PDA-mediated Mild Photothermal Therapy Combined with Autophagy Inhibitors Kill Breast Cancer Cells / 肿瘤防治研究

Objective To explore whether inhibiting autophagy can enhance the sensitivity of photothermal treatment under mild photothermal conditions. Methods CQ@PLGA@PDA NPs were prepared by an improved double emulsification method and a PDA-based surface modification method. After basic characterization, CCK-8 method was used to detect the cytotoxicity of nanoparticles; the near-infrared laser irradiation nanoparticle solution was used to detect the heating effect; CCK-8 method and live-dead cell staining were used to detect the killing effect of tumor cells; Western blot was used to detect the expression of autophagy-related proteins. Results The CQ@PLGA@PDA NPs were successfully prepared, with a particle size of 253.10±2.39 nm, a zeta potential of -22.57±0.80 mV, uniform particle size and good dispersion. The temperature of nanoparticle solution increased to 45℃ after the near-infrared laser irradiation for 10 min. CQ@PLGA@PDA NPs had no obvious toxicity to cells. The survival rates of breast cancer cell MDA-MB-231 and mouse embryonic fibroblast NIH-3T3 cell were above 95%. The inhibition of autophagy under mild photothermal conditions could improve the sensitivity of photothermal therapy. Conclusion The prepared CQ@PLGA@PDA NPs have good photothermal performance and high biological safety; by inhibiting autophagy, they can effectively kill tumor cells under mild photothermal conditions(< 50℃).

Influência da laserterapia de baixa intensidade no processo inflamatório e na imunomarcação da interleucina­23 e do fator indutor de hipóxia 1-alfa no tecido pulpar de dentes clareados / Influence of low-level laser therapy on the inflammatory process and immunolabeling of interleukin-23 and hypoxia-inducing factor 1-alpha in the pulp tissue of bleached teeth

Introdução: O uso do laser infravermelho (LIV) através da fotobiomodulação, tem demonstrado efeitos benéficos aos tecidos. Objetivos: Avaliar a influência do LIV sobre o processo inflamatório, por meio da imunomarcação da interleucina próinflamatória IL-23, e sobre a angiogênese, por meio da imunomarcação do fator induzível por hipóxia-1 alfa (HIF-1α), no tecido pulpar de dentes clareados. Materiais e métodos: Quarenta ratos Wistar foram distribuídos aleatoriamente em 4 grupos de 20 hemi-maxilas cada: Grupo Controle ­ recebeu o tratamento com o gel placebo; Grupo Cla - recebeu 30 minutos do gel clareador H2O2 a 35%; Grupo LIV ­ recebeu uma aplicação de LIV (808 nm, 30 segundos, 3J); Grupo Cla-LIV ­ imediatamente após a aplicação do H2O2 a 35%, recebeu uma aplicação de LIV, como descrito no grupo LIV. Após 2 e 30 dias (n = 10), os animais foram eutanasiados e as maxilas removidas e processadas para avaliação histológica (H.E.) e imunoistoquímica. Forma de análise: Os cortes teciduais seriados e com espessura de 5 µm corados em H.E. foram avaliados por escores atribuídos à inflamação, e a análise imunoistoquímica foi realizada através de escores atribuídos à imunomarcação. Os dados foram submetidos aos testes de Wilcoxon e Mann Whitney (p < 0,05). Resultados: Aos 2 dias, houve inflamação severa e necrose nos terços oclusal e médio do tecido pulpar no grupo Cla, diferente do grupo Cla-LIV com inflamação leve à moderada (p < 0,05). No terço cervical, houve inflamação moderada a severa no grupo Cla, e leve no grupo Cla-LIV (p < 0,05). Aos 30 dias, houve ausência de inflamação e os grupos clareados apresentaram deposição de dentina terciária. Em relação à IL-23, aos 2 dias foi observada imunomarcação severa no grupo Cla e moderada no grupo Cla-LIV (p < 0,05); aos 30 dias, houve redução na imunomarcação de IL-23 nos grupos clareados, onde o grupo Cla apresentou imunomarcação moderada, e o grupo Cla-LIV leve imunomarcação, sem diferença significante (p > 0,05). HIF-1α foi mais presente aos 2 dias no grupo Cla, sem diferença significativa com Cla-LIV (p > 0,05). Foi observada diferença entre os grupos clareados e seus respectivos controles, que não apresentaram imunomarcação (p < 0,05); aos 30 dias, o grupo Cla apresentou redução na imunomarcação para HIF-1α, enquanto o grupo Cla-LIV apresentou aumento da imunomarcação, mas a diferença permaneceu apenas entre os grupos clareados e seus controles (p > 0,05). Conclusão: O laser infravermelho minimizou o infiltrado inflamatório e a imunomarcação de IL-23 no tecido pulpar após a clareação dentária, mas não influenciou a imunomarcação de HIF-1α(AU)

Introduction: The use of infrared laser (IRL) through photobiomodulation has shown beneficial effects on tissues. Objectives: To evaluate the influence of LLL on the inflammatory process, by immunolabeling IL-23 pro-inflammatory interleukin, and on angiogenesis, by immunolabeling hif-1 alpha inducible factor (HIF-1α) in the pulp tissue of bleached teeth (HIF-1α). Materials and methods: Forty Wistar rats were randomly divided into 4 groups of 20 hemimaxilla each: control group - received treatment with placebo gel; Ble group - received 30 minutes of 35% H2O2 bleaching gel; IRL group - received one application of IRL (808 nm, 30 seconds, 3 J); Ble-IRL group - immediately after application of 35% H2O2, received an application of IRL, as described in the IRL group.. After 2 and 30 days (n = 10), the rats were euthanized and the jaws removed and processed for histological evaluation (H.E.) and immunohistochemistry. Form of analysis: Serial tissue sections with thickness of 5 µm stained in H.E. were evaluated by scores attributed to inflammation, and immunohistochemical analysis was performed by scores attributed to immunolabeling. The data were submitted to the Wilcoxon and Mann Whitney tests (P < 0.05). Results: At 2 days, there was a severe inflammation and the presence of necrosis in the occlusal and middle thirds of the pulp tissue in the Ble group, different compared to the Ble-IRL group with moderate to mild inflammation (P < 0.05). In the cervical third, there was moderate to severe inflammation in the Ble group, and mild in the Ble-IRL group (P < 0.05). At 30 days, there was absence of inflammation and bleached groups had an extensive deposition of tertiary dentin. Regarding IL-23, at 2 days was observed severe immunolabeling in the Ble group and moderate in the Ble-IRL group (P < 0.05); at 30 days, there was a reduction in IL-23 immunolabeling in the bleached groups, where Ble group had moderate immunolabeling, and Ble-IRL group had mild immunolabeling, without significant difference (P > 0.05). HIF-1α was more evident at 2 days in the Ble group, without significant difference with Ble-IRL (P > 0.05). The difference was observed between the bleached groups and their respective controls, which had no immunolabeling (P < 0.05); at 30 days, the Ble group had a reduction in HIF-1α immunolabeling, while the Ble-IRL group had an increase in immunolabeling from moderate to severe; however, the difference remained only between the bleached groups and their controls (P > 0.05). Conclusion: Infrared laser minimized the inflammatory infiltrate and IL-23 immunolabeling in the pulp tissue after dental bleaching, but did not influenced HIF-1α immunolabeling(AU)

Tejido tiroideo y láser infrarrojo: un estudio morfométrico / Thyroid tissue and infrared laser: a morphometric study

La glándula tiroides posee gran importancia debido a la síntesis y secreción de hormonas, las cuales desempeñan funciones fundamentales para la mantención de la fisiología animal. En este contexto, el objetivo del presente trabajo consistió en determinar parámetros morfométricos de estructuras tiroideas sometidas a estimulaciones con láser infrarrojo (LIR). Para ello, 10 ratas Sprague Dawley de 3 meses de vida y peso aproximado de 200 g, fueron divididas en dos grupos de 5 animales cada uno: grupo control y grupo experimental. Estos últimos recibieron estimulaciones infrarrojas en la tiroides con dosis de 16 J/cm2 durante 15 días seguidos. Una vez sacrificadas las ratas, se extrajeron las glándulas tiroides las que fueron procesadas para microscopía óptica obteniéndose placas histológicas y micrografías con aumentos finales de hasta 1000 X. Se efectuaron estudios morfométricos para determinar en 40 placas, variaciones tisulares generadas por las inducciones infrarrojas, con especial énfasis en la disposición coloidal y dimensiones de folículos y células tiroideas. El análisis de las 40 placas histológicas generados por las inducciones del láser infrarrojo comparados con los controles, reveló que existen marcadas diferencias en todos los componentes del tejido tiroideo analizado, lo cual podría otorgar antecedentes de diferentes funcionalidades en el metabolismo de las glándulas.

The thyroid gland is of great importance because of the synthesis and secretion of hormones which play key roles in the maintenance of animal physiology. In this context, the aim of the present study was to determine morphometric parameters of thyroid structures subjected to infrared laser stimulation (ILS) and for this purpose, 10 Sprague Dawley rats, 3 months of age and weighing approximately 200 grams, were divided into two groups of 5 animals each: the control group and the remaining 5 animals constituting the experimental group received infrared stimulation in the thyroid with doses of 16 J/cm2 for 15 consecutive days. After the rats were sacrificed, the respective thyroids were removed and processed for optical microscopy. Histological plates and micrographs were obtained with final increases of up to 1000 X. Morphometric studies were carried out to determine the tissue variations generated by infrared inductions, with special emphasis on the colloidal arrangement and dimensions of follicles and thyroid cells. Our results revealed that there are marked differences in all the components of the analyzed thyroid tissue which could give antecedents of different functionalities in the metabolism of thyroid glands.

Efecto del láser infrarrojo sobre células tiroideas / Infrared laser effect on thyroid cells

Diez ratas Sprague Dawley de 4 meses de vida y peso aproximado de 250 g fueron divididas en dos grupos de 5 animales cada uno, el grupo A se mantuvo como control y los animales del grupo B recibieron estimulaciones con láser infrarrojo en la tiroides con dosis de 16 J/cm2 durante 15 días consecutivos. Posteriormente las ratas fueron sacrificadas, se extrajeron las respectivas tiroides siendo procesadas para microscopía óptica y se obtuvieron placas histológicas y micrografías de tiroides con aumentos finales de hasta 1000X, las cuales fueron sometidas a estudios morfométricos para determinar en 100 células foliculares: número, áreas y perímetro tanto celular como nuclear, además de disposición coloidal y presencia de vasos sanguíneos. El análisis de los resultados entre las 100 células foliculares pertenecientes a tiroides normal y estimulada revela que existen marcadas diferencias en todos los componentes analizados los que se podría traducir en distintas funcionalidades en el metabolismo de las respectivas glándulas.

Ten 4-month-old Sprague Dawley rats weighing approximately 250 g were divided into two groups of 5 animals each. Group A was the control and the animals in group B received thyroid stimulation with infrared laser in a dose of 16 J/cm2 for 15 consecutive days. Subsequently, rats were euthanized and thyroids were removed and processed for optical microscopy. From both cell types thyroid histological slides and micrographs were obtained with final increases of 400 and 1000X. Morphometric analysis determined the number, areas and cell perimeter as well as colloidal dispersion and presence of blood vessels in 100 follicular cells. Analysis of the results among the 100 follicular cells belonging to normal and stimulated thyroids revealed marked differences in all the analyzed components, which could translate into different functionalities in the metabolism of the respective glands.

Inhibitory effect of gold nanoparticles conjugated with interferon gamma and methionine on breast cancer cell line

Objective: To develop a gold nanoparticles complex conjugated with interferon-gamma (IFN-γ) and methionine along with application of hyperthermia using near-infrared laser beams for the treatment of cancer cells. Methods: Gold nanorods (10 nm) were conjugated with IFN-γ and methionine using carbodiimide family and characterized after purification by dialysis bags. Breast cancer cells were cultured and incubated with gold nanorods at different concentrations followed by irradiation with near-infrared laser beam. Samples were then evaluated for their viability in order to determine the effect of treatment and variables by MTT assy. Results: Zetasizer results confirmed the conjugation of gold nanorods with methionine and IFN-γ. The median percentage of cell viability in 0.30 μg/mL concentration of gold nanorods was 82%. The cell viability reached to 85% at the same concentration of gold nanorods, which existed in the assayed complex. The results of MTT assay showed that the 0.60 μg/mL concentration of gold nanoparticles complex was toxic on tumor cells (P < 0.05). After exposure to hyperthermia, the viability of cells at 6 min decreased to 77% in 0.30 μg/mL concentration of gold nanorods complex. Conclusions: The size and concentration of gold nanorods was not cytotoxic. However, their presence during irradiation near-infrared laser increased the number of dead cells during the treatment of cells.

Characteristics and impact factors of ocular damage effects induced by transitional near-infrared laser:a review / 军事医学

The transitional near-infrared (NIR) laser was defined as ranging from 1.3μm to 1.4μm, within which the most sensitive tissue to laser damage changed from the retina to the cornea.The ocular damage effect has attracted much attention due to the increased varieties and output power of laser in this spectrum region in recent years.Compared with visible and mid-and-far infrared wavelengths, the ocular damage effect induced by transitional NIR wavelengths has many peculiarities and impact factors due to the bulk absorptionby ocular media.This paper reviews the existing ocular damage threshold data and analyzes the characteristics, impact factors and unresolved issues relating to ocular effects induced by laser radiation over the transition zone.

El retículo endoplasmático liso en hepatocitos estimulados con distintas dosis de láser infrarrojo / The smooth endoplasmic reticulum in hepatocytes stimulated with different doses of infrared laser

Veinticuatro ratas hembras Sprague Dawley de 4 meses de vida con peso aproximado de 250 g, fueron divididas en cuatro grupos (A, B, C y D), donde el grupo A (control) no recibió estimulación infrarroja, B se irradió con láser infrarrojo 4 J/cm², C con dosis de 8 J/cm² y D con 16 J/cm². La estimulación infrarroja se realizó diariamente, por 15 días ininterrumpidos. Las ratas fueron sacrificadas y se extrajeron muestras tanto de hígado normal (control) como estimulado con las distintas dosis infrarrojas, las que fueron procesadas para microscopía electrónica de transmisión. De los hepatocitos normales y estimulados, se obtuvieron microfotografías con aumentos finales de hasta 36.500 X, que fueron sometidas a estudios morfométricos para determinar fracciones volumétricas con especial énfasis en el retículo endoplásmico liso (REL) y de los siguientes componentes celulares: retículo endoplasmático rugoso (RER), mitocondrias, glicógeno, eu y heterocromatina. De igual manera se cuantificaron las áreas celulares y nucleares. Del análisis de los resultados entre hepatocitos normales y estimulados con diferentes dosis infrarrojas, se visualiza que existen notables diferencias en todos los componentes celulares cuantificados particularmente el REL. Se concluye que las estimulaciones infrarrojas provocan una drástica transformación en la ultraestructura y morfología de los hepatocitos, lo que provocaría una variación funcional, representando de esta manera el efecto que estas estimulaciones provocan en este tipo celular.

A total of 24 female Sprague-Dawley rats aged 4 months and weighing approximately 250 g, were divided into four groups labeled A, B, C and D. Group A received no infrared stimulation and served as control. Group B was radiated with a dose of 4 J/cm² of infrared laser, Group C with doses of 8 J/cm² and Group D with 16 J/cm². This infrared stimulation was carried out daily for 15 days uninterrupted. The rats were then sacrificed and samples of both normal-control liver and liver stimulated with the different infrared doses were extracted for immediate processing via transmission electron microscopy. Transmission electron microphotographs were obtained at magnifications of 21300X from both normal and stimulated hepatocytes; these were subjected to morphometric studies to determine volumetric fractions with special emphasis on the smooth endoplasmic reticulum (SER) and the following cell components: rough endoplasmic reticulum (RER), mitochondria, glycogen, eu and heterochromatin. Likewise, cell and nuclear areas were quantified. Analysis of the results of normal and stimulated hepatocytes with different infrared doses showed considerable differences in all the quantified cell components and particularly from the SER it is concluded that the effects of these stimulations bring about a drastic transformation in the ultrastructure and morphology of the hepatocytes, which may ultimately translate into a functional variation, thus representing the effect that these stimulations cause in this cell type.

Persistencia de los efectos del láser infrarrojo 6 J/cm² en hepatocitos de rata / Persistence of the effects of 6 J/cm2 infrared laser in rat hepatocytes

Hígados de ratas Sprague Dawley fueron irradiados con dosis diarias de 6 J/cm2 emitida por el láser AsGa equivalente a 904 nm durante 15 días De estos animales previamente anestesiados fueron sacrificados transcurridos 5, 10, 30, 45 y 60 días post irradiación para posteriormente obtener quirúrgicamente muestras de hígado y ser procesadas para microscopía electrónica de transmisión, aplicando técnicas morfométricas utilizando aumentos de 8.500 X con especial énfasis en cuantificar fracciones volumétricas de componentes celulares con el objetivo de precisar la duración de las estimulaciones infrarrojas. El análisis de los resultados entre hepatocitos controles e irradiados con dosis de 6 J/cm2 y tiempo de estimulación infrarroja revela que existen marcadas diferencias entre las fracciones volumétricas de componentes celulares determinantes de funcionalidad celular e involucrados en síntesis proteica, cuantificación que demuestra claramente que el efecto del láser infrarrojo persiste hasta los 30 días post estimulación, evidenciándose modificaciones de organelos que revelan alta funcionalidad, mientras que sobre este tiempo es observada una notable inhibición de dicha funcionalidad, concluyéndose entonces que los efectos de radiación infrarroja persisten en tiempos precisos provocando en los hepatocitos una drástica transformación en sus componentes y por ende en su funcionalidad. en estas células de elevado metabolismo.

Livers of Sprague Dawley rats were irradiated with daily doses of 6 J/cm2 emitted by a laser AsGa, equivalent to 904 nm during 15 days. Experiment animals were anaesthetised and killed after 5, 10, 30, 45 and 60 days post irradiation, in order to obtain samples of liver by surgery. These were processed for transmission electron microscopy, and morphometric techniques were applied using 8,500 X magnification with special emphasis on measuring the volumetric fractions of cell components in order to determine the duration of infrared stimulation. Analysis of the results between control hepatocytes and those irradiated with doses of 6 J/cm2 and by period after infra-red stimulation revealed the existence of marked differences between the volumetric fractions of cell components which determine cell function or are involved in protein synthesis. The measurements show clearly that the effect of the infrared laser persists up to 30 days post stimulation, with evidence of modifications of organelles revealing high functioning, while after 30 days a notable inhibition of this functioning is observed. It is therefore concluded that the effects of infrared radiation persist for precise times, provoking a drastic transformation in hepatocyte components, and thus the functioning of these high-metabolism cells.

Estudio Morfométrico del Efecto de Láser Infrarrojo 16 J/cm2 sobre Hepatocitos de Rata / Morphometric Study of Effect 16 J/cm2 Infrared Laser in Rat Hepatocytes

Hígados de ratas Sprague Dawley fueron irradiados durante 15 días con dosis diarias de 8 y 16 J/cm2 proveniente del láser AsGa equivalente a 904 nm. De estos animales previamente anestesiados, fueron quirúrgicamente obtenidas muestras de hígado y posteriormente procesadas para microscopía electrónica de transmisión, siendo estudiadas y sometidas a técnicas morfométricas utilizando aumentos de 8.500 X con especial énfasis en cuantificar fracciones volumétricas de componentes celulares. El análisis de los resultados entre hepatocitos irradiados con dosis de 8 y 16 J/cm2 revela que existen marcadas diferencias entre las fracciones volumétricas de componentes celulares determinantes de funcionalidad celular e involucrados en síntesis proteica, cuantificación que demuestra claramente que dosis de 8 J/cm2 logra un óptimo de estimulación para lograr alta funcionalidad, mientras que 16 J/cm2 evidencia notable inhibición de dicha funcionalidad, concluyéndose entonces que los efectos de dosis específicas de radiación infrarroja provoca en los hepatocitos una drástica transformación en sus componentes y por ende en su funcionalidad representando el efecto de estas estimulaciones sobre este tipo celular de elevado metabolismo.

Sprague Dawley rat livers were irradiated with 8 and 16 J/cm2 daily doses during 15 days from the AsGA laser, equivalent to 904 nm. Liver samples were surgically removed from rats previously anesthetized, then processed for electronic microscopic transmission and subsequently studied and subjected to morphometric techniques using 8.500 X enlargements with special emphasis on quantifying cellular component volumetric fractions. The result analysis of the hepatocytes irradiated with 8 and 16J/cm2 doses revealed that there were marked differences between the volumetric fractions of cellular components that determine cellular functionality and protein synthesis, a quantification that clearly demonstrates that with a dose of 8 J/cm2, the optimum stimuli is achieved in order to obtain high functionality, while with a dose of 16 J/cm2 a notable reduction of that functionality is noted, concluding that the effects of specific infrared radiation doses provoke a drastic transformation of its components in hepatocytes, and therefore its functionality, activating or inhibiting it, and representing the effect of these stimulations over this elevated metabolism cellular type.

Análisis morfométrico del efecto de estimulaciones de láser infrarrojo sobre núcleos de hepatocitos de rata / Morphometric analysis of the effect of infrared laser stimulation on hepatocyte nuclei in rats

De ratas Sprague Dawley tanto normales como irradiadas con dosis diarias de 1, 2, 4, 8, y 16 J/cm2 durante 15 días emitidas por el láser AsGa equivalente a 904 nm previamente anestesiadas, fueron quirúrgicamente obtenidas muestras de hígado, las que posteriormente fueron procesadas para microscopía óptica, siendo estudiadas y sometidas a técnicas morfométricas utilizando aumentos de 1000X, con especial énfasis en cuantificar áreas de núcleos y nucleolos. El análisis de los resultados entre hepatocitos normales e irradiados revela que existen marcadas diferencias entre sus áreas tanto nucleares como nucleolares, concluyéndose que los efectos de estas dosis de radiación infrarroja provoca en los hepatocitos una drástica transformación en sus componentes y por ende en su funcionalidad, principalmente en la relativa a la síntesis proteica, representando el efecto de estas estimulaciones sobre este tipo celular de elevado metabolismo.

Liver samples were taken from previously anaesthetised Sprague Dawley rats, both normal and irradiated with daily doses of 1, 2, 4, 8, and 16 J/cm2 applied over 15 days by AsGa laser equivalent to 904 nm. These samples were then processed for optical microscopy. They were studied and subjected to morphometric techniques using 1000X magnification, placing special emphasis on the quantification of the areas of nuclei and nucleoli. An analytic comparison of the results between normal and irradiated hepatocytes reveals the existence of significant differences between both the nuclear and nucleolar areas studied, from which it is concluded that the effect of these doses of infrared radiation is to provoke a drastic transformation in the components of the hepatocytes, and therefore in their functioning, principally with respect to protein synthesis, and that this would be the effect of stimulation of this nature on this type of high-metabolism cell.