The authors thank Ms. Kaylee Smith, Ms. Lauren Kwok, and Mr. Alexander Floru for proofreading the manuscript. H.F. acknowledges grant support from the NIH (CA134743 and CA215059), the American Cancer Society (RSG-17-204 01-TBG), and the St. Baldrick&#39;s Foundation. F.J.F.L. acknowledges a fellowship from Boston University Innovation Center-BUnano Cross-Disciplinary Training in Nanotechnology for Cancer (XTNC). I.S acknowledges NSF support (grant CBET 1605405) and NIH R41AI142890.

All animal procedures were approved by the Institutional Animal Care and Use Committee (IACUC) at Boston University School of Medicine under the protocol #: PROTO201800543.
1. Generation of Casper zebrafish embryos
<ol>
	<li>Choose adult Casper fish that are at least 3 months of age for natural breeding to generate transparent Casper zebrafish embryos.</li>
	<li>Fill two-chamber mating tanks with fish water in the evening, separate the upper tanks using dividers, p...

<ol>
	<li>Dadwal, A., Baldi, A., Kumar Narang, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanoparticles+as+carriers+for+drug+delivery+in+cancer.">Nanoparticles as carriers for drug delivery in cancer.</a> Artificial Cells, Nanomedicine, Biotechnology. 46 (Suppl 2), 295-305 (2018).</li><li>Cho, K., Wang, X., Nie, S., Chen, Z. G., Shin, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Therapeutic+nanoparticles+for+drug+delivery+in+cancer.">Therapeutic nanoparticles for drug delivery in cancer.</a> Clinical Cancer Research. 14 (5), 1310-1316 (2008).</li><li>Senapati, S., Mahanta, A. K., Kumar, S., Maiti, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlled+drug+delivery+vehicles+for+cancer+treatment+and+their+performance.">Controlled drug delivery vehicles for cancer treatment and their performance.</a> Signal Transduction and Target Therapy. 3, 7 (2018).</li><li>Chinen, A. B., 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=Nanoparticle+Probes+for+the+Detection+of+Cancer+Biomarkers,+Cells,+and+Tissues+by+Fluorescence.">Nanoparticle Probes for the Detection of Cancer Biomarkers, Cells, and Tissues by Fluorescence.</a> Chemal Reviews. 115 (19), 10530-10574 (2015).</li><li>Palantavida, S., Guz, N. V., Woodworth, C. D., Sokolov, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrabright+fluorescent+mesoporous+silica+nanoparticles+for+prescreening+of+cervical+cancer.">Ultrabright fluorescent mesoporous silica nanoparticles for prescreening of cervical cancer.</a> Nanomedicine. 9 (8), 1255-1262 (2013).</li><li>Singh, M., Murriel, C. L., Johnson, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetically+engineered+mouse+models:+closing+the+gap+between+preclinical+data+and+trial+outcomes.">Genetically engineered mouse models: closing the gap between preclinical data and trial outcomes.</a> Cancer Research. 72 (11), 2695-2700 (2012).</li><li>Sharkey, F. E., Fogh, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Considerations+in+the+use+of+nude+mice+for+cancer+research.">Considerations in the use of nude mice for cancer research.</a> Cancer Metastasis Reviews. 3 (4), 341-360 (1984).</li><li>Vargo-Gogola, T., Rosen, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modelling+breast+cancer:+one+size+does+not+fit+all.">Modelling breast cancer: one size does not fit all.</a> Nature Reviews Cancer. 7 (9), 659-672 (2007).</li><li>Minn, A. J., 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=Genes+that+mediate+breast+cancer+metastasis+to+lung.">Genes that mediate breast cancer metastasis to lung.</a> Nature. 436 (7050), 518-524 (2005).</li><li>Soares, K. C., 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=A+preclinical+murine+model+of+hepatic+metastases.">A preclinical murine model of hepatic metastases.</a> Journal of Visualized Experiments. (91), e51677 (2014).</li><li>Etchin, J., Kanki, J. P., Look, A. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+as+a+model+for+the+study+of+human+cancer.">Zebrafish as a model for the study of human cancer.</a> Methods in Cell Biology. 105, 309-337 (2011).</li><li>Liu, S., Leach, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+models+for+cancer.">Zebrafish models for cancer.</a> Annual Reviews in Pathology. 6, 71-93 (2011).</li><li>Kimmel, C. B., Ballard, W. W., Kimmel, S. R., Ullmann, B., Schilling, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stages+of+embryonic+development+of+the+zebrafish.">Stages of embryonic development of the zebrafish.</a> Developmental Dynamics. 203 (3), 253-310 (1995).</li><li>White, R. 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=Transparent+adult+zebrafish+as+a+tool+for+in+vivo+transplantation+analysis.">Transparent adult zebrafish as a tool for in vivo transplantation analysis.</a> Cell Stem Cell. 2 (2), 183-189 (2008).</li><li>Lam, S. H., Chua, H. L., Gong, Z., Lam, T. J., Sin, Y. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+maturation+of+the+immune+system+in+zebrafish,+Danio+rerio:+a+gene+expression+profiling,+in+situ+hybridization+and+immunological+study.">Development and maturation of the immune system in zebrafish, Danio rerio: a gene expression profiling, in situ hybridization and immunological study.</a> Developmental and Comparative Immunology. 28 (1), 9-28 (2004).</li><li>Westerfield, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> THE ZEBRAFISH BOOK. , (2007).</li><li>Anderson, N. 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=The+TCA+cycle+transferase+DLST+is+important+for+MYC-mediated+leukemogenesis.">The TCA cycle transferase DLST is important for MYC-mediated leukemogenesis.</a> Leukemia. 30 (6), 1365-1374 (2016).</li><li>Peerzade, S., 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=Ultrabright+fluorescent+silica+nanoparticles+for+in+vivo+targeting+of+xenografted+human+tumors+and+cancer+cells+in+zebrafish.">Ultrabright fluorescent silica nanoparticles for in vivo targeting of xenografted human tumors and cancer cells in zebrafish.</a> Nanoscale. 11 (46), 22316-22327 (2019).</li><li>Peng, B., 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=Ultrabright+fluorescent+cellulose+acetate+nanoparticles+for+imaging+tumors+through+systemic+and+topical+applications.">Ultrabright fluorescent cellulose acetate nanoparticles for imaging tumors through systemic and topical applications.</a> Materials Today (Kidlington). 23, 16-25 (2019).</li><li>Peng, B., 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=Data+on+ultrabright+fluorescent+cellulose+acetate+nanoparticles+for+imaging+tumors+through+systemic+and+topical+applications.">Data on ultrabright fluorescent cellulose acetate nanoparticles for imaging tumors through systemic and topical applications.</a> Data in Brief. 22, 383-391 (2019).</li><li>Masters, J. R., Stacey, G. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changing+medium+and+passaging+cell+lines.">Changing medium and passaging cell lines.</a> Nature Protocols. 2 (9), 2276-2284 (2007).</li><li>Rao, P. N., Engelberg, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hela+Cells:+Effects+of+Temperature+on+the+Life+Cycle.">Hela Cells: Effects of Temperature on the Life Cycle.</a> Science. 148 (3673), 1092-1094 (1965).</li><li>Avdesh, 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=Regular+care+and+maintenance+of+a+zebrafish+(Danio+rerio)+laboratory:+an+introduction.">Regular care and maintenance of a zebrafish (Danio rerio) laboratory: an introduction.</a> Journal of Visualized Experiments. (69), e4196 (2012).</li><li>Spence, R., Gerlach, G., Lawrence, C., Smith, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+behaviour+and+ecology+of+the+zebrafish,+Danio+rerio.">The behaviour and ecology of the zebrafish, Danio rerio.</a> Biological Reviews of the Cambridge Philosophical Society. 83 (1), 13-34 (2008).</li></ol>

I.S. declares interest in NanoScience Solutions, LLC (recipient of STTR NIH R41AI142890 grant). All other authors declare no conflicts of interest.

The protocol described here utilizes the zebrafish as an in vivo system to test the ability of nanoparticles to recognize and target metastatic human cancer cells. Several factors can impact the successful execution of the experiments. First, embryos need to be fully developed at 48 hpf. The correct developmental stage of the embryos enables them to endure and survive the transplantation of human cancer cells. Embryos younger than 48 hpf have a significantly lower survival rate compared to older and more developed embryo...

The development of nanoparticles that are capable of detecting, targeting, and destroying cancer cells is of great interest to both physicists and biomedical researchers. The emergence of nanomedicine led to the development of several nanoparticles, such as those conjugated with targeting ligands and/or chemotherapeutic drugs1,2,3. The added properties of nanoparticles enable their interaction with the biological system, sensing and monitoring biological events with high efficiency and accuracy along with therapeutic applications. Gold and iron oxide nanoparticles are primarily used in computed tomography and magnetic resonance imaging applications, respectively. While the enzymatic activities of gold and iron oxide nanoparticles allow the detection of cancer cells through colorimetric assays, fluorescent nanoparticles are well suited for in vivo imaging applications4. Among them, ultrabright fluorescent nanoparticles are particularly beneficial, due to their ability to detect cancers early with fewer particles and reduced toxicities5.
Despite these advantages, nanoparticles require experimentation using in vivo animal models for the selection of suitable nanomaterials and optimization of the synthesis process. Additionally, just like drugs, nanoparticles rely on animal models for preclinical testing to determine their efficacy and toxicities. The most widely used preclinical model is the mouse, which is a mammal whose upkeep comes at a relatively high cost. For cancer studies, either genetically engineered mice or xenografted mice are typically used6,7. The length of these experiments often spans from weeks to months. In particular, for cancer metastasis studies, cancer cells are directly injected into the circulatory system of the mice at locations such as tail veins and spleens8,9,10. These models only represent the end stages of metastasis when tumor cells extravasate and colonize distant organs. Moreover, due to visibility issues, it is particularly challenging to monitor tumor cell migration and nanoparticle targeting of tumor cells in mice.
The zebrafish (Danio rerio) has become a powerful vertebrate system for cancer research due to its high fecundity, low cost, rapid development, optical transparency, and genetic conservations11,12. Another advantage of the zebrafish over the mouse model is the fertilization of the fish eggs ex utero, which allows the embryos to be monitored throughout their development. Embryonic development is rapid in zebrafish, and within 24 hours postfertilization (hpf), the vertebrate body plane has already formed13. By 72 hpf, eggs are hatched from the chorion, transitioning from the embryonic to the fry stage. The transparency of the zebrafish, the Casper strain in particular14, provides a unique opportunity to visualize the migration of cancer cells and their recognition and targeting by nanoparticles in a living animal. Finally, zebrafish develop their innate immune system by 48 hpf, with the adaptive immune system lagging behind and only becoming functional at 28 days postfertilization15. This time gap is ideal for the transplantation of various types of human cancer cells into zebrafish embryos without experiencing immune rejections.
Described here is a method that takes advantage of the transparency and rapid development of zebrafish to demonstrate the recognition and targeting of human cancer cells by fluorescent nanoparticles in vivo. In this assay, human cervical cancer cells (HeLa cells) genetically engineered to express a red fluorescent protein were injected into the vascularized area in the perivitelline cavity of 48 hpf embryos. After 20-24 h, HeLa cells had already spread throughout the embryos through the fish circulatory system. Embryos with apparent metastasis were microinjected with ~0.5 nL of a nanoparticle solution directly behind the eye, where the rich capillary bed is located. Using this technique, the ultrabright fluorescent silica nanoparticles can target HeLa cells as quickly as 20-30 min postinjection. Due to its simplicity and effectiveness, the zebrafish represents a robust in vivo model to test a variety of nanoparticles for their ability to target specific cancer cells.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Agarose</td>
			<td>KSE scientific</td>
			<td>BMK-A1705</td>
			<td></td>
		</tr>
		<tr>
			<td>Borosilicate glass capillaries</td>
			<td>World Precision Instruments</td>
			<td>1.0 mm O.D. x 0,78 mm</td>
			<td></td>
		</tr>
		<tr>
			<td>Computer and monitor</td>
			<td>ThinkCentre</td>
			<td>X000335</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM (Dulbecco&#39;s Modified Eagle&#39;s Medium)</td>
			<td>Corning</td>
			<td>10-013-CV</td>
			<td>sold by Fisher</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Sigma-Aldrich</td>
			<td>F0926</td>
			<td></td>
		</tr>
		<tr>
			<td>Fish incubator</td>
			<td>VWR</td>
			<td>35960-056</td>
			<td></td>
		</tr>
		<tr>
			<td>Hemocytometer</td>
			<td>Fishersci brand</td>
			<td>02-671-51B</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnetic stand</td>
			<td>World Precision Instruments</td>
			<td>M10</td>
			<td></td>
		</tr>
		<tr>
			<td>Microloader tip</td>
			<td>Eppendorf</td>
			<td>E5242956003</td>
			<td>sold by Fisher</td>
		</tr>
		<tr>
			<td>Micromanipulator</td>
			<td>Applied Scientific Instrumentation</td>
			<td>MMPI-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Needle Puller</td>
			<td>Sutter instruments</td>
			<td>P-97</td>
			<td></td>
		</tr>
		<tr>
			<td>Olympus MVX-10 fluorescent microscope</td>
			<td>Olympus</td>
			<td>MVX-10</td>
			<td></td>
		</tr>
		<tr>
			<td>P200 tip</td>
			<td>Fishersci brand</td>
			<td><a href="https://www.fishersci.com/shop/products/corning-universal-fit-pipet-tips-bulk-packed-6/07200293?keyword=true" target="_blank">07-200-293</a></td>
			<td></td>
		</tr>
		<tr>
			<td>PBS (Dulbecco&#39;s Phosphate-Buffered Salt Solution 1X)</td>
			<td>Corning</td>
			<td>21-030-CV</td>
			<td>sold by Fisher</td>
		</tr>
		<tr>
			<td>Petri dish</td>
			<td>Corning</td>
			<td>SB93102</td>
			<td>sold by Fisher</td>
		</tr>
		<tr>
			<td>Plastic pipette</td>
			<td>Fishersci brand</td>
			<td>50-998-100</td>
			<td></td>
		</tr>
		<tr>
			<td><a href="https://www.addgene.org/113726/" target="_blank">pLenti6.2_miRFP670</a></td>
			<td>Addgene</td>
			<td>13726</td>
			<td></td>
		</tr>
		<tr>
			<td>Pneumatic pico pump</td>
			<td>World Precision Instruments</td>
			<td>SYSPV820</td>
			<td></td>
		</tr>
		<tr>
			<td>Pronase</td>
			<td>Roche-Sigma-Fisher</td>
			<td>50-100-3275</td>
			<td>Roche product made by Sigma- sold by Fisher</td>
		</tr>
		<tr>
			<td>Razor blade</td>
			<td>Fishersci brand</td>
			<td>12-640</td>
			<td></td>
		</tr>
		<tr>
			<td>SZ51 dissection microscope</td>
			<td>Olympus</td>
			<td>SZ51</td>
			<td></td>
		</tr>
		<tr>
			<td>Tricaine methanesulfonate</td>
			<td>Western Chemicals</td>
			<td>NC0872873</td>
			<td>sold by Fisher</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA</td>
			<td>Corning</td>
			<td>MT25053CI</td>
			<td>sold by Fisher</td>
		</tr>
		<tr>
			<td>Tweezer</td>
			<td>Fishersci brand</td>
			<td>12-000-122</td>
			<td></td>
		</tr>
	</tbody>
</table>

in vivo targeting of xenografted human cancer cells with functionalized fluorescent silica nanoparticles in zebrafish

Developing nanoparticles capable of detecting, targeting, and destroying cancer cells is of great interest in the field of nanomedicine. In vivo animal models are required for bridging the nanotechnology to its biomedical application. The mouse represents the traditional animal model for preclinical testing; however, mice are relatively expensive to keep and have long experimental cycles due to the limited progeny from each mother. The zebrafish has emerged as a powerful model system for developmental and biomedical research, including cancer research. In particular, due to its optical transparency and rapid development, zebrafish embryos are well suited for real-time in vivo monitoring of the behavior of cancer cells and their interactions with their microenvironment. This method was developed to sequentially introduce human cancer cells and functionalized nanoparticles in transparent Casper zebrafish embryos and monitor in vivo recognition and targeting of the cancer cells by nanoparticles in real time. This optimized protocol shows that fluorescently labeled nanoparticles, which are functionalized with folate groups, can specifically recognize and target metastatic human cervical epithelial cancer cells labeled with a different fluorochrome. The recognition and targeting process can occur as early as 30 min postinjection of the nanoparticles tested. The whole experiment only requires the breeding of a few pairs of adult fish and takes less than 4 days to complete. Moreover, zebrafish embryos lack a functional adaptive immune system, allowing the engraftment of a wide range of human cancer cells. Hence, the utility of the protocol described here enables the testing of nanoparticles on various types of human cancer cells, facilitating the selection of optimal nanoparticles in each specific cancer context for future testing in mammals and the clinic.

The protocol schematic in Figure 1&#160;illustrates the overall procedures for this study. Transparent Casper male and female adult fish were bred to generate embryos (section 1). RFP+ HeLa cells were injected into the vascularized area under the perivitelline cavity of the zebrafish embryos at 48 hpf, with uninjected embryos as controls (section 3). For individuals experienced in microinjection, the survival rate of embryos is often high, with at le...

Watch this Scientific Journal Video about In Vivo Targeting of Xenografted Human Cancer Cells with Functionalized Fluorescent Silica Nanoparticles in Zebrafish at JoVE.com

In Vivo Targeting of Xenografted Human Cancer Cells with Functionalized Fluorescent Silica Nanoparticles in Zebrafish

Described here is a method for utilizing zebrafish embryos to study the ability of functionalized nanoparticles to target human cancer cells in vivo. This method allows for the evaluation and selection of optimal nanoparticles for future testing in large animals and in clinical trials.

Developing nanoparticles capable of detecting, targeting, and destroying cancer cells is of great interest in the field of nanomedicine. In vivo animal ...

This procedure allows researchers to evaluate and select optimal nano-particles for studies in large animals and clinical trials. This simple method helps determine whether the nanoparticle can target the human cancer cells in vivo and if so, how efficient the targeting is. Demonstrating the procedures will be Dr.Xiodan Qin, a postdoc, and Mr.Andrew Lam, a research technician from our lab.Fill two chamber mating tanks with fish water in the evening and separate the upper tanks using dividers. Place a male zebrafish into one side of the chamber and one or two females zebrafish into another side of the chamber. Remove the dividers at 8:00 AM the following morning.Add artificial enrichment plants and tilt the top chamber slightly to create a shallow area of water. Allow the zebrafish to breed for three to four hours and collect the eggs from the bottom chamber by pouring the water through a mesh net. Transfer the eggs to a Petri dish with no more than 200 eggs per dish.Remove any dead or unfertilized eggs. Fill the dish two thirds full with fresh fish water, and place it in the incubator. After 24 hours, remove the dish from the incubator and remove as much water as possible from the dish.Add a few drops of one milligram per milliliter pronase solution and gently swirl the dish. When the chorions show signs of disintegration, pipette the embryos up and down a few times to break down the chorions and release the embryos. Once the majority of the embryos are out of the chorions, immediately add fresh fish water to terminate the process.Rinse the embryos with fish water three times to remove the floating chorions Return the embryos to the incubator. Make a 3%agarose solution by adding three grams of electrophoresis-grade agarose to 100 milliliters of autoclaved fish water. Microwave the agarose and water until the agarose is completely dissolved.To create the injection plate, pour hot agarose solution into a Petri dish until it is three quarters full and place the micro injection mold on the agarose. After the agarose has solidified, carefully remove the mold. Fill the resulting injection plate with autoclaved fish water and store it at four degrees Celsius.To make the injection needles, pull borosilicate glass capillaries one millimeter outer diameter by 0.78 millimeters on a pipette puller. Store the injection needles on putty in a large Petri dish that has been wiped with an ethanol towel. Before harvesting the HeLa cells, rewarm the injection plate and fish water in the incubator at 35.5 degrees Celsius.30 minutes to an hour before transplantation, harvest the HeLa cells. Working in a tissue culture hood, remove the medium from the flask by aspiration. Rinse the cells with sterile PBS to remove all traces of the medium.Add three milliliters of sterile trypsin EDTA to the flask and incubate it at 37 degrees Celsius for three to five minutes. Observe the flask under the microscope to monitor the progress of enzymatic digestion. When approximately 80%of the cells are suspended, add six to eight milliliters of growth medium to the flask.Collect the cells by pipetting gently and transfer them to a 15 milliliter sterile tube. Centrifuge the tube at 135 times G for five minutes. Aspirate the supernatant and resuspend the HeLa cells in three milliliters of complete growth medium.After repeating the wash steps twice, resuspend the cells in one milliliter of complete growth medium. Then count the cells under the microscope using a hemocytometer. After centrifuging the cells again, remove the supernatant.Resuspend the cells in a 1.5 milliliter microcentrifuge tube at a concentration of five times 10 to the seventh cells per milliliter. Keep the cells warm by holding the tube in one hand when transporting to the fish facility. Using a plastic pipette, place the zebrafish embryos on the injection plate.Lay embryos on their sides with the anterior facing forward aligned to the grooves on the plate. Turn on the air source and the micro injector. After retrieving the HeLa cells from the incubator, pipette the cells up and down 20 to 30 times using a P200.Using a gel loading tip with a cut end, immediately load three microliters of the HeLa cell suspension into one of the injection needles. Insert the needle into the needle holder and place the injection plate under the microscope. Inject the cell mixture into the embryo at the vascularized area under the perivitelline cavity by pressing the foot pedal.Then pipette a few drops of sterile fish water onto the embryo. To continue the injection process, use the non-dominant hand to move the injection plate to the next embryo, and then use the dominant hand to extend and retract the injector while simultaneously pressing the foot pedal. Finish injection of all embryos on the plate and wash the embryos with sterile fish water into a sterile Petri dish.Immediately incubate the dish at 35.5 degrees Celsius. After three hours, examine the injected embryos and remove the dead ones. On the next day, make injection needles for the nano-particles and store them on putty in a large Petri dish.Working under a fluorescent microscope, carefully pick up embryos with tail metastases and place them on a new injection plate with sterile fish water. For each zebrafish with a tail metastasis, inject either nano-particle solution or water behind the eye. Also inject nano-particle solution behind the eyes of the zebrafish in the control group which did not receive HeLa transplants and incubate all the injected embryos at 35.5 degrees Celsius.Immediately after injection and again at 30 to 60 minute intervals, transfer two to three embryos to a Petri dish and immobilize them by adding five drops of diluted MS222 solution. Once the embryos stop swimming, remove most of the water to allow the embryos to lie on their sides. Using a pipette with a thin soft brush attached to its end, align the embryos so they lie with the anterior facing forward and only one eye visible.Using 2X magnification, capture images of the whole embryo on red, blue, and Brightfield channels. To capture the tail area, image the embryos at 6.4X magnification. Focus the embryo under the red channel to avoid the bleeding of nano-particles.Immediately after imaging, add fresh fish water to the embryos and return them to the incubator. Injected and control zebrafish embryos were imaged using a fluorescent microscope. The red dots are metastatic human cervical cancer cells while the blue dots represent the nano-particles.In one control group, zebrafish that were injected with cancer cells but not nano-particles, fluorescent HeLa cells were visible in the red channel and no specific fluorescent signals were detected in the blue channel. In the other control group, zebrafish were injected with nano-particles, but not with cancer cells. Blue fluorescent nano-particles were distributed throughout the circulatory system.No specific red florescence was visible. When zebrafish with tail metastases were injected with nano-particles, overlaid images from the red and blue channels show co-localization of HeLa cells and nano-particles. After performing this method, we can analyze tumor burden and cancer cell survival, the animals with and without injecting nano-particles to adjust their ability of killing cancer cells.This technique may facilitate the selection of the optimal nanoparticle that can specifically recognize the cancer cell in vivo leading to early detection of cancer and cancer metastasis.

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Research

JoVE Journal

Cancer Research

4.5K visualizações.  Boston University School of Medicine. Este procedimento permite que os pesquisadores avaliem e selecionem nanopartículas ideais para estudos em animais de grande porte e ensaios clínicos. Este método simples ajuda a determinar se a nanopartícula pode atingir as células cancerígenas humanas in vivo e, se sim, quão eficiente é o alvo. Demonstrando os procedimentos estarão o Dr. Xiodan Qin, um pós-doutor, e o Sr. Andrew Lam, um técnico de pesquisa do nosso laboratório.Encha dois tanques de acasalamento de câmara ...