The Nano-bio Interactions Of Nanocarriers And Its Mechanisms

Nanoparticles have been used to deliver drug,which have greatly expanded the range of drug carriers.However,there is growing evidence that nanoparticles are not just inert drug delivery vehicles when interacting with biological systems,but often have cellular biological effects,these interactions may affect biocompatibility of nanomedicines,but may also induce beneficial effects.However,little is known about the cellular biological effects of most nanoparticles,which greatly limits the clinical transformation of nanomedicines.Therefore,it is of great means to study the mechanism of the nano-bio interactions and to discover new biological effects and toxicity mechanisms of nanoparticles.Carboxyl polystyrene nanoparticles（CPS）are stable and easy to be prepared,are generally considered of good biocompatibility,and are widely used for delivery of biological macromolecules such as antibodies and peptides,however,our knowledge about the biological effect and the nano-bio interactions of CPS nanoparticles is still very limited,so we chose CPS as a model nanoparticle in this study.This study has an important finding:CPS of 60 nm（including those larger than 60 nm）can induce vacuoles and reduce cellular ROS and inhibit ferroptosis,the mechanism of which is that CPS induced nuclear transcription factor（TFEB）to transfer into the nucleus,stimulating the synthesis of lysosome related proteins and superoxide dismutase（SOD）,and thus decreased the intracellular ROS and inhibited ferroptosis.After co-incubation of 60 nm CPS with cells overnight in vitro,small"light spots"could be observed in cells under light microscope,while the dispersion media of nanoparticles could not induce similar phenomena.The results of confocal laser scanning electron microscopy（CLSM）and scanning electron microscopy（SEM）show that the"light spots"are vacuoles in the cytoplasm rather than depressions or holes on the cell membrane.Furthermore,such vacuoles were also observed in HK2,Hepg2,B16F10,DC2.4,THP-1 and Hela cells,but the size of CPS induced vacuoles（CIVs）varied in different cells,indicating different sensitivity to CIVs in different cells.After CPS was injected intravenously into mice,vacuoles were observed in the liver of mice after two days.Vacuoles were also observed in macrophages isolated from mouse abdominal cavity 2 days after intraperitoneal injection of CPS into mice.Further studies showed a positive correlation between the size of CIVs and the concentration of nanoparticles.The growth and formation of CIVs were observed by live-cell images.It was found that the growth of CIVs started from a"small spot"of about 0.5μm in diameter,expanded continuously with the fusion of vacuoles,and eventually disintegrated by a sudden burst.Interestingly,these"small spots"were0.5-1μm in diameter and filled with CPS.The results of SEM and CLSM showed that there were a large number of CPS attached to the cavity wall,and the size of vacuoles ranged from 0.5 to 8μm.Next,we study the uptake mechanism of CPS by cells.It was reported that nanoparticles are internalized mainly through endocytosis,including clathrin-mediated endocytosis,caveolin-mediated endocytosis,and macropinocytosis.Interestingly,clathrin-coated vesicles（100-200 nm）and caveosomes（60-100 nm）are smaller than macropinosomes,which is generally bigger than 0.5μm.TEM results show that the CPS were surrounded by pinocytosis cups（the hallmark structure of macropinocytosis）on the cell membrane,and the size of pinocytosis cups ranged from 0.5 to 1μm,and the size of endosomes formed after endocytosis was similar.Furthermore,we found that Cytochalasin D,an inhibitor of macropinocytosis,significantly inhibited the formation of CIVs and endocytosis of CPS.In contrast,other inhibitors of the endocytosis pathway could not inhibit endocytosis of CPS and the formation of CIVs.In addition,cells were labeled with Dextran-FITC（70k D）,a tracker of macropinocytosis,and we found that Dextran was enriched in CIVs.Flow cytometry also showed that the uptake of Dextran by cells was increased after CPS treatment,and the inhibition of macropinocytosis could eliminate this effect.These results suggest that CPS was internalized by cell mainly through macropinocytosis.Furthermore,we found that the size of the pinocytic cups formed during the endocytosis of CPS by the cells was relatively stable（0.5-1μm）.Therefore,although scanning microscopy（SEM）results show a large number of nanoparticles（300 nm CPS）adsorbed on the surface of cell membrane,only up to three 300 nm-CPS can be captured by the pinocytic cup and enter into the macropinosome.In contrast,a large number of nanoparticles could be found in each endosome in cells treated with smaller（60,80,or 120 nm）CPS.Since intracellular endocytosis of CPS is a prerequisite for CIVs formation,the efficiency of intracellular endocytosis may explain why vacuoles could not be induced by 300 nm-CPS and 1000 nm-CPS,whereas smaller CPS nanoparticles are more likely to induce CIVs.These results indicate that the size of pinocytosis cup determines the endocytosis efficiency of nanoparticles,and the size of pinocytosis cup is 0.5-1μm.Having settled the internalization mechanism,we then investigated the growth mechanism of CIVs.As nanoparticles usually accumulate in lysosomes,we first tested whether CIVs were lysosomes.The results of Lysosensor Green DND-189 and Neutral Red staining showed that the p H of CIVs was 6-6.8,which was lower than that of cytoplasm but higher than that of lysosomes.CLSM results show that LAMP1and Cathepsin B and Cathepsin D were distributed in CIVs.Western Blot（WB）results also showed that the expression of these proteins increased significantly after CPS treatment,and the CIVs inhibitors mentioned above and below could attenuate this effect.These results suggest that CIVs is an abnormal lysosomal structure and can be regarded as a marker of lysosomal stress.Next,we explored whether lower p H in CIVs is associated with the formation of CPS.V-ATPase is reported to be the main H⁺ion channel in endocytosomes.In fact,we found that Bafilomycin A1,an inhibitor of V-ATPase,could completely inhibit the formation of vacuoles.The results of TEM and CLSM showed that Bafilomycin A1did not affect the uptake of CPS.CPS was still aggregated in the macropinosome after endocytosis,and its size was 0.5-1μm.These results indicate that the formation of CIVs depended on the action of V-ATPase.Living-cell images show that the vacuoles continued to grow after the fusion of vacuoles and lysosomes.Therefore,it appears that the p H gradient of the vacuole promotes the entry of water into the vacuole to expand CIVs.AQPs was reported to be the major water transporter in the endosomes of cells,and immunofluorescence staining results show that AQPs was aboundant on the margins of CIVs.In fact,the inhibitors of AQPs,phloretin or Hg Cl₂,significantly inhibited the formation of vacuoles.These results suggest that AQPs transport water molecules to promote CIVs expansion.CLSM showed that many lysosomes surrounded the vacuoles and the WB results also showed that the expression of lysosomal-related proteins increased significantly after CPS treatment.It was reported that mammalian target of rapamycin C1（m TORC1）was capable of sensing lysosomal stress,and it is inactivated when a large number of lysosomes are required in cells,which reduced the phosphorylation of the nuclear transcription factor EB（TFEB）and facilitated nuclear transfer of TFEB.Therefore,we examined the TFEB nuclear translocation and m TORC1 activity in cells.Results showed CPS treatment indeed triggered TFEB nuclear translocation and raised the TFEB level in whole cells,and the TFEB translocation could be reduced by inhibitors of CIVs.In addition,CPS could also induce nuclear transfer of TFEB in some hepatocytes in vivo.The phosphorylation of 4-EBP1 is a marker of m TORC1activation.We found that the phosphorylation of 4-EBP1 was significantly decreased after CPS treatment,indicating that the activity of m TORC1 was inhibited by CIVs.Since it has been reported that nuclear transfer of TFEB is associated with the regulation of superoxide dismutase（SOD）and reactive oxygen species（ROS）,we then investigated whether CPS can affect SOD expression and ROS level in the cells.Our study found that CPS significantly increased the expression of SOD and decreased the level of ROS in cells.Moreover,there was a negative correlation between the degree of ROS reduction by CPS and the size of CPS.All the CIVs inhibitors mentioned above were able to significantly reduce the effect of CPS on cellular ROS,which was consistent with their role in decreasing the nuclear transfer of TFEB.In TFEB knockout cells（using CRISPR/CAS-9）,CPS did not increase intracellular SOD and decrease intracellular ROS.Furthermore,the basal SOD decreased,basal ROS increased,and CIVs became smaller in the TFEB-knocked-out cells.These results suggest that CPS increased intracellular SOD and decreases intracellular ROS in a TFEB-dependent manner.Since ROS and lipid peroxidation play an important role in the mechanism of ferroptosis,and increased SOD expression has been reported to reduce lipid peroxidation in vivo.Here,we found that overexpression of SOD1 did reduce ROS and inhibit ferroptosis both in RAW264.7 and HK2 cells.In addition,the SOD mimic molecule Tempol was reported to neutralize intracellular ROS,and we found that it could reduce cellular ROS and inhibit ferroptosis effectively,whereas inhibition of SOD1 with ATN-224 attenuated the inhibition effect of Tempol on ferroptosis.As CPS could increase cellular SOD,we speculated that it can also inhibit ferroptosis.Results show that CPS significantly inhibited the increase of ROS and lipid peroxidation induced by Erastin in cells,and the inhibitory effect of CPS on ferroptosis was even better than that of desferrioxamine（DFO）.In addition,the effect of CPS on ferroptosis is negatively correlated with the size of nanoparticles and can be attenuated by the CIVs inhibitors,which is consistent with their effect on cellular ROS.Further studies show that CPS is no longer able to inhibit the increased lipid peroxidation and inhibit ferroptosis in cells knocked out TFEB.In fact,the TFEB-knocked-out cells are more sensitive to ferroptosis.These results suggest that CPS inhibit ferroptosis in a TFEB-dependent way.Our further study showed that CPS did not affect intracellular levels of Glutathione Peroxidase 4（GPX4）and glutathione and transferrin receptors,which are reported to be involved in the regulation of ferroptosis.In addition,we found that CPS also inhibited the cytotoxicity of other ROS-inducing chemicals such as Rotenone,but did not affect the cytotoxicity of Doxorubicin,which induces apoptosis,suggesting that CPS could also inhibit other kinds of cell death triggered by increased ROS but not limited to ferroptosis.Taken together,we found that CPS cells were enveloped and endocytosis by a relatively stable pinocytic cups（0.5-1μm）on the cell membrane.Then,Then the macropinosome that contained CPS grew into large vacuoles in V-ATPase-and AQP-dependent manners.This vacuole is a marker of lysosomal stress and promotes nuclear transfer in TFEB by inhibiting the activity of m TORC1.The nuclear translocation of TFEB promoted the expression of SOD,which decreased cellular ROS and inhibited ferroptosis.The pathways of CPS regulating ROS and inhibiting ferroptosis revealed in this study have important implications for understanding the biological effects of polystyrene nanoparticles,and designing new nanoparticles or small molecular drugs against ferroptosis.It also suggests that the biological effects of nanoparticles are more complex than we thought,and we need to do more research in this area.