In Vivo Real-Time Imaging of Extracellular Vesicles in Liver

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In Vivo Real-Time Imaging of Extracellular Vesicles in Liver Regeneration via Aggregation-Induced Emission Luminogens Hongmei Cao, Zhiwei Yue, Heqi Gao, Chao Chen, Kaige Cui, Kaiyue Zhang, Yuanqiu Cheng, Guoqiang Shao, Deling Kong, Zongjin Li, Dan Ding, and Yuebing Wang ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.8b09776 • Publication Date (Web): 07 Mar 2019 Downloaded from http://pubs.acs.org on March 8, 2019

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In Vivo Real-Time Imaging of Extracellular Vesicles in Liver Regeneration via Aggregation-Induced Emission Luminogens

Hongmei Cao, †, ∞ Zhiwei Yue, †, ∞ Heqi Gao, ‡ Chao Chen, ‡ Kaige Cui, † Kaiyue Zhang, †

Yuanqiu Cheng, † Guoqiang Shao, § Deling Kong,

‡

Zongjin Li, *, † Dan Ding, *, ‡

Yuebing Wang, *, †

† Nankai ‡

University School of Medicine, Tianjin 300071, China

The Key Laboratory of Bioactive Materials, Ministry of Education, Nankai University,

The College of Life Science, Tianjin 300071, China §

Department of Nuclear Medicine, Nanjing First Hospital, Nanjing Medical University,

Nanjing 210006, China

Corresponding Authors: *E-mail: [email protected] (Yuebing Wang) *E-mail: [email protected] (Dan Ding) *E-mail: [email protected] (Zongjin Li)

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ABSTRACT: Extracellular vesicles (EVs) attract much attention in liver pathology because they regulate cell-cell communication and a lot of pathophysiological events via transferring their cargos. Monitoring and understanding the in vivo fate and therapeutic capacity of these EVs is critical for the development and optimization of EV-based diagnosis and therapy. Herein, we demonstrate the use of an aggregationinduced emission luminogen (AIEgen), DPA-SCP, for the real-time tracking of EVs derived from human placenta-derived mesenchymal stem cells (hP-MSCs) and their therapeutic effects in a mouse acute liver injury (ALI) model. In vitro, DPA-SCP does not alter the inherent characteristics of MSC-derived EVs and shows extremely low toxicity. Moreover, DPA-SCP exhibited superior labeling efficiency and tracking capability to the most popular commercial EV trackers, PKH26 and DiI. In vivo, DPASCP precisely and quantitatively tracked the behaviors of EVs for 7 days in the mouse ALI model without influencing their regenerative capacity and therapeutic efficacy. The therapeutic effects of EVs may attribute to their ability for reducing inflammatory cell infiltration, enhancing cell survival and antiapoptotic effects. In conclusion, DPASCP with an AIE signature serves as a favorable and safe tracker for in vivo real-time imaging of EVs in liver regeneration.

KEYWORDS: extracellular vesicles, aggregation-induced emission, human placentaderived mesenchymal stem cells, liver regeneration, fluorescence imaging

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Although the liver is renowned for its attractive regenerative capacity, the morbidity and mortality of liver disease remain high and are increasing globally.1 Chronic liver disease has emerged as one of the leading causes of global health burden.2 Lifestyle issues such as alcohol abuse and obesity, environmental factors and host factors can all result in liver injury, even liver failure and cirrhosis.3 In severe inflammatory liver conditions, the liver regeneration capacity becomes compromised, and no current treatments other than liver transplantation are effective.4 However, a major challenge of liver transplantation is the scarcity of organ donors compared with the growing number of transplant candidates. Moreover, extended survival after liver transplantation is hindered by graft-vs-host disease and the side effects of lifelong administration of immunosuppression.5 Mesenchymal stem cells (MSCs) provide an alternative to organ or hepatocyte transplantation.6-9 Increasing clinical trials of MSC-based treatments for liver disease are ongoing.10 Several lines of evidence have revealed that the immunosuppressive properties and various paracrine factors secreted by MSCs play key therapeutic roles in regenerating and repairing damaged liver tissues.11-13 Consequently, MSC-derived extracellular vesicles (MSC-EVs) attract great attention in the cell-free treatment of liver disease. EVs, which are secreted from all types of cells, are small vesicles of multivesicular bodies released upon endocytic fusion with the plasma membrane. Because circulating EVs are composed of specific proteins, mRNAs or miRNAs, they are considered to be robust molecular biomarkers for early detection of liver disease.1417

In addition, EVs derived from stem cells, especially MSCs, have shown positive 3


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effects in a variety of animal models of liver disease such as liver fibrosis.18-20 However, to effectively utilize EVs as diagnostic or therapeutic tools, noninvasive, continuous and precise tracking of their in vivo fate and behaviors must be established. EVs are inherent nano-size and possess a highly complex membrane structure, it remains a great challenge to track them efficiently. EV tracking methods are mainly direct and indirect labeling. Fluorescence labeling is a strategy extensively used for the direct imaging EVs. EVs have been fluorescently labeled with lipophilic dyes such as PKH26 or with carbocyanine dyes such as DiI in the visible region to track cellular interaction and uptake.21, 22 However, such dyes form dye aggregates or micelles in aqueous solutions, potentially giving misleading uptake results. Indirect labeling is performed by labeling parent cells to obtain labeled EVs. Examples include fluorescent protein, luciferase or cell metabolite reporting systems.23, 24 However, these indirect methods require the construction of stable overexpression cell lines, which may change the EV composition, thus affect the EV function. Multimodal imaging of EVs has also been attempted by method such as the simultaneous use of magnetic nanoparticles and CdSe quantum dots (QDs).25, 26Although these strategies can achieve EV tracking, they suffer from a high quenching coefficient; moreover, the EV membrane structure was seriously damaged; thus, the physiological properties and function of EVs were badly influenced. Luminescent materials with aggregation-induced emission characteristics (AIEgens) have shown fantastic results in many biomedical areas since their discovery by Tang in 2001.27 In contrast to traditional aggregation-caused quenching (ACQ) fluorescein, 4


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AIEgens are highly emissive in the aggregated state as a result of restriction of intermolecular motion but are not emissive in the solution state.28, 29 Because of the smart properties of superior resistance to photobleaching, excellent biocompatibility and high signal-to-noise ratios, AIEgens have exhibited powerful abilities from monitoring biological processes to disease diagnosis and theranostics.30,

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Consequently, the superior performance of AIEgens motivates us to exploit them in EV monitoring and investigation. In the present study, we report the application of an AIEgen, DPA-SCP, as an in vivo EV tracker. We compared the brightness and labeling efficiency of DPA-SCP with those of PKH26 and DiI, which are the most popular commercial EV trackers. Furthermore, we assessed the therapeutic efficiency of MSC-EVs using DPA-SCP in a mouse acute liver injury model (ALI) with fluorescence imaging and explored the underlying mechanism of MSC-EVs in liver regeneration.

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RESULTS AND DISCUSSION Characterization of AIEgens. DPA-SCP used for EV tracking was synthesized as described in our previous publication.32 The synthetic route of DPA-SCP is shown in Figure S1. The chemical structure of DPA-SCP, which has been demonstrated to exhibit the AIE signature, is shown in Figure 1A. The chemical structure of DPA-SCP was verified by standard spectroscopic techniques including nuclear magnetic resonance (NMR) and high resolution mass spectrometry (HRMS) (Figure S2, 3).

Characterization of EVs. EVs were isolated from medium conditioned by human placenta-derived MSCs (hP-MSCs) via sequential ultracentrifugation. Prior to extraction, we determined the phenotypic properties of hP-MSCs by flow cytometry. As shown in Figure S4, MSCs maintained the expression of surface markers CD90 and CD44, which demonstrated the phenotypic and multidifferentiation features of hPMSCs used in the present study. EV surface has an overall negative charge because of the abundance of proteoglycans and sialic acids.33 Recent publication revealed that EV membrane is also rich in negatively charged lipids, and this advantage was utilized to attain an effective cytosolic delivery of biomacromolecules.34 Similarly, we achieve reliable EV labeling by attaching the positively charged DPA-SCP to the negatively charged EV membrane. EVs derived from hP-MSCs were labeled with DPA-SCP. Afterwards, we characterized both the unlabeled EVs and AIEgen labeled EVs (AIE-EVs) to determine whether DPA-SCP influenced the physiological properties of EVs. 6


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Under transmission electron microscopy (TEM), both EVs and AIE-EVs appear as round and cup-shaped vesicles of approximately 120 nm in diameter, exhibiting clear membrane structures. Furthermore, in contrast to unlabeled EVs, we observed the clear attachment of DPA-SCP to the EV surface (Figure 1B). Western blot results confirmed the expression of EV surface markers (CD9 and CD63) and cytosolic protein (ALIX and TSG101) in analyzed EVs; however, GM130 was negatively expressed (Figure 1C). The nanoparticle tracking analysis (NTA) results showed that the diameter of the AIE-EVs was unchanged compared to that of the unlabeled EVs (Figure 1D). The zeta potential became slightly more positive due to the attachment of positively charged AIEgens to the EV surface (Figure 1E). The above results indicated the successful labeling of EVs with DPA-SCP; moreover, the DPA-SCP labeling did not affect the physiological properties of the EVs. Subsequently, the photophysical properties of AIE-EVs were investigated. The absorption spectrum of AIE-EVs had a peak centered at 460 nm, which was the same as that of DPA-SCP (Figure 1F). As shown in Figure 1G, AIEgens are weakly fluorescent in phosphate buffered saline (PBS) solution; however, the AIE-EVs had a significant fluorescence in PBS solution under 620 nm emission, displaying the AIEgen fluorescence “turn-on” property in the presence of EVs.

In Vitro Fluorescence Imaging of EVs. After characterization of both the AIEgens and EVs, we examined whether the AIEgens could precisely track EVs using confocal laser scanning microscopy (CLSM). EVs labeled with different concentrations of 7


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AIEgens were incubated with human umbilical vein endothelial cells (HUVECs) for 48 hours. The supernatant was then removed, and the cells were washed with PBS thrice to remove the uninternalized AIE-EVs. Upon excitation at 488 nm, obvious red fluorescence was observed in the HUVEC cytoplasm and nucleus, indicating that AIEEVs were internalized into the cells (Figure 2A, C). Moreover, as shown in Figure 2B, the intensity of the fluorescence signals was dependent on the concentration of the AIEgens. Notably, there was no significant difference in the fluorescence intensity between EVs labeled with 4 μM and 8 μM AIEgens; therefore, a concentration of 4 μM AIEgens was selected for the following experiments. As cluster of differentiation 31 (CD31) is a glycoprotein particularly expressed on HUVEC cell membrane, we then performed CD31 immunofluorescence staining to further confirm the internalizing of AIE-EVs.35 As shown in Figure 2D, red fluorescence signals of AIE-EVs were not observed in the cell membrane, which did not colocalize with green fluorescence signals of CD31. All the above results confirmed that DPA-SCP can precisely and specifically label the MSC-EVs in vitro. As a control, AIEgens (4 μM) were incubated with HUVECs for 2 hours. Under the imaging conditions, red fluorescence was observed only in the cytoplasm (Figure S5). The in vitro toxicity of AIE-EVs was finally examined using MTT cell viability assays. Figure 2E revealed that EVs labeled with AIEgen concentrations ranging from 1 μM to 8 μM showed no significant cytotoxicity and exhibited good biocompatibility with the HUVECs.

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Labeling Efficiency and Photostability of AIEgens. We next evaluated the labeling efficiency of DPA-SCP for EVs using the most popular commercial EV tracker PKH26 and DiI as the references. 36, 37 EVs labeled with 4 μM of DPA-SCP, PKH26 or DiI were first examined by NTA. NTA analysis in the presence of a low-pass 500 nm filter showed the majority (~86.9%) of AIE-EVs were fluorescent (Figure S6), whereas no fluorescence was observed when only DPA-SCP existed (Figure S7). In contrast to DPA-SCP, only 50.7% PKH26-EVs and 39.7% DiI-EVs were fluorescent (Figure S6). Flow cytometry was then performed to verify the labeling efficiency of DPA-SCP at designated time intervals. The data from Figure 3A revealed that the labeling efficiency of DPA-SCP for EVs was 85.3% at 1 hour after labeling, which was in consistent with the NTA results. Quantitative analysis showed that the labeling rate slowly decreased as the time elapses (Figure 3B). On day 1, the labeling efficiency was up to 73.1%, suggesting that DPA-SCP is firmly bound with the EVs. Notably, the labeling rate of DPA-SCP was above 60% before 3 days. On day 5 after labeling, the labeling efficiency remained 24.9%. We also compared the tracking capacity of DPA-SCP with PKH26 and DiI using CLSM. EVs were incubated with 4 μM of AIEgens, PKH26 or DiI for different time, followed by incubation with HUVECs for additional 48 hours. The fluorescence signals were subsequently observed by CLSM. On day 1 after labeling, the fluorescence signals were observed for all the internalized EVs (Figure 3C). In contrast to DPA-SCP, PKH26 and DiI formed obvious dye aggregates. Quantitative analyses of the CLSM images on day 1 indicated that the mean fluorescence intensity from AIE-EVs was ~1.59


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and ~4.5-fold higher than that from PKH26-EVs and DiI-EVs, respectively (Figure 3D). As shown in Figure S8, the results of CD31 immunostaining also showed that AIE-EVs exhibited highest fluorescence intensity in the cytoplasm and nucleus than that from PKH26-EVs and DiI-EVs. On day 5 after labeling, only very weak fluorescence was detectable in the PKH26-EVs and DiI-EVs, whereas intense and homogeneous fluorescence signals were still observed for AIE-EVs (Figure 3C). All these results demonstrated that DPA-SCP possesses a favorable labeling efficiency and excellent photostability for EV tracking in vitro. Non-covalent incorporating lipophilic dyes such as PKH26 and carbocyanine dyes such as DiI are most commonly used to label EVs. However, these dyes suffer from a high quenching coefficient, resulting in a short half-life and easy quenching. Additionally, they formed dye aggregates in aqueous solutions similar to EVs, resulting in errors in EV uptake experiments. DPASCP labeling offered a simple labeling procedure, good biocompatibility, high labeling efficiency and negligible photobleaching, which circumvented the inherent limitation of commercial probes.

In Vivo Distribution and Toxicity of AIE-EVs. Basing on the preferential characteristics of DPA-SCP labeling for EVs in vitro, we next addressed the distribution and toxicity of AIE-EVs in vivo. First, 0.1 mL of 100 μg AIE-EVs was injected through the tail vein into healthy FVB mice, followed by monitoring the distribution at designated time intervals. As shown in Figure 4A and B, the AIE-EVs were mainly enriched in the liver and exhibited a time-dependent distribution profile in the healthy 10


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mice. The fluorescence signals were observed 1 hour post-injection and peaked on day 1 in the liver. Then the intensity of fluorescence signals gradually decreased over time, but still clearly distinguishable in the liver until day 6. On day 12 post-injection, almost no detectable fluorescence signals were observed in the liver but existed in the kidneys. Ex vivo imaging of organs dissected after the in vivo experiments revealed the highest fluorescence signals in the liver, followed by the spleen, lungs, kidneys, and heart (Figure 4C, D). In contrast, 0.1 mL AIEgens injected through the tail vein into healthy FVB mice exhibited a different signal distribution (Figure S9A). As shown in Figure S9B, the fluorescence intensity in the liver peaked on day 1, but the fluorescence signals were lower than those of AIE-EVs, and clear fluorescence signals were still observed in the liver on day 12 post-injection. There was no noticeable body weight loss for either AIEgen-treated mice or AIEEV-treated mice relative to the untreated control group (Figure S10). Subsequently, histological analyses were carried out on important normal organs including the heart, liver, spleen, lungs and kidneys from each group. As shown in Figure 4E, H&E-stained slices assessed by three independent pathologists suggested that the AIE-EVs barely caused any significant lesions in the normal organs. Finally, a series of blood chemistry tests to determine liver function, kidney function, hemoglobin albumin globulin and albumin ratio were performed after sacrifice on day 12. As shown in Figure S11, there was no distinctive difference between the AIE-EV-treated and untreated groups. These data demonstrated that DPA-SCP can precisely and quantitatively report the fate of EVs for 12 days and is very safe for in vivo applications. 11


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In Vivo Tracking of AIE-EVs in ALI Mice. ALI will develop into end-stage liver disease if not treated promptly and appropriately.38 There are no effective treatments for end-stage liver disease except orthotropic liver transplantation (OLT) or artificial liver therapies. However, the shortage of available donor livers and multiple postoperative complications limit their use. Recently, MSC-derived EVs have shown great potential in the treatment of liver disease. Compared with MSCs, MSC-derived EVs have a much lower propensity to trigger immune responses and a reduced risk of ectopic engraftment.39-42 Therefore, we continued to examine the tracking ability of DPA-SCP for hP-MSC-derived EVs in a mouse ALI model utilizing noninvasive fluorescence imaging techniques. As shown in Figure 5A, fluorescence signals were observed in the liver regions on day 1 in both AIE-EV and AIEgen groups. However, more clustered and stronger signals were detected in the AIE-EV group, and scattered signals were observed in the AIEgen group. Subsequent signal analysis of the liver revealed that the fluorescence signal of the AIE-EV groups was approximately 2.5-fold stronger than that of the AIEgen group (Figure 5B). Ex vivo imaging of the dissected organs also revealed that the highest fluorescence signals in the liver were all observed in the AIE-EV group on day 1 and day 7 (Figure 5C). Quantitative analyses of the fluorescence intensity in the liver on day 7 revealed a signal approximately 1.5-fold larger in the AIE-EV groups than in the AIEgen groups (Figure 5D). Furthermore, on day 7 post-administration, mice were sacrificed, and the liver tissues were dissected and sliced for CLSM observation. As shown in Figure 5E, AIE-EVs were still clearly 12


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detected as punctate in the perinuclear region of the liver cells on day 7, indicating the stable EV labeling property of DPA-SCP in the ALI model. In contrast to AIE-EVs, AIEgens seriously self-aggregate in the liver. Collectively, these results indicated that EVs derived from hP-MSCs specifically targeted the liver injury sites. Moreover, DPASCP realized the real-time, long-term and precise monitoring of EVs in the ALI mice without influencing the therapeutic efficacy of EVs.

Inhibited Inflammatory and Enhanced Proliferative Effects of AIE-EVs In Vivo. Compared with the AIEgen- or PBS-treated groups, the elevation of serum AST and ALT levels was significantly reduced by AIE-EV treatment (Figure S12). We next explored the underlying mechanism of the positive effects of AIE-EVs. MSC-EVs have been proposed to play a curial role in tissue regeneration by modulating cell inflammation and promoting cell proliferation;43-45 therefore, we next investigated the anti-inflammatory effect of AIE-EVs in the mouse ALI model. The histopathology of liver tissues was analyzed. Figure 6A showed that the liver of both the AIEgen and the PBS group exhibited dilation of the sinusoidal space with inflammatory cell infiltration; however, the liver of the AIE-EV group showed an almost normal architecture with no necrosis, similar to those of the control mice. Immunostaining of TNF-α was performed to evaluate infiltrating inflammatory cells. TNF-α expression was significantly decreased in the AIE-EV group compared with the other groups (Figure 6B, C). Similar results were obtained by measuring the gene expression of the pro- and anti-inflammatory cytokines, including TNF-α, IL-1β, IL-10 13


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and IL-4 by using qRT-PCR analysis (Figure 6D). These findings consistently indicated an anti-inflammatory effect of AIE-EVs on the mouse ALI model. Next, we examined whether AIE-EVs could promote the proliferation of liver cells, and Ki67 (cell nuclear antigen) immunostaining was performed. The number of Ki67+ cells was markedly higher in mice injected with AIE-EVs than in those injected with PBS or AIEgens (Figure 7A, B). Consistent results were observed in qRT-PCR analysis of the proliferation-related genes HGF, EGF IGF-1 and FGF-2 (Figure 7C). Furthermore, the liver cell apoptosis genes Bad, Bax, Caspase 8 and Fas were assessed by qRT-PCR analysis. As shown in Figure 7D, AIE-EV administration inhibited the expression of cell apoptosis genes compared with the PBS and AIEgen groups, which was in accordance with the Western bolt results of Caspase 3 and 8 (Figure S13). Liver fibrosis is a direct consequence of injury and results in a progressive loss of liver function. Masson staining demonstrated a significant reduction in the fibrotic area of the AIE-EV treatment group compared with the other groups (Figure S14). All these data suggested that DPA-SCP did not influence the regenerative capacity and therapeutic efficacy of MSC-EVs in vivo; moreover, AIE-EVs could protect against liver injury by modulating inflammation, promoting cell proliferation and inhibiting apoptosis. MSC transplantation has emerged as a promising strategy for treating liver disease through tissue repair and immune regulation.46, 47 An increasing number of results have shown that MSCs possess therapeutic effects on ameliorating acute and chronic liver damage.48, 49 In addition, some studies have shown that MSC therapies can prevent the 14


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progression of end-stage liver disease and treat advanced fibrosis liver disease.46, 50 However, MSC transplantation inevitably faces many potential risks, such as ethical issues and immune responses. The surgical donor procedure in most commonly used autologous MSC transplantation can injure the donor and result in limited cell number harvested, restricting the practical clinical application. Recently, several studies have shown that MSC-derived EVs exhibit similar functions to their parent MSCs, including homing, modulating inflammation and repairing tissue damage. MSC-EVs can provide beneficial effects parallel to those of MSC transplantation. In the present study, we successfully demonstrated the homing of MSC-EVs to the liver injury sites. We also verified that the transplantation of MSC-EVs alleviated hepatic inflammation, enhancing cell survival and prevented the development of liver fibrosis in CCl4-induced ALI model. It is worthy mention that AIEgens provide an effective means for comprehensively tracing and evaluating the therapeutic efficiency of MSC-EVs throughout the liver regenerative process.

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CONCLUSIONS In conclusion, we have developed an AIEgen, DPA-SCP, to directly label EVs without influencing their native biological structures and functions. The biocompatible DPA-SCP allows real-time visualizing and monitoring EV trafficking and exhibits superior brightness, negligible photobleaching, excellent photostability to the most popular commercial EV trackers. We also demonstrated that DPA-SCP can safely, precisely and quantitively report the fate and therapeutic effects of EVs in liver regeneration. Furthermore, AIE-EVs facilitate live regeneration by promoting cell survival, inhibiting inflammatory responses and attenuating liver fibrosis. Our favorable labeling strategy enables non-invasive, real-time and precise tracking of EV fate and behaviors in live systems, serves as an efficient alternative to the commercial probes, and providing much insight for both basic and applied research of EVs.

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METHODS Materials. All chemicals and reagents were purchased from Sigma-Aldrich unless otherwise specified and used without further purification. Dulbecco's modified eagle's medium (DMEM)/F12, fetal bovine serum (FBS), antibiotics (penicillin-streptomycin) and phosphate buffered saline (PBS) were purchased from Gibco (Grand Island, NY). Endothelial growth medium-2 (EGM-2) was purchased from Lonza (Walkersville, MD). Distilled deionized water was obtained from a Millipore Milli-DI water purification system.

AIEgen Characterization. The AIEgen, namely, DPA-SCP, was synthesized as we previously described.32 The chemical structure of DPA-SCP was verified by NMR (Bruker AV 400) and HRMS (GCT Premier CAB 048). Absorption spectra were measured using a Varian Cary 50 UV-Vis spectrophotometer. Steady-state fluorescence spectra were recorded on a Perkin-Elmer LS 55 spectrofluorometer with a xenon discharge lamp excitation.

Cell Culture. Human placenta-derived MSCs (hP-MSCs) and human umbilical vein endothelial cells (HUVECs) were harvested and cultured as we previously described.51 The hP-MSCs were cultured in complete DMEM/F12 with 100 U/ml penicillin-streptomycin and 10% FBS at 37 °C in a humidified incubator with 5% CO2. hP-MSCs between passages 3 and 8 were used for subsequent experiments. HUVECs were cultured in EGM-2. EV-depleted FBS was prepared as follows: FBS was filtered 17


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using a 100 nm filter (Millipore, Merck, Germany), then centrifuged at 100,000 g for 16 hours and filtered again using a 100 nm filter.

Flow Cytometric Characterization of hP-MSCs. The hP-MSCs were dissociated with 0.25% trypsin-EDTA (Gibco, Grand Island, NY) and then washed with PBS containing 2% FBS. Afterwards, the cell suspensions were incubated with fluorescence conjugated antibodies including CD44 and CD90 (Abcam, Cambridge, MA) or unstained control for 30 min at room temperature. Then the cells were washed with PBS and resuspended in FACS buffer. The FACS analysis was performed using a FACS CaliburTM flow cytometer (BD Biosciences), and the data were analyzed using the Cell Quest Pro software (BD Biosciences).

EV Isolation. EVs were isolated as we previously described.51 Supernatant was collected from hP-MSCs that were cultured in medium containing EV-depleted 10% FBS for 48 hours and was subsequently subjected to sequential centrifugation steps at 500 ×g for 10 minutes to remove cells, at 2,000 ×g for 20 minutes to remove apoptotic bodies and at 5,000 ×g for 30 minutes to remove cell debris. The resulting supernatant was then filtered using 0.2 μm filters (Millipore, Merck, Germany), and EVs were harvested by centrifugation at 130,000 ×g for 2 hours in a SW32 Ti rotor (Beckman Coulter, L-100XP Ultracentrifuge, CA). The pellet was resuspended in PBS and subsequently ultracentrifuged at 130,000 ×g for another 2 hours to remove the contaminating proteins. 18


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EV Labeling with AIEgens. Different concentrations (1 μM, 2 μM, 4 μM and 8 μM) of AIEgens were added to the isolated EVs suspended in PBS. After 2 hours of incubation at 37 °C, the mixture was ultracentrifuged at 130,000 ×g in PBS for another 2 hours. The supernatant containing the unbound AIEgens was removed, and then the obtained pellet (AIE-EVs) was dissolved in PBS again. These AIE-EVs were subsequently used for in vitro and in vivo assays.

EV Characterization. TEM. TEM (Talos F200C, Thermo Fisher, MA) was performed at 200 kV to visualize and examine the morphology of EVs and AIE-EVs. Samples were deposited on copper grids covered with a carbon support film (Zhongjingkeyi Technology, Beijing, China) and dried for 2 minutes at room temperature. The excess fluid was removed with a piece of filter, and the samples were negatively stained with 2% uranyl acetate for 30 seconds. These samples were air-dried for 60 minutes, and images were captured. NTA. The size, concentration and zeta potential of EVs and AIE-EVs were determined using NTA (Particle Metrix, Germany). The measurement parameters were set using 100 nm polystyrene-latex beads as standards. EVs and AIE-EVs were diluted 4000- and 2000-fold, respectively, using distilled deionized water, to achieve between 20 and 100 objects per frame. Each sample was measured in triplicate at the camera setting with an acquisition time of 60 seconds. 19


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Western Blot. The protein concentrations of EVs and AIE-EVs were determined using a bicinchoninic acid (BCA) Protein Assay Reagent (Pierce, Rockford, IL) according to the manufacturer’s instructions. EVs were lysed with ice-cold lysis buffer [150 mM NaCl, 50 mM Tris-Cl (pH 7.5), 1 mM EDTA, 1 % NP-40, 0.1 % SDS, 0.25 % sodium deoxycholate, 1 mM PMSF, 1 mM β-glycerophosphate, 1 mM NaF, 1 mM Na3VO4 and protease inhibitor cocktail tablets (Roche Molecular BioChemicals, Indianapolis, IN). Aliquots containing 30 μg of protein were mixed with loading buffer, followed by denaturation at 95 °C for 5 minutes. Proteins were separated by 12 % sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE; Invitrogen, Carlsbad, CA) and transferred to polyvinylidene difluoride (PVDF; Millipore, Watford, UK) membranes. The membranes were blocked with 5 % nonfat milk in Tris-BufferedSaline/Tween-20 (TBST) buffer (20 mM Tris-HCl, pH 7.6, 136 mM NaCl and 0.1 % Tween-20) and then probed overnight with primary antibodies: CD63 (1:1000; Abcam, Cambridge, MA), CD9 (1:1000; Abcam, Cambridge, MA), ALIX (1:1000; Abcam, Cambridge, MA), TSG101 (1:1000; Abcam, Cambridge, MA) and GM130 (1:1000; Abcam, Cambridge, MA). The membranes were then incubated with horseradish peroxidase-conjugated secondary antibody (1:5000; System Bioscience, Mountain View, CA) for 2 hours at room temperature. Protein signals were visualized using the West Pico Chemiluminescent Substrate Kit (Pierce, Rockford, IL).

Cytotoxicity Assay. HUVECs were seeded at a density of 5 x103 cells/well into 96well plates in EGM-2. Then, 100 μg of EVs, either unlabeled or labeled with different 20


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concentrations of AIEgens (1 μM, 2 μM, 4 μM and 8 μM), was added to the HUVECs. After 48 hours of incubation, 20 μL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) solution (0.5 mg/ml) was added to each well and cultured for an additional 4 hours. Afterwards, the supernatant was removed, and dimethyl sulfoxide (DMSO) was added to each well to dissolve the formazan crystals. The absorbance of MTT at 490 nm was measured using a microplate reader (Biotek, VT).

EV Tracking In Vitro. HUVECs were seeded at a density of 1 x105 cells/well into cell climbing slices in EGM-2. Then, 100 μg of EVs labeled with different concentrations of AIEgens (1 μM, 2 μM, 4 μM and 8 μM) was added and incubated in the HUVECs for 48 hours at 37 °C. The supernatant was then removed, and the cells were washed with PBS three times. Afterwards, the cells were fixed using 4% paraformaldehyde (PFA; Sangon Biotech, Shanghai, China) for 10 minutes and then washed with PBS three times. The fixed cells were incubated with primary antibody against CD31 (1:200; Abcam, Cambridge, MA) overnight at 4°C, then incubated with Alexa Fluor 488 goat anti-mouse IgG or Alexa Fluor 594 goat anti-mouse IgG (Invitrogen, Grand Island, NY). Subsequently, the cell nuclei were counterstained with 4,6-diamidino-2-phenylindole (DAPI) for 10 minutes and imaged using CLSM; Leica TSC SP8, Germany) with excitation at 488 nm and signal collection from 500 to 700 nm. Commercial dyes DiI (4 μM; λex = 551 nm, λem = 567 nm) and PKH26 (4 μM; λex = 565 nm, λem = 594 nm) were assessed following the same procedures.

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Flow Cytometric Analysis of Labeling Efficiency. The labeling efficiencies of DPA-SCP for EVs at different time were quantified by a FACSCalibur flow cytometer (BD Biosciences, NY). The background noise of buffer solution that was used to dissolve the EVs was first evaluated. AIEgens were then used as a negative control for gating. Unlabeled EVs were used to set the voltages and thresholds for measurements, as well as to provide references for gating in the forward-scatter (FSC) and side-scatter (SSC) channels. To measure the percentage of AIEgen-positive EVs among the total EVs, AIE-EVs were analyzed with flow cytometry at time points of 1 hour, 1 day, 2 days, 3 days and 5 days. Illumination was provided by a standard 488 nm red laser, and fluorescence was collected through a FITC filter. Data were processed with FACSDiva software.

Animals. All mouse experiments were performed according to our experimental protocols approved by the Tianjin Committee for the Use and Care of Laboratory Animals, and overall project protocols were approved by the Animal Ethics Committee of Nankai University. Female FVB mice (8-10 weeks old) were purchased from the Laboratory Animal Center of the Academy of Military Medical Sciences (Beijing, China). Mice were labeled with numbers on their ear tags or tails using permanent markers and randomized into control and treatment groups. All animals and resulting samples were assigned a number that did not reveal the group allocation, allowing analyses to be performed by blinded investigators.

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EV Distribution and Toxicity In Vivo. The mice were anesthetized and placed on an animal plate heated to 37 °C. A total of 0.1 mL containing 100 μg AIE-EVs was injected through the tail vein into each healthy FVB mouse (n = 5). The time-dependent biodistribution of the AIE-EVs in the mice was imaged using an IVIS Lumina imaging system (Xenogen Corporation, Hopkinto, MA). Light with a central wavelength of 488 nm was selected as the excitation source. In vivo spectral imaging from 500 nm to 700 nm (10 nm step) was carried out with an exposure time of 200 ms for each image frame. As a control, AIEgens were also injected through the tail vein into FVB mice following the same procedure. The intensity of fluorescence signals was quantified by average radiance from a fixed-area region of interest (ROI) over the liver. Furthermore, at designated time intervals, the AIE-EV-injected mice were sacrificed, and the main organs (heart, liver, spleen, lungs, kidneys) were isolated, and ex vivo fluorescence imaging was performed using the imaging system. The mice were weighed every two days to observe the dynamic changes in weight in each group. For the in vivo toxicity study, on day 14 post-administration, all of the mice were sacrificed, and the vital organs, including the heart, liver, spleen, lungs and kidneys, were excised for histology analyses. Blood samples were harvested for blood chemistry analyses using a biochemistry analyzer (Randox-RX-DaytonU98a).

Real-time Quantitative Polymerase Chain Reaction (RT-qPCR). Total RNA was extracted from tissues using Trizol (Invitrogen, Grand Island, NY) according to the manufacturer’s manuals. RNA concentrations were determined using a UV 23


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spectrophotometer at an absorbance ratio of 260 to 280 nm (A260/280). Reversetranscription was performed using 1 μg of total RNA in a 25-µl reaction volume at 42 °C for 60 min using a PCR machine (Bio-rad Laboratories). All qPCR experiments were performed on a real-time PCR machine (Bio-Rad Laboratories) with the QuantiTect SYBR Green PCR Kit and gene-specific primers. Gene expression was quantified by the comparative cycle threshold (Ct) method. The relative amounts of target gene mRNA were determined by subtracting the Ct values for these genes from the Ct value for the housekeeping gene GAPDH (△Ct). The data are presented as 2(△△CT).

The primer sequences used in this study are listed in Table S1.

Animal Model. Mice were housed in a temperature-controlled sterile animal facility with 12-hour light/dark cycles and free access to food and water. An acute liver injury (ALI) model was established as previously described.52 In brief, carbon tetrachloride (CCL4) (2 ml/kg diluted 1:4 in mineral oil; Sigma-Aldrich) was intraperitoneally injected twice in one week, with the control mice receiving mineral oil vehicle only.

AIE-EV Tracking in ALI Mice. The ALI mice were randomly assigned to three groups and injected through the tail vein with PBS (n = 5), AIEgens (n = 5) or AIEEVs (n = 5). 100 μg AIE-EVs suspended in 100 μL PBS was injected into the AIE-EV group. Injection with 100 μL PBS or AIEgens served as controls. The IVIS Lumina imaging system was utilized by placing the anesthetized mice on the equipped platform 24


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(λex = 488 nm, signal collection: 500 to 700 nm (10 nm step), exposure time = 200 ms, scans: day 0, 1, 3, 5 and 7 post-injection, respectively). The intensity of fluorescence signals was quantified by average radiance from a fixed-area ROI over the liver. Furthermore, ex vivo imaging was performed on days 1 and 7. On day 7 postadministration, the mice were sacrificed, and the livers from each group were isolated for histological observations and RT-qPCR analyses of inflammation-, proliferationand apoptosis-related genes.

Histology and Immunohistochemistry. On day 7 or 12 post-administration, all mice were sacrificed, and the livers were excised. Some liver tissues were immediately fixed in 4% PFA overnight. Afterwards, these tissues were embedded in paraffin, sectioned at 5 μm and then subjected to hematoxylin and eosin (H&E) or Masson’s trichrome staining. The slices obtained were examined with an optical microscope (OLYMPUS BX51, Japan). Furthermore, another fraction of liver tissue was embedded into OCT compound (Sakura Finetek, Japan) and cut into micron-thick frozen sections (10 μm) for immunofluorescence staining. The sections were incubated with primary antibody against Ki67 (1:200; BD Biosciences, NY) or TNF-α (1:200; BD Biosciences, NY overnight at 4℃, then incubated with Alexa Fluor 488 goat anti-mouse IgG or Alexa Fluor 488 goat anti-rat IgG (Invitrogen, Grand Island, NY). Cell nuclei were counterstained

with

DAPI.

ImageJ

software

was

used

to

binarize

the

immunofluorescence images taken with the same excitation, gain and exposure settings as previously described.53 The numbers of Ki67- and TNF-α-positive cells were 25


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counted by a blinded investigator in ten randomly selected areas using a fluorescence microscope (200 x).

Statistical Analysis. All data are expressed as the mean ± S.D. (standard deviation). An independent t-test was used for two-group comparisons, and one-way ANOVA was used for multiple-group comparisons with a suitable post hoc test. Significant differences were defined as P

In Vivo Real-Time Imaging of Extracellular Vesicles in Liver

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