Real-Time Imaging of Endocytosis and Intracellular Trafficking of

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Real-time imaging of endocytosis and intracellular trafficking of semiconducting polymer dots Yuping Han, Xiaoming Li, Haobin Chen, Xingjie Hu, Yao Luo, Ting Wang, Zejun Wang, Qian Li, Chunhai Fan, Jiye Shi, Lihua Wang, Yun Zhao, Changfeng Wu, and Nan Chen ACS Appl. Mater. Interfaces, Just Accepted Manuscript • Publication Date (Web): 06 Jun 2017 Downloaded from http://pubs.acs.org on June 7, 2017

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ACS Applied Materials & Interfaces

Real-time Imaging of Endocytosis and Intracellular Trafficking of Semiconducting Polymer Dots ⊥

Yuping Han†, ‡, Xiaoming Li§, ‡, Haobin Chen , Xingjie Hu#, Yao Luo†, #, Ting Wang†, #, ‡

Zejun Wang#, Qian Li#, Chunhai Fan#,§, Jiye Shi#, , Lihua Wang#, Yun Zhao†,*, Changfeng Wu⊥,*, 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 ⊥

Department of Biomedical Engineering, Southern University of Science and

Technology, Shenzhen, Guangdong 518055, China §

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

China ‡

UCB Pharma, 208 Bath Road, Slough, SL1 3WE, UK

KEYWORDS: Fluorescent polymer dots, Endocytosis, Intracellular trafficking, Theranostic nanoparticles, Live cell imaging

ACS Paragon Plus Environment


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ABSTRACT

Semiconducting polymer dots (Pdots) have shown great promise in biomedical applications, including biosensing, drug delivery and live imaging of cells and bio-molecules. Insight into the mechanism and regulation of cellular uptake and intracellular metabolism of Pdots is important for the development of superior Pdots-based theranostic nano-conjugates. Herein, we performed real-time imaging of endocytosis and intracellular trafficking of a type of fluorescent Pdots that showed excellent biocompatibility in various types of cells. The endocytic routes and kinetics of Pdots were differently regulated in distinct cell types. Following endocytosis, Pdots were transported in vesicles along microtubule and destined for lysosomes. Furthermore, our results revealed exosome-mediated extracellular release of Pdots and have tracked the dynamic process at single particle level. These results provide new insight into the design of more effective and selective imaging probes as well as drug carriers.

Introduction

Fluorescent imaging techniques play increasing role in biological study and provide detailed information on multiple cellular processes ranging from gene regulation to protein interactions and signaling transduction.1 Fluorescent proteins (FPs) serve as powerful tools to observing the behavior of bio-molecules and tracking cell fate within the living organisms.2-3 This technique requires genetic encoding of fusion protein with a fluorescent tag. Moreover, the brightness and photostability of natural


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fluorescent proteins are not satisfactory for applications in long-term observation and single particle tracking.4 To overcome these limitations, various fluorescent nanoparticles were developed and applied in bioanalysis and bioimaging.5-10 For example, semiconductor quantum dots (Qdots) exhibit broad absorption bands and narrow emissions; Qdots also have superior fluorescence brightness and photostability compared to FPs.11-13 Despite their effective performance in biological applications, heavy metal (e.g. Cadmium) in the composition of Qdots caused serious concerns about toxicity and prevented their further application.14-15 In response to this challenge, researchers explored alternative materials for the synthesis of fluorescent NPs with less risk of bio-toxicity.4, 16 Among these NPs, fluorescent semiconducting polymer dots have attracted much interest for their outstanding optical properties and encouraging results in biosensing and cell imaging.17-19 These organic nanoparticles possess good photon budget, photostability, and biocompatibility.20-21 Pdots have been utilized for cell labelling, immunolabeling of proteins via conjugated antibodies, bioorthogonal labeling of glycoproteins on the cell membrane, and single particle tracking.21-22 Moreover, Pdots with improved property of photoblinking have also shown the potential as fluorescent probes for super-resolution cell imaging.23-25

In addition to bioimaging, Pdots have also been explored for drug and gene delivery. Hydrophobic conjugated polymer nanoparticles provide an ideal system for encapsulating therapeutic agents that have poor solubility in water.26 Hydrophilic Pdots have been utilized to form stable complexes with plasmid or small interfering RNA (siRNA) and modulate gene expression in target cells.27 Importantly, the



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intrinsic fluorescence of Pdots provides unparalleled advantage for monitoring the targeting efficiency of cargo-loaded NPs and simultaneously tracking the release of drugs. These multifunctional Pdots-based nanocomplexes have shown great potential in theranostics of diseases.28

Despite the promising prospects of Pdots in biomedical applications, current knowledge about the mechanism and regulation of cellular uptake and intracellular destination of Pdots is limited. The processes of endocytosis and trafficking of fluorescent nanoparticles such as Qdots and fluorescent nanodiomands (NDs) have been investigated and depicted in detail.29-31 In contrast, comprehensive study on the membrane trafficking of Pdots is in demand. It has been reported that Pdots could enter macrophage-like cells efficiently and swiftly via macropinocytosis.32 Since macrophages are responsible for non-specific engulfing of exogenous materials, other cell types such as epithelial cells that can develop into carcinoma may have distinct mechanism for Pdots uptake and metabolism.33 Herein, we investigated endocytosis and intracellular trafficking of poly (9, 9-dioctylfluorenyl-2, 7-diyl)-co-(1, 4-benzo-{2, 1’, 3}-thiadiazole) PFBT fluorescent polymer dots. The endocytic pathways of PFBT Pdots were differently regulated in epithelial cells and macrophages. Following endocytosis, Pdots were transported in vesicles along microtubule and destined for lysosomes. Furthermore, we identified that Pdots could be released from cells through exocytosis and successfully tracked the movement of Pdots-containing exosomes at single particle level. These results provide new insight into the design of Pdots-based imaging probes as well as drug carriers with improved efficiency and selectivity.


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Results and Discussion

Biocompatibility and cellular uptake of Pdots

Pdots made from a highly fluorescent semi-conducting polymer (PFBT) were chosen for the investigation of their cellular uptake and trafficking behavior. PFBT conjugated nanoparticles were synthesized as previously described.34 According to the results of transmission electron microscopy (TEM) and dynamic light scattering (DLS) analysis, diameter of PFBT Pdots was about 20 nm (Figure 1a & 1b). Pdots were well dispersed in MEM medium. Analysis of the absorption spectrum for PFBT dots indicated a peak around 460 nm; a maximum fluorescence emission around 540 nm was detected (Figure 1c). Having confirmed the fluorescent brightness and monodispersibility of Pdots, we examined whether they have adverse effects on cell viability. Four different cell types, including Raw 264.7 murine macrophage cells, MCF-7 human breast cancer cells, HeLa human cervical cancer cells and human umbilical mesenchymal stem cells (HUMSC), were incubated with Pdots at various concentrations for 48 hours respectively. Cell viability was determined by MTT assay. Pdots showed no toxicity to any of these cell lines at concentrations up to 20 µg/mL (Figure 1d). Biocompatibility of Pdots was further verified with analysis of apoptosis by flow cytometry (Figure 1e). Less than 10% apoptotic HeLa cells were detected after 48-hour incubation with 20 µg/mL Pdots. These data indicated that Pdots possessed superior biocompatibility, and therefore had great potential in biomedical applications, including live cell imaging and long-term cell tracing.



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Distinct uptake kinetics of Pdots in different cell lines Having confirmed their biocompatibility, we set out to determine the endocytic mechanism of Pdots. It has previously been shown that fluorescent Pdots could efficiently enter cells. However, details about the processes of endocytosis and intracellular transportation have not been elucidated. Firstly, we compared cellular uptake and accumulation of Pdots in murine macrophage-like (Raw 264.7) cells and epithelial (HeLa) cells to test whether endocytosis was determined by cell type. Both cells were incubated with Pdots and intracellular fluorescent signals were observed using a confocal fluorescence microscope and measured using a flow cytometer. Pdots were rapidly ingested by Raw 264.7 cells. After 2-hour incubation, significant intensity of fluorescent signals was detected, which peaked at 4-6 hours and slightly declined afterwards (Figure 2a & 2b, Figure S1 in the Supporting Information). HeLa cells, in contrast, exhibited a much slower process of fluorescence accumulation. Fluorescent signals were hardly detectable before 6 hours, and gradually increased over time (Figure 2a & 2c, Figure S2 in the Supporting Information). Notably, Raw 264.7 cells exhibit much more efficient uptake of Pdots than HeLa cells. In the above experiment, Raw 264.7 cells were incubated with 5 µg/mL Pdots and HeLa cells were incubated with 20 µg/mL Pdots whereas the former demonstrated stronger fluorescence intensity. In addition, the time-dependent uptake kinetics of Pdots was distinct in these two cell lines; internalization of Pdots in Raw 264.7 cells was much faster than that in HeLa cells. To have a detailed look at the early stage of the endocytic process in Raw 264.7 cells, we examined the fluorescence at 5 min, 30 min


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and 60 min. Strikingly, after only 5-min incubation, signals of Pdots were already detectable inside Raw 264.7 cells, suggesting macrophages ingested NPs immediately after they met each other. In the following 60 min, amount of intracellular Pdots increased linearly with time (Figure 3, Figure S1 in the Supporting Information). We also noticed that the fluorescent dots had a tendency to form bigger puncta with prolonged incubation time, suggesting fusion of vesicles following endocytosis.

Distinct endocytic pathways of Pdots in different cell lines

Since Raw 264.7 cells and HeLa cells showed completely different uptake kinetics of Pdots, we suspected that these two types of cells employed distinct pathways for endocytosis of Pdots. Therefore, we examined endocytic processes in both cells with selective inhibitors of different routes. First, we checked the energy dependence of cellular uptake. At 4℃, all active energy-dependent endocytic processes are stalled. When we incubated Raw 264.7 or HeLa cells with Pdots at 4℃, intracellular fluorescent signals were undetectable in both types of cells, indicating that Pdots were internalized through energy-dependent endocytosis (Figure 4). Next, to elucidate which endocytic pathway was responsible for internalization of Pdots; cells were pre-incubated with selective pharmaceutical inhibitors targeting distinct endocytic pathways.35 Methyl-β-cyclodextrin (mβCD) could disrupt lipid raft on the cell membrane by cholesterol depletion and block caveolae-mediated endocytosis. Chlorpromazine (CPZ) was applied to block clathrin-dependent endocytosis. 5-(N-Ethyl-N-isopropyl)-amiloride (EIPA), an inhibitor of micropinocytosis was also



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included.36 Compared to untreated cells, pretreatment of Raw 264.7 cells with EIPA greatly reduced intracellular signals of Pdots by more than 80%, suggesting Raw 264.7 cells ingested Pdots mainly via micropinocytosis (Figure 4a & 4b, Figure S3 in the Supporting Information). This observation was consistent with previous report that macrophage-like J774A.1 cells ingested Pdots by micropinocytosis.32 In contrast, pretreating HeLa cells with EIPA hardly changed the accumulation of Pdots, implying different endocytic mechanism. CPZ treatment did not change fluorescent signals in either cell lines, suggesting clathrin-dependent endocytosis were not involved in the trafficking of Pdots. Significantly, mβCD treatment resulted in a strong reduction (more than 65%) of internalized Pdots in HeLa cells, while it has no effect on Raw 264.7 cells (Figure 4a & 4c, Figure S3 in the Supporting Information). Taken together, these data revealed that Pdots enter HeLa cells through caveolae-mediated endocytosis and Raw 264.7 cells uptook Pdots via macropinocytosis, which well explained the striking difference in the speed of Pdots internalization between two cell lines. Unlike receptor-mediated endocytosis, macropinocytosis is nonspecific for cargos.36 Circulating macrophages mainly rely on pinocytosis or phagocytosis to engulf invading particles or pathogens. Therefore, Pdots that applied by intravenous administration most likely will enter macrophages efficiently in short time and function as imaging and tracing probes. This finding also provided new thought for cell-specific targeting of Pdots-based nano-conjugates. 37

Intracellular trafficking of Pdots in the endo-lysosomal compartments


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Having elucidated the endocytic mechanisms of Pdots, we then extended our analysis to

their

intracellular trafficking

process.

Substances

that

internalized

by

macropinocytosis will be targeted to lysosome, and it has been demonstrated for Pdots in macrophage-like J774A.1 cells.32 However, intracellular trafficking of Pdots in epithelial cells has not been elucidated. Therefore, we first examined intracellular distribution of Pdots in HeLa cells. We labeled endosomes and lysosomes by corresponding RFP-fused protein markers and performed colocalization analysis with Pdots of at different time points. Rab family of small GTPases is the main coordinator of intracellular vesicle traffic.38 Rab5 is the first GTPase encountered following endocytosis and serves as a marker of early endosomes.39 We observed that colocalization between Pdots and Rab5-RFP increased with prolonged incubation time and peaked at 12 hours (Figure 5a & 5c), suggesting that the uptake and traffic of Pdots are continuous processes in HeLa cells. We then examined the colocalization between Pdots and RFP-tagged LAMP1, a marker for lysosomes.40 58% of intracellular Pdots were colocalized with LAMP1-RFP after 6-hour incubation and the ratio increased to about 80% after 12 hours (Figure 5b & 5c). We concluded that following endocytosis, Pdots progressed through the endo-lysosomal pathway in epithelial cells. This information is important for Pdots-mediated intracellular delivery. Since majority of the Pdots were destined for acidic lysosomes, design of PH-responsive Pdots would facilitate the release of drugs. On the other hand, for cargos that are sensitive to the enzymes in lysosomes (e.g. proteins), it is necessary to modify the Pdots to achieve endosomal escape to ensure its functionality.



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Intracellular transportation of vesicles containing Pdots

Distribution of Pdots in the endo-lysosomal system indicated that they were transported via membrane-coated vesicles. Therefore, we inspected the intracellular trafficking of Pdots-containing vesicles. Time-lapse movies of living cells were acquired using a confocal microscope (Video S1 in the Supporting Information, ∆t = 3s, total time = 225s). We randomly selected 2 vesicles and analyzed their movement and mobility by generating trajectories of these vesicles. As shown in Figure 6a, these particles moved with a highest moving speed up to 0.5 µm/s. It has been well established that cargo-filled vesicles were attached with specialized motor proteins and travelled along cytoskeleton filaments.41 To evaluate individual contribution of microtubules and actin filaments to the transportation of Pdots-containing vesicles, we separately employed selective pharmaceutical inhibitors to depolymerize one of the two components of cytoskeleton. In HeLa cells pretreated with cytochalasin B, which depolymerizes actin filaments, the motility of representative vesicles was hardly changed (Figure 6a and Video S2 in the Supporting Information, ∆t = 3s, total time = 225s). However, movement of Pdots-containing vesicles was confined and the average moving speed was dramatically reduced to < 0.1 µm/s in HeLa cells pretreated with nocodazole, which depolymerizes tubulins (Figure 6a and Video S3 in the Supporting Information, ∆t = 3s, total time = 225s).42 These data strongly suggested that microtubule-dependent motors were responsible for the trafficking of Pdots inside the endo-lysosomal compartments. To further prove this, we performed immunostaining against tubulin in HeLa cells and inspected the localization of Pdots


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(Figure 6b). Significant amount of green Pdots-containing vesicles were located right on

red microtubules (merged in

yellow),

inferring microtubule-dependent

transportation of Pdots. Taken together, these results demonstrated that following caveolae-dependent endocytosis, intracellular traffic of Pdots required microtubules and associated motor proteins like kinesin and dynein.

Exocytosis of Pdots

The superior photostability of Pdots made them suitable for long-term cell imaging and tracing. Sufficient amount of intracellular nanoparticles was necessary for cell detection. We then investigated the dynamic equilibrium between uptake and secretion of Pdots. Although the internalization of various NPs has been extensively studied, mechanistic insight into exocytosis has been lacking.43-44 Therefore, we sought to investigate whether Pdots could be released via exocytosis by assessing intracellular retention of Pdots in HeLa cells. Cells were preload with Pdots for sufficient time (24 hours) to label majority of the population. Then cells were washed thoroughly and incubated in serum-free culture media without Pdots. Intracellular fluorescence intensity was determined at different time points by flow cytometry analysis. The fluorescent signals in the cells gradually declined with prolonged incubation time and reduced to about 25% by 48 hours (Figure 7a, Figure S4 in the Supporting Information). Since Pdots have proven to be stable in cellular environment, it is unlikely that this reduction was caused by degradation of Pdots. Serum starvation prevented cell division and dilution of fluorescent Pdots. Therefore, we postulated



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that this decrease was caused by excretion of Pdots out of HeLa cells. Next, we tested the hypothesis that Pdots could leave cells via exocytosis. An important route of exocytosis was the fusion of multivesicular bodies (MVBs) with the plasma membrane when vesicles called exosomes were released to the extracellular space. We checked whether Pdots were located inside exosomes. To label exosomes, a DNA construct encoding RFP-CD9 was expressed in HeLa cells. CD9 is a well-established marker of exosomes.45 Cells expressing RFP-CD9 were incubated with Pdots and examined under a confocal microscope. Clear colocalization of Pdots with RFP-CD9 was observed. We then traced the dynamic movement of green vesicles that colocalized with RFP-CD9 via live cell imaging. Time-lapse movie was acquired (Video S4 in the Supporting Information, ∆t = 3s, total time = 249s). Indeed, we observed that co-localized signals for RFP-CD9 and Pdots moved together in the cytoplasm. Moving speed and trajectories of a representative particle (indicated by a blue circle) that moved towards the edge of cell were shown (Figure 7b & 7c). These data confirmed that Pdots could be exported out of HeLa cells. Our work provided the first evidence of exocytosis of Pdots, which could be exploited in the future design of nanovehicles that left cells after unloading of their cargos.

Conclusion

Wide ranges of NPs have shown great potential for theranostic applications.46-50 Insight into the interaction between NPs and cells is critical for the improvement and further applications.51-55 While semiconductor polymer dots represent one of the


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major types of fluorescent NPs and 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.56 In this study, we explored the endocytosis, exocytosis and intracellular trafficking of PFBT Pdots. HeLa cells internalized Pdots via caveolae-mediated endocytosis. Majority of internalized Pdots resided in endocytic vesicles and fused with endo-lysosomal compartments. These vesicles were transported along microtubules. We also observed that Pdots were targeted to exosomes and transported towards the cell membrane, indicating that Pdots could be released by HeLa cells. More importantly, we have compared cellular uptake behavior of Pdots in HeLa cells and Raw 264.7 macrophage cells. Accumulation of Pdots in HeLa cells increased steadily for 48 hours, implying relatively slow interaction between Pdots and receptors on the plasma membrane. In contrast, Pdots entered macrophages within minutes. Experiments with selective inhibitor revealed that Raw 264.7 uptook Pdots through macropinocytosis, which was completely different from HeLa cells. Such cell type-dependent regulation of endocytic pathways and kinetics of Pdots provides a new approach for targeting specific cell populations. These new findings not only shed light on the interaction between cells and Pdots, but also facilitate design of Pdots-based imaging probes or drug vehicles with improved selectivity and safety.

Experimental Section Reagents and cell lines



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Poly (styrene-co-maleic anhydride) (PSMA, Mn = 1700) and anhydrous tetrahydrofuran

(THF,

99.9%)

were

purchased from

Sigma-Aldrich.

Poly

[(9,9-dioctylfluorenyl-2,7-diyl)-co-(1,4-benzo-{2,1′,3}-thiadiazole)] (PFBT, MW = 10 000, polydispersity 1.7) was obtained from ADS Dyes (Quebec, Canada). HeLa and Raw 264.7 cell lines were purchased from Cell Bank of Chinese Academy of Sciences (Shanghai). Minimum Essential Media (MEM), Dulbecco’s Modified Eagle’s Medium (DMEM) and fetal bovine serum (FBS) was from Gibco, Invitrogen. Thiazolyl

blue

tetrazolium

bromide

(MTT),

chlorpromazine

(CPZ),

methyl-β-cyclodextrin (mβCD), EIPA, cytochalasin B and nocodazole were purchased from Sigma-Aldrich (USA). mCherry-CD9 was a gift from Michael Davidson (Addgene plasmid # 55013). Preparation and Characterization of Pdots Pdots were synthesized using a modified precipitation method. THF solution (5 mL) containing conjugated polymers (0.5 mg) and PSMA (0.2 mg) was quickly injected into 10 mL deionized water and the mixture was subsequently sonicated for 2 min. THF was removed by partial vacuum evaporation. The resulting solution was concentrated by continuous heating, followed by filtration through a 0.22 µm filter to remove fraction of aggregates. The apparent hydrodynamic size and the zeta potential of nanoparticles were measured using a Zetasizer (nano ZS90, Malvern Instruments). For transmission electron microscopy, solution containing Pdots was dropped onto carbon coated copper grids to evaporate excess solvent, and examined with TEM (Jeol 2010, 200KV).


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Cell culture Hela and Raw 264.7 cell lines were grown in MEM or DMEM respectively supplemented with 10 % heat-inactivated FBS and antibiotics (100 g/mL of streptomycin and 100 g/mL of penicillin) at 37 °C with humidified atmosphere (5 % CO2). MTT assay

Cells were seeded in 24-well plates at a concentration of 4 × 105 cells/well one day before treatment. Varying concentrations of Pdots (0-20 µg/mL) were incubated with cells for 48 h at 37 °C. 5 mg/mL MTT (diluted in MEM medium) solution was then added to each well, followed by 4 h incubation at 37 °C. Cells were lysed with 10% acid SDS solution (pH 2~3). After centrifugation, the absorbance of supernatant was determined at 570 nm using a microplate reader (Bio-Rad 680, USA).

Apoptosis analyses

Apoptosis analysis was carried out using the Annexin V-PE Apoptosis Detection kit (BD Pharmingen, San Jose, CA) according to the manufacturer's protocol. Cells were seeded in 6-well cell culture plates and treated with different concentration of Pdots for 48 hours. All cells were harvested at indicated time points, washed twice with cold PBS and suspended in 1× binding buffer. A 100µl aliquot of the cell suspension was transferred to a culture tube, to which 5µl of Annexin V-PE and 5µl of 7-AAD were added, and the mixture were incubated for 15 minutes at room temperature in the dark. Apoptosis was then estimated by flow cytometry.



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Flow Cytometry

Before measurements, cell media was removed and cells were washed for three times with PBS. Next, 0.2 mL trypsin (Invitrogen) was added to each sample and incubated for 1 min at 37 ℃ before 0.5 mL MEM was added. Cell suspensions were transferred into tubes before analyzed using a FACS Calibur flow cytometer (Amnis ImageStream MarkⅡ, MERCK MILLIPORE, USA). Consistent gating based on cell size and granularity (forward and side scatter) was applied to select the fluorescence signals of counted cells. At least 10000 cells were counted for each sample and experiments were performed in triplicates.

Endocytosis and membrane trafficking studies HeLa cells (1×106) were transfected with 1µg of the different expression plasmids (RFP-Rab5, RFP-LAMP1) using lipofectamine 3000 (Invitrogen) according to the manufacturer’s instructions. After incubating with 20 µg/mL Pdots for indicated time, cells were imaged by confocal microscopy (Leica SP8). For inhibitor treatment, cells were pre-treated with low tempreture (4℃), 5 µg/ml CPZ, 10 µg/ml EIPA, 7 mM mβCD, 5 µM cytochalasin B and 60 µM nocodazole respectively.

Immunostaining

HeLa cells were incubated with 20 µg/mL Pdots for 24 h, washed with PBS buffer, and then fixed in 4% (w/v) paraformaldehyde at room temperature for 20 min. After incubation with PBS containing 6% (w/v) BSA and 0.25% (v/v) Triton X-100 for 45


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min, cells were stained with anti-α-tubulin Mouse IgG (B-7) (sc-5286, Santa Cruz) followed by Goat anti-mouse Alexa 647 IgG (H&L) (A21237, Thermo Fisher), then washed for confocal imaging. Confocal fluorescence microscopy Cells expressing fluorescent protein markers or cells immunostained for microtubule were imaged using a Leica SP8 confocal microscope. Alexa 647 were excited with a 633 nm HeNe laser, RFP were excited with a 561 nm helium-neon laser, the PFBT and green-labeled tubulin were excited with a 488 nm Ar-Kr laser, and Hoechst 33258-labeled nuclei were excited with a 405 nm diode laser, respectively. The imaging channels were set at 650-670, 570-620, 500-550, and 450-500 nm, respectively.

Live cell imaging

HeLa cells were transfected with plasmids encoding mCherry-CD9 using Lipofectamine 3000 reagent (Invitrogen). After transfection for 24 h, cells were incubated with 20 µg/mL Pdots. 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 oil-immersion 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



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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-S4 and Video S1-S4 with accompanying figure legends.

AUTHOR INFORMATION

Corresponding Author

*[email protected]

*[email protected]

*[email protected]

Author Contributions

‡These authors contributed equally.

ACKNOWLEDGMENT

This work was supported by National Natural Science Foundation of China (31470970, 61335001 and 21505148), National Basic Research Program of China


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(2016YFA0201200

and

2013CB932800),

the

Youth

Innovation

Promotion

Association from Chinese Academy of Sciences (2015211) and the Key Research Program of Frontier Sciences, CAS (QYZDJ-SSW-SLH031, QYZDJ-SSW-SLH019). Wu C. acknowledges financial support from “Thousand Young Talents Program”.

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Figure 1. Physical properties and biocompatibility of Pdots. (a) TEM images of PFBT polymer dots. Chemical structure of PFBT polymers was shown in the inset. (b) Hydrodynamic diameter of Pdots was measured by dynamic light scattering. (c) Spectra of Pdots were determined in water. (d) HeLa, Raw 264.7, MCF-7 or MSC cells were incubated with Pdots for 48 hours at indicated concentrations and then analyzed for viability by MTT assays. (e) Cells were stained for Annexin V and 7-AAD and apoptotic cells were measured by FACS.



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Figure 2. Distinct kinetics of Pdots internalization for Raw264.7 and HeLa cells. (a) Raw264.7 cells were incubated with 5 µg/mL Pdots and HeLa cells were incubated with 20 µg/mL Pdots for up to 48 hours. At indicated time points, intracellular fluorescence was observed using confocal microscopy. Lower panel show magnified images of the square region in the upper panel. (b) & (c) Fluorescence of internalized Pdots were quantified using flow cytometry analysis.


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Figure 3. Fast accumulation of Pdots in Raw264.7 cells. (a) Confocal images of Raw264.7 cells incubated with 5 µg/mL Pdots for 5, 30 or 60 mins. Lower panel show magnified images of the square region in the upper panel. (b) Fluorescence of internalized Pdots was quantified using flow cytometry analysis.



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Figure 4. Endocytosis pathways of Pdots in different cell lines. (a) Confocal images of Raw264.7 or HeLa cells treated with indicated pharmacological inhibitors. Cells were pretreated with low temperature (4℃), mβ-CD, EIPA or CPZ for 30 minutes, followed by incubation with 20 µg/mL Pdots. Lower panel show magnified images of the square region in the upper panel. (b) Intracellular fluorescent signals of Raw264.7 cells treated in the presence of different inhibitors were quantified using the ImageJ software (20 cells analyzed). (c) Intracellular fluorescent signals of HeLa cells treated in the presence of different inhibitors were quantified using the ImageJ software (20 cells analyzed).


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Figure 5. Colocalization of Pdots with endo-lysosomal compartments. (a) HeLa cells expressing RFP-Rab5 were incubated with 20 µg/mL Pdots and imaged by confocal microscope at indicated time points. The colocalization between Rab5 and Pdots were analyzed by line profiling fluorescence intensity of RFP-Rab5 (red) and Pdots (green) along the line selected in the upper panel. (b) The colocalization between LAMP1 and Pdots were examined by confocal microscopy and line profiling of fluorescence intensities of RFP-LAMP1 (red) and Pdots (green). (c) Colocalization ratio of Pdots with Rab5 (red) or LAMP1 (blue) was quantified using the ImageJ software (20 cells analyzed).



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Figure 6. Microtubule-dependent transport of Pdots. (a) Confocal images of HeLa cells incubated with Pdots in the absence or presence of cytochalasin B or nocodazole. Representative trajectories of 2 randomly selected Pdots were shown (see also Video S1-S3 in the Supporting Information). Lower panel showed the instantaneous speed of selected Pdots particles. (b) Confocal images of Pdots (green) colocalized with Alexa 647-stained microtubule (red).


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Figure 7. Exocytosis of Pdots. (a) HeLa cells were incubated with 20 µg/mL Pdots for 24 hours, followed by incubation with serum-free media with no Pdots. Intracellular fluorescence of Pdots was determined at indicated time points using flow cytometry analysis. (b) & (c) Confocal images and analysis of colocalized Pdots (green) with RFP-CD9 (red). A representative particle was indicated with an orange circle. The instantaneous speed of this particle was shown in (b). Trajectories at different time points (one frame per 3 seconds, with a total of 249 seconds; also see Video S4) were shown in (c).



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Real-Time Imaging of Endocytosis and Intracellular Trafficking of

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