate Chlorin e6 internalization by Leishmania major and Leishmania braziliensis promastigotes and its viability and effects on mitochondrial activity. Control groups were kept in the dark, while PDT groups received fluence of 10J/cm(2) (660nm). Chlorin internalization was evaluated using confocal microscopy after one hour of incubation for both species. RESULTS: The mitochondrial activity was evaluated by MTT assay, and viability was measured by the Trypan blue exclusion test. Giemsa staining was used to observe morphological changes. PS was internalized in both species and mitochondrial activity changed in all groups. However, the obtained MTT and Trypan results indicated that despite the change in mitochondrial activity in the dark groups, their viability was not affected, whereas the PDT treated groups had significantly reduced viability. Morphology was drastically altered in PDT treated groups, while groups kept in the dark exhibited the standard morphology. CONCLUSIONS: This study demonstrates that Chlorin has great potential for being used in PDT as a treatment for cutaneous leishmaniasis, although more studies are needed to determine in vivo application protocols.

In vitro evaluation of photodynamic therapy using curcumin on Leishmania major and Leishmania braziliensis.

Cutaneous leishmaniasis is an infectious disease caused by the Leishmania protozoan. The conventional treatment is long-lasting and aggressive, in addition to causing harmful effect. Photodynamic therapy has emerged as a promising alternative treatment, which allows local administration with fewer side effects. This study investigated the photodynamic activity of curcumin on Leishmania major and Leishmania braziliensis promastigote. Both species were submitted to incubation with curcumin in serial dilutions from 500 µg/ml up to 7.8 µg/ml. Control groups were kept in the dark while PDT groups received a fluency of 10 J/cm(2) at 450 nm. Mitochondrial activity was assessed by MTT assay 18 h after light treatment, and viability was measured by Trypan blue dye exclusion test. Morphological alterations were observed by Giemsa staining. Confocal microscopy showed the uptake of curcumin by both tested Leishmania species. Mitochondrial activity was inconclusive to determine viability; however, Trypan blue test was able to show that curcumin photodynamic treatment had a significant effect on viability of parasites. The morphology of promastigotes was highly affected by the photodynamic therapy. These results indicated that curcumin may be a promising alternative photosensitizer, because it presents no toxicity in the dark; however, further tests in co-culture with macrophages and other species of Leishmania should be conducted to determine better conditions before in vivo tests are performed.

Biochemical imaging of normal, adenoma, and colorectal adenocarcinoma tissues by Fourier transform infrared spectroscopy (FTIR) and morphological correlation by histopathological analysis: preliminary results

Introduction The colorectal cancer is a major health problem worldwide. Histology is considered the gold standard for differential diagnosis. However, it depends on the observer's experience, which can lead to discrepancies and poor results. Spectroscopic imaging by Fourier transform infrared (FTIR) is a technique that may be able to improve the diagnosis, because it is based on biochemical differences of the structural constituents of tissue. Therefore, the main goal of this study was to explore the use of FTIR imaging technique in normal colon tissue, colorectal adenoma, and adenocarcinoma in order to correlate their morphological structures with their biochemical imaging. Methods Samples were collected from normal (n = 4), adenoma (n = 4), and adenocarcinoma human colorectal tissue (n = 4) from patients undergoing colonoscopy or surgical resection of colon lesions. The samples were sectioned with a cryostat in sequential sections; the first slice was placed on CaF2 slide and the second slice was placed on glass slide for histological analysis (HE staining). The cluster analyses were performed by the software Cytospec (1.4.02)®. Results In normal samples, biochemical analysis classified six different structures, namely the lamina propria of mucous glands (epithelial cells and goblet cells), central lumen of the gland, mucin, and conjunctive tissue. In samples with adenoma and adenocarcinoma, altered regions could also be identified with high sensitivity and specificity. Conclusion The results of this study demonstrate the potential and viability of using infrared spectroscopy to identify and classify colorectal tissues.

Confocal Raman spectroscopy: determination of natural moisturizing factor profile related to skin hydration

INTRODUCTION: Skin health and skin care to reduce the effects of aging are the main interests of many researchers. The skin is very important because it protects the body from various effects of the external environment, and studies of the largest organ of the human body have been conducted since antiquity. In skin, aging effects are severe enough to promote changes in cell structure and biochemical composition. In this study, we quantitatively analyzed the water content and natural moisturizing factor of human facial skin in vivo and in real time by confocal Raman spectroscopy. This non-invasive technique is capable of providing detailed information on the biochemical composition at different depth profiles in the skin. METHODS: We studied 10 volunteers, phototype II (40 and 50 years old), using a confocal Raman system to examine the skin surface down to 25 µm. Raman spectra were obtained before product use (T0), and after 30 days of continuous use of cosmetics (T30). RESULTS: The results show a significant increase of 6.4% in water content in the surface layer of the facial skin after the cosmetic use. The amounts of natural moisturizing factor (NMF) compounds were also increased. Urocanic acid underwent a greater change in relation to carboxylic acid pyrrolidone, with a 38.5% increase in the stratum corneum. CONCLUSION: Confocal Raman spectroscopy identified changes in the biochemical composition of the superficial layers of the epidermis, which suggests the anti-aging efficacy of the formulation.

Study of advanced rheumatoid arthritis

INTRODUCTION: Rheumatic diseases are considered public health problems affecting millions of people worldwide resulting in high and rising health-care costs. In this work, Fourier Transform Infrared spectroscopy associated to Partial Least Square regression (PLS) analysis was used to diagnose rheumatoid arthritis (RA) from human serum. METHODS: The sera of 94 individuals were collected, which included 47 from rheumatic patients and 47 from healthy individuals. The results from PLS analysis were compared to standard clinical trials such as anti-citrullinated peptide antibodies, C- Reactive protein, and Rheumatoid factor. RESULTS: For clinical diagnosis, the anti-citrullinated peptide antibodies of second generation proved to be the most specific to diagnosis rheumatoid arthritis even after long periods of drug therapy. CONCLUSIONS: The qualitative PLS analysis has shown higher values of IgM of RA group, but the difference was very small. The RA patients were under medication, which interfered with the IgM concentration.

Cicatrização de feridas decorrentes da hanseníase utilizando laser de baixa intensidade / Wound healing leprosy induced by low intensity laser

A hanseníase é causada pelo bacilo Mycobacterium leprae(M. leprae), apresentando grande capacidade de infectar vários indivíduos com contágio pelas vias aéreas superiores. A hanseníase considerada um problema de saúde pública, principalmente nos países subdesenvolvidos e nos em desenvolvimento, devido à presença de incapacidades e estigma social...

Leprosy is caused by the bacillus Mycobacterium leprae(M. leprae), presenting the capacity to infect multiple individuals with infection of the upper airways. Hansen considered a public health problem, especially in under developed countries and developing countries, due to the presence of disabilities and social stigma...