Study On The Function Monitoring Of Three-dimensional Embryoid Body Based On PLGA Nano-fluorescence Sensor

With the industrial production and useage in common life,persistent organic pollutants(POPs)seriously threaten human health and cause global ecological and environmental problems due to its chemical properties and stability,persistence,bioaccumulation,long distance mobility,semi volatility,and high toxicity.For example,poly brominated diphenyl ethers(PBDE)enter human body mainly through air,water and food.As an environmental endocrine disrupting compound,it has the effect similar to estrogen,and can produce serious side effects on the endocrine system,nervous system,thyroid,brain,kidney,liver and other organs.Therefore,it is very important to establish an effective screening platform for in vitro toxicity of organic compounds.At present,the main detection methods are based on molecular,cellular and animal models.Compared with the time-consuming and high-cost animal experiments,in vitro cell experiments have become a rapid screening method for toxicity test of toxic substances and have been an indispensable part of toxicity data.However,there are still some disadvantages in the current cell based toxicity assays.For example,the end-point assays used in traditional toxicity studies failed to pinpoint the subtle changes of cellular function at low exposure dosage in real time.Cancer cell line used in toxicity assays usually suffer from accumulating mutations,leading to different biological response to that of primary cells.Besides,compared with the traditional two-dimensional(2D)cell culture,the three-dimensional(3D)cell culture mimics the growth conditions of cells in vivo,and exhibites more realistic mass transfer process in the extracellular matrix and other microenvironment than 2D cell culture does.Therefore,the toxicity data obtained by 3D cell culture are more credible and more related to those of organism.To address the aforementioned chanllenges,this paper designed and synthesized a long-retained intracellular polymeric fluorescence nanosensor.Nanosensor labeled mouse embryonic stem cells(ESCs)can form cell aggregation morphology of embryoid body under 3D culture condition.The engineered EB provide real-time cellular function and can be used as a toxicity screening platform.The work of this paper is mainly divided into the following aspects:1.The biodegradable polylactic acid glycolic acid copolymer(PLGA)was used as a carrier to load fluorescent molecular probes to prepare intracellular sensors.The prepared PLGA sensor has a size of about 1 micrometer,can stay in the embryonic body for a long time(about 15 days),and release the probe into the cytoplasm as it degrades,which can effectively resist the signal attenuation of the fluorescent probe due to exocytosis.The two nanosensors PLGA @Calcein and PLGA @CellROX were pre-labeled with m ESC,and then induced m ESC to form EB.Under long-term exposure of different doses of polybrominated diphenyl ether(PBDE-47),the changes of cytosolic esterase(cell activity)and reactive oxygen species in EB were monitored.The study found that the low-dose 0.1μM PBDE-47 exposure did not significantly change the cell activity,and the active oxygen increased to 100% of the initial value.The high-dose 10 μM PBDE-47 exposed cell activity was significantly reduced,and active oxygen showed an overall downward trend,but the experimental group was significantly higher than the control group about 1 times in 11-15 days.2.Design and synthesis of intracellular nanosensors PLGA@β-actin MB and PLGA@PAX6 MB based on Pax6 and β-actin molecular beacons(MB),and real-time monitoring of Pax6 gene expression under long-term exposure of different doses of PBDE-47.The results showed that PBDE-47 significantly inhibited the expression of the neural differentiation gene Pax6,and the decrease was maintained at about 15-20%.The expression of Pax6 at the high dose of 10 μM PBDE-47 was significantly lower than that of 0.1 μM,and the highest decrease was 23%.3.Designed and synthesized DNA-AuNPs molecular probe modified by DNA molecular chain.The method based on salt aging has greatly improved the stability of the probe,and the detection limit in vitro has also been improved.The results of in vitro target pairing show that the fluorescence produced by the synthetic molecular probes varies linearly with the concentration of the target chain.The probe can work stably in the cell and maintain specificity.In summary,a toxicity screening platform based on engineered EB was developed by the combination of intracellular polymeric nanosensor and 3D cell culture.It provides a new method for the monitoring of the celluar function in a 3D organoid under the exposure of toxic substances.