Live-cell imaging is a powerful tool to visualize dynamic processes. The examination of fixed cells provides only static pictures, which can lead to misinterpretation and confusion about the process. This work presents a method to study uptake, drug release, and intracellular localization of liposomal nanoparticles in living cells.
Conventional imaging techniques can provide detailed information about cellular processes. However, this information is based on static images in an otherwise dynamic system, and successive phases are easily overlooked or misinterpreted. Live-cell imaging and time-lapse microscopy, in which living cells can be followed for hours or even days in a more or less continuous fashion, are therefore very informative. The protocol described here allows for the investigation of the fate of chemotherapeutic nanoparticles after the delivery of doxorubicin (dox) in living cells. Dox is an intercalating agent that must be released from its nanocarrier to become biologically active. In spite of its clinical registration for more than two decades, its uptake, breakdown, and drug release are still not fully understood. This article explores the hypothesis that lipid-based nanoparticles are taken up by the tumor cells and are slowly degraded. Released dox is then translocated to the nucleus. To prevent fixation artifacts, live-cell imaging and time-lapse microscopy, described in this experimental procedure, can be applied.
The ability to follow a biological process, such as cell-cell interactions, inter- and intracellular transport, cytotoxic compound uptake, and protein-protein interaction of single molecules in living cells and tissues, has gained great interest over the last decade, as it provides the extra dimension of "real time." In this manuscript, a method to follow the intracellular fate of free dox and dox incorporated in nanoparticles (Dox-NP) is described.
Nanoparticles have been used for decades to treat certain cancers. Dox-NP has been approved for the treatment of AIDS-related Kaposi sarcoma, advanced ovarian and breast cancer, and multiple myeloma1. It was one of the first liposomal nanoparticles developed and introduced in the clinical setting. The free form, dox, is vastly cleared from the blood circulation, limiting its capacity to reach the tumor site in sufficient amounts. Furthermore, treatment is accompanied by severe and dose-limiting side-effects, such as stomatitis and heart failure. When encapsulated into pegylated liposomal nanoparticles the circulation time increases from minutes to days2. This type of cholesterol-containing liposome is quite stable and this stability determines the pharmacokinetics of encapsulated drug.
However, this rigidity has a downside. The cytotoxic ability of a compound towards tumor cells is first evaluated in vitro, and it has been shown that dox is more potent than Dox-NP in cytotoxic assays3,4. It was hypothesized that the stability of the carrier prevents the release and cellular uptake of the drug, and it is worthwhile to investigate this phenomenon further. The intrinsic liposomal properties, built to last the aggressive elements in the blood stream and to reach the tumor site intact, resulted in the impaired bioavailability of the cytotoxic component (i.e., dox) at the target site (i.e., the tumor cell nucleus). Although Dox-NP hardily improved the response compared to the free form, it was still of clinical value, as encapsulation significantly reduced the cardiotoxic effects5. In the following years, other compounds were encapsulated in nano-carriers6. Also, modifying the carriers resulted in triggered drug release (i.e., at the tumor site) but maintained stability in the blood circulation7,8. Despite years of investigation and clinical use, the intratumoral fates of nanocarriers like Dox-NP are still not entirely clear. In a recent publication, it was shown that the nanoparticle is taken up as a whole while the dox remains trapped in the lysosomes9.
Cellular uptake of a nanoparticle and drug release are dynamic processes that can best be monitored using live-cell imaging. Moreover, classical histology, in which cells are incubated and thereafter fixed, can give false results if the fixation destroys the liposomal carrier and introduces artifacts. The main requirement for live-cell imaging is visualization, preferably by fluorescence, of the desired process. Certain compounds, like dox, have an intrinsic red fluorescence. Also, fluorescent markers can be introduced into the carrier, and cell organelles can be visualized using live-cell markers. In this way, an array of parameters, like uptake of the carrier, drug release, and cellular localization of the carrier and drug, can be imaged and analyzed. Here, a method is described in which the intercellular fate of dox and Dox-NP in several tumor cells is followed in real-time. Also, this method can be easily adapted to create the ideal conditions for targeted (e.g., charged nanoparticles10) and triggered (e.g., hyperthermia7) drug release.
As observed in the past, fixation can destroy liposomal nanocarriers almost immediately and DXR released form these destroyed liposomes still had the time to enter the nucleus, making the interpretation of data very difficult and even misleading. With some microscopic adjustments, the chemotherapeutic fate in living cells and tissues can be routinely investigated to confirm or overthrow histological observations. A recent paper showed the uptake, release, and localization of DXR in cells treated with free and encapsulated dox using live-cell time-lapse imaging9. This work describes the experimental setup in more detail. Using this model, it is possible to visualize the immediate transportation of dox to the nucleus, where it continuously accumulates until the cell eventually dies. In contrast, when Dox-NP is used, only small amounts of released dox are found in the nucleus. Although continuous exposure results in continuous DXR buildup, the fluorescent signal is much lower in Dox-NP- versus dox-treated cells, indicating a difference in cellular concentration and subsequent cytotoxicity. Furthermore, by using live-cell markers, it was shown that, when exposing cells to Dox-NP, DXR and the nanocarrier are located in the lysosome. This indicates that the complete liposome is taken up by the cell and demonstrates the involvement of an endocytic pathway during the uptake of these nanoparticles. The DXR in the nucleus is clearly from released dox. However, using this method, as both the DXR and carrier co-localize and seem to be entrapped in lysosomes, it is still inconclusive whether this is encapsulated or released dox. It is possible that the intrinsic stability of the nanoparticle prevents dox release when localized in the lysosome.
In this protocol, live-cell imaging is demonstrated using a specific microscopic setup, with a custom-made stage holder (the specifications can be given on request). However, most confocal microscope manufactures offer a range of incubators that perform live-cell imaging. Preserving cell culture conditions (i.e., temperature, CO2, pH, humidity, and sterility) is the main criterion of these incubators and requires some preliminary testing to determine the proper conditions, which is further explained. Automated data acquisition programs can also be delivered by the same manufactures. A "Multi location" option makes it possible to image several positions in the same chamber, refining statistical analysis. However, this option in the macro mentioned in this manuscript requires fixed XYZ positions, by which the possibility to correct for Z-drift is lost. Scanning the Z-dimension can be included in this macro and uses a focus plane for the analysis. However, this requires extra scanning, increasing the possibility of phototoxicity and bleaching.
As with all fluorescent measurements, overexposure is a problem, especially when comparing two compounds with a different cellular uptake. The fluorescent intensity is not only dependent upon the intrinsic signal of the compound, but also upon the rate of uptake, the concentration, and the drug exposure time. As shown in Figure 3, the nuclear DXR content of dox-treated cells is already visible, whereas no signal is seen in Dox-NP-treated cells. Only by increasing the gain can the DXR in Dox-NP-treated cells be visualized, leading to overexposure in dox-treated cells. Moreover, even intracellularly, Dox-NP is heterogeneously distributed, which can result in unavoidable under- or overexposure. Especially when investigating the cellular DXR entrapment, the gain was reduced significantly to distinguish individual organelles (Figures 5B and 5C). Thus, the measurements represented by pixel density must be accompanied by the representative images for the proper interpretation of results.
Phototoxicity, bleaching, and a low signal-to-noise ratio are also important issues in fluorescent microscopy, and a compromise between the quality of the image and the image acquisition must be established. However, even when the microscope setup is optimal, the image quality is also largely determined by the quality of the cells and the added compound. DXR gives a strong signal and, with the proper precautions, bleaching was not observed, even after longer periods (up to 72 h). However, the reduction of the fluorescent signal can also be a result of efflux. Efflux is an important phenomenon in drug resistance15 and occurs when cells pump out the drug from the intracellular site of action. A decrease of the fluorescent signal over time can therefore also be a result of dox efflux9. Comparing the fluorescent signal of a non-scanned location at the end of the experiment with the last image from the time-lapse series will demonstrate bleaching (i.e., the signal will be higher in the non-scanned location) or efflux (i.e., both signals will be identical). Several correctional options (e.g., background, drift, bleaching, etc.) are available in programs like ImageJ16. Also, as the fluorescent intensity increases over time, the configuration settings can be pre-evaluated in a pilot experiment to minimize overexposure at the end of the time-lapse (step 3.21). Alternatively, an imaging chamber with cells already exposed to the drug for the desired time-period can be used to initialize the settings. Finally, for this type of analysis, toxic drug concentrations (step 3.22) should be avoided and pre-established using conventional bio-assays.
Cells are continuously cultured and maintained in medium without phenol red. Phenol red fluoresces in the red part of the spectrum and can be observed in cellular organelles, most likely lysosomes, presenting false-positive information (step 1.2). To allow for the monitoring of individual cells, cell confluence should not exceed 70% (step 3.1.). The CO2 flow-speed (step 3.5) needs to be regulated in a validation experiment when the equipment is purchased or after maintenance. When the speed is too high, medium will evaporate. When the speed is too low, the pH of the medium will increase, which can affect cell growth. As the medium is without the phenol red pH indicator changes in pH cannot be observed and are therefore easily overlooked. Once the CO2 flow is validated, these settings can be used in all future experiments.
Using the method described here, the fate of nanoparticles can easily be investigated using live-cell imaging and time-lapse microscopy. In this procedure, commercially available nanoparticles were used. However, the fate of thermosensitive17, cationic liposomes10; liposomes enriched with short-chain shingolipids18; and nanoparticles encapsulating other drugs8,19 were also investigated in this manner in tumor and normal cells.
The authors have nothing to disclose.
The microscopy facilities used are part of the Erasmus Optical Imaging Center, and we would like to thank the staff of the OIC for their services.
DMEM (no phenol-red) | Sigma | D1145 | Supplemented with 1 mM L-glutamine and 10 % FCS |
Trypsin-versene (EDTA) | Lonza | BE17-161E | |
Fetal calf serum | Sigma | F7524 | Heat inactivate for 30 min at 55 ºC |
L-Glutamin | Lonza | BE17-605E | |
Gelatin from bovine skin | Sigma | G9391 | Make a 0,1% solution in PBS, sterile |
Phosphate buffered saline (PBS) | Sigma | D8537 | |
Stericup 0,22 µm filter – 500 ml | Millipore | SCGPU05RE | |
Trypan-blue | Sigma | T8154 | |
Cells: Lewis lung carcimoma | ATCC | CRL-1642 | |
Cells: B16BL6 | Dr. P. Brouckaert, Ghent University | donated | Ref.10 |
Cells: BLM and 1F6 | Dr. van Muijen, University of Nijmegen | donated | Ref.11 |
Petri dish | Greiner | 664160 | |
Doxorubicin | Actavis | mentioned as dox in the manuscript | |
Doxil/Caelyx | Janssen-Cilag | mentioned as Dox-NP in the manuscript | |
Syringe filter 0.2 µm | VWR international | 10462200 | |
Lysotracker-green DND-26 | Invitrogen | L7526 | mentioned as lyosomal marker (LM)-green in manuscript |
Lysotracker-red DND-99 | Invitrogen | L7528 | mentioned as lyosomal marker (LM)-red in manuscript |
Attofluor cell ring | Invitrogen | A7816 | |
Cover glass 25 mm #1 | Thermo scientific | CB00250RA1 | |
Stage holder + sealing lid | Custom made | ||
ZEISS LSM 510 microscope | Zeiss | ||
Software LSM 510 version 3.2 SP2 | Zeiss | ||
Image J | NIH | https://imagej.nih.gov/ij/index.html |