| Nanoparticles has been widely used in the development of drug delivery,biomedical diagnostic and therapeutic agents,due to its excellent physical and chemical properties.However,its application in vivo is obstacled for various biological barriers and the low efficiency problem of delivery.In order to improve the bioavailability of nanoparticles,we replenish the basic research on the interaction between nanoparticles and organisms with some new ideas and means.The concept of interaction between nanoparticles with cells or tissues is promising to help researches to optimize design and preparation of nanoparticles,further holding new opportunities for effectively improve the utilization of nanoparticles in vivo.The intestinal epithelium,one of the most important barrier system closely related with uptake efficiency,has highly selective permeability for foreign substances after a long period of evolution.At present,although the study on the efficiency of uptake,transport of nanoparticles and related mechanism through intestinal epithelial barrier is sufficient,most of them are restricted to drug permeability rather than the carrier of drug,i.e,the nanoparticle itself.To a large extent,these studies cannot truly reveal the efficiency of nanoparticle permeability across intestinal epithelial barrier.Indeed,it's necessary and imperative to focus on the bio-behavior of nanoparticle itself and carry out more research about these questions based on the interaction of nanoparticle and biology.The barrier function of intestinal epithelium mainly depends on epithelial cells,which account for more than 90%of the total epithelium surface area.The pathway among cells is sealed under the presence of intact epithelial layer.The interval distance is just enlarged to about 20 nm with the exogenous stimulation to open tight junction.It is extremely difficult for nanoparticles to transit.Under physiological conditions,the cell membrane is impermeable to most hydrophilic substances without specific transporters.However,once epithelial cells damaged,the expression of some related proteins would be changed.Actually,in our study,we explored the efficiency of nanoparticles uptake in intestinal epithelial cells and the related mechanism in inflammatory conditions,which plays an important role to understand intestinal epithelial barrier uptake and transport of nanoparticles.It may provide further insight into design and develop nanoparticles applied in vivo for researchers.In this paper,we established a rapid,simple and label-free method for detection of intracellular nanoparticles for the first time.The amount of nanoparticles in intestinal epithelial cells was studied by establishing a proper enteritis model in vivo.Besides,we studied the amount of nanoparticles in intestinal epithelial cells,and explored the relevant mechanism,revealed the biological influence of the uptake of nanoparticles by intestinal epithelial cells,with the inflammatory model in vitro.1.Establishment of a method for the detection of intracellular nanoparticles by flow cytometric light scattering.In this study,flow cytometric light scattering method was developed to accurately detect intracellular non-fluorescent nanoparticles.We used the flow cytometric light scattering detected the principle of size-dependence by analyzing cellular uptake of gold nanoparticles and carbon nanomaterials by Caco-2 cells,A549 cells and mouse peritoneal macrophages.The results showed the uptake effect of mouse peritoneal macrophages and Caco-2 cells with gold nanoparticles was consistent with gold standards(transmission electron microscope detection).The uptake of carbon nanoparticles by A549 cells was in accordance with concentration dependence and size effects,consistent with those obtained by transmission electron microscopy.In addition,we also detected the effect of several common inhibitors on cellular uptake of nanoparticles by flow cytometric light scattering technique,the results also reflected the mechanisms of cellular uptake of nanoparticles accurately.Above all,this work established a method for accurate quantification of label-free metal nanoparticles and non-metallic nanoparticles(carbon nanoparticles)in mammalian cells using flow cytometric light scattering.2.Study on the efficiency and mechanism of Fe3O4 nanoparticles uptake in intestinal epithelial cells under inflammatory conditionsIn this study,for investigating the efficiency and mechanism of uptake of Fe3O4 nanoparticles in intestinal epithelial cells under inflammatory conditions,we used dextran sulfate(DSS)-induced enteritis model mice to study the amount changes of the Fe3O4 nanoparticles through the intestinal epithelium.Caco-2 cell monolayers stimulated with inflammation factor interleukin-1 beta(IL-1?)were used to quantitatively study uptake of Fe3O4 nanoparticles by intestinal epithelial cells and the related mechanism.The flow cytometric light scattering and inductively coupled plasma mass spectrometry were used to quantify the amount of Fe3O4 nanoparticles in cells.To explore pathways possibly related to uptake efficiency,several cellular uptake inhibitors were used to treat Caco-2 cell monolayers in inflammatory conditions to examine the subsequent expression change of transport-related proteins by molecular biology techniques.The results revealed that stimulated intestinal epithelial cells uptook more Fe3O4 nanoparticles than control group.The study found that,Caco-2 cells were always dependent on clathrin mediated pathway to uptake Fe3O4 nanoparticles both under inflammatory and normal conditions.For the first time,we found that expression of clathrin was up-regulated in IL-1? induced Caco-2 cells.Overall,this study revealed the efficiency and mechanism of the uptake of Fe3O4 nanoparticles in intestinal epithelial cells under inflammatory conditions.3.Study on the efficiency and mechanism of PLGA nanoparticles and gold nanoparticles uptake and transport in intestinal epithelial cells under inflammatory conditionsIn this study,for further investigating the efficiency and mechanism of uptake and transport of gold nanoparticles and PLGA nanoparticles in intestinal epithelial cells under inflammatory conditions,we still used DSS-induced enteritis model mice and IL-1? induced Caco-2 cell monolayers to study the changes about the amount of Fe3O4 nanoparticles.Flow cytometric light scattering technique,inductively coupled plasma mass spectrometry and fluorescence spectrum detection technique used in the quantitative study on the cellular uptake of gold nanoparticles and PLGA nanoparticles.The uptake and transport mechanisms of gold nanoparticles and PLGA nanoparticles in intestinal epithelial cells were investigated using an IL-1? treated Caco-2 cell monolayers in vitro.The amount of gold nanoparticles in the blood of mice under inflammatory conditions was determined.The corresponding expression of related uptake pathway proteins was also examined by molecular biology technique.The results showed that inflamed intestinal epithelial cells uptake more gold nanoparticles but not for PLGA nanoparticles in vitro,however inflamed intestinal epithelial cells uptake both more gold nanoparticles and PLGA nanoparticles in vivo.The results may be one of the reason why the amount of gold nanoparticles in mice blood both under inflammatory conditions and control condition,is a million percent level.This is likely to be related to the "easy enter hard out" phenomenon in the cellular transport of nanoparticles.We also found that upregulation of clathrin expression in IL-1? induced Caco-2 cells and downregulation of caveolin-1 expression.Therefore,this study provided some help for further understanding the efficiency and mechanism of intestinal epithelial cells uptake of different nanoparticles under inflammatory conditions.In summary,we studied the efficiency of nanoparticles and the related mechanisms on the uptake of intestinal epithelial cells in inflammatory conditions using flow cytometric light scattering established in this study.We analyzed nanoparticles in cells in vitro and in animal models in vivo,revealed the amount change and mechanism of intestinal epithelial cells uptaking Fe3O4 nanoparticles,gold nanoparticles and PLGA nanoparticles.To some extent,the efficiency and related mechanisms of intestinal epithelial cells transporting gold nanoparticles and PLGA nanoparticles under inflammatory conditions were revealed.The cellular uptake efficiency of nanoparticles and the expression of related proteins in intestinal epithelial cells under inflammatory conditions were revealed.It is expected that the studies shown will further yield fundamental highlights and novel opportunities for researchers to optimize the design and preparation of nanoparticles with high bioavailability and accelerating the clinical translation of nanoparticles from bench to bedside... |
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