Research Article www.acsami.org
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*,‡ †
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, United Kingdom
ACS Appl. Mater. Interfaces 2017.9:21200-21208. Downloaded from pubs.acs.org by UNIV OF KANSAS on 01/05/19. For personal use only.
‡
S Supporting Information *
ABSTRACT: Semiconducting polymer dots (Pdots) have shown great promise in biomedical applications, including biosensing, drug delivery, and live imaging of cells and biomolecules. Insight into the mechanism and regulation of cellular uptake and intracellular metabolism of Pdots is important for the development of superior Pdots-based theranostic nanoconjugates. 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 the single particle level. These results provide new insight into the design of more effective and selective imaging probes as well as drug carriers. KEYWORDS: fluorescent polymer dots, endocytosis, intracellular trafficking, theranostic nanoparticles, live cell imaging
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fluorescent NPs with less risk of biotoxicity.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 labeling, 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
INTRODUCTION Fluorescent imaging techniques play an increasing role in biological studies 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 for observing the behavior of biomolecules and tracking cell fate within a living organisms.2,3 This technique requires genetic encoding of fusion protein with a fluorescent tag. Moreover, the brightness and photostability of natural 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 metals (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 © 2017 American Chemical Society
Received: April 23, 2017 Accepted: June 6, 2017 Published: June 6, 2017 21200
DOI: 10.1021/acsami.7b05662 ACS Appl. Mater. Interfaces 2017, 9, 21200−21208
Research Article
ACS Applied Materials & Interfaces
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 h 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.
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 h. 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 and c) Fluorescence of internalized Pdots were quantified using flow cytometry analysis.
with plasmid or small interfering RNA (siRNA) and modulate gene expression in target cells.27 Importantly, the intrinsic fluorescence of Pdots provides unparalleled advantage for monitoring the targeting efficiency of cargo-loaded NPs and simultaneously tracking the release of drugs. These multifunc-
tional 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 21201
DOI: 10.1021/acsami.7b05662 ACS Appl. Mater. Interfaces 2017, 9, 21200−21208
Research Article
ACS Applied Materials & Interfaces
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 min. Lower panel show magnified images of the square region in the upper panel. (b) Fluorescence of internalized Pdots was quantified using flow cytometry analysis.
after 48-h 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. 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. First, 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-h incubation, significant intensity of fluorescent signals was detected, which peaked at 4−6 h and slightly declined afterward (Figure 2a,b, 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 h, and gradually increased over time (Figure 2a,c, 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, 30, 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, the 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
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, a 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 nonspecific engulfing of exogenous materials, other cell types such as epithelial cells that can develop into carcinoma may have a 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 Pdots. The endocytic pathways of PFBT Pdots were differently regulated in epithelial cells and macrophages. Following endocytosis, Pdots were transported in vesicles along a 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 the 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 semiconducting 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, the diameter of PFBT Pdots was about 20 nm (Figure 1a,b). 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 h, 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). The biocompatibility of Pdots was further verified with analysis of apoptosis by flow cytometry (Figure 1e). Less than 10% apoptotic HeLa cells were detected 21202
DOI: 10.1021/acsami.7b05662 ACS Appl. Mater. Interfaces 2017, 9, 21200−21208
Research Article
ACS Applied Materials & Interfaces
pinocytosis, 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 nanoconjugates.37 Intracellular Trafficking of Pdots in the Endolysosomal Compartments. 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 by 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 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 h (Figure 5a,c), suggesting that the uptake and traffic of Pdots are continuous processes in HeLa cells. We then examined the colocalization between Pdots and RFPtagged LAMP1, a marker for lysosomes.40 A total of 58% of intracellular Pdots were colocalized with LAMP1-RFP after 6-h incubation, and the ratio increased to about 80% after 12 h (Figure 5b,c). We concluded that, following endocytosis, Pdots progressed through the endolysosomal pathway in epithelial cells. This information is important for Pdots-mediated intracellular delivery. Since the majority of the Pdots were destined for acidic lysosomes, the 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. Intracellular Transportation of Vesicles Containing Pdots. Distribution of Pdots in the endolysosomal 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, Δt = 3 s, total time = 225 s). We randomly selected two 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 1.1 μm/s. It has been well established that cargo-filled vesicles were attached with specialized motor proteins and traveled 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, Δt = 3 s, total time = 225 s). However, movement of Pdots-containing vesicles was confined, and the average
cells with selective inhibitors of different routes. First, we checked the energy dependence of cellular uptake. At 4 °C, all active energy-dependent endocytic processes are stalled. When we incubated Raw 264.7 or HeLa cells with Pdots at 4 °C, intracellular fluorescent signals were undetectable in both types of cells, indicating that Pdots were internalized through energydependent endocytosis (Figure 4). Next, to elucidate which
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 °C), mβ-CD, EIPA, or CPZ for 30 min, followed by incubation with 20 μg/mL Pdots. (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).
endocytic pathway was responsible for internalization of Pdots; cells were preincubated 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 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,b, Figure S3 in the Supporting Information). This observation was consistent with a 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 a different endocytic mechanism. CPZ treatment did not change fluorescent signals in either cell lines, suggesting clathrindependent 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,c, 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 macro21203
DOI: 10.1021/acsami.7b05662 ACS Appl. Mater. Interfaces 2017, 9, 21200−21208
Research Article
ACS Applied Materials & Interfaces
Figure 5. Colocalization of Pdots with endolysosomal compartments. (a) HeLa cells expressing RFP-Rab5 were incubated with 20 μg/mL Pdots and imaged by confocal microscope at the indicated time points. The colocalization between Rab5 and Pdots was 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 was 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).
moving speed was dramatically reduced to
