- Email: [email protected]
Real-Time Assessment of Extracellular Vesicles by Intravital Microscopy Imaging
10th IFAC Symposium on Biological and Medical Systems São Paulo, Brazil, September 3-5, 2018 10th Paulo, IFAC Symposium on Biological and Medical Systems São Brazil, September 3-5, 2018 10th IFAC Symposium on Biological and Medical Systems Available online at www.sciencedirect.com São Paulo, Brazil, September 3-5, 2018 10th IFAC Symposium on Biological and Medical Systems São Paulo, Brazil, September 3-5, 2018 São Paulo, Brazil, September 3-5, 2018
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PapersOnLine 51-27 (2018) 22–23 Real-Time Assessment ofIFAC Extracellular Vesicles by Intravital Microscopy Imaging Real-Time Assessment of Extracellular Vesicles by Intravital Microscopy Imaging Real-Time Vesicles by Intravital Microscopy Imaging Real-Time Assessment Assessment of of Extracellular Extracellular Vesicles Horacio Cabral* by Intravital Microscopy Imaging Real-Time Assessment of Extracellular Vesicles Horacio Cabral* by Intravital Microscopy Imaging
Horacio Cabral* Horacio Cabral* * Department of Bioengineering, Graduate School of Engineering, The University of Tokyo, Horacio Cabral* * Department of Bioengineering, Graduate School of Engineering, The University of Tokyo, Tokyo, Japan (Tel: +81-3-5841-7138; e-mail: [email protected]). * Department Bioengineering, Graduate School of Engineering, The University of Tokyo, Tokyo, of Japan (Tel: +81-3-5841-7138; e-mail: [email protected]). * Department of Bioengineering, Graduate School of Engineering, The University of Tokyo, Tokyo, Japan (Tel: +81-3-5841-7138; e-mail: [email protected]). * Department Bioengineering, Graduate School of Engineering, The University of Tokyo, Tokyo, of Japan (Tel: +81-3-5841-7138; e-mail: [email protected]). Tokyo, Japanvesicles (Tel: +81-3-5841-7138; e-mail: [email protected]). Abstract: Extracellular (EVs), such as exosomes, are essential players in cell-cell Abstract: Extracellular vesicles (EVs), such as exosomes, are essential players in cell-cell communication, which are vesicles being considered developing novel drug applications. there Abstract: Extracellular (EVs), for such as exosomes, are delivery essential players inThus, cell-cell communication, which are being considered for developing novel drug delivery applications. there Abstract: Extracellular vesicles (EVs), such as exosomes, are essential players inThus, cell-cell is much interest in developing reliable methods for tracing EVs in biological settings in real-time. communication, which are vesicles being considered for developing novel drug delivery applications. Thus, there Abstract: Extracellular (EVs), such as exosomes, are essential players in cell-cell is much interest in developing reliable methods for tracing EVs in biological settings in real-time. communication, which are being considered for developing novel drug delivery applications. Thus, there Current methods are based on lipophilic fluorescent dyes,inthough this labeling not been is much standard interestwhich in developing reliable methods for tracing EVs biological settings has inThus, real-time. communication, are being considered for developing novel drug delivery applications. there Current methods are based on lipophilic fluorescent dyes, this labeling has not been is much standard interest in result developing reliable methods for EVs inthough biological settings in real-time. validated and may in biased understanding of tracing EVs. Herein, we developed labeling methods Current standard methods are based on lipophilic fluorescent dyes, though this labeling has not been is much interest in developing reliable methods for tracing EVs in biological settings in real-time. validated and may result inthebiased understanding of EVs. Herein, we developed labeling methods Current standard methods are based on lipophilicthe fluorescent dyes, though this labeling has not been directed to faithfully follow EVs for assessing fate in biological settings both in vitro and in vivo. validated may result inthebiased understanding of EVs. Herein, we developed methods Current standard methods are based on lipophilic fluorescent dyes, though this labeling been directed toand faithfully follow EVs for assessing the fate in biological settings both inlabeling vitrohas and in vivo. validated and may result in biased understanding of EVs. Herein, developed labeling methods Our results showed that by installing fluorescent probes on thebiological surface ofwe EVs byboth conjugation to not the directed to faithfully follow the EVs for assessing the fate in settings in vitro and in EVs’ vivo. validated and may result in biased understanding of EVs. Herein, we developed labeling methods Our results showed that by installing fluorescent probes on the surface of EVs by conjugation to the EVs’ directed to faithfully follow the EVs for assessing the fate in biological settings both in vitro and in vivo. amino groups, it is possible to preserve the EVs concentration and stability. we found that the Our results showed that by installing fluorescent probes on in thebiological surface ofsettings EVsMoreover, byboth conjugation to the directed to faithfully follow thepreserve EVs for assessing the fate in we vitro and in EVs’ vivo. amino groups, itour is possible to the EVsbloodstream, concentration and stability. Moreover, found that the Our results showed that by installing fluorescent probes on the surface of EVs by conjugation to the EVs’ EVs labeled by method were stable in the while the EVs labeled with the conventional amino groups, itour is possible to preserve the EVsbloodstream, concentration and stability. Moreover, we found that the Our results showed that by installing fluorescent probes on the surface of EVs by conjugation to the EVs’ EVs labeled by method were stable in the while the EVs labeled with the conventional amino groups, it is possible to preserve the EVs concentration and stability. Moreover, we found that lipophilic dyes were immediately removed frombloodstream, circulation, while highlythe accumulating inwith the lungs. Finally, the by EVs labeled by our method were stable in the EVs labeled the conventional amino groups, is possible to preserve the EVs concentration and the stability. Moreover, we that4T1 the lipophilic dyes immediately removed from circulation, highly accumulating inwith thebreast lungs. Finally, by EVs labeled byitwere our method were stable in the bloodstream, while EVs labeled thefound conventional using our new labeling method for imaging EVs, we found that EVs from murine cancer lipophilic dyes were immediately removed from circulation, highly accumulating inwith thebreast lungs. Finally,4T1 by EVs labeled by our method were stable in the bloodstream, while the EVs labeled the conventional using our new labeling method for imaging EVs, we found that EVs from murine cancer lipophilic dyes were immediately removed from circulation, highly accumulating in the lungs. Finally, by accumulated along the brain capillaries, which correlate withthat theEVs incidence of brainbreast metastasis from using our new labeling method for imaging EVs, we found from murine cancer 4T1 lipophilic dyes were immediately removed from circulation, highly accumulating in the lungs. Finally, by accumulated the brain capillaries, which correlate withthat theEVs incidence of brainbreast metastasis using our newalong labeling method for imaging EVs, we found from murine cancer from 4T1 breast tumors. accumulated along the brain capillaries, which correlate with the incidence of brain metastasis from using our new labeling method for imaging EVs, we found that EVs from murine breast cancer 4T1 breast tumors.along the brain capillaries, which correlate with the incidence of brain metastasis from accumulated breast accumulated along the brain capillaries, which correlate with the by incidence of brain metastasis from © 2018,tumors. IFAC (International Federation Automatic Control)technology; Hosting Elsevier Ltd. All rights reserved. Keywords: Pharmacokinetics and drugofdelivery; Medical Biomedical imaging systems and breast tumors. Keywords: Pharmacokinetics and drug delivery; Medical technology; Biomedical imaging systems and breast tumors. image processing Keywords: Pharmacokinetics and drug delivery; Medical technology; Biomedical imaging systems and image processing Keywords: Pharmacokinetics and drug delivery; Medical technology; Biomedical imaging systems and image processing Keywords: Pharmacokinetics and drug delivery; Medical technology; Biomedical imaging systems and image processing image processing Cell Microvesicular 1. INTRODUCTION Body Cell Microvesicular 1. INTRODUCTION Cell Microvesicular Body Cell Microvesicular 1. INTRODUCTION Body Extracellular vesicles (EVs; Fig. 1), such as exosomes, are 1. INTRODUCTION Body Cell Microvesicular Extracellular vesicles (EVs; Fig. 1), such as exosomes, are 1. as INTRODUCTION gaining much vesicles interest mediators of cell-cell interactions, as Body Extracellular (EVs; Fig. 1), such as exosomes, are gaining much interest as mediators of cell-cell interactions, as Extracellular vesicles (EVs; Fig. 1), such as exosomes, are well as potential drug carriers. While EVs naturally contain gaining much interest mediators of cell-cell interactions, as Extracellular (EVs; Fig. 1), such as exosomes, are well askinds potential drugas carriers. While EVsthey naturally gaining much interest as mediators of cell-cell interactions, as many ofvesicles proteins and nucleic acids, can be contain further well as potential drug carriers. While EVs naturally contain gaining much interest as mediators of cell-cell interactions, as many of proteins and nucleic acids, can be contain further well askinds potential drugbioactive carriers. While EVsthey naturally loaded with different molecules for diagnostic and many kinds of proteins and nucleic acids, they can be further well as potential drug carriers. While EVs naturally contain loaded with applications different molecules for diagnostic and many kinds of proteinsbioactive and nucleic they can be further (Conlan, etacids, al. 2017). Thus, major therapeutic loaded with applications different bioactive molecules for diagnostic and many kinds of proteins and nucleic acids, they can be further (Conlan, ettrace al. 2017). Thus, major therapeutic loaded with different bioactive molecules for diagnostic and Microvesicles Exosomes efforts have been dedicated to EVs in biological (Conlan, et al. 2017). Thus, major therapeutic applications loaded with different bioactive molecules for diagnostic and Microvesicles Exosomes efforts have been dedicated to EVs in biological (Conlan, ettrace al.in2017). Thus, major therapeutic applications 100-1000 nm 40-120 nm systems for evaluating their function situ. In particular, Microvesicles Exosomes efforts have been dedicated to ettrace EVs in biological 100-1000 nm 40-120 nm (Conlan, al. 2017). Thus, major therapeutic applications systems for evaluating their function in situ. In particular, Microvesicles Exosomes efforts have been dedicated to trace EVs in biological Extracellular Vesicles (EVs) lipophilic fluorescent dyes, such as PKH and DiR, are used 100-1000 nm 40-120 nm systems for evaluating theirsuch function in EVs situ. In particular, Microvesicles Exosomes efforts have been dedicated to trace in biological 100-1000 nm 40-120 nm Extracellular Vesicles (EVs) lipophilic fluorescent dyes, as PKH and DiR, are used systems for evaluating their function in situ. In particular, for tracking EVs in vivo (Hoshino, et al. 2015). However, Extracellular Vesicles (EVs) lipophilic fluorescent dyes, such as PKH and DiR, are used 100-1000 nm 40-120(EVs) nm systems for evaluating their function in situ. In particular, for tracking EVs in vivo (Hoshino, et al. 2015). However, Extracellular Vesicles lipophilic fluorescent dyes, such as PKH and DiR, are used theirtracking validityEVs remains unknown. Moreover, these However, dyes are for in vivo (Hoshino, et al. 2015). Extracellular Vesicles (EVs) Fig. 1. Extracellular vesicles lipophilic fluorescent dyes, such as PKH and DiR, are used their validity remains unknown. these dyes are for tracking EVs vivo (Hoshino, et al. 2015). However, usually dissolved ininethanol, whichMoreover, may affect the stability of Fig. 1. Extracellular vesicles their validity remains unknown. Moreover, these dyes are for tracking EVs in vivo (Hoshino, et al. 2015). However, usually dissolved in ethanol, which may affect the stability of Fig. 1. Extracellular vesicles their validity remains unknown. Moreover, these dyes are (Goldstein 1986). Also, lipophilic dyes bind to EV EVs usually dissolved in ethanol, which may affect thebind stability of Fig. 1. Extracellular vesicles their validity remains unknown. Moreover, these dyes are 1986). Also, lipophilic dyes to EV EVs (Goldstein usually dissolved in ethanol, which may affect the stability of Fig. 1. Extracellular vesicles 2. METHODS membrane by non-covalent partitioning, so they may be (Goldstein 1986). Also, lipophilic dyes bind to EV EVs usually dissolved in ethanol, which mayetaffect the stability of 2. METHODS membrane by non-covalent partitioning, so2012). they may be 1986). Also, lipophilic dyes bind to EV EVs (Goldstein easily detached from membrane (Li, al. Thus, to 2. METHODS membrane by non-covalent partitioning, so2012). they may be (Goldstein 1986). Also, lipophilic dyes bind to EV EVs easily detached from membrane (Li, et al. Thus, to 2. METHODS membrane by non-covalent so they may be avoid detached interference from the partitioning, labeling method, developing easily from membrane (Li, et al. 2012). Thus, to 2.1 Isolation and labeling of EVs 2. METHODS membrane by non-covalent partitioning, so they may be avoid interference from the labeling method, developing easily detached from membrane (Li, et al. 2012). Thus, to proper labeling strategies willlabeling be keymethod, for advancing the 2.1 Isolation and labeling of EVs avoid interference from the developing easily detached membrane (Li,key et method, al. Thus, the to 2.1 Isolation and labeling of EVs proper labeling strategies willlabeling be for2012). advancing avoid interference from the developing understanding of from EVs in biological and labeling of EVs breast cancer 4T1 cells or proper labeling strategies willlabeling besettings. keymethod, for advancing the 2.1 EVsIsolation were isolated from murine avoid interference from the developing understanding of EVs in biological settings. proper labeling strategies will be key for advancing the 2.1 Isolation and labeling of EVs breast cancer 4T1 cells or In this research, we aimed to develop an appropriate labeling EVs were isolated from murine understanding ofwe EVs in biological U87-MG differential proper labeling strategies besettings. key for advancing the human In this research, aimed towill develop aninappropriate labeling EVs were glioblastoma isolated from murine breast using cancer 4T1 cells or understanding of EVs in biological settings. method capable of tracking EVs in vitro and in vivo human glioblastoma U87-MG using differential EVs were isolated from murine breast cancer 4T1 cells or In this research, we aimed to develop an appropriate labeling ultracentrifugation. Nanoparticle Tracking Analysis (NTA) understanding of EVs in biological settings. human glioblastoma U87-MG using differential method capable of aimed tracking EVs in vitro andbelabeling inhighly vivo EVs In this research, we to develop aninappropriate were isolated from murine breast cancer 4T1 cells or settings in real-time. Such labeling method would ultracentrifugation. Nanoparticle Tracking Analysis (NTA) human glioblastoma U87-MG using differential method capable of tracking EVs in in vitro and in vivo performed to measure the size distribution of EVs. For In this research, we to develop aninappropriate ultracentrifugation. Nanoparticle Tracking Analysis (NTA) settings in real-time. Such labeling method would belabeling highly method capable of aimed tracking EVs in vitro and inorganvivo was human glioblastoma U87-MG using differential beneficial for studying the factors responsible for was performed to measure the size distribution of EVs. For ultracentrifugation. Nanoparticle Tracking Analysis (NTA) settings real-time. Such method would beinorganhighly labeling EVs, we used sulfo-Cy5-NHS ester by targeting method in capable of tracking EVs Thus, in in vitro and vivo performed to measure the size distribution of EVs. For beneficial for studying thelabeling factors responsible for settings in real-time. Such labeling method would be highly ultracentrifugation. Nanoparticle Tracking Analysis (NTA) specific accumulation of EVs. we focused our was labeling EVs, we used sulfo-Cy5-NHS ester by targeting was performed to measure the size distribution of EVs. For beneficial for studying the factors responsible for organamines on their surface. We incubated the mixture them settings in real-time. Such labeling method would be highly labeling EVs, we used sulfo-Cy5-NHS ester by targeting specific accumulation of EVs. Thus, we focused our beneficial for studying the factors responsible for organperformed to surface. measure the size distribution of EVs. For approach on using controlled covalent conjugation of was amines on theirwe We incubated the mixture them labeling EVs, used sulfo-Cy5-NHS ester by targeting specific accumulation of EVs. Thus, we focused our overnight, and removed the free dyes by ultrafiltration. We beneficial for studying the EVs. factors responsible for organamines on theirwe surface. We incubated the mixture them approach on using controlled covalent of specific accumulation of Thus, weconjugation focused our labeling EVs, used sulfo-Cy5-NHS ester by targeting fluorescent probes to amine moieties on the EVs surface in overnight, and removed the free dyes by ultrafiltration. We amines on their surface. We incubated the mixture them approach on using controlled covalent conjugation of used fluorescence correlation spectrometry (FCS) to count specific accumulation of EVs. Thus, weconjugation focused our and removed theWe free dyes by the ultrafiltration. We fluorescent probes to amine moieties onvalidated the EVs surface in overnight, approach on using controlled covalent of amines on their surface. incubated mixture them aqueous settings. The strategy was then in cellular used fluorescence correlation spectrometry (FCS) to count overnight, and removed the free dyes by ultrafiltration. We fluorescent probes to amine moieties onvalidated the conjugation EVs in surface in the number of dyes correlation on each EV spectrometry surface. approach on using controlled covalent of used fluorescence (FCS) to count aqueous settings. The strategy was then cellular fluorescent probes to amine moieties on the EVs surface in overnight, and removed the free dyes by ultrafiltration. We and animal models. Moreover, as one application of this new the number of dyes on each EV surface. used fluorescence correlation spectrometry (FCS) to count aqueous settings. The strategymoieties wasone then cellular fluorescent probes toMoreover, amine onvalidated the EVscancer surface in the and animal models. as application ofin this new number of dyes correlation on each EV spectrometry surface. aqueous settings. The strategy was then validated in cellular used fluorescence (FCS) to count labeling, we studied the role of EVs from breast cells the number of dyes on each EV surface. and animal models. Moreover, as one application ofinthis new aqueous settings. The strategy was then validated cellular labeling, we studied the role of EVs from breast cancer cells and animal models. Moreover, one application of this new the 2.2.number Intravital of imaging dyes on each EV surface. to target brain capillaries, asas breast tumors show high labeling, we studied the role of EVs from breast cancer cells and animal models. Moreover, as one application of this new 2.2. Intravital imaging to target we brain capillaries, asEVs breast tumors show cells high labeling, studied the role (Tominaga, of fromet breast cancer al. 2015). incidence of brain metastasis to target of brain capillaries, asEVs breast tumors show cells high 2.2. Intravital imaging labeling, studied the role (Tominaga, of fromet breast cancer al. 2015). incidence brain metastasis to target we brain capillaries, as breast tumors show high 2.2. Intravital imaging (Tominaga, et al. 2015). incidence of brain metastasis to target of brain as breast et tumors show high 2.2. Intravital imaging al. 2015). incidence braincapillaries, metastasis (Tominaga, Copyright © 2018 IFAC 22 incidence of brain metastasis (Tominaga, et al. 2015). 2405-8963 © 2018, IFAC (International Federation of Automatic Control) Hosting by Elsevier Ltd. All rights reserved. Copyright © 2018 IFAC 22 Peer review©under of International Federation of Automatic Copyright 2018 responsibility IFAC 22 Control. Copyright © 2018 IFAC 22 10.1016/j.ifacol.2018.11.601 Copyright © 2018 IFAC 22
IFAC BMS 2018 São Paulo, Brazil, September 3-5, 2018
Horacio Cabral et al. / IFAC PapersOnLine 51-27 (2018) 22–23
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occurring. The results showed the immediate formation of aggregates in blood for the PKH26-labeled EVs, and the mechanical trapping in the blood vessels in lungs, indicating the inadequacy of PKH26-labeling for tracing EVs (Fig. 4). Finally, we injected 4T1 derived EVs labeled with our method into mice and observed brain blood vessels directly in vivo. We observed these EVs gradually accumulated along the brain vasculature. This observation could be correlated with the high incidence of brain metastasis from breast tumors and the recent findings showing that exosomes from breast cancer disrupt the blood-brain barrier (Tominaga, et al. 2015).
For in vivo studies, sulfo-Cy5-labeled EVs and PKH26labeled EVs were intravenously injected into mice to check their stabilities in blood and their bio-distribution. In vivo tracking of EVs was performed by an intravital confocal micro-videography using a Nikon R1 microscope with fast scanning lasers. The blood circulation and the accumulation of EVs in the living tissues was then analyzed and quantified by analysis with ImageJ. 3. RESULTS 3.1 Isolation and labeling of EVs. EVs from 4T1 cells were isolated by differential ultracentrifugation. The particles size of 4T1-derived EVs was 185 ± 86 nm (Fig. 2), as determined by NTA. Moreover, round-shaped vesicles around 100 nm in diameter were confirmed by TEM (Fig. 2). Next, these EVs were labeled with sulfo-Cy5 NHS ester. These NHS ester dyes can bind to amines on EV surface by covalent bonds. FCS measurement showed 4 dyes bound to each EV. This labeling was mediated by covalent bond, and we hypothesized that it may show higher stability in blood than PKH lipophilic dyes. EVs were also labeled with PKH26 and used as control in our subsequent studies.
Fig. 4. In vivo imaging in lungs after injection of EVs-labeled with PKH26 (green) or sulfo-Cy5 (red). Dextran-blue was used as control (blue). Image at t = 0 s is just before injection 5. CONCLUSIONS We developed a method for stably labeling EVs by using sulfo-Cy5 NHS ester. While the conventional labeling method caused aggregates of EVs in blood, leading to their instantaneous seizing in lung capillaries, our labeling method allowed the EVs to stably circulate in the bloodstream without the formation of aggregates. By using our labeling method, we found that 4T1 cells-derived EVs accumulated along the vasculature in brain. Taken together, the labeling we developed here will promote the proper understanding of EVs’ behavior, and facilitate the use of EVs for therapy and diagnosis.
Fig. 2. Size distribution determined by NTA (left panel) and TEM image (right panel) of EVs. Scale bar 100 nm.
3.2 Intravital imaging of labeled EVs. The differently labeled EVs were injected into mice, and their fate was followed in real time by using intravital microscopy. PKH26-labeled EVs disappeared at 10 min post-injection, while EVs- labeled with sulfo-Cy5 circulated in blood for more than 2 h (Fig. 3). We also checked the bio-distribution of labeled EVs at 2 h post-injection, and found that high liver and spleen distribution for both labeling methods (data not shown). However, only PKH26-labeled EVs accumulated in lungs.
REFERENCES Conlan, R. S., Pisano, S., Oliveira, M. I., Ferrari, M. and Pinto, I. M. (2017). Exosomes as reconfigurable therapeutic systems. Trends in molecular medicine, 23(7), 636-650. Goldstein, D. B. (1986). Effect of alcohol on cellular membranes. Annals of Emergency Medicine, 15(9), 1013-1018. Hoshino, A., Costa-Silva, B., Shen, T. L., Rodrigues, G., Hashimoto, A., Mark, M. T. and Singh, S. (2015). Tumour exosome integrins determine organotropic metastasis. Nature, 527(7578), 329. Li, P., Zhang, R., Sun, H., Chen, L., Liu, F. and Jiang, X. (2012). PKH26 can transfer to host cells in vitro and vivo. Stem cells and development, 22(2), 340-344. Tominaga, N., Kosaka, N., Ono, M., Katsuda, T., Yoshioka, Y., Tamura, K. and Ochiya, T. (2015). Brain metastatic cancer cells release microRNA-181c-containing extracellular vesicles capable of destructing blood–brain barrier. Nature communications, 6, 6716.
Fig. 3. Blood circulation of EVs labeled with different methods. Fluorescent dextran was used as internal standard. We checked the real-time micro-distribution of sulfo-Cy5and PKH26-labeled EVs in lungs to appreciate what was 23
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PapersOnLine 51-27 (2018) 22–23 Real-Time Assessment ofIFAC Extracellular Vesicles by Intravital Microscopy Imaging Real-Time Assessment of Extracellular Vesicles by Intravital Microscopy Imaging Real-Time Vesicles by Intravital Microscopy Imaging Real-Time Assessment Assessment of of Extracellular Extracellular Vesicles Horacio Cabral* by Intravital Microscopy Imaging Real-Time Assessment of Extracellular Vesicles Horacio Cabral* by Intravital Microscopy Imaging
Horacio Cabral* Horacio Cabral* * Department of Bioengineering, Graduate School of Engineering, The University of Tokyo, Horacio Cabral* * Department of Bioengineering, Graduate School of Engineering, The University of Tokyo, Tokyo, Japan (Tel: +81-3-5841-7138; e-mail: [email protected]). * Department Bioengineering, Graduate School of Engineering, The University of Tokyo, Tokyo, of Japan (Tel: +81-3-5841-7138; e-mail: [email protected]). * Department of Bioengineering, Graduate School of Engineering, The University of Tokyo, Tokyo, Japan (Tel: +81-3-5841-7138; e-mail: [email protected]). * Department Bioengineering, Graduate School of Engineering, The University of Tokyo, Tokyo, of Japan (Tel: +81-3-5841-7138; e-mail: [email protected]). Tokyo, Japanvesicles (Tel: +81-3-5841-7138; e-mail: [email protected]). Abstract: Extracellular (EVs), such as exosomes, are essential players in cell-cell Abstract: Extracellular vesicles (EVs), such as exosomes, are essential players in cell-cell communication, which are vesicles being considered developing novel drug applications. there Abstract: Extracellular (EVs), for such as exosomes, are delivery essential players inThus, cell-cell communication, which are being considered for developing novel drug delivery applications. there Abstract: Extracellular vesicles (EVs), such as exosomes, are essential players inThus, cell-cell is much interest in developing reliable methods for tracing EVs in biological settings in real-time. communication, which are vesicles being considered for developing novel drug delivery applications. Thus, there Abstract: Extracellular (EVs), such as exosomes, are essential players in cell-cell is much interest in developing reliable methods for tracing EVs in biological settings in real-time. communication, which are being considered for developing novel drug delivery applications. Thus, there Current methods are based on lipophilic fluorescent dyes,inthough this labeling not been is much standard interestwhich in developing reliable methods for tracing EVs biological settings has inThus, real-time. communication, are being considered for developing novel drug delivery applications. there Current methods are based on lipophilic fluorescent dyes, this labeling has not been is much standard interest in result developing reliable methods for EVs inthough biological settings in real-time. validated and may in biased understanding of tracing EVs. Herein, we developed labeling methods Current standard methods are based on lipophilic fluorescent dyes, though this labeling has not been is much interest in developing reliable methods for tracing EVs in biological settings in real-time. validated and may result inthebiased understanding of EVs. Herein, we developed labeling methods Current standard methods are based on lipophilicthe fluorescent dyes, though this labeling has not been directed to faithfully follow EVs for assessing fate in biological settings both in vitro and in vivo. validated may result inthebiased understanding of EVs. Herein, we developed methods Current standard methods are based on lipophilic fluorescent dyes, though this labeling been directed toand faithfully follow EVs for assessing the fate in biological settings both inlabeling vitrohas and in vivo. validated and may result in biased understanding of EVs. Herein, developed labeling methods Our results showed that by installing fluorescent probes on thebiological surface ofwe EVs byboth conjugation to not the directed to faithfully follow the EVs for assessing the fate in settings in vitro and in EVs’ vivo. validated and may result in biased understanding of EVs. Herein, we developed labeling methods Our results showed that by installing fluorescent probes on the surface of EVs by conjugation to the EVs’ directed to faithfully follow the EVs for assessing the fate in biological settings both in vitro and in vivo. amino groups, it is possible to preserve the EVs concentration and stability. we found that the Our results showed that by installing fluorescent probes on in thebiological surface ofsettings EVsMoreover, byboth conjugation to the directed to faithfully follow thepreserve EVs for assessing the fate in we vitro and in EVs’ vivo. amino groups, itour is possible to the EVsbloodstream, concentration and stability. Moreover, found that the Our results showed that by installing fluorescent probes on the surface of EVs by conjugation to the EVs’ EVs labeled by method were stable in the while the EVs labeled with the conventional amino groups, itour is possible to preserve the EVsbloodstream, concentration and stability. Moreover, we found that the Our results showed that by installing fluorescent probes on the surface of EVs by conjugation to the EVs’ EVs labeled by method were stable in the while the EVs labeled with the conventional amino groups, it is possible to preserve the EVs concentration and stability. Moreover, we found that lipophilic dyes were immediately removed frombloodstream, circulation, while highlythe accumulating inwith the lungs. Finally, the by EVs labeled by our method were stable in the EVs labeled the conventional amino groups, is possible to preserve the EVs concentration and the stability. Moreover, we that4T1 the lipophilic dyes immediately removed from circulation, highly accumulating inwith thebreast lungs. Finally, by EVs labeled byitwere our method were stable in the bloodstream, while EVs labeled thefound conventional using our new labeling method for imaging EVs, we found that EVs from murine cancer lipophilic dyes were immediately removed from circulation, highly accumulating inwith thebreast lungs. Finally,4T1 by EVs labeled by our method were stable in the bloodstream, while the EVs labeled the conventional using our new labeling method for imaging EVs, we found that EVs from murine cancer lipophilic dyes were immediately removed from circulation, highly accumulating in the lungs. Finally, by accumulated along the brain capillaries, which correlate withthat theEVs incidence of brainbreast metastasis from using our new labeling method for imaging EVs, we found from murine cancer 4T1 lipophilic dyes were immediately removed from circulation, highly accumulating in the lungs. Finally, by accumulated the brain capillaries, which correlate withthat theEVs incidence of brainbreast metastasis using our newalong labeling method for imaging EVs, we found from murine cancer from 4T1 breast tumors. accumulated along the brain capillaries, which correlate with the incidence of brain metastasis from using our new labeling method for imaging EVs, we found that EVs from murine breast cancer 4T1 breast tumors.along the brain capillaries, which correlate with the incidence of brain metastasis from accumulated breast accumulated along the brain capillaries, which correlate with the by incidence of brain metastasis from © 2018,tumors. IFAC (International Federation Automatic Control)technology; Hosting Elsevier Ltd. All rights reserved. Keywords: Pharmacokinetics and drugofdelivery; Medical Biomedical imaging systems and breast tumors. Keywords: Pharmacokinetics and drug delivery; Medical technology; Biomedical imaging systems and breast tumors. image processing Keywords: Pharmacokinetics and drug delivery; Medical technology; Biomedical imaging systems and image processing Keywords: Pharmacokinetics and drug delivery; Medical technology; Biomedical imaging systems and image processing Keywords: Pharmacokinetics and drug delivery; Medical technology; Biomedical imaging systems and image processing image processing Cell Microvesicular 1. INTRODUCTION Body Cell Microvesicular 1. INTRODUCTION Cell Microvesicular Body Cell Microvesicular 1. INTRODUCTION Body Extracellular vesicles (EVs; Fig. 1), such as exosomes, are 1. INTRODUCTION Body Cell Microvesicular Extracellular vesicles (EVs; Fig. 1), such as exosomes, are 1. as INTRODUCTION gaining much vesicles interest mediators of cell-cell interactions, as Body Extracellular (EVs; Fig. 1), such as exosomes, are gaining much interest as mediators of cell-cell interactions, as Extracellular vesicles (EVs; Fig. 1), such as exosomes, are well as potential drug carriers. While EVs naturally contain gaining much interest mediators of cell-cell interactions, as Extracellular (EVs; Fig. 1), such as exosomes, are well askinds potential drugas carriers. While EVsthey naturally gaining much interest as mediators of cell-cell interactions, as many ofvesicles proteins and nucleic acids, can be contain further well as potential drug carriers. While EVs naturally contain gaining much interest as mediators of cell-cell interactions, as many of proteins and nucleic acids, can be contain further well askinds potential drugbioactive carriers. While EVsthey naturally loaded with different molecules for diagnostic and many kinds of proteins and nucleic acids, they can be further well as potential drug carriers. While EVs naturally contain loaded with applications different molecules for diagnostic and many kinds of proteinsbioactive and nucleic they can be further (Conlan, etacids, al. 2017). Thus, major therapeutic loaded with applications different bioactive molecules for diagnostic and many kinds of proteins and nucleic acids, they can be further (Conlan, ettrace al. 2017). Thus, major therapeutic loaded with different bioactive molecules for diagnostic and Microvesicles Exosomes efforts have been dedicated to EVs in biological (Conlan, et al. 2017). Thus, major therapeutic applications loaded with different bioactive molecules for diagnostic and Microvesicles Exosomes efforts have been dedicated to EVs in biological (Conlan, ettrace al.in2017). Thus, major therapeutic applications 100-1000 nm 40-120 nm systems for evaluating their function situ. In particular, Microvesicles Exosomes efforts have been dedicated to ettrace EVs in biological 100-1000 nm 40-120 nm (Conlan, al. 2017). Thus, major therapeutic applications systems for evaluating their function in situ. In particular, Microvesicles Exosomes efforts have been dedicated to trace EVs in biological Extracellular Vesicles (EVs) lipophilic fluorescent dyes, such as PKH and DiR, are used 100-1000 nm 40-120 nm systems for evaluating theirsuch function in EVs situ. In particular, Microvesicles Exosomes efforts have been dedicated to trace in biological 100-1000 nm 40-120 nm Extracellular Vesicles (EVs) lipophilic fluorescent dyes, as PKH and DiR, are used systems for evaluating their function in situ. In particular, for tracking EVs in vivo (Hoshino, et al. 2015). However, Extracellular Vesicles (EVs) lipophilic fluorescent dyes, such as PKH and DiR, are used 100-1000 nm 40-120(EVs) nm systems for evaluating their function in situ. In particular, for tracking EVs in vivo (Hoshino, et al. 2015). However, Extracellular Vesicles lipophilic fluorescent dyes, such as PKH and DiR, are used theirtracking validityEVs remains unknown. Moreover, these However, dyes are for in vivo (Hoshino, et al. 2015). Extracellular Vesicles (EVs) Fig. 1. Extracellular vesicles lipophilic fluorescent dyes, such as PKH and DiR, are used their validity remains unknown. these dyes are for tracking EVs vivo (Hoshino, et al. 2015). However, usually dissolved ininethanol, whichMoreover, may affect the stability of Fig. 1. Extracellular vesicles their validity remains unknown. Moreover, these dyes are for tracking EVs in vivo (Hoshino, et al. 2015). However, usually dissolved in ethanol, which may affect the stability of Fig. 1. Extracellular vesicles their validity remains unknown. Moreover, these dyes are (Goldstein 1986). Also, lipophilic dyes bind to EV EVs usually dissolved in ethanol, which may affect thebind stability of Fig. 1. Extracellular vesicles their validity remains unknown. Moreover, these dyes are 1986). Also, lipophilic dyes to EV EVs (Goldstein usually dissolved in ethanol, which may affect the stability of Fig. 1. Extracellular vesicles 2. METHODS membrane by non-covalent partitioning, so they may be (Goldstein 1986). Also, lipophilic dyes bind to EV EVs usually dissolved in ethanol, which mayetaffect the stability of 2. METHODS membrane by non-covalent partitioning, so2012). they may be 1986). Also, lipophilic dyes bind to EV EVs (Goldstein easily detached from membrane (Li, al. Thus, to 2. METHODS membrane by non-covalent partitioning, so2012). they may be (Goldstein 1986). Also, lipophilic dyes bind to EV EVs easily detached from membrane (Li, et al. Thus, to 2. METHODS membrane by non-covalent so they may be avoid detached interference from the partitioning, labeling method, developing easily from membrane (Li, et al. 2012). Thus, to 2.1 Isolation and labeling of EVs 2. METHODS membrane by non-covalent partitioning, so they may be avoid interference from the labeling method, developing easily detached from membrane (Li, et al. 2012). Thus, to proper labeling strategies willlabeling be keymethod, for advancing the 2.1 Isolation and labeling of EVs avoid interference from the developing easily detached membrane (Li,key et method, al. Thus, the to 2.1 Isolation and labeling of EVs proper labeling strategies willlabeling be for2012). advancing avoid interference from the developing understanding of from EVs in biological and labeling of EVs breast cancer 4T1 cells or proper labeling strategies willlabeling besettings. keymethod, for advancing the 2.1 EVsIsolation were isolated from murine avoid interference from the developing understanding of EVs in biological settings. proper labeling strategies will be key for advancing the 2.1 Isolation and labeling of EVs breast cancer 4T1 cells or In this research, we aimed to develop an appropriate labeling EVs were isolated from murine understanding ofwe EVs in biological U87-MG differential proper labeling strategies besettings. key for advancing the human In this research, aimed towill develop aninappropriate labeling EVs were glioblastoma isolated from murine breast using cancer 4T1 cells or understanding of EVs in biological settings. method capable of tracking EVs in vitro and in vivo human glioblastoma U87-MG using differential EVs were isolated from murine breast cancer 4T1 cells or In this research, we aimed to develop an appropriate labeling ultracentrifugation. Nanoparticle Tracking Analysis (NTA) understanding of EVs in biological settings. human glioblastoma U87-MG using differential method capable of aimed tracking EVs in vitro andbelabeling inhighly vivo EVs In this research, we to develop aninappropriate were isolated from murine breast cancer 4T1 cells or settings in real-time. Such labeling method would ultracentrifugation. Nanoparticle Tracking Analysis (NTA) human glioblastoma U87-MG using differential method capable of tracking EVs in in vitro and in vivo performed to measure the size distribution of EVs. For In this research, we to develop aninappropriate ultracentrifugation. Nanoparticle Tracking Analysis (NTA) settings in real-time. Such labeling method would belabeling highly method capable of aimed tracking EVs in vitro and inorganvivo was human glioblastoma U87-MG using differential beneficial for studying the factors responsible for was performed to measure the size distribution of EVs. For ultracentrifugation. Nanoparticle Tracking Analysis (NTA) settings real-time. Such method would beinorganhighly labeling EVs, we used sulfo-Cy5-NHS ester by targeting method in capable of tracking EVs Thus, in in vitro and vivo performed to measure the size distribution of EVs. For beneficial for studying thelabeling factors responsible for settings in real-time. Such labeling method would be highly ultracentrifugation. Nanoparticle Tracking Analysis (NTA) specific accumulation of EVs. we focused our was labeling EVs, we used sulfo-Cy5-NHS ester by targeting was performed to measure the size distribution of EVs. For beneficial for studying the factors responsible for organamines on their surface. We incubated the mixture them settings in real-time. Such labeling method would be highly labeling EVs, we used sulfo-Cy5-NHS ester by targeting specific accumulation of EVs. Thus, we focused our beneficial for studying the factors responsible for organperformed to surface. measure the size distribution of EVs. For approach on using controlled covalent conjugation of was amines on theirwe We incubated the mixture them labeling EVs, used sulfo-Cy5-NHS ester by targeting specific accumulation of EVs. Thus, we focused our overnight, and removed the free dyes by ultrafiltration. We beneficial for studying the EVs. factors responsible for organamines on theirwe surface. We incubated the mixture them approach on using controlled covalent of specific accumulation of Thus, weconjugation focused our labeling EVs, used sulfo-Cy5-NHS ester by targeting fluorescent probes to amine moieties on the EVs surface in overnight, and removed the free dyes by ultrafiltration. We amines on their surface. We incubated the mixture them approach on using controlled covalent conjugation of used fluorescence correlation spectrometry (FCS) to count specific accumulation of EVs. Thus, weconjugation focused our and removed theWe free dyes by the ultrafiltration. We fluorescent probes to amine moieties onvalidated the EVs surface in overnight, approach on using controlled covalent of amines on their surface. incubated mixture them aqueous settings. The strategy was then in cellular used fluorescence correlation spectrometry (FCS) to count overnight, and removed the free dyes by ultrafiltration. We fluorescent probes to amine moieties onvalidated the conjugation EVs in surface in the number of dyes correlation on each EV spectrometry surface. approach on using controlled covalent of used fluorescence (FCS) to count aqueous settings. The strategy was then cellular fluorescent probes to amine moieties on the EVs surface in overnight, and removed the free dyes by ultrafiltration. We and animal models. Moreover, as one application of this new the number of dyes on each EV surface. used fluorescence correlation spectrometry (FCS) to count aqueous settings. The strategymoieties wasone then cellular fluorescent probes toMoreover, amine onvalidated the EVscancer surface in the and animal models. as application ofin this new number of dyes correlation on each EV spectrometry surface. aqueous settings. The strategy was then validated in cellular used fluorescence (FCS) to count labeling, we studied the role of EVs from breast cells the number of dyes on each EV surface. and animal models. Moreover, as one application ofinthis new aqueous settings. The strategy was then validated cellular labeling, we studied the role of EVs from breast cancer cells and animal models. Moreover, one application of this new the 2.2.number Intravital of imaging dyes on each EV surface. to target brain capillaries, asas breast tumors show high labeling, we studied the role of EVs from breast cancer cells and animal models. Moreover, as one application of this new 2.2. Intravital imaging to target we brain capillaries, asEVs breast tumors show cells high labeling, studied the role (Tominaga, of fromet breast cancer al. 2015). incidence of brain metastasis to target of brain capillaries, asEVs breast tumors show cells high 2.2. Intravital imaging labeling, studied the role (Tominaga, of fromet breast cancer al. 2015). incidence brain metastasis to target we brain capillaries, as breast tumors show high 2.2. Intravital imaging (Tominaga, et al. 2015). incidence of brain metastasis to target of brain as breast et tumors show high 2.2. Intravital imaging al. 2015). incidence braincapillaries, metastasis (Tominaga, Copyright © 2018 IFAC 22 incidence of brain metastasis (Tominaga, et al. 2015). 2405-8963 © 2018, IFAC (International Federation of Automatic Control) Hosting by Elsevier Ltd. All rights reserved. Copyright © 2018 IFAC 22 Peer review©under of International Federation of Automatic Copyright 2018 responsibility IFAC 22 Control. Copyright © 2018 IFAC 22 10.1016/j.ifacol.2018.11.601 Copyright © 2018 IFAC 22
IFAC BMS 2018 São Paulo, Brazil, September 3-5, 2018
Horacio Cabral et al. / IFAC PapersOnLine 51-27 (2018) 22–23
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occurring. The results showed the immediate formation of aggregates in blood for the PKH26-labeled EVs, and the mechanical trapping in the blood vessels in lungs, indicating the inadequacy of PKH26-labeling for tracing EVs (Fig. 4). Finally, we injected 4T1 derived EVs labeled with our method into mice and observed brain blood vessels directly in vivo. We observed these EVs gradually accumulated along the brain vasculature. This observation could be correlated with the high incidence of brain metastasis from breast tumors and the recent findings showing that exosomes from breast cancer disrupt the blood-brain barrier (Tominaga, et al. 2015).
For in vivo studies, sulfo-Cy5-labeled EVs and PKH26labeled EVs were intravenously injected into mice to check their stabilities in blood and their bio-distribution. In vivo tracking of EVs was performed by an intravital confocal micro-videography using a Nikon R1 microscope with fast scanning lasers. The blood circulation and the accumulation of EVs in the living tissues was then analyzed and quantified by analysis with ImageJ. 3. RESULTS 3.1 Isolation and labeling of EVs. EVs from 4T1 cells were isolated by differential ultracentrifugation. The particles size of 4T1-derived EVs was 185 ± 86 nm (Fig. 2), as determined by NTA. Moreover, round-shaped vesicles around 100 nm in diameter were confirmed by TEM (Fig. 2). Next, these EVs were labeled with sulfo-Cy5 NHS ester. These NHS ester dyes can bind to amines on EV surface by covalent bonds. FCS measurement showed 4 dyes bound to each EV. This labeling was mediated by covalent bond, and we hypothesized that it may show higher stability in blood than PKH lipophilic dyes. EVs were also labeled with PKH26 and used as control in our subsequent studies.
Fig. 4. In vivo imaging in lungs after injection of EVs-labeled with PKH26 (green) or sulfo-Cy5 (red). Dextran-blue was used as control (blue). Image at t = 0 s is just before injection 5. CONCLUSIONS We developed a method for stably labeling EVs by using sulfo-Cy5 NHS ester. While the conventional labeling method caused aggregates of EVs in blood, leading to their instantaneous seizing in lung capillaries, our labeling method allowed the EVs to stably circulate in the bloodstream without the formation of aggregates. By using our labeling method, we found that 4T1 cells-derived EVs accumulated along the vasculature in brain. Taken together, the labeling we developed here will promote the proper understanding of EVs’ behavior, and facilitate the use of EVs for therapy and diagnosis.
Fig. 2. Size distribution determined by NTA (left panel) and TEM image (right panel) of EVs. Scale bar 100 nm.
3.2 Intravital imaging of labeled EVs. The differently labeled EVs were injected into mice, and their fate was followed in real time by using intravital microscopy. PKH26-labeled EVs disappeared at 10 min post-injection, while EVs- labeled with sulfo-Cy5 circulated in blood for more than 2 h (Fig. 3). We also checked the bio-distribution of labeled EVs at 2 h post-injection, and found that high liver and spleen distribution for both labeling methods (data not shown). However, only PKH26-labeled EVs accumulated in lungs.
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Fig. 3. Blood circulation of EVs labeled with different methods. Fluorescent dextran was used as internal standard. We checked the real-time micro-distribution of sulfo-Cy5and PKH26-labeled EVs in lungs to appreciate what was 23