Study On The Interaction Between Noble Metal Nanomaterials And Living Cells Based On Dark Field Imaging

With the improvement of synthetic methods and advancement of therapeutic strategies,the application of nanomaterials in the fields of biomedicine and diagnostic therapy become extensive and profound.The high efficiency and safety of nanomaterials are very important prerequisites for their further clinical translation.In order to gain an in-depth understanding of how nanomaterials interact with biological interfaces in complex biological environments,nanoparticles and living cells needs to be accurately and quantitatively studied at the level of single cells and single particles.This interaction process can be monitored directly and continuously through imaging.However,the commonly used fluorescence imaging methods have limitations such as photobleaching and phototoxicity,and it is difficult to meet the needs of long-term,single-particle resolution imaging.With the help of the unique optical properties of noble metal nanoparticles,the dark field microscope can be used to observe the dynamic processes of these nanoparticles engulfed by the cells,as well as their movement and aggregation inside cells,to provide important visual evidence for nano-bio interface and molecular mechanisms.In this thesis,gold and silver nanoparticles were selected as plasmonic nanoprobes,and dark field imaging was used as the major research method.The interaction between nanoparticles and living cells and possible regulatory mechanisms were investigated in the following parts.First,we used a gold nanoprobe to study the targeting mechanism of nanoparticles coated with cell membranes.The biomimetic strategy of directly coating the cell membrane on the surface of the nanoparticles can give the material better biocompatibility and targeting capability,and has been proven to promote medical applications including tumor targeted therapy.However,the cell membrane composition is complex,and the mechanism of regulating the targeted uptake of nanoparticles is not yet clear.To solve this problem,we constructed a gold nanoprobe?AuNP@CCM?camouflaged with cancer cell membranes,and quantitatively studied the cellular uptake and intracellular behavior of AuNP@CCM at the single cell level under dark field microscopy.We have also developed a program that allows quantitative analysis of nanoparticles in based on single cell dark field images.The results show that the cell membrane can improve the uptake efficiency of gold nanoparticles by 7 times,and the intracellular aggregation speed of nanoparticles in the cell can be accelerated by more than 90 times.In addition,we found that integrin?_V?₃,which is generally highly expressed in tumor cells,is essential for the cell targeting of AuNP@CCM.This part of the work provided new insights into the regulation mechanism of the cell membrane coating strategy.Secondly,we used silver nanoprobes to study the movement and agglomeration of nanomaterials in cells.Intracellular aggregation is universal across various nanoparticles.At present,the dynamic process and biological effects of intracellular nanomaterial aggregation are still unclear,due to the lack of reliable and sensitive methods for quantitative monitoring of single particle and single cell levels.We used spherical nucleic acids containing nano-silver cores as multiplex plasma probes?plas-SNA?to investigate this problem.The color change of the probe can reflect the different aggregation states of the nanoparticles under dark field microscopy.We have developed an automated image processing and data analysis program to identify,classify,and count plasmonic signals in the original dark-field image.The results reveal the dynamic process of nano-aggregation in the cell.We analyzed the spatial distribution and motion patterns of single particles,small clusters and large aggregates by single particle tracking.In addition,we found that nucleic acid modification attenuated the aggregation degree of silver nanoparticles while alleviating their cytotoxicity.This finding demonstrated the correlation between intracellular aggregation of nanomaterials and their biological effects.This part of the work provides quantitative information on the intracellular aggregation of nanoparticles,which helps to make safer and more effective nanodesigns.Finally,we used gold nanoprobe to study the elimination pathway of nanomaterials.The mechanism of nanomaterials clearance is crucial for their translation in clinical medicine.However,there is a lack of direct evidence for whether nanomaterials will be excluded during cell migration.Therefore,in this study,AuNPs was utilized as a plasmonic imaging probe,and dark field microscopy was used to observe and analyze the movement and exocytosis of gold nanoparticles within the retraction fibers in real time during cell migration.The results showed that some of the gold nanoparticles engulfed by the cells would remain in the retraction fibers,and the movement rate of these particles decreased corresponding to the increase of the distance from the cell body.When the retraction fibers were disconnected from the cell body,the gold nanoparticles were apart from cell.This part of the work proved that exogenous nanomaterials can be eliminated during cell migration.The results of this study help to design safer and more efficient nanomaterials for diagnosis and treatment.