Size-Dependent Regulation of Intracellular Trafficking of Polystyrene

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Size-dependent regulation of intracellular trafficking of polystyrene nanoparticle-based drug delivery carriers Ting Wang, Lu Wang, Xiaoming Li, Xingjie Hu, Yuping Han, Yao Luo, Zejun Wang, Qian Li, Ali Aldalbahi, Lihua Wang, Shiping Song, Chunhai Fan, Yun Zhao, Maolin Wang, and Nan Chen ACS Appl. Mater. Interfaces, Just Accepted Manuscript • Publication Date (Web): 12 May 2017 Downloaded from http://pubs.acs.org on May 13, 2017

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ACS Applied Materials & Interfaces

Size-dependent Regulation of Intracellular Trafficking of Polystyrene Nanoparticle-based Drug Delivery Carriers Ting Wang†‡, Lu Wang#‡, Xiaoming Liǁ, Xingjie Hu#, Yuping Han†, Yao Luo†, Zejun Wang#, Qian Li#, Ali Aldalbahi§, Lihua Wang#, Shiping Song#, Chunhai Fan#, Yun Zhao†, Maolin Wang†*, Nan Chen#* †

College of Life Sciences, Sichuan University, Chengdu 610064, China

#

Division of Physical Biology and Bioimaging Center, Shanghai Synchrotron Radiation Facility,

CAS Key Laboratory of Interfacial Physics and Technology, Shanghai Institute of Applied Physics, Chinese Academy of Sciences, Shanghai 201800, China ‖

§

School of Life Science and Technology, ShanghaiTech University, Shanghai 201210, China

Chemistry Department, King Saud University, Riyadh 11451, Saudi Arabia

*Emails: [email protected], [email protected];

ABSTRACT

Nanoparticles (NPs) have shown great promise as intracellular imaging probes or nanocarriers and play increasing role in biomedical applications. A detailed understanding of how NPs get “in

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and out” of cells is important for developing new nanomaterials with improved selectivity and less cytotoxicity. Both physical and chemical characteristics have proven to regulate the cellular uptake of nanoparticles. However, the exocytosis process and its regulation are less explored. Herein, we investigated the size-regulated endocytosis and exocytosis of carboxylated polystyrene (PS) nanoparticles. PS nanoparticles with a smaller size were endocytosed mainly through clathrin-dependent pathway while PS nanoparticles with a larger size preferred caveolaemediated endocytosis. Furthermore, our results revealed exocytosis of larger PS nanoparticles and tracked the dynamic process at single particle level. These results indicated that particle size is a key factor for the regulation of intracellular trafficking of nanoparticles and provide new insight into the development of more effective cellular nanocarriers.

Keywords: nanoparticles, imaging, exocytosis, endocytosis, intracellular trafficking

Introduction Nanoparticles (NPs) play essential role in the rapidly developing field of nanomedicine.1-4 For example, various kinds of fluorescent NPs have been widely used in biological imaging, ranging from fixed cells to living cells and model organisms.5-9 NPs also provide new tools for drug delivery and gene therapy.10-12 Ideally, NPs in such applications should efficiently enter target cells and leave after fulfilling their function to avoid adverse health effects.13-15 Therefore, it is necessary to understand the mechanisms by which NPs are internalized by cells, as well as their trafficking routes and final destinations.16-18 Cellular uptake of various types of NPs has been extensively studied, revealing active endocytosis processes and pathways involved in their internalization.19-21 Almost all cells can internalize NPs by pinocytosis. 22-23 Four basic pinocytic


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mechanisms are currently known, macropinocytosis, clathrin-mediated endocytosis, caveolaemediated endocytosis and mechanisms independent of clathrin and caveolin.24 Endocytosis of NPs can be regulated by their physical and chemical properties including the size and shape of NPs, surface charge and modifications.25-27 In addition, cell type and biomolecules in the environment were also identified as strongly modulating the cellular uptake efficiency.28-29 Following endocytosis, majority of the NPs entered the endosomes and was transported into lysosomes.30-31 In contrast to endocytosis, investigations regarding exocytosis of NPs are rather rare.32-33 In most studies, exocytosis was assumed to be negligible.34-35 In fact, exocytosis functions as an important mechanism to reduce intracellular accumulation of unwanted NPs.36 A detailed understanding of exocytosis can greatly assist in the engineering of NPs that leave cells to avoid adverse effects.37-38 In the present study, we investigated processes of both endocytosis and exocytosis. Carboxylated polystyrene (PS) nanoparticles with fluorescence were chosen as a type of representative NPs, which have found widespread application in cell imaging and drug delivery for their good biocompatibility, ease of synthesis and high stability.39 It has been proposed that particle size played a critical role in dynamic interaction between nano-bio interfaces. Therefore we focused on the size-dependent regulation and chose PS nanoparticles of 40 nm and 150 nm diameters for comparison of their trafficking behaviors. The results indicated that 40 nm PS nanoparticles were mainly internalized through clathrin-mediated endocytosis pathway, whereas the uptake of 150 nm PS nanoparticles relied on the caveolae-mediated endocytosis. More importantly, we detected significantly more colocalization of 150 nm PS nanoparticles with exosomes than 40 nm PS nanoparticles. We also observed the exocytosis process of 150 nm NPs with single particle tracking analysis. These results demonstrate that


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particle size is important for the regulation of cellular trafficking of NPs and should be exploited in design of functional nanomaterials.

Results and Discussion Distinct endocytic mechanisms for PS nanoparticles with different sizes Carboxylate-modified polystyrene (PS-COOH) NPs have proven to enter cells efficiently.40-41 In this study, carboxylated PS nanoparticles (green fluorescence) with diameters of 40 nm and 150 nm were chosen as model NPs to investigate whether the process of endocytosis was sizedependent. Both PS nanoparticles were well-dispersed in Phosphate Buffered Saline (PBS) and complete MEM medium supplemented with 10% FBS. They also exhibited similar zeta potential, inferring same surface modification. Dynamic light scattering (DLS) and transmission electron microscope (TEM) analysis verified particle sizes (Figure S1 in the Supporting Information). PS nanoparticles of different sizes were incubated with HeLa cells for 18 hours. Cells were then imaged with confocal microscopy to detect intracellular fluorescent signals. To elucidate which endocytosis mechanism was responsible for cellular uptake of PS nanoparticles, cells were pre-incubated with selective pharmaceutical inhibitors targeting distinct endocytic pathways. Methyl-β-cyclodextrin (MβCD) could disrupt the structure and function of lipid raft by cholesterol depletion and was utilized to block caveolae-mediated endocytosis. Chlorpromazine (CPZ) was applied to block the formation of clathrin-coated vesicles.42 Compared to untreated cells, pretreatment of HeLa cells with CPZ significantly decreased intracellular signals of 40 nm PS nanoparticles whereas it only slightly reduced the accumulation of 150 nm PS nanoparticles. To the contrary, pretreating cells with MβCD resulted in a strong


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reduction of internalized 150 nm PS nanoparticles, while hardly inhibiting the uptake of 40 nm PS nanoparticles (Figure 1a). Subsequently, the mean intracellular fluorescence intensities of HeLa cells were measured using flow cytometry to determine the amount of internalized NPs. In consistent with imaging observations, MβCD pretreatment reduced the internalization of 150 nm PS nanoparticles to 33% and CPZ blocked more than 88% of 40 nm PS nanoparticles uptake (Figure 1c, and Figure S2 in the Supporting Information). Importantly, the effects of both inhibitors were quite selective. These results suggested that endocytosis of PS nanoparticles was size-dependent. Caveolae-mediated endocytosis was the major pathway for 40 nm PS nanoparticles, while 150 nm PS nanoparticles were internalized predominantly via clathrinmediated endocytosis. One limitation of chemical inhibitors is that they often resulted in multi-targeted regulation of cellular pathways. Variations of drug concentration applied in individual experiment could also result in inconsistent observations.41, 43 To rule out this possibility, we verified the dependence of selective endocytosis pathways by means of gene regulation. Small interfering RNAs (siRNA) specifically targeting caveolin-1 or clathrin were utilized to knock down the expression of key proteins in each endocytic pathway, respectively (Figure S3 in the Supporting Information). As shown in Figure 1b, knockdown of the clathrin-1 heavy chain by CHC-1 siRNA strikingly blocked internalization of 40 nm PS nanoparticles, which was very similar to the effect of CPZ. The internalization of 150 nm PS nanoparticles was decreased to 85% by CHC-1 siRNA, while it was reduced to 24% by CAV-1siRNA treatment (Figure1d, and Figure S4 in the Supporting Information), confirming that uptake of 150 nm PS nanoparticles was caveolae-dependent. To further confirm the size-dependent regulation of cellular internalization, we performed selective inhibition of endocytic pathway in MCF-7 breast cancer cells and examined intracellular


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fluorescent signals. Very similar to the effects in HeLa cells, uptake of 40 nm PS was mostly inhibited by CPZ, while the uptake of 150 nm PS was blocked by MβCD in MCF-7 cells (Figure S5 in the Supporting Information). Taken together, these data demonstrated that particle size played pivotal role in regulation of cellular uptake of NPs. Clearly size is not the only parameter that regulates endocytic process of NPs; cationic or non-ionic particles may have different mechanism to enter cells. For PS nanoparticles with anionic surface modification, caveolaemediated and clathrin-mediated pathways are responsible for internalization of large or small particles, respectively. Therefore, it is possible to achieve selective cellular targeting and delivery through controlling size of NPs. Dynamics of intracellular trafficking of PS nanoparticles Following internalization, NPs normally enter early endosomes, which function as sorting compartments to further destinations of recycling, degradation or exocytosis.44 Since we have observed that PS nanoparticles with different sizes enter cells through different routes, we then inspected the kinetics of colocalization between early endosomes and NPs. To mark early endosomes in living cells, a DNA construct encoding red fluorescent protein (RFP)-fused Rab5 was transfected into HeLa cells.45 The small GTPase Rab5 is a key component and commonly used as a protein marker of early endosomes.30 Cells expressing RFP-Rab5 were incubated with 40 nm or 150 nm NPs and imaged at 1, 2, 4, 6, 8, 18 and 24 h, respectively. And the colocalization between RFP-Rab5 and PS nanoparticles with different sizes were compared. Clear colocalization between 40 nm NPs and RFP-Rab5 was observed after 1-hour incubation (Figure 2a). The ratio of 40 nm NPs that localized to early endosome reached a peak value of 27% at 4 h and gradually declined afterwards. In contrast, intracellular accumulation of 150 nm NPs was slower compared to their smaller counterparts. Colocalization was detectable after 4-


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hour incubation and peaked around 8 hour, with a ratio of 17% (Figure 2b). These experiments revealed that endocytosis of 40 nm PS nanoparticles via clathrin-mediated pathway were relatively faster than caveolae-mediated uptake of 150 nm PS nanoparticles. Nevertheless, NPs internalized through both routes entered early endosomes. Having compared the endocytic mechanism and kinetics of NPs with different sizes, we then extended our analysis to their duration of stay within cells. HeLa cells were incubated with two types of PS nanoparticles and total mounts of intracellular NPs were examined using confocal microscope and quantified with flow cytometry at various time points. As shown in Figure 3a, at the earlier stage of cellular uptake (1 hour), both NPs were distributed surrounding cell membrane. 40 nm PS nanoparticles started to be internalized by cells. 150 nm PS-NPs, on the contrary, were hardly detectable within cells. For 40 nm NPs, fluorescence intensity increased with prolonged incubation time (Figure 3b & Figure S6 in the Supporting Information). Fluorescent signals were scattered in the cytoplasm after 4-hour incubation and aggregation dots were observed near the nucleus at 24 hours, indicating dynamic transportation and vesicle fusion during intracellular trafficking process. Unlike 40 nm NPs-treated cells, fluorescence intensity peaked at 4 hours in cells incubated with 150 nm PS nanoparticles, followed by a significant decrease at 8 hours (Figure 3c & Figure S7 in the Supporting Information). Since PS nanoparticles have proven to be stable in cellular environment, it is unlikely that the reduction was caused by degradation of NPs. Therefore, we postulated that this decrease might be caused by excretion of 150 nm NPs out of cells. Exocytosis of 150 nm PS nanoparticles Next we tested the hypothesis that 150 nm PS nanoparticles could leave cells through exocytosis.


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Basically, exocytosis is the reverse of endocytosis. As an important form of exocytosis, vesicles called exosomes were transported to the plasma membrane and "secreted" to the extracellular space. It is an important way for cells to release bio-active molecules including hormones, neurotransmitters and microRNAs.36 Whether exogenous particles can escape in this fashion is still under investigation. To label exosomes, a DNA construct encoding RFP-CD9 was transfected into HeLa cells. CD9 is an established protein marker of exosomes.46 Cells expressing RFP-CD9 were incubated with 40 nm or 150 nm NPs and imaged at 4 and 24 hours, respectively (Figure 4a). Colocalization between 40 nm NPs and RFP-CD9 was hardly detectable at 4 hours (5%), which increased to 22% at 24 hours. Significantly, the ratio of colocalization between 150 nm NPs and RFP-CD9 was much higher at both 4 hours (49%) and 24 hours (68%) (Figure 4b). The perfect colocalization between 150 nm PS nanoparticles and CD9 (Figure 4c) suggested an active exocytotic process. Furthermore, we validated this phenomenon in MCF-7 cells. As shown in Figure S8, the colocalization ratio between RFP-CD9 with 150 nm PS was much higher than 40 nm PS. We then traced the dynamic movement of 150 nm PS particles that colocalized with CD9 via live cell imaging. Time-lapse movie was acquired using confocal microscope (Figure 5a; Video S1 ∆t = 3s, total time = 465s). Indeed, we found that some of the colocalized signals of RFP-CD9 and 150 nm NPs moved quickly in the cytoplasm. Trajectories of a representative particle (indicated by a blue circle) that moved towards the edge of cell were shown in Figure 5b. Taken together, these data indicated that 150 nm PS nanoparticles were more likely to be exported out of the cells than their smaller counterparts and the exocytosis of NPs was clearly related with their diameters. This sizedependent regulation of exocytosis can be exploited in the future design of nanovehicles that leave cells after unloading of their cargos.


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Conclusion While nanoparticles have been widely utilized in a variety of biomedical applications, specific mechanisms of their interaction with living cells still need to be investigated in depth. In this study we explored the endocytosis and exocytosis of two types of polystyrene NPs with identical properties, except for particle size. Studies with selective inhibitory drug and siRNAs revealed that uptake of PS nanoparticles were controlled by their diameters. 40 nm PS nanoparticles was internalized mainly through clathrin-mediated endocytosis pathway, whereas the uptake of 150 nm PS nanoparticles proceeded predominantly via caveolae-mediated endocytosis. Our results revealed details of distinct receptor-mediated interaction between cells and NPs with different sizes, which provided new thought for the design of drug carriers targeting selective receptors on the cell membrane. We also investigated the behavior of HeLa cells to exocytose PS nanoparticles. In

contrast to smaller NPs, which accumulated intracellularly after internalization, larger PS nanoparticles were targeted to exosomes and transported towards the cell membrane, indicating that they were exocytosed by the cell. The observed selective extracellular exportation of larger NPs implied that cellular retention of drug carriers might be regulated by particle size. These new findings not only shed light on the interaction between cells and NPs, but will assist in the engineering of NPs with improved selectivity and safety.

Experimental Section Materials Methyl-β-cyclodextrin (MβCD), chlorpromazine (CPZ) and Hoechst 33258 were purchased from


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Sigma-Aldrich. HeLa cells and MCF-7 cells were purchased from Cell Bank, Shanghai Institutes for Biological Sciences, CAS. Minimum Essential Media (MEM), RPMI 1640 Media and fetal bovine serum (FBS) were from Gibco, Invitrogen. Streptomycin, Penicillin were purchased from Beyotime Biotechnology. mRFP-Rab5 was a gift from Ari Helenius (Addgene plasmid # 14437). mRFP-CD9 was a gift from Michael Davidson (Addgene plasmid # 55013). Characterization of nanoparticles Fluorescent (Excitation/Emission wavelengths: 505/515) carboxylated polystyrene nanoparticles were purchased from Invitrogen (40 nm) and Hugebiotech, Shanghai (150 nm) and used without further modification. Before experiments, particle suspensions were dispersed by sonication. The mean hydrodynamic diameter and zeta potential of NPs were measured using a Zetasizer (nano ZS90, Malvern Instruments). Mean hydrodynamic diameter was determined by number distribution. Solution containing NPs was dropped onto carbon coated copper grids to evaporate excess solvent, and examined with TEM (Jeol 2010, 200KV). Endocytosis determination HeLa cells and MCF-7 cells were grown in complete MEM or RPMI 1640 media supplemented with 10% heat inactivated FBS, 100 U/ml penicillin, 100 mg/ml streptomycin, and 2 mM Lglutamine at 37℃ in the humidified atmosphere with 5% CO2. Cells were plated and cultured overnight before uptake experiments. For inhibitory study, cells were pre-treated with the CPZ (3 µg/ml) or MβCD (5 mg/ml) for 30 min, respectively. Cells were then incubated with media contain both inhibitor and NPs. After incubation for desired time, media was removed and cells were washed three times with PBS, fixed with 4% paraformaldehyde (PFA) for 30 min. The nucleus was stained with Hoechst 33258. Images were obtained using a Leica SP8 confocal


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microscope. Alternatively, mean fluorescence intensities were analyzed by a flow cytometry (FACS Calibur, BD Biosciences). At least 5000 cells were counted for each sample and experiments were performed in triplicates. Cellular trafficking studies Typically, HeLa cells (1×106) were transfected with 1µg of the different expression plasmids (RFP-Rab5, RFP-CD9) 24 h before incubating 0.1 mM 40 nm PS-NPs or 150 nm PS-NPs for indicated time courses using the Amaxa electroporation system according to the manufacturer’s instructions. Following fixation and nucleus staining (see above), cells were imaged by confocal microscopy (Leica SP8). siRNA transfection siRNAs were synthesized by Shanghai GenePharma Biotechnology. Sequences used in the experiments were shown in Table 1. Transfection of siRNA was carried out using an electroporation system (Amaxa Nucleofector 2b, Lonza) following to the manufacturer’s instruction. Table 1. Sequences of siRNA used in knock-down experiments. Clathrin Heavy Chain siRNA Caveolin-1 siRNA Negative siRNA

Sense strand 5′-GCAGAAGAAUCAACGUUAUTT-3′ Antisense strand 5′-TTAUAACGUUGAUUCUUCAGC-3′ Sense strand 5′-GCAUCAACUUGCAGAAAGATT-3′ Antisense strand 5′-GCAUCAACUUGCAGAAAGATT-3′ Sense strand 5′-ATGTCTGUGTTAUGGCATCUUTT-3′ Antisense strand 5′-ATGTCTGUGTTAUGGCATCUU-3′

Live cell imaging


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HeLa cells were transfected with plasmids encoding mRFP-CD9 using Lipofectamine 3000 reagent (Invitrogen). After transfection for 24 h, cells were incubated with 0.1 mM PS. At indicated time points, images were acquired using a laser confocal microscope (Leica TCS SP8, Germany) equipped with a live cell incubator and collected with a HC×PL APO 63×, 1.4 NA oilimmersion objective. RFP were excited with a 561 nm helium-neon laser and PS nanoparticles were excited with a 488 nm Ar-Kr laser. Image analysis Fluorescence images were analyzed using ImageJ software (US National Institutes of Health). To quantify the co-localization ratio of two fluorescent signals, tMr values (the thresholded Mander's coefficients) indicating the percentage of green signals colocalized with red signals in merged images was calculated. Values represent mean ± SE based on analysis of randomly selected 20 cells. For single particle tracking, the trajectories of red signals and green signals were built by pairing spots in each frame using single-particle tracking plug-in of ImageJ. Supporting Information Figures S1-S8: Characterization of PS nanoparticles, original flow cytometry charts of intracellular fluorescence of HeLa cells, knockdown of expression levels of caveolin-1 or clathrin heavy chain-1 by RNAi, comparison of endocytosis and exocytosis of 40 nm and 150 nm PS nanoparticles in MCF-7 cells. Video S1: Tracking of a representative particle containing colocalized CD9-RFP and 150 nm PS in live cell (AVI).


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AUTHOR INFORMATION Corresponding Author *[email protected] *[email protected] Author Contributions ‡These authors contributed equally. ACKNOWLEDGMENT This work was supported by National Natural Science Foundation of China (31470970, 21505148 and 21373260), The National Basic Research Program of China (2016YFA0201200 and 2013CB932800) and the Youth Innovation Promotion Association from Chinese Academy of Sciences (2015211) and the Key Research Program of Frontier Sciences, CAS (QYZDJ-SSWSLH019). Ali Aldalbahi would like to extend his sincere appreciation to the Deanship of Scientific Research at King Saud University for funding this work (RG-1436-005). REFERENCES (1) Karimi, M.; Ghasemi, A.; Sahandi Zangabad, P.; Rahighi, R.; Moosavi Basri, S.M.; Mirshekari, H.; Amiri, M.; Shafaei Pishabad, Z.; Aslani, A.; Bozorgomid, M.; Ghosh, D.; Beyzavi, A.; Vaseghi, A.; Aref, A.R.; Haghani, L.; Bahrami, S.; Hamblin, M.R., Smart Micro/Nanoparticles in Stimulus-Responsive Drug/Gene Delivery Systems. Chem. Soc. Rev. 2016, 45, 1457-1501. (2) Ding, H.; Yang, D.; Zhao, C.; Song, Z.; Liu, P.; Wang, Y.; Chen, Z.; Shen, J., Protein-Gold Hybrid Nanocubes for Cell Imaging and Drug Delivery. ACS Appl. Mater. Interfaces 2015, 7, 4713-4719. (3) Faklaris, O.; Joshi, V.; Irinopoulou, T.; Tauc, P.; Sennour, M.; Girard, H.; Gesset, C.; Arnault,


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J.C.; Thorel, A.; Boudou, J.P.; Curmi, P.A.; Treussart, F., Photoluminescent Diamond Nanoparticles for Cell Labeling: Study of the Uptake Mechanism in Mammalian Cells. ACS nano 2009, 3, 3955-3962. (4) Cui, X.H.; Chen, H.Y.; Yang, T., Research Progress on the Preparation and Application of Nano-Sized Molybdenum Disulfide. Acta Chim. Sin. (Engl. Ed.) 2016, 74, 392-400. (5) Zhang, H.; Peng, C.; Yang, J.; Lv, M.; Liu, R.; He, D.; Fan, C.; Huang, Q., Uniform Ultrasmall Graphene Oxide Nanosheets with Low Cytotoxicity and High Cellular Uptake. ACS Appl. Mater. Interfaces 2013, 5, 1761-1767. (6) Sykes, E.A.; Dai, Q.; Sarsons, C.D.; Chen, J.; Rocheleau, J.V.; Hwang, D.M.; Zheng, G.; Cramb, D.T.; Rinker, K.D.; Chan, W.C., Tailoring Nanoparticle Designs to Target Cancer Based on Tumor Pathophysiology. Proc. Natl. Acad. Sci. U.S.A. 2016, 113, 1142-1151. (7) Nishimura, H.; Ritchie, K.; Kasai, R.S.; Goto, M.; Morone, N.; Sugimura, H.; Tanaka, K.; Sase, I.; Yoshimura, A.; Nakano, Y.; Fujiwara, T.K.; Kusumi, A., Biocompatible Fluorescent Silicon Nanocrystals for Single-Molecule Tracking and Fluorescence Imaging. J. Cell Biol. 2013, 202, 967-983. (8) Feng, X.; Lv, F.; Liu, L.; Tang, H.; Xing, C.; Yang, Q.; Wang, S., Conjugated Polymer Nanoparticles for Drug Delivery and Imaging. ACS Appl. Mater. Interfaces 2010, 2, 2429-2435. (9) Wang, S.P.; Deng, S.H.; Cai, X.Q.; Hou, S.G.; Li, J.J.; Gao, Z.S.; Li, J.; Wang, L.H.; Fan, C.H., Superresolution Imaging of Telomeres with Continuous Wave Stimulated Emission Depletion (STED) Microscope. Sci. China Chem. 2016, 59, 1519-1524. (10) Yi, H.; Wang, Z.; Li, X.; Yin, M.; Wang, L.; Aldalbahi, A.; El-Sayed, N.N.; Wang, H.; Chen, N.; Fan, C.; Song, H., Silica Nanoparticles Target a Wnt Signal Transducer for Degradation and Impair Embryonic Development in Zebrafish. Theranostics 2016, 6, 1810-1820. (11) Huo, S.; Jin, S.; Ma, X.; Xue, X.; Yang, K.; Kumar, A.; Wang, P.C.; Zhang, J.; Hu, Z.; Liang, X.J., Ultrasmall Gold Nanoparticles as Carriers for Nucleus-Based Gene Therapy Due to SizeDependent Nuclear Entry. ACS Nano 2014, 8, 5852-5862. (12) Sun, J.; Chao, J.; Huang, J.; Yin, M.; Zhang, H.; Peng, C.; Zhong, Z.; Chen, N., Uniform Small Graphene Oxide as an Efficient Cellular Nanocarrier for Immunostimulatory CpG Oligonucleotides. ACS Appl. Mater. Interfaces 2014, 6, 7926-7932. (13) Zarschler, K.; Rocks, L.; Licciardello, N.; Boselli, L.; Polo, E.; Garcia, K.P.; De Cola, L.; Stephan, H.; Dawson, K.A., Ultrasmall Inorganic Nanoparticles: State-of-the-Art and


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Perspectives for Biomedical Applications. Nanomedicine 2016, 12, 1663-1701. (14) Chen, N.; Wang, H.; Huang, Q.; Li, J.; Yan, J.; He, D.; Fan, C.; Song, H., Long-Term Effects of Nanoparticles on Nutrition and Metabolism. Small 2014, 10, 3603-3611. (15) Xu, H.; Gao, S.; Yang, Q.; Pan, D.; Wang, L.; Fan, C., Amplified Fluorescent Recognition of G-Quadruplex Folding with a Cationic Conjugated Polymer and DNA Intercalator. ACS Appl. Mater. Interfaces 2010, 2, 3211-3216. (16) Mu, Q.; Jiang, G.; Chen, L.; Zhou, H.; Fourches, D.; Tropsha, A.; Yan, B., Chemical Basis of Interactions between Engineered Nanoparticles and Biological Systems. Chem. Rev. 2014, 114, 7740-7781. (17) Chen, N.; Li, J.; Song, H.; Chao, J.; Huang, Q.; Fan, C., Physical and Biochemical Insights on DNA Structures in Artificial and Living Systems. Acc. Chem. Res. 2014, 47, 1720-1730. (18) Treuel, L.; Jiang, X.; Nienhaus, G.U., New Views on Cellular Uptake and Trafficking of Manufactured Nanoparticles. J. R. Soc. Interface 2013, 10, 20120939. (19) Vranic, S.; Boggetto, N.; Contremoulins, V.; Mornet, S.; Reinhardt, N.; Marano, F.; BaezaSquiban, A.; Boland, S., Deciphering the Mechanisms of Cellular Uptake of Engineered Nanoparticles by Accurate Evaluation of Internalization Using Imaging Flow Cytometry. Part. Fibre. Toxicol. 2013, 10, 2. (20) Lesniak, A.; Salvati, A.; Santos-Martinez, M.J.; Radomski, M.W.; Dawson, K.A.; Aberg, C., Nanoparticle Adhesion to the Cell Membrane and Its Effect on Nanoparticle Uptake Efficiency. J. Am. Chem. Soc. 2013, 135, 1438-1444. (21) Canton, I.; Battaglia, G., Endocytosis at the Nanoscale. Chem. Soc. Rev. 2012, 41, 27182739. (22) Linares, J.; Concepcion Matesanz, M.; Vila, M.; Jose Feito, M.; Goncalves, G.; Vallet-Regi, M.; Marques, P.A.A.P.; Teresa Portoles, M., Endocytic Mechanisms of Graphene Oxide Nanosheets in Osteoblasts, Hepatocytes and Macrophages. ACS Appl. Mater. Interfaces 2014, 6, 13697-13706. (23) Zhang, W.; Ji, Y.; Wu, X.; Xu, H., Trafficking of Gold Nanorods in Breast Cancer Cells: Uptake, Lysosome Maturation, and Elimination. ACS Appl. Mater. Interfaces 2013, 5, 98569865. (24) Kumari, S.; Mg, S.; Mayor, S., Endocytosis Unplugged: Multiple Ways to Enter the Cell. Cell Res. 2010, 20, 256-275.


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(25) Voigt, J.; Christensen, J.; Shastri, V.P., Differential Uptake of Nanoparticles by Endothelial Cells through Polyelectrolytes with Affinity for Caveolae. Proc. Natl. Acad. Sci. U.S.A. 2014, 111, 2942-2947. (26) Salvati, A.; Pitek, A.S.; Monopoli, M.P.; Prapainop, K.; Bombelli, F.B.; Hristov, D.R.; Kelly, P.M.; Aberg, C.; Mahon, E.; Dawson, K.A., Transferrin-Functionalized Nanoparticles Lose Their Targeting Capabilities When a Biomolecule Corona Adsorbs on the Surface. Nat. Nanotechnol. 2013, 8, 137-143. (27) Choi, C.H.; Hao, L.; Narayan, S.P.; Auyeung, E.; Mirkin, C.A., Mechanism for the Endocytosis of Spherical Nucleic Acid Nanoparticle Conjugates. Proc. Natl. Acad. Sci. U.S.A. 2013, 110, 7625-7630. (28) Walkey, C.D.; Chan, W.C., Understanding and Controlling the Interaction of Nanomaterials with Proteins in a Physiological Environment. Chem. Soc. Rev. 2012, 41, 2780-2799. (29) Monopoli, M.P.; Aberg, C.; Salvati, A.; Dawson, K.A., Biomolecular Coronas Provide the Biological Identity of Nanosized Materials. Nat. Nanotechnol. 2012, 7, 779-786. (30) Sandin, P.; Fitzpatrick, L.W.; Simpson, J.C.; Dawson, K.A., High-Speed Imaging of Rab Family Small GTPases Reveals Rare Events in Nanoparticle Trafficking in Living Cells. ACS nano 2012, 6, 1513-1521. (31) Liang, L.; Li, J.; Li, Q.; Huang, Q.; Shi, J.; Yan, H.; Fan, C., Single-Particle Tracking and Modulation of Cell Entry Pathways of a Tetrahedral DNA Nanostructure in Live Cells. Angew. Chem. Int. Ed. Engl. 2014, 53, 7745-7750. (32) Jiang, X.; Rocker, C.; Hafner, M.; Brandholt, S.; Dorlich, R.M.; Nienhaus, G.U., Endo- and Exocytosis of Zwitterionic Quantum Dot Nanoparticles by Live HeLa Cells. ACS nano 2010, 4, 6787-6797. (33) Strobel, C.; Oehring, H.; Herrmann, R.; Forster, M.; Reller, A.; Hilger, I., Fate of Cerium Dioxide Nanoparticles in Endothelial Cells: Exocytosis. J. Nanopart. Res. 2015, 17, 206. (34) Tree-Udom, T.; Seemork, J.; Shyou, K.; Hamada, T.; Sangphech, N.; Palaga, T.; Insin, N.; Pan-In, P.; Wanichwecharungruang, S., Shape Effect on Particle-Lipid Bilayer Membrane Association, Cellular Uptake, and Cytotoxicity. ACS Appl. Mater. Interfaces 2015, 7, 2399324000. (35) Zhang, Y.; Tekobo, S.; Tu, Y.; Zhou, Q.; Jin, X.; Dergunov, S.A.; Pinkhassik, E.; Yan, B., Permission to Enter Cell by Shape: Nanodisk vs Nanosphere. ACS Appl. Mater. Interfaces 2012,


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4, 4099-4105. (36) Li, W.H.; Li, D., Fluorescent Probes for Monitoring Regulated Secretion. Curr. Opin. Chem. Biol. 2013, 17, 672-681. (37) Kah, J.C.; Yeo, E.L.; Koh, W.L.; Poinard, B.E.; Neo, D.J., Nanoparticle Interface to Biology: Applications in Probing and Modulating Biological Processes. Crit. Rev. Bioeng. 2013, 41, 323-341. (38) Cheng, R.; Meng, F.; Deng, C.; Klok, H.A.; Zhong, Z., Dual and Multi-Stimuli Responsive Polymeric Nanoparticles for Programmed Site-Specific Drug Delivery. Biomaterials 2013, 34, 3647-3657. (39) Frohlich, E., Cellular Targets and Mechanisms in the Cytotoxic Action of NonBiodegradable Engineered Nanoparticles. Curr. Drug Metab. 2013, 14, 976-988. (40) Varela, J.A.; Bexiga, M.G.; Aberg, C.; Simpson, J.C.; Dawson, K.A., Quantifying SizeDependent Interactions between Fluorescently Labeled Polystyrene Nanoparticles and Mammalian Cells. J. Nanobiotechnol 2012, 10, 39. (41) Jiang, X.; Musyanovych, A.; Rocker, C.; Landfester, K.; Mailander, V.; Nienhaus, G.U., Specific Effects of Surface Carboxyl Groups on Anionic Polystyrene Particles in Their Interactions with Mesenchymal Stem Cells. Nanoscale 2011, 3, 2028-2035. (42) Dos Santos, T.; Varela, J.; Lynch, I.; Salvati, A.; Dawson, K.A., Effects of Transport Inhibitors on the Cellular Uptake of Carboxylated Polystyrene Nanoparticles in Different Cell Lines. PloS one 2011, 6, e24438. (43) Ekkapongpisit, M.; Giovia, A.; Follo, C.; Caputo, G.; Isidoro, C., Biocompatibility, Endocytosis, and Intracellular Trafficking of Mesoporous Silica and Polystyrene Nanoparticles in Ovarian Cancer Cells: Effects of Size and Surface Charge Groups. Int. J. Nanomed 2012, 7, 4147-4158. (44) Conner, S.D.; Schmid, S.L., Regulated Portals of Entry into the Cell. Nature 2003, 422, 3744. (45) Vonderheit, A.; Helenius, A., Rab7 Associates with Early Endosomes to Mediate Sorting and Transport of Semliki Forest Virus to Late Endosomes. PLoS Biol. 2005, 3, e233. (46) Mazurov, D.; Barbashova, L.; Filatov, A., Tetraspanin Protein CD9 Interacts with Metalloprotease CD10 and Enhances Its Release Via Exosomes. FEBS J. 2013, 280, 1200-1213.


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Figure 1. Endocytosis pathways of PS nanoparticles with different sizes. (a) HeLa cells were incubated with 0.1 mM PS nanoparticles (40 nm or 150 nm) for 18 h in the presence or absence of selective inhibitor of either endocytosis pathways. (b) HeLa cells transfected with siRNA targeting caveolin-1 or clathrin were incubated with 0.1 mM PS nanoparticles (40 nm or 150 nm) for 18 h. Internalized NPs were imaged by confocal microscope. Scale bar: 30 µm. (c) Amounts of internalized NPs in (a) were quantified using flow cytometry and normalized to cells without inhibitors. (d) Amounts of internalized NPs in (b) were quantified using flow cytometry and normalized to cells transfected with negative siRNAs. In (c) and (d), fluorescence intensity of un-treated cells was normalized to 100%. Error bars represent standard deviation (SD) of three independent experiments.


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Figure 2. Kinetics of colocalization between early endosomes and PS nanoparticles. (a) HeLa cells expressing RFP-Rab5 (red) were incubated with NPs (40 nm and 150 nm) and imaged by confocal microscope at indicated time points. (b) Percentages of PS nanoparticles (green) colocalized with the early endosome marker Rab5 (red) were quantified using the ImageJ software. (20 cells analyzed). Scare bar: 10 µm


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Figure 3. Dynamics of accumulation of 40 nm and 150 nm PS nanoparticles in HeLa cells. (a) Confocal images of HeLa cells incubated with PS nanoparticles for indicated time (1 h, 4 h and 24 h), the upper panel represents 40 nm NPs and the lower panel represents 150 nm NPs. Scale bar: 30 µm. (b) & (c) Fluorescence of internalized PS nanoparticles were quantified using flow cytometry analysis.


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Figure 4. 150 nm PS nanoparticles colocalized with CD9. (a) HeLa cells expressing RFP-CD9 were incubated with NPs (40 nm and 150 nm) and imaged by confocal microscope at indicated time points. (b) Percentages of PS nanoparticles (green) colocalized with CD9 (red) were quantified using the ImageJ software. (c) The colocalization between CD9 and PS nanoparticles were analyzed by line profiling fluorescence intensity of RFP-CD9 (red) and PS (green) along the line selected in (a). Scale bar: 10 µm.


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Figure 5. Single particle tracking of 150 nm PS nanoparticles in live cells. (a) Snapshots of the start (0 s) and end (465 s) point in live cell imaging of HeLa cells expressing RFP-CD9 (red) with internalized 150 nm PS nanoparticles (green). Scare bar: 10 µm. (b) Enlarged view of the square region in (a) showed the trajectory of a representative particle (blue circle) containing colocalized CD9 and PS at different time points (one frame per 3 s, with a total of 465 s; also see Video S1). Scare bar: 10 µm.


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Size-Dependent Regulation of Intracellular Trafficking of Polystyrene

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