Investigating The Effect Of Pericellular Matrix Of Cells On The Uptake Of Gold Nanparticles By Dark Field Imaging Technique

Previous works have shown that the cellular uptake of nanoparticles (NPs) can bedepend on many factors, including the size and/or shape、surface chemistry、sedimentation oflarge NPs and cell cycle. However, an implied assumption in almost all previous study is thatthe first-contact between an invading NP and the cell membrane, which is considered as theoutmost boundary between the intracellular and extracellular environment. The pericellularmatrix (PCM) which around the cell membrane was always be ignored or only considered asbarrier for transporting substances. One has demonstrated that the cellular uptake efficiency ofsmall molecule could be enhanced when degraded the PCM. Here, by using dark fiedmicroscopy and gold nanoparticles (AuNPs) as probes, we have investigated the effect of thePCM on the cellular uptake process and uptake efficiency of AuNPs. The main works wereintroduced as follows:(1) In chapter2, by using optical sectioning dark field microscopy, we obtained verticaldistribution of diffusion coefficients of single AuNP in the extracellular solution space ofliving cells. It was identified that before reaching the plasma membrane surface during theintracellular uptake process, AuNPs must diffuse through a viscous pericellular “b ffer zone”several microns thick where their motion is retarded significantly. The pericellular layer existsin two different cell types and is unrelated to the surface chemistry of AuNPs.(2) In order to demonstrate the effect of the PCM on the cellular uptake efficiency ofAuNPs directly, in chapter3, MG-63cells having a thick PCM layer was used as model cells.Then the hyaluronidase (HA`ase) degraded the PCM can serve as a control cell with thinPCM layer. Dark filed microscopy can be used to semi-quantification of the cellular uptake ofAuNPs in the thick PCM cells and thin PCM cells. Results of dark field images showed thatthe large concentrated NPs in the extracellular and intracellular environment of thick PCMcells, but in thin PCM cells, there has little AuNPs in the cells, indicating cells with thickPCM would enhance the cellular uptake efficiency of AuNPs. The quantification of cellularuptake of AuNPs in thick PCM cells and thin PCM cells were studied by ICP-AES. Resultsshowed that the thick PCM cells have higher cellular uptake efficiency of AuNPs in reality,the uptake amount decreased significantly when the PCM has been collapsed.(3) The mechanism of that how could the thick PCM facilite the cellular uptake ofAuNPs was studied in chapter4. With a laser scanning confocal microscope (LSCM), weobtained optical sections of AuNP scattering of the thick PCM and thin PCM cells, findingthat cells with thick PCM has a wide pericellular zone concentrated with AuNPs outside the cell membrane, but few AuNPs can be observed beyond the cell membrane of the thin PCMcell. By keeping the optical focal plane of the LSCM at the PCM region of a normal MG-63cell, the AuNP trapping process was recorded in real time. With increasing incubation time,the regions around the cells accumulated more and more particles. Therefore, one possiblereason for the PCM promote the cellular uptake efficiency was that it could capture largeamout of AuNPs in the region and enchance the interaction concentration between AuNPsand cell membrane. Moreover, by performing single particle tracking (SPT) analysis on thediffusion behavior of individual AuNP near the cell membrane surface of both thick PCM andthin PCM cells. The diffusion coefficients of AuNPs were slowed in the thick PCM to nearly0.1m2/s which have a similarly value to the diffusion rate of membrane receptors. But in thinPCM, the AuNPs have relatively large diffusion coefficients. Thererfore, another possiblereason for the PCM promote the cellular uptake efficiency was that it could slow the diffusionrate of AuNPs and facilitating its binding with the membrane receptors. Further, we foundthat PCM accumulation of AuNPs also consumes energy and is synchronized with receptormediated endocytosis. Moreover, the PCM accumulation of AuNPs was also affected by theendocytosis inhibitors, suggesting that the structural organization of the PCM network ischanging dynamically and is regulated by receptor mediated endocytosis.(4) In previous works for cellular uptake studies, NPs properties such as size、shape、surface chemistry have been demonstrated palying important role on cellular uptakeefficiency. Here, the relationship between the PCM promoting the cellular uptake efficiencyand NPs properties with different size、surface chemistry were studied in chaper5. No matterthe size of AuNPs from50nm to120nm, the cellular uptake efficiency was higher in thickPCM cells. However, the PCM promoting the cellular uptake efficiency was significantlydependent on the surfaces chemistry of AuNPs. The PCM does not grab AuNPs withoutdiscrimination, and at least a certain degree of proteins coverage is needed before the AuNPscan pass its screening.(5) To find out if the same phenomenon occurs in other cell lines with a less abundantPCM, we performed similar experiments on HeLa cells whose PCM thickness is smilar to thatof the HA`ase treated MG-63cells (chapter6). Previous works reported that adding AA intothe cell growth media could enhance the density and viscosity of the PCM. Here, for cellularuptake studies, HeLa cells served as control cells with normal PCM formation, AA treatedHeLa cells served as model cells with thick PCM formation, and HA`ase treated HeLa cellsserved as molde cells with thin PCM formation. Dark field results showed the celluar uptakeof AuNPs in HeLa cells after different treating. Compared to normal HeLa cells, depleting thePCM by using HA`ase leads to less AuNP uptake and enhancing the PCM by adding AA leads to more AuNP uptake.(5) On the base of the works of studying parameters which could affect on the cellularuptake efficiency, we also have investigated the celluar responses of HeLa cells induced byinternalization of different sizes of AuNPs. Results indicating that the cellular responsesshoud be considered when the further bioapplication with NPs in future due to the cellvability assay is not enough to test the cytotoxicity of NPs. Moreover, effects of AuNPs onthe differentiation of stem cells have observed by using P19cells as model stem cells, findingthat the differentiation could be enhanced after AuNPs treated (chaper7).