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

The overall goal of this procedure is to quantify the uptake of nanoparticles into tumors using intra vital imaging. This is accomplished by first des shelling and culturing avian embryos. The second step is to establish xenograft tumors in the chorio elan toic membrane or cam, and allow them to grow for approximately one week.

The third step of the procedure is to intravenously inject nanoparticles into the cam and perform real-time intravital confocal imaging of the xenograft tumor. The final step of the procedure is to excise and section the tumor. Ultimately, the uptake of nanoparticles by the tumor is quantified from the images and secondary tumor analysis can be performed.

The main advantage of this technique over existing methods is that it allows for the generation of high resolution images to assess the dynamics of nanoparticle accumulations in tumors at the microscopic level. Furthermore, using this model system, it is possible for a single researcher to analyze a very large number of experimental groups within two weeks. Additionally, the Cory Alloid membrane of the Shells embryos are easily accessible for physical manipulation and non-invasive imaging.

My laboratory is focused on the development of nanoparticles for targeted molecular imaging and drug delivery in cancer. Our search for a rapid and inexpensive xenograft model in which to perform detailed uptake analyses led us to the shells chicken embryo. Here we use high resolution intravital imaging to evaluate nanoparticle uptake in human tumor xenografts in a modified shells chicken embryo model.

For this protocol, prepare, shell list fertilized chicken embryos at day four. Using the standard method, ensure that the embryos are fertilized and viable by checking for a heartbeat and incubate them at 38 degrees Celsius with at least 60%relative humidity until embryonic. Day nine, the embryos are then ready for tumor cell inoculation.

Assemble a micro injector by connecting an 18 gauge needle to a one milliliter syringe with a five to six inch piece of tigon tubing. Carefully insert the bevel of the needle into the tubing. Approximately four to five inches of tubing should extend from the tip of the needle.

Now, draw the tumor cell suspension through the tubing into the syringe and invert. Carefully tap the side of the syringe and depress the plunger to remove any air bubbles. Next, insert a microinjection glass needle at the end of the tubing and depress the plunger until the cell suspension reaches the tip of the needle.

Now, under a dissection microscope, slowly and carefully inject 10, 000 to 100, 000 cancer cells into the interstitial space within the cam. The cells should form a visible bolus within the cam cells deposited onto the cam surface can be removed by lightly dabbing with a kim wipe or cotton applicator. Now, return the embryos to an incubator at 38 degrees Celsius with at least 60%humidity.

Allowed the tumor to grow and vascularize for up to seven days, reaching a diameter of at least five millimeters before imaging nanoparticles must be labeled with a fluorescent dye, compatible with the intravital microscope and stored at four degrees Celsius in PBS where they are stable for at least one year. Then spin down the nanoparticles for one minute to pellet any aggregates before being drawn into the micro injector. Now using PBS to dilute the nanoparticles, prepare a working concentration of one milligram per milliliter to ultimately inject 50 micrograms of nanoparticles in a volume of 50 microliters.

Now proceed with injections on day 16 of embryonic development. Check that the tumors are sufficiently vascularized to select the best tumor bearing embryos intravenously. Inject a low molecular weight fluorescent dextrin spectrally, distinct from the tumor cells and nanoparticles.

Then visualize the dextrin under a fluorescent microscope and select the embryos with the most vascularized tumors. Once the embryos have been selected, use a prepared micro injector to drop at least 200 microliters of the prepared nanoparticles. Invert the syringe.

Carefully tap the side and depress the plunger to remove any air bubbles. Now, insert a long tapered glass microinjection needle into the end of the tubing and depress the plunger until the nanoparticle solution reaches the tip of the needle. A blunt needle will not pierce through the ectoderm and an overly sharp and thin needle will break when inserted into the tissue.

Next intravenously, inject between 30 and 200 microliters of nanoparticles into embryos distal from the desired side to be visualized immediately after injecting the nanoparticles image. The tumors as described in the next section begin by preparing the embryo imaging unit. First, apply a thin layer of vacuum grease around the circumference of the imaging port on the underside of the lid.

Then fit an 18 millimeter glass cover slip onto the port. Now load the embryo into the unit so the cover slip is above the desired area for imaging. Slowly lower the unit's lid until the cover slip just makes contact with the embryo and screw the lid in place.

Now fill the dish containing the embryo into imaging unit with 37 degrees Celsius water. This unit will hold the embryo in place under the spinning disc confocal microscope at the spinning disc confocal fluorescence microscope. Check that the environmental chamber is 37 degrees Celsius.

Now, position the imaging unit on the stage so that the cover slip window is directly underneath the microscope. Objective and proceed with imaging. Acquire high resolution image stacks of the tumor and surrounding vasculature immediately after the injection and at hourly intervals.

Thereafter, images will reflect detailed structural changes occurring in the tumor vasculature. The embryo may be imaged continuously for up to 36 hours with no need to change the water bath. To quantify the uptake of viral nanoparticles from the images, use a software package such as velocity.

First, select the tumor and delineate it from the surrounding stroma. The selection is facilitated by labeling the tumor with fluorescent dextrin. Now calculate the mean fluorescent signal from the nanoparticles in the tumor and in representative areas of the adjacent stroma.

Next, calculate the ratio of the mean signal. Strengthen the tumor selection to the mean signal. Strengthen the stromal selection.

A ratio greater than one indicates that the nanoparticles are preferentially localized to the tumor. HT 29 colon cancer cells were injected to form a bolus of approximately one millimeter in diameter within the cam of day nine. Chicken embryos after seven days of incubation embryos were injected intravenously with a low molecular weight dextrin to confirm tumor vascularization.

Intravenous administration of CPMV LOR 6 47 or pegylated CPMV LOR 6 47 nanoparticles, followed by high resolution real-time confocal imaging revealed that both CPMV and pegylated CPMV nanoparticles rapidly labeled the entire vasculature. Within 12 hours after injection quantification of fluorescent signal in tumor versus stroma regions revealed the uptake of pegylated CPMV into the tumor was approximately three times higher than that of CPMV. After watching this video, you should have a good understanding of how to perform high resolution intravital imaging to evaluate nanoparticle uptake in tumor xenografts in an avian embryo model from preparation of the avian embryos to xog grafting of tumor cells, intravenous injection of nanoparticles, and assessment of nanoparticle uptake in tumors by real-time intravital confocal imaging.