Evaluación de la respuesta inmunológica en monocitos diferenciados tratados con nanopartículas de quitosano comercial y purificado.
Bibliographic record
Abstract
El quitosano es un polímero biodegradable proveniente del exoesqueleto de los crustáceos, el cual es biocompatible y no toxico, presenta actividad inmunogénica y se ha utilizar en la síntesis de nanopartículas con actividad biológica, la cual puede depender del grado de purificación del quitosano. En este trabajo se sintetizaron y caracterizaron nanopartículas de quitosano (CtsNps) purificado y no purificado, obtenidas a partir de dos casas comerciales (Sigma-Aldrich® y Canada \nBioscience) con el fin de evaluar si estas diferentes nanopartículas pueden generar la misma actividad inmunológica en monocitos aislados de sangre periférica mediante el análisis de perfil de citocinas, actividad fagocítica y marcadores de diferenciación. Las muestras de quitosano fueron purificadas mediante hidrolisis química y posteriormente se analizaron mediante FT-IR \nobservándose la generación de nuevos grupos OH y la detección de grupos NH2 a una menor longitud de onda en comparación con las muestras no purificadas. Las nanopartículas de quitosano presentaron un tamaño menor a 200 nm con forma esférica y una carga superficial menor a +34 mV. Los monocitos fueron aislados a partir de sangre periférica obtenida de pacientes aparentemente sanos, estas células fueron cultivadas en placas de 6 pozos y posteriormente fueron tratadas con dos concentraciones diferentes de CtsNps (50 y 300 µg) por un tiempo de 72 horas. El medio de cultivo fue recuperado y almacenado a -40°C para el posterior análisis de citocinas. Los monocitos al ser tratados con diferentes CtsNps presentaron cambios morfológicos visibles al microscopio, los niveles de CD14 y CD86 se vieron disminuidos en comparación con los monocitos que no fueron tratados con CtsNps, a la vez estar partículas indujeron la alta producción de IL-8, baja producción de IL-10, IL-12p70 y TNF y la moderada producción de IL-1β e IL-6 dando un indicio de que estas nanopartículas son capaces de activar una respuesta inmunológica primaria en las células. La capacidad fagocítica de los monocitos diferenciados se vio disminuido en comparación con los monocitos no tratados. Finalmente, para determinar el fenotipo celular obtenido una vez que los monocitos se diferenciaban se realizó una inmunofluorescencia para \ndeterminar arginasa e iNOS las cuales son enzimas presentes en macrófagos antiinflamatorios y proinflamatorios respectivamente, mediante este ensayo se determinó que la mayoría de los monocitos diferenciados pertenecían a un fenotipo proinflamatorio, debido a que se detectó una alta fluorescencia perteneciente a la enzima iNOS. Con este trabajo de investigación damos a \nconocer que las nanopartículas de quitosano obtenidas a partir de quitosano purificado presentan una mayor actividad inmunológica en comparación con el quitosano comercial; por lo cual su uso como agente encapsulante, terapéutico o de diferenciación dependerá del tipo de partícula que se utilice. \n \nAbstract \nChitosan is a biodegradable polymer from the exoskeleton of crustaceans, which is biocompatible and non-toxic, has immunogenic activity and has been used in the synthesis of nanoparticles with biological activity, which may depend on the degree of purification of chitosan. In this work, purified and unpurified chitosan nanoparticles (CtsNps) were synthesized and characterized, obtained from two commercial houses (Sigma-Aldrich® and Canada Bioscience) in order to assess whether these different nanoparticles can generate the same immunological activity in Monocytes isolated from peripheral blood by analysis of cytokine profile, phagocytic activity and \ndifferentiation markers. The chitosan samples were purified by chemical hydrolysis and subsequently analyzed by FT-IR, observing the generation of new OH groups and the detection of NH2 groups at a shorter wavelength compared to the unpurified samples. The chitosan \nnanoparticles presented a size smaller than 200 nm with a spherical shape and a surface charge less than +34 mV. Monocytes were isolated from peripheral blood obtained from apparently healthy patients, these cells were cultured in 6-well plates and subsequently treated with two different concentrations of CtsNps (50 and 300 µg) for a time of 72 hours. The culture medium was recovered and stored at -40 ° C for subsequent cytokine analysis. Monocytes when treated with different CtsNps showed visible morphological changes under a microscope, the levels of CD14 and CD86 were decreased compared to monocytes that were not treated with CtsNps, while \nbeing particles induced high production of IL-8, Low production of IL-10, IL-12p70 and TNF and moderate production of IL-1β and IL-6 giving an indication that these nanoparticles are capable of activating a primary immune response in cells. The phagocytic capacity of differentiated monocytes was decreased compared to untreated monocytes. Finally, to determine the cellular \nphenotype obtained once the monocytes were differentiated, an immunofluorescence was performed to determine arginase and iNOS which are enzymes present in anti-inflammatory and proinflammatory macrophages, respectively, by means of this test it was determined that most of the differentiated monocytes belonged to a proinflammatory phenotype, because a high fluorescence belonging to the iNOS enzyme was detected. With this research work we announce that chitosan nanoparticles obtained from purified chitosan have a higher immunological activity compared to commercial chitosan; Therefore, its use as an encapsulating, therapeutic or \ndifferentiation agent will depend on the type of particle used.
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.001 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.000 |
| Insufficient payload (model declined to judge) | 0.001 | 0.000 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".