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.

1. Preparation
<ol>
	<li>Assemble the imaging chamber, which consists of two parts.
	<ol>
		<li>Place a 25-mm cover glass (Figure 1A-1) at the bottom part of the chamber (Figure 1A-2) and screw the top part into the bottom part (Figure 1A-3). Do not tighten too hard, as the glass can break. Autoclave the entire chamber (Figure 1A-4).</li>
	</ol>
	</li>
	<li>Prepare cell culture medium by supplementing Dulbecco&#39;s Modified Eagle&#39;s Medium (DMEM) without phenol red with 10% heat-inactivated fetal calf serum (FCS) and 1 mM L-glutamine under sterile conditions. Store at 4 &#176;C and warm to 37 &#176;C before use.</li>
	<li>Maintain the cells with cell culture medium in culturing flasks using standard cell culture techniques. 
	NOTE: Here, two mouse cell lines, melanoma B16BL611 and Lewis lung carcinoma (LLC), and two human melanomas, BLM and 1F612, were used.</li>
	<li>Make 0.1% gelatin by weighing and dissolving gelatin in phosphate-buffered saline (PBS) at 37 &#176;C overnight. Sterilize the solution by filtering it through a 0.22-&#181;m filter and store it at 4 &#176;C.</li>
</ol>
2. Plate Cells
<ol>
	<li>Remove the imaging chamber from the sterilization bag in a laminar flow cabinet, carefully tighten the chamber, and place it in a petri dish. 
	NOTE: To avoid contamination, keep the imaging chamber in a petri dish until the chamber can be placed under the microscope.</li>
	<li>Coat the glass of the imaging chamber with 1 mL of 0.1% sterile gelatin for 20 min at 37 &#176;C and 5% CO2. 
	NOTE: This will allow better cell attachment.</li>
	<li>Remove the medium from the cell culture flasks (see steps 1.2 and 1.3), wash once with 1x PBS, and detach the cells using 0.25% trypsin. Inactivate the trypsin by adding cell culture medium, collect the cells, spin them down at 1,100 x g for 5 min, and resuspend the pellet in 5 mL of cell culture medium.</li>
	<li>Dilute 20 &#181;L of cell suspension with 20 &#181;L of trypan blue (which will be taken up by dead cells). Count the number of living and dead cells using a hemocytometer; the number of dead cells should not exceed 10%.</li>
	<li>Aspirate the gelatin and plate the cells in a dilution that allows for a maximum of 70% coverage within 24 h. For example, use a dilution of 5 x 104 or 10 x 104 cells in 1 mL. Allow the cells to adhere at 5% CO2 and 37 &#176;C for 24 h.</li>
</ol>
3. Time-lapse Microscopy (Figure 2)
NOTE: Here, a confocal microscope with a custom-made stage holder was used, although most manufactures offer a range of incubators for live-cell imaging that are also suitable to investigate nanoparticulated drug delivery.
<ol>
	<li>Evaluate the cells under a microscope for confluency, cellular morphology, and contamination. Place the chamber back in the CO2 incubator. 
	NOTE: The confluency should not exceed 70%. If the cellular morphology is inconsistent (i.e., the cells do not adhere) or contamination is detected, discard the imaging chamber.</li>
	<li>Switch on the confocal microscope (Figure 1E-1), fluorescent light (Figure 1E-2), and computer (Figure 1E-3).</li>
	<li>Connect the lens heater to the objective and set it to 35 &#176;C (Figure 1E-4 + insert).</li>
	<li>Prepare the incubation unit, as shown in Figure 1B. 
	NOTE: This unit consists of a heated stage (Figure 1B-1), a flow stage with a connector for the CO2 tubes (Figure 1B-2) and CO2 probe (Figure 1B-3), and a red closing patch (Figure 1B-4). This patch is used to temporarily close the unit until the imaging chamber is placed.</li>
	<li>Place the incubation unit on the microscope stage and connect the in- and out-flow CO2 tubes (Figure 1E-5). Open the main CO2 valve (Figure 1E-6) and switch on the CO2 controller (Figure 1E-7). Check that the controller is set to 5% CO2. 
	NOTE: For humidity, CO2 passes through a humidifier (Figure 1E-8). For hypoxia experiments, a separate O2 regulator (Figure 1E-9) is included in the setup.</li>
	<li>Set the stage temperature to 37 &#176;C (Figure 1E-10). For hyperthermia experiments, set the temperature to 42 &#176;C. Allow the incubation unit to reach the selected temperature and CO2 percentage.</li>
	<li>Take the imaging chamber from the main CO2 incubator and transport the chamber in a petri dish to the microscopy room.</li>
	<li>Place the imaging chamber (Figure 1A-4) in a custom-made, 35 mm-diameter stage holder (Figure 1A-5) and close the chamber with a custom-made sealing lid (Figure 1A-6). 
	NOTE: The lid allows CO2 to pass and prevents evaporation and contamination.
	<ol>
		<li>Open the incubation unit, replace the red patch with the imaging chamber, and close the unit (Figure 1C). Do this as fast as possible to prevent the cooling down of the cells. Make sure that no cables are stuck between stage and the microscope.</li>
	</ol>
	</li>
	<li>Leave the unit to acclimatize for 30 min.</li>
	<li>Open the software and switch on the lasers. 
	NOTE: Here, 488 nm argon, 543 nm helium-neon, and 633-nm helium-neon lasers were used.</li>
	<li>Make a new database by clicking &#34;New&#34; under the File tab. Name and save the database.</li>
	<li>Set the configuration by clicking &#34;Config&#34; under the Acquire tab. Select &#34;Channel mode&#34; and &#34;Single track&#34; to evaluate a single fluorophore or &#34;Multi track&#34; for several fluorophores. Select the appropriate band-pass filters matching the fluorophore (e.g., 560 to 615 nm band-pass filter for dox and Dox-NP; see the Representative Results for more examples). Select the bright-field channel in one of the tracks.</li>
	<li>Set the scan control by clicking &#34;Scan&#34; under the Acquire tab. Set the frame size (1,024 x 1,024), scan speed (optimal), scan direction (8-bit, single), and scan average (4) by clicking &#34;Mode.&#34; Set the pinhole (200 &#181;m), gain and offset (e.g., 580, 700, and 835; see Figure 3), and laser power (488 = 10%, 543 = 100%, 633 = 100%) by clicking &#34;Channels.&#34;</li>
	<li>Save these settings by clicking &#34;Config&#34; in the Configuration tab and save them in the configuration database.</li>
	<li>Set the time-lapse by clicking &#34;Multi Time X&#34; (Figure 1D) in the Macro tab. Click &#34;Single location&#34;.</li>
	<li>To keep the cells in focus, select the automated focus in the macro by clicking &#34;Auto Focus.&#34; 
	NOTE: Autofocus is achieved with the 633-nm laser using the reflection of the glass as a reference point.</li>
	<li>Apply the saved configuration by clicking &#34;Single track&#34; or &#34;Multi track,&#34; scrolling to the required configuration in the configuration database, setting the time interval (e.g., 15 min), number of scans (e.g., 97) and clicking &#34;Load Config.&#34; 
	Note: Using these settings, a time-series of 97 x 15 min = 24 h will be made.</li>
	<li>Name the time-lapse in the &#34;Base File Name&#34; and select the database (made in step 3.11) by clicking &#34;Image Data Base&#34; to save the time-lapse. Set a temporary folder by clicking &#34;Tmp Image Folder.&#34; 
	NOTE: If the system stops or crashes for any reason, the images are located in this folder.</li>
	<li>Open the incubating unit, lift the imaging ring, add a drop of immersion oil to the objective, and close the unit as fast as possible. Focus and select a position in the imaging chamber. 
	NOTE: This position contains cells in a monolayer, with a minimum of 70% confluency, allowing for the imaging of individual cells.</li>
	<li>Check the configuration settings for the background and correct if necessary. 
	NOTE: The background can be corrected by reducing the gain or laser power. Any change in the configuration needs to be saved (see step 3.17) and loaded (see step 3.18) again in the macro.</li>
	<li>In the laminar flow cabinet, dilute free drug or nanoparticles at a concentration of 5 &#181;g/mL drug or 0.05 &#181;mol of lipids in cell culture medium. Filter through a 0.22-&#181;m syringe filter if the drug/nanoparticles are not sterile.</li>
	<li>Open the incubating chamber, lift the sealing lid, remove the medium from the cells, add 1 mL of diluted drug/nanoparticles, and close the unit as fast as possible.</li>
	<li>Focus on the cells and start the time-lapse by clicking &#34;Start Time&#34; in the Multi Time X macro.</li>
	<li>Check regularly to see if the cells are still in focus or use the autofocus (see step 3.16). The program automatically saves the time-lapse in the database; do not close the database while the program is running.</li>
</ol>
4. Data Analysis
<ol>
	<li>Depending on the original research question, use software programs such as ImageJ for data analysis (see the Results section for examples).</li>
</ol>

<ol>
	<li>Duggan, S. T., Keating, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pegylated+liposomal+doxorubicin:+a+review+of+its+use+in+metastatic+breast+cancer,+ovarian+cancer,+multiple+myeloma+and+AIDS-related+Kaposi's+sarcoma.">Pegylated liposomal doxorubicin: a review of its use in metastatic breast cancer, ovarian cancer, multiple myeloma and AIDS-related Kaposi's sarcoma.</a> Drugs. 71 (18), 2531-2558 (2011).</li><li>Gabizon, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prolonged+circulation+time+and+enhanced+accumulation+in+malignant+exudates+of+doxorubicin+encapsulated+in+polyethylene-glycol+coated+liposomes.">Prolonged circulation time and enhanced accumulation in malignant exudates of doxorubicin encapsulated in polyethylene-glycol coated liposomes.</a> Cancer Res. 54 (4), 987-992 (1994).</li><li>Ten Hagen, T. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low-dose+tumor+necrosis+factor-alpha+augments+antitumor+activity+of+stealth+liposomal+doxorubicin+(DOXIL)+in+soft+tissue+sarcoma-bearing+rats.">Low-dose tumor necrosis factor-alpha augments antitumor activity of stealth liposomal doxorubicin (DOXIL) in soft tissue sarcoma-bearing rats.</a> Int J Cancer. 87, 829-837 (2000).</li><li>Hoving, S., Seynhaeve, A. L., van Tiel, S. T., Eggermont, A. M., Ten Hagen, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Addition+of+low-dose+tumor+necrosis+factor-alpha+to+systemic+treatment+with+STEALTH+liposomal+doxorubicin+(Doxil)+improved+anti-tumor+activity+in+osteosarcoma-bearing+rats.">Addition of low-dose tumor necrosis factor-alpha to systemic treatment with STEALTH liposomal doxorubicin (Doxil) improved anti-tumor activity in osteosarcoma-bearing rats.</a> Anticancer Drugs. 16 (6), 667-674 (2005).</li><li>Gabizon, A. A., Lyass, O., Berry, G. J., Wildgust, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+safety+of+pegylated+liposomal+doxorubicin+(Doxil/Caelyx)+demonstrated+by+endomyocardial+biopsy+in+patients+with+advanced+malignancies.">Cardiac safety of pegylated liposomal doxorubicin (Doxil/Caelyx) demonstrated by endomyocardial biopsy in patients with advanced malignancies.</a> Cancer Invest. 22 (5), 663-669 (2004).</li><li>Muggia, F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liposomal+encapsulated+anthracyclines:+new+therapeutic+horizons.">Liposomal encapsulated anthracyclines: new therapeutic horizons.</a> Curr Oncol Rep. 3 (2), 156-162 (2001).</li><li>Li, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Triggered+content+release+from+optimized+stealth+thermosensitive+liposomes+using+mild+hyperthermia.">Triggered content release from optimized stealth thermosensitive liposomes using mild hyperthermia.</a> J Control Release. 143 (2), 274-279 (2010).</li><li>Lu, T., Lokerse, W. J., Seynhaeve, A. L., Koning, G. A., ten Hagen, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formulation+and+optimization+of+idarubicin+thermosensitive+liposomes+provides+ultrafast+triggered+release+at+mild+hyperthermia+and+improves+tumor+response.">Formulation and optimization of idarubicin thermosensitive liposomes provides ultrafast triggered release at mild hyperthermia and improves tumor response.</a> J Control Release. 220, 425-437 (2015).</li><li>Seynhaeve, A. L., Dicheva, B. M., Hoving, S., Koning, G. A., Ten Hagen, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intact+Doxil+is+taken+up+intracellularly+and+released+doxorubicin+sequesters+in+the+lysosome:+Evaluated+by+in+vitro/in+vivo+live+cell+imaging.">Intact Doxil is taken up intracellularly and released doxorubicin sequesters in the lysosome: Evaluated by in vitro/in vivo live cell imaging.</a> J Control Release. 172 (1), 330-340 (2013).</li><li>Dicheva, B. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cationic+Thermosensitive+Liposomes:+A+Novel+Dual+Targeted+Heat-Triggered+Drug+Delivery+Approach+for+Endothelial+and+Tumor+Cells.">Cationic Thermosensitive Liposomes: A Novel Dual Targeted Heat-Triggered Drug Delivery Approach for Endothelial and Tumor Cells.</a> Nano Lett. 13 (6), 2324-2331 (2012).</li><li>Brouckaert, P. G., Leroux-Roels, G. G., Guisez, Y., Tavernier, J., Fiers, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+anti-tumour+activity+of+recombinant+human+and+murine+TNF,+alone+and+in+combination+with+murine+IFN-gamma,+on+a+syngeneic+murine+melanoma.">In vivo anti-tumour activity of recombinant human and murine TNF, alone and in combination with murine IFN-gamma, on a syngeneic murine melanoma.</a> Int J Cancer. 38 (5), 763-769 (1986).</li><li>Van Muijen, G. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antigen+expression+of+metastasizing+and+non-metastasizing+human+melanoma+cells+xenografted+into+nude+mice.">Antigen expression of metastasizing and non-metastasizing human melanoma cells xenografted into nude mice.</a> Clin Exp Metastasis. 9 (3), 259-272 (1991).</li><li>Das, A. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+TIMP3+expression+affects+tumor+progression+and+angiogenesis+in+melanomas+through+regulation+of+directionally+persistent+endothelial+cell+migration.">Differential TIMP3 expression affects tumor progression and angiogenesis in melanomas through regulation of directionally persistent endothelial cell migration.</a> Angiogenesis. 17 (1), 163-177 (2013).</li><li>Brouckaert, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+necrosis+factor-alpha+augmented+tumor+response+in+B16BL6+melanoma-bearing+mice+treated+with+stealth+liposomal+doxorubicin+(Doxil)+correlates+with+altered+Doxil+pharmacokinetics.">Tumor necrosis factor-alpha augmented tumor response in B16BL6 melanoma-bearing mice treated with stealth liposomal doxorubicin (Doxil) correlates with altered Doxil pharmacokinetics.</a> Int J Cancer. 109 (3), 442-448 (2004).</li><li>Chen, V. Y., Posada, M. M., Zhao, L., Rosania, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+doxorubicin+efflux+from+the+nucleus+of+drug-resistant+cancer+cells+following+extracellular+drug+clearance.">Rapid doxorubicin efflux from the nucleus of drug-resistant cancer cells following extracellular drug clearance.</a> Pharm Res. 24 (11), 2156-2167 (2007).</li><li>Parslow, A., Cardona, A., Bryson-Richardson, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sample+drift+correction+following+4D+confocal+time-lapse+imaging.">Sample drift correction following 4D confocal time-lapse imaging.</a> J Vis Exp. (86), (2014).</li><li>Li, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mild+hyperthermia+triggered+doxorubicin+release+from+optimized+stealth+thermosensitive+liposomes+improves+intratumoral+drug+delivery+and+efficacy.">Mild hyperthermia triggered doxorubicin release from optimized stealth thermosensitive liposomes improves intratumoral drug delivery and efficacy.</a> J Control Release. 168 (2), 142-150 (2013).</li><li>Pedrosa, L. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improving+intracellular+Doxorubicin+delivery+through+nanoliposomes+equipped+with+selective+tumor+cell+membrane+permeabilizing+short-chain+sphingolipids.">Improving intracellular Doxorubicin delivery through nanoliposomes equipped with selective tumor cell membrane permeabilizing short-chain sphingolipids.</a> Pharm Res. 30 (7), 1883-1895 (2013).</li><li>Pedrosa, L. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Short-chain+glycoceramides+promote+intracellular+mitoxantrone+delivery+from+novel+nanoliposomes+into+breast+cancer+cells.">Short-chain glycoceramides promote intracellular mitoxantrone delivery from novel nanoliposomes into breast cancer cells.</a> Pharm Res. 32 (4), 1354-1367 (2015).</li></ol>

The authors have nothing to disclose.

As observed in the past, fixation can destroy liposomal nanocarriers almost immediately&#160;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 &#34;Multi location&#34; 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 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 &#34;real time.&#34; 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.

<table><tbody><tr><td>DMEM (no phenol-red)</td><td>Sigma</td><td>D1145</td><td>Supplemented with 1 mM L-glutamine and 10 % FCS</td></tr><tr><td>Trypsin-versene (EDTA)</td><td>Lonza</td><td>BE17-161E</td><td></td></tr><tr><td>Fetal calf serum</td><td>Sigma</td><td>F7524</td><td>Heat inactivate for 30 min at 55 &amp;ordm;C</td></tr><tr><td>L-Glutamin</td><td>Lonza</td><td>BE17-605E</td><td></td></tr><tr><td>Gelatin from bovine skin</td><td>Sigma</td><td>G9391</td><td>Make a 0,1% solution in PBS, sterile</td></tr><tr><td>Phosphate buffered saline (PBS)</td><td>Sigma</td><td>D8537</td><td></td></tr><tr><td>Stericup 0,22 &amp;micro;m filter - 500 ml&amp;nbsp;</td><td>Millipore</td><td>SCGPU05RE</td><td></td></tr><tr><td>Trypan-blue</td><td>Sigma</td><td>T8154</td><td></td></tr><tr><td>Cells: Lewis lung carcimoma</td><td>ATCC</td><td>CRL-1642</td><td></td></tr><tr><td>Cells: B16BL6</td><td>Dr. P. Brouckaert, Ghent University</td><td>donated</td><td>Ref.10</td></tr><tr><td>Cells: BLM and 1F6</td><td>Dr. van Muijen, University of Nijmegen</td><td>donated</td><td>Ref.11</td></tr><tr><td>Petri dish</td><td>Greiner</td><td>664160</td><td></td></tr><tr><td>Doxorubicin</td><td>Actavis&amp;nbsp;</td><td></td><td>mentioned as dox in the manuscript</td></tr><tr><td>Doxil/Caelyx</td><td>Janssen-Cilag</td><td></td><td>mentioned as Dox-NP in the manuscript</td></tr><tr><td>Syringe filter 0.2 &amp;micro;m</td><td>VWR international</td><td>10462200</td><td></td></tr><tr><td>Lysotracker-green DND-26</td><td>Invitrogen</td><td>L7526</td><td>mentioned as lyosomal marker (LM)-green in manuscript</td></tr><tr><td>Lysotracker-red DND-99</td><td>Invitrogen</td><td>L7528</td><td>mentioned as lyosomal marker (LM)-red in manuscript</td></tr><tr><td>Attofluor cell ring</td><td>Invitrogen</td><td>A7816</td><td></td></tr><tr><td>Cover glass 25 mm #1</td><td>Thermo scientific</td><td>CB00250RA1</td><td></td></tr><tr><td>Stage holder + sealing lid</td><td>Custom made</td><td></td><td></td></tr><tr><td>ZEISS LSM 510 microscope</td><td>Zeiss</td><td></td><td></td></tr><tr><td>Software LSM 510 version 3.2 SP2</td><td>Zeiss</td><td></td><td></td></tr><tr><td>Image J</td><td>NIH</td><td></td><td>https://imagej.nih.gov/ij/index.html</td></tr></tbody></table>

using in vitro live-cell imaging to explore chemotherapeutics delivered by lipid-based nanoparticles

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.

Labeling liposomal carriers with different markers has been previously described9. Carriers labeled with far-red dioctadecyl tetramethylindotricarbocyanine perchlorate (DiD; Ex = 644 nm; Em = 665 nm) or green 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(carboxyfluorescein) (CF-PE; Ex = 490 nm; Em = 515 nm) are presented in this manuscript. To show intercellular similarities and differences in liposomal uptake, release, and intracellular localization, several tumor types were studied. These include two mouse lines, the B16BL6 melanoma11 and the Lewis lung carcinoma (LLC), and two human melanomas, a highly metastatic (BLM) and a non-metastatic (1F6) melanoma12,13. All experiments were performed using living cells. In all experiments, a concentration of 5 &#181;g/mL dox, 5 &#181;g/mL Dox-NP, or 0.05 &#181;mol of nanoparticles were administered for 3 or 24 h. Measured and analyzed dox (in free, released, encapsulated, sequestered, or intercalated form) were referred to as DXR. A 40X (numerical aperture: 1.3) oil objective lens was used, and imaging was performed with a 488 nm argon laser (10%/0.2 mW power) and a 505 to 550 nm band-pass filter for CF-PE and lysosomal marker (LM)-green. A 543 nm helium-neon laser (100%/ 0.2 mW power) and a 560 to 615 nm band-pass filter was used for dox, Dox-NP, and lysosomal marker-red. A 633 nm helium-neon laser (100%/0.5 mW power) and a 640 nm long-pass filter was used for DiD.
Because of its encapsulation, the in vitro cytotoxicity, bioavailability, and pharmacokinetics of dox were significantly altered3,9,14. The difference between the bioavailability of the drug when administered in free and encapsulated form is demonstrated in Figure 3. Dox is an intercalating agent and must enter the cell nucleus to become cytotoxic. Figure 3 and related Movies 1 and 2 show a 3 h time-lapse of BLM cells treated with dox or Dox-NP under identical conditions. Within minutes of dox exposure, nuclear DXR can be observed (Figure 3A, Movie 1) and, using a lower gain (insert), can be seen intercalating in the nucleus. In contrast, when exposed to Dox-NP, intracellular DXR is hardly visible within this time-frame (Figure 3B, Movie 2). Only by increasing the photomultiplier gain, DXR in the cytoplasm can be observed (insert). Using ImageJ, the pixel densities of cytoplasmic and nuclear DXR were analyzed. As cells are in motion during time-lapse evaluation, it is important to combine fluorescent with bright-field images. This presents the possibility of tracking individual cells and stabilizing the XY-drift using specific plugins in ImageJ. In the analysis presented in Figure 3, the bright-field image was used to draw a region of interest (ROI) around the cytoplasm (solid line) and nucleus (dotted line). The ROI manager in ImageJ can be found in the menu item Analyze &#62; Tools &#62; ROI manager. These ROIs are used in the fluorescent image to analyze pixel density by using the menu item Analyze &#62; Measure. The difference between the pixel density in the nucleus (Figure 3C) and cytoplasm (Figure 3D) of dox- or Dox-NP-treated cells confirms what is seen in the images. Also, cells were incubated for 24 h with dox or Dox-NP, after which the compound was replaced by medium and immediately imaged with increased sensitivity of the photomultiplier gain (Figure 4). Other settings, such as laser power, offset, pinhole, and image display, were identical. The low DXR signal in Dox-NP-treated compared to dox-treated cells is striking. Using a gain of 700, DXR in the Dox-NP-treated cells becomes visible, whereas the image of dox is already overexposed. This simple in vitro experiment visualizes a significant difference in the bioavailability of free versus encapsulated drug.
When cells are continuously exposed to Dox-NP for 24 h, DXR builds up over time, as shown in Figure 5A and Movies 3 and 4. The signal increases in the cytoplasm, and later, DXR is also found intercalated in the nucleus. These observations are confirmed by the pixel density graphs (Figure 5B). The measured cytoplasmic pixel density is lower in BLM compared to 1F6 cells because of the difference in intracellular distribution. Whereas DXR in 1F6 cells is distributed throughout the cell, DXR in BLM cells is more concentrated in an organelle close to the nucleus (Figure 5C).
Therefore, the localization of drug and carrier in different cell lines (Figure 6) was investigated. Cells were incubated for 24 h with Dox-NP/DiD and imaged. Using the bright-field image, an overlay of the cell membrane (solid line) and nucleus (dotted line) was drawn in the fluorescent image of three different cell lines. It is demonstrated here that the carrier, labeled with DiD, follows the same cytoplasmic pattern as DXR: concentrated in an organelle close to the nucleus in BLM, around the entire nucleus in LLC, and randomly scattered throughout the cytoplasm in B16BL6 cells. In the nucleus, only DXR and no DiD can be seen, indicating released dox. By reducing the photomultiplier gain, individual cellular vesicles containing DXR and carrier, labelled with DiD, as well as with CF-PE (Figures 6B and 6C), can be seen.
Cytoplasmic DXR sequesters in small vesicles, and cells were labeled with a live-cell lysosomal marker in green or red. The movement of these lysosomes can be followed in the cells (Movie 5). Cytoplasmic DXR and the nanocarrier colocalize within these structures, and the intercellular difference in the DXR pattern, as seen in Figure 6, is explained here. Lysosomes are randomly distributed throughout the cytoplasm in B16BL6 and are located next to the nucleus in BLM cells (Figure 7A). Using ImageJ, the colocalization between DXR and carrier in the lysosome was calculated (Figure 7B). The color images were made binary (Process &#62; Binary &#62; Make Binary) and, using the colocalization plug-in (Analyze &#62; Colocalization), a colocalization image was made of DXR with DiD. This creates a green-red-white colored image, with white pixels representing the colocalized pixels of both images. Via the menu item Process &#62; Binary &#62; Make Binary, the white pixels will be separated and converted to black pixels, from which the pixel density is measured (Analyze &#62; Measure). Thereafter, a colocalization image was made between this DXR-DiD image and the binary image of the lysosomal marker (LM) and pixel density measured. The colocalization data is the percentage of pixels positive for DXR-DiD and DXR-DiD/lysosomal marker (Figure 7C). That the carrier, labeled with CF-PE as well as DiD, is located in the lysosome is also shown in Figure 7D.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/55405/55405fig1.jpg" /> 
Figure 1: Instruments and equipment setup. A) Imaging chamber + necessities. 25-mm #1 cover glass (1), bottom part of the chamber (2), top part of the chamber (placed upside down) and O-ring (3), stage holder (4), and sealing lid (5). Scale bar: 2 cm. B) Incubation unit. Heated microscope stage (1), flow stage with in- and out-flow for CO2 (2), a CO2 probe (3), and a closing patch (4). Scale bar: 2 cm. C) Imaging chamber in the incubator unit, as seen under the microscope. D) Multi-time series macro. E) Equipment for imaging. Inverted microscope (1), fluorescent light (2), computer (3), ring heater (4, insert) and controller (4, main figure), CO2 flow tubing (5), CO2 valve (6), CO2 controller (7), CO2 humidifier (8), O2 valve and controller (9), and stage temperature controller (10). <a href="http://cloudflare.jove.com/files/ftp_upload/55405/55405fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/55405/55405fig2.jpg" /> 
Figure 2: Schematic of the microscope parameters detailed in Protocol section 3. <a href="http://cloudflare.jove.com/files/ftp_upload/55405/55405fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/55405/55405fig3.jpg" /> 
Figure 3: Investigating the short-term uptake of dox and Dox-NP.&#160; BLM cells were continuously exposed to dox or Dox-NP for 3 h, and a time-lapse was taken. (A) Still pictures of the time-lapse of cells incubated with dox. (B) Still pictures of the time-lapse of cells incubated with Dox-NP. Scale bar = 50 &#181;m. (C) Pixel density increase of nuclear DXR in dox- and Dox-NP-treated cells. (D) Pixel density increase of cytoplasmic DXR in dox- and Dox-NP-treated cells. The data represent the mean &#177; SD of 3 cells per field. <a href="http://cloudflare.jove.com/files/ftp_upload/55405/55405fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/55405/55405fig4.jpg" /> 
Figure 4: Investigating the long-term uptake of dox and Dox-NP in different cell lines. Cells were exposed to dox or Dox-NP and imaged 24 h later. Images were taken immediately after the removal of the drugs, with 3 different gains. Scale bar = 50 &#181;m. <a href="http://cloudflare.jove.com/files/ftp_upload/55405/55405fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/55405/55405fig5.jpg" /> 
Figure 5: Investigating the cellular uptake and release of liposomal agents. Cells were continuously exposed to Dox-NP for 24 h, and a time-lapse was made. (A) Still pictures of the time-lapse. Scale bar = 50 &#181;m. (B) Pixel density increase of DXR in the nucleus and cytoplasm. The data represent the mean &#177; SD of 3 cells per field. (C) Images demonstrating the localization of DXR within the cell. Solid line: cell membrane, dotted line: nucleus. <a href="http://cloudflare.jove.com/files/ftp_upload/55405/55405fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/55405/55405fig6.jpg" /> 
Figure 6: Investigating the localization of drug and carrier in different cell lines. (A) Cells were exposed to Dox-NP/DiD for 24 h and imaged. Scale bar = 50 &#181;m. (B) Cells were exposed to Dox-NP/DiD/CF-PE for 24 h and imaged with a lower intensity gain to distinguish individual vesicles. (C) Bright-field and DXR overlay showing DXR in cytoplasmic vesicles (arrow). <a href="http://cloudflare.jove.com/files/ftp_upload/55405/55405fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/55405/55405fig7.jpg" /> 
Figure 7: Investigation of the intracellular localization of the drug and carrier. Cells were exposed to Dox-NP/DiD or CF-PE/DiD for 24 h, and lysosomes are visualized using the live-cell lysosomal marker (LM) in green or red. (A) Intracellular localization of DXR and the carrier (DiD). The arrows represent 3 examples of DXR and DiD localized in the lysosome. Scale bar = 50 &#181;m. (B) Co-localization analysis of DXR and the carrier (DiD) in lysosomes (LM) using ImageJ. (C) Percentage of co-localization of DXR and the carrier in lysosomes. The data represent the mean &#177; SEM of 3 individual experiments. (D) Lysosomal sequestering of the carrier visualized using two different markers. Scale bar = 50 &#181;m. <a href="http://cloudflare.jove.com/files/ftp_upload/55405/55405fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Movie 1" src="/files/ftp_upload/55405/55405mov1.jpg" /> 
Movie 1: Related to Figure 3: BLM cells were incubated with dox for 3 h. Time-lapse: 19 images, with an interval of 10 min. Frame rate: 3 fps. Scale bar = 50 &#181;m. <a href="http://cloudflare.jove.com/files/ftp_upload/55405/Movie_1_BLM_Doxorubicin_for_3h.avi" target="_blank">Please click here to view this video. (Right-click to download.)</a>
<img alt="Movie 2" src="/files/ftp_upload/55405/55405mov2.jpg" /> 
Movie 2: Related to Figure 3: BLM cells were incubated with Dox-NP for 3 h. Time-lapse: 19 images, with an interval of 10 min. Frame rate: 3 fps. Scale bar = 50 &#181;m. <a href="http://cloudflare.jove.com/files/ftp_upload/55405/Movie_2_BLM_Doxil_for_3h.avi" target="_blank">Please click here to view this video. (Right-click to download.)</a>
<img alt="Movie 3" src="/files/ftp_upload/55405/55405mov3v3.jpg" /> 
Movie 3: Related to Figure 5: BLM cells were incubated with Dox-NP for 24 h. Time-lapse: 96 images, with an interval of 15 min. Frame rate: 8 fps. Scale bar = 50 &#181;m. <a href="http://cloudflare.jove.com/files/ftp_upload/55405/Movie_3,_BLM_Doxil_for_24h_correct2.avi" target="_blank">Please click here to view this video. (Right-click to download.)</a>
<img alt="Movie 4" src="/files/ftp_upload/55405/55405mov4.jpg" /> 
Movie 4: Related to Figure 5: 1F6 cells were incubated with Dox-NP for 24 h. Time-lapse: 96 images, with an interval of 15 min. Frame rate: 8 fps. Scale bar = 50 &#181;m. <a href="http://cloudflare.jove.com/files/ftp_upload/55405/Movie_4_1F6_Doxil_for_24h.avi" target="_blank">Please click here to view this video. (Right-click to download.)</a>
<img alt="Movie 5" src="/files/ftp_upload/55405/55405mov5.jpg" /> 
Movie 5: Related to Figure 7: Lysosomes of B16BL6 cells were stained with a live-cell marker. Time-lapse: 10 images, with an interval of 10 s. Frame rate: 3fps. Scale bar = 50 &#181;m. <a href="http://cloudflare.jove.com/files/ftp_upload/55405/Movie_5_B16BL6_lysotracker.avi" target="_blank">Please click here to view this video. (Right-click to download.)</a>

Watch this Scientific Journal Video about Using In Vitro Live-cell Imaging to Explore Chemotherapeutics Delivered by Lipid-based Nanoparticles at JoVE.com

Using In Vitro Live-cell Imaging to Explore Chemotherapeutics Delivered by Lipid-based Nanoparticles

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.

Using In Vitro Live-cell Imaging to Explore Chemotherapeutics Delivered by Lipid-based Nanoparticles

Conventional imaging techniques can provide detailed information about cellular processes. However, this information is based on static images in an ...

using-vitro-live-cell-imaging-to-explore-chemotherapeutics-delivered

Research

JoVE Journal

Medicine

8.4K Views.  Erasmus MC. Live-cel imaging is een krachtig hulpmiddel voor het visualiseren van dynamische processen. Het onderzoek van vaste cellen biedt alleen statische afbeeldingen, die tot verkeerde interpretaties en verwarring over het proces leiden kunnen. Dit werk presenteert een methode om te studeren opname, drug release en intracellulaire lokalisatie van liposomaal nanodeeltjes in levende cellen.

Video: Using In Vitro Live-cell Imaging to Explore Chemotherapeutics Delivered by Lipid-based Nanoparticles

A Comprehensive Procedure to Evaluate the In Vivo Performance of Cancer Nanomedicines

Inspired by the success of previous cancer nanomedicines in the clinic, researchers have generated a large number of novel formulations in the past decade. However, only a small number of nanomedicines have been approved for clinical use, whereas the majority of nanomedicines under clinical development have produced disappointing results. One major obstacle to the successful clinical translation of new cancer nanomedicines is the lack of an accurate understanding of their in vivo performance. This article features a rigorous procedure to characterize the in vivo behavior of nanomedicines in tumor-bearing mice at systemic, tissue, single-cell, and subcellular levels via the integration of positron emission tomography-computed tomography (PET-CT), radioactivity quantification methods, flow cytometry, and fluorescence microscopy. Using this approach, researchers can accurately evaluate novel nanoscale formulations in relevant mouse models of cancer. These protocols may have the ability to identify the most promising cancer nanomedicines with high translational potential or to aid in the optimization of cancer nanomedicines for future translation.

A Comprehensive Procedure to Evaluate the In Vivo Performance of Cancer Nanomedicines

The poor understanding of the in vivo performance of nanomedicines stymies their clinical translation. Procedures to evaluate the in vivo behavior of cancer nanomedicines at systemic, tissue, single-cell, and subcellular levels in tumor-bearing immunocompetent mice are described here. This approach may help researchers to identify promising cancer nanomedicines for clinical translation.

Inspired by the success of previous cancer nanomedicines in the clinic, researchers have generated a large number of novel formulations in the past ...

Cancer Research

Evaluation of Nanoparticle Uptake in Tumors in Real Time Using Intravital Imaging

Current technologies for tumor imaging, such as ultrasound, MRI, PET and CT, are unable to yield high-resolution images for the assessment of nanoparticle uptake in tumors at the microscopic level1,2,3, highlighting the utility of a suitable xenograft model in which to perform detailed uptake analyses. Here, we use high-resolution intravital imaging to evaluate nanoparticle uptake in human tumor xenografts in a modified, shell-less chicken embryo model. The chicken embryo model is particularly well-suited for these in vivo analyses because it supports the growth of human tumors, is relatively inexpensive and does not require anesthetization or surgery 4,5. Tumor cells form fully vascularized xenografts within 7 days when implanted into the chorioallantoic membrane (CAM) 6. The resulting tumors are visualized by non-invasive real-time, high-resolution imaging that can be maintained for up to 72 hours with little impact on either the host or tumor systems. Nanoparticles with a wide range of sizes and formulations administered distal to the tumor can be visualized and quantified as they flow through the bloodstream, extravasate from leaky tumor vasculature, and accumulate at the tumor site. We describe here the analysis of nanoparticles derived from Cowpea mosaic virus (CPMV) decorated with near-infrared fluorescent dyes and/or polyethylene glycol polymers (PEG) 7, 8, 9,10,11. Upon intravenous administration, these viral nanoparticles are rapidly internalized by endothelial cells, resulting in global labeling of the vasculature both outside and within the tumor7,12. PEGylation of the viral nanoparticles increases their plasma half-life, extends their time in the circulation, and ultimately enhances their accumulation in tumors via the enhanced permeability and retention (EPR) effect 7, 10,11. The rate and extent of accumulation of nanoparticles in a tumor is measured over time using image analysis software. This technique provides a method to both visualize and quantify nanoparticle dynamics in human tumors.

We present a novel approach to quantify nanoparticle localization in the vasculature of human xenografted tumors using dynamic, real-time intravital imaging in an avian embryo model.

Current technologies for tumor imaging, such as ultrasound, MRI, PET and CT, are unable to yield high-resolution images for the assessment of nanoparticle ...

Long-term Live-cell Imaging to Assess Cell Fate in Response to Paclitaxel

Live-cell imaging is a powerful technique that can be used to directly visualize biological phenomena in single cells over extended periods of time. Over the past decade, new and innovative technologies have greatly enhanced the practicality of live-cell imaging. Cells can now be kept in focus and continuously imaged over several days while maintained under 37 &#176;C and 5% CO2 cell culture conditions. Moreover, multiple fields of view representing different experimental conditions can be acquired simultaneously, thus providing high-throughput experimental data. Live-cell imaging provides a significant advantage over fixed-cell imaging by allowing for the direct visualization and temporal quantitation of dynamic cellular events. Live-cell imaging can also identify variation in the behavior of single cells that would otherwise have been missed using population-based assays. Here, we describe live-cell imaging protocols to assess cell fate decisions following treatment with the anti-mitotic drug paclitaxel. We demonstrate methods to visualize whether mitotically arrested cells die directly from mitosis or slip back into interphase. We also describe how the fluorescent ubiquitination-based cell cycle indicator (FUCCI) system can be used to assess the fraction of interphase cells born from mitotic slippage that are capable of re-entering the cell cycle. Finally, we describe a live-cell imaging method to identify nuclear envelope rupture events.

Live-cell imaging provides a wealth of information on single cells or whole populations that is unattainable by fixed cell imaging alone. Here, live-cell imaging protocols to assess cell fate decisions following treatment with the anti-mitotic drug paclitaxel are described.

Live-cell imaging is a powerful technique that can be used to directly visualize biological phenomena in single cells over extended periods of time. Over ...

Viral Nanoparticles for In vivo Tumor Imaging

The use of nanomaterials has the potential to revolutionize materials science and medicine. Currently, a number of different nanoparticles are being investigated for applications in imaging and therapy. Viral nanoparticles (VNPs) derived from plants can be regarded as self-assembled bionanomaterials with defined sizes and shapes. Plant viruses under investigation in the Steinmetz lab include icosahedral particles formed by Cowpea mosaic virus (CPMV) and Brome mosaic virus (BMV), both of which are 30 nm in diameter. We are also developing rod-shaped and filamentous structures derived from the following plant viruses: Tobacco mosaic virus (TMV), which forms rigid rods with dimensions of 300 nm by 18 nm, and Potato virus X (PVX), which form filamentous particles 515 nm in length and 13 nm in width (the reader is referred to refs. 1 and 2 for further information on VNPs).
From a materials scientist's point of view, VNPs are attractive building blocks for several reasons: the particles are monodisperse, can be produced with ease on large scale in planta, are exceptionally stable, and biocompatible. Also, VNPs are "programmable" units, which can be specifically engineered using genetic modification or chemical bioconjugation methods 3. The structure of VNPs is known to atomic resolution, and modifications can be carried out with spatial precision at the atomic level4, a level of control that cannot be achieved using synthetic nanomaterials with current state-of-the-art technologies.
In this paper, we describe the propagation of CPMV, PVX, TMV, and BMV in Vigna ungiuculata and Nicotiana benthamiana plants. Extraction and purification protocols for each VNP are given. Methods for characterization of purified and chemically-labeled VNPs are described. In this study, we focus on chemical labeling of VNPs with fluorophores (e.g. Alexa Fluor 647) and polyethylene glycol (PEG). The dyes facilitate tracking and detection of the VNPs 5-10, and PEG reduces immunogenicity of the proteinaceous nanoparticles while enhancing their pharmacokinetics 8,11. We demonstrate tumor homing of PEGylated VNPs using a mouse xenograft tumor model. A combination of fluorescence imaging of tissues ex vivo using Maestro Imaging System, fluorescence quantification in homogenized tissues, and confocal microscopy is used to study biodistribution. VNPs are cleared via the reticuloendothelial system (RES); tumor homing is achieved passively via the enhanced permeability and retention (EPR) effect12. The VNP nanotechnology is a powerful plug-and-play technology to image and treat sites of disease in vivo. We are further developing VNPs to carry drug cargos and clinically-relevant imaging moieties, as well as tissue-specific ligands to target molecular receptors overexpressed in cancer and cardiovascular disease.

Viral Nanoparticles for In vivo Tumor Imaging

Plant viral nanoparticles (VNPs) are promising platforms for applications in biomedicine. Here, we describe the procedures for plant VNP propagation, purification, characterization, and bioconjugation. Finally, we show the application of VNPs for tumor homing and imaging using a mouse xenograft model and fluorescence imaging.

The use of nanomaterials has the potential to revolutionize materials science and medicine. Currently, a number of different nanoparticles are being ...

Bioengineering

Live Imaging of Drug Responses in the Tumor Microenvironment in Mouse Models of Breast Cancer

The tumor microenvironment plays a pivotal role in tumor initiation, progression, metastasis, and the response to anti-cancer therapies. Three-dimensional co-culture systems are frequently used to explicate tumor-stroma interactions, including their role in drug responses. However, many of the interactions that occur in vivo in the intact microenvironment cannot be completely replicated in these in vitro settings. Thus, direct visualization of these processes in real-time has become an important tool in understanding tumor responses to therapies and identifying the interactions between cancer cells and the stroma that can influence these responses. Here we provide a method for using spinning disk confocal microscopy of live, anesthetized mice to directly observe drug distribution, cancer cell responses and changes in tumor-stroma interactions following administration of systemic therapy in breast cancer models. We describe procedures for labeling different tumor components, treatment of animals for observing therapeutic responses, and the surgical procedure for exposing tumor tissues for imaging up to 40 hours. The results obtained from this protocol are time-lapse movies, in which such processes as drug infiltration, cancer cell death and stromal cell migration can be evaluated using image analysis software.

We describe a method for imaging response to anti-cancer treatment in vivo and at single cell resolution.

The tumor microenvironment plays a pivotal role in tumor initiation, progression, metastasis, and the response to anti-cancer therapies. Three-dimensional ...

Fluorescence-quenching of a Liposomal-encapsulated Near-infrared Fluorophore as a Tool for In Vivo Optical Imaging

Optical imaging offers a wide range of diagnostic modalities and has attracted a lot of interest as a tool for biomedical imaging. Despite the enormous number of imaging techniques currently available and the progress in instrumentation, there is still a need for highly sensitive probes that are suitable for in vivo imaging. One typical problem of available preclinical fluorescent probes is their rapid clearance in vivo, which reduces their imaging sensitivity. To circumvent rapid clearance, increase number of dye molecules at the target site, and thereby reduce background autofluorescence, encapsulation of the near-infrared fluorescent dye, DY-676-COOH in liposomes and verification of its potential for in vivo imaging of inflammation was done. DY-676 is known for its ability to self-quench at high concentrations. We first determined the concentration suitable for self-quenching, and then encapsulated this quenching concentration into the aqueous interior of PEGylated liposomes. To substantiate the quenching and activation potential of the liposomes we use a harsh freezing method which leads to damage of liposomal membranes without affecting the encapsulated dye. The liposomes characterized by a high level of fluorescence quenching were termed Lip-Q. We show by experiments with different cell lines that uptake of Lip-Q is predominantly by phagocytosis which in turn enabled the characterization of its potential as a tool for in vivo imaging of inflammation in mice models. Furthermore, we use a zymosan-induced edema model in mice to substantiate the potential of Lip-Q in optical imaging of inflammation in vivo. Considering possible uptake due to inflammation-induced enhanced permeability and retention (EPR) effect, an always-on liposome formulation with low, non-quenched concentration of DY-676-COOH (termed Lip-dQ) and the free DY-676-COOH were compared with Lip-Q in animal trials.

Fluorescence-quenching of a Liposomal-encapsulated Near-infrared Fluorophore as a Tool for In Vivo Optical Imaging

The use of fluorophores for in vivo imaging can be greatly limited by opsonization, rapid clearance, low detection sensitivity and cytotoxic effects on the host. Encapsulation of fluorophores in liposomes by film hydration and extrusion leads to fluorescence quenching and protection which enables in vivo imaging with high detection sensitivity.

Optical imaging offers a wide range of diagnostic modalities and has attracted a lot of interest as a tool for biomedical imaging. Despite the enormous ...

Solid Lipid Nanoparticles (SLNs) for Intracellular Targeting Applications

Nanoparticle-based delivery vehicles have shown great promise for intracellular targeting applications, providing a mechanism to specifically alter cellular signaling and gene expression. In a previous investigation, the synthesis of ultra-small solid lipid nanoparticles (SLNs) for topical drug delivery and biomarker detection applications was demonstrated. SLNs are a well-studied example of a nanoparticle delivery system that has emerged as a promising drug delivery vehicle. In this study, SLNs were loaded with a fluorescent dye and used as a model to investigate particle-cell interactions. The phase inversion temperature (PIT) method was used for the synthesis of ultra-small populations of biocompatible nanoparticles. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylphenyltetrazolium bromide (MTT) assay was utilized in order to establish appropriate dosing levels prior to the nanoparticle-cell interaction studies. Furthermore, primary human dermal fibroblasts and mouse dendritic cells were exposed to dye-loaded SLN over time and the interactions with respect to toxicity and particle uptake were characterized using fluorescence microscopy and flow cytometry. This study demonstrated that ultra-small SLNs, as a nanoparticle delivery system, are suitable for intracellular targeting of different cell types.

Solid Lipid Nanoparticles SLNs for Intracellular Targeting Applications

In this study, a method for synthesizing ultra-small populations of biocompatible nanoparticles was described, as well as several in vitro methods by which to assess their cellular interactions.

Nanoparticle-based delivery vehicles have shown great promise for intracellular targeting applications, providing a mechanism to specifically alter ...

Super-resolution Imaging of the Natural Killer Cell Immunological Synapse on a Glass-supported Planar Lipid Bilayer

The glass-supported planar lipid bilayer system has been utilized in a variety of disciplines. One of the most useful applications of this technique has been in the study of immunological synapse formation, due to the ability of the glass-supported planar lipid bilayers to mimic the surface of a target cell while forming a horizontal interface. The recent advances in super-resolution imaging have further allowed scientists to better view the fine details of synapse structure. In this study, one of these advanced techniques, stimulated emission depletion (STED), is utilized to study the structure of natural killer (NK) cell synapses on the supported lipid bilayer. Provided herein is an easy-to-follow protocol detailing: how to prepare raw synthetic phospholipids for use in synthesizing glass-supported bilayers; how to determine how densely protein of a given concentration occupies the bilayer's attachment sites; how to construct a supported lipid bilayer containing antibodies against NK cell activating receptor CD16; and finally, how to image human NK cells on this bilayer using STED super-resolution microscopy, with a focus on distribution of perforin positive lytic granules and filamentous actin at NK synapses. Thus, combining the glass-supported planar lipid bilayer system with STED technique, we demonstrate the feasibility and application of this combined technique, as well as intracellular structures at NK immunological synapse with super-resolution.

We describe here a combination of the glass-supported lipid bilayer technique of forming immunological synapses with the super-resolution imaging technique of stimulated emission depletion (STED) microscopy. The goal of this protocol is to provide users with the instructions necessary to successfully carry out these two techniques.

The glass-supported planar lipid bilayer system has been utilized in a variety of disciplines. One of the most useful applications of this technique has ...

Immunology and Infection

Targeted Plasma Membrane Delivery of a Hydrophobic Cargo Encapsulated in a Liquid Crystal Nanoparticle Carrier

The controlled delivery of drug/imaging agents to cells is critical for the development of therapeutics and for the study of cellular signaling processes. Recently, nanoparticles (NPs) have shown significant promise in the development of such delivery systems. Here, a liquid crystal NP (LCNP)-based delivery system has been employed for the controlled delivery of a water-insoluble dye, 3,3&#8242;-dioctadecyloxacarbocyanine perchlorate (DiO), from within the NP core to the hydrophobic region of a plasma membrane bilayer. During the synthesis of the NPs, the dye was efficiently incorporated into the hydrophobic LCNP core, as confirmed by multiple spectroscopic analyses. Conjugation of a PEGylated cholesterol derivative to the NP surface (DiO-LCNP-PEG-Chol) enabled the binding of the dye-loaded NPs to the plasma membrane in HEK 293T/17 cells. Time-resolved laser scanning confocal microscopy and F&#246;rster resonance energy transfer (FRET) imaging confirmed the passive efflux of DiO from the LCNP core and its insertion into the plasma membrane bilayer. Finally, the delivery of DiO as a LCNP-PEG-Chol attenuated the cytotoxicity of DiO; the NP form of DiO exhibited ~30-40% less toxicity compared to DiOfree delivered from bulk solution. This approach demonstrates the utility of the LCNP platform as an efficient modality for the membrane-specific delivery and modulation of hydrophobic molecular cargos.

A liquid crystal nanoparticle (LCNP) nanocarrier is exploited as a vehicle for the controlled delivery of a hydrophobic cargo to the plasma membrane of living cells.

The controlled delivery of drug/imaging agents to cells is critical for the development of therapeutics and for the study of cellular signaling processes. ...

Preparation, Administration, and Assessment of In Vivo Tissue-Specific Cellular Uptake of Fluorescent Dye-Labeled Liposomes

There is a growing interest in using liposomes to deliver compounds in vivo particularly for targeted treatment approaches. Depending on the liposome formulation, liposomes may be preferentially taken up by different cell types in the body. This may influence the efficacy of the therapeutic particle as progression of different diseases is tissue- and cell-type-specific. In this protocol, we present one method for synthesizing and fluorescently labeling liposomes using DSPC, cholesterol, and PEG-2000 DSPE and the lipid dye DiD as a fluorescent label. This protocol also presents an approach for delivering liposomes in vivo and assessing cell-specific uptake of liposomes using flow cytometry. This approach can be used to determine the types of cells that take up liposomes and quantify the distribution and proportion of liposome-uptake across cell types and tissues. While not mentioned in this protocol, additional assays such as immunofluorescence and single-cell fluorescence imaging on a cytometer will strengthen any findings or conclusions made as they permit assessment of intracellular staining. Protocols may also need to be adapted depending on the tissue(s) of interest.

The goal of this protocol is to synthesize fluorescently-labeled liposomes and use flow cytometry to identify in vivo localization of liposomes at a cellular level.

There is a growing interest in using liposomes to deliver compounds in vivo particularly for targeted treatment approaches. Depending on the liposome ...

Using In Vitro Live-cell Imaging to Explore Chemotherapeutics Delivered by Lipid-based Nanoparticles

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