The overall goal of this procedure is to evaluate the in vivo behavior of cancer nanomedicines at systemic, tissue, single cell, and subcellular levels in tumor-bearing, immunocompetent mice. This method can help answer the key question in the cancer nanomedicine field. How do nanomedicines accumulate quantitatively in different tissues of living animals over time?
The main advantage of this technique is that this dual-labeling approach allows for in vivo nanomedicine characterization from the macroscopic to the microscopic level. We choose liposomes as example to demostrate our procedure because liposomes are currently in clinical use, and they're also a common platform for experimental nanomedicines. To begin the procedure, dissolve 20 milligrams of a lipid mixture, which include 0.1%by mole of a lipophilic cationic fluorescent dye and two milliliters of chloroform.
Then, dry the lipids with a rotary evaporator. Add 20 milliliters of phosphate-buffered saline to the dried lipids. Sonicate the mixture on ice with a 3.8 millimeter probe sonicator at 30 watts for 30 minutes, keeping the mixture between four and 20 degrees Celsius throughout.
Centrifuge the resulting liposomal nanoparticle suspension at 4, 000 G for 10 minutes. Discard the pellet and wash the liposomes with PBS, using a 100 kilodalton MWCO centrifugal filter unit at 4000 G.Transfer the concentrated liposomes from the top chamber to a new centrifuge tube. The liposomes may be stored in PBS in a fridge for up to one week.
Perform dynamic light scattering on a mixture of 50 microliters of the purified liposome suspension and 950 microliters of PBS. Measure the particle sizes and determine the size distribution. To begin the radiolabeling procedure, transfer to a 1.5 milliliter tube, the needed volume of purified liposomes and PBS, to obtain two milligrams of lipids.
Add to this one millicurie of zurconium-89 oxalate for a final volume of 100 microliters and a pH of 6.9 to 7.1. Stir the mixture at 37 degrees Celsius for two hours. Then, remove free zirconium-89 by centrifugal filtration with a 100 kilodalton filter unit at 4, 000 G.Wash the retentate with sterile PBS three times at 4, 000 G.Then, dilute the retentate to about three microcurie per microliter for injection.
Use size-exclusion HPLC paired with a radiation detector to determine the radiochemical purity of the radiolabeled liposomes. If the radiochemical purity is below 95%reformulate the dose. Use dynamic light scattering to confirm that the particle size and distribution values are both similar to those of the non-radiolabeled liposomes.
To begin the imaging procedure, inject about 300 microcurie of radiolabeled liposomes into the tail vein of a restrained melanoma mouse. Two minutes after injection, use a 28 gauge insulin syringe to collect 10 to 20 microliters of blood from the tail vein of the mouse. Transfer the blood sample to a pre-weighed counting tube.
Weigh the blood sample and measure the activity with a gamma counter. Calculate the radioactivity concentration as percentage of injected dose per gram. Fifteen minutes after injection, collect and measure another blood sample in the same way.
Then, anesthetize the mouse by administration of 2%isoflurane and oxygen via a nosecone. Collect and measure a third blood sample one hour after injection. Then, allow the mouse to recover in a recovery cage until normal mobility has been regained.
Collect additional samples four, eight, 24, and 48 hours after injection. Allow the mouse to recover fully between injections. House the mouse in a room designated for animals to which radioactive materials have been administered.
At 24 or 48 hours after liposome injection, anesthetize the mouse with 2%isoflurane and oxygen. Place the mouse on its belly on a small animal microPET-CT scanner bed. Continue delivering 2%isoflurane and oxygen via a nose cone.
Apply vet ointment to the mouse's eyes to prevent them from drying. Then, move the scanner bed into the instrument. Perform a 15 minute whole-body PET static scan with at least 50 million coincident events, with an energy of 250 to 700 kiloelectron volts, and a coincidence-timing window of six nanoseconds.
Next, perform a five minute CT scan with 120 rotational steps over 220 degrees, with approximately 145 milliseconds per frame. After scan completion, immediately sacrifice the mouse by carbon dioxide asphyxiation. Confirm death by pinching the paw of the animal, and then perform cervical dislocation.
Perfuse the tissues with PBS to remove blood, and harvest the relevant tissue samples. Weigh the collected tissues and measure their activity. Calculate the radiolabeled liposome accumulation in tissue as percentage of injected dose per gram.
Store the tumor tissue in cold PBS. Perform ex vivo flow cytometry and immunoflurescence imaging on the harvested tumor tissue to measure accumulation with respect to cell populations. PET/CT imaging of tumor-bearing mice injected with nanoparticles labeled with zirconium-89 showed significant nanaparticle accumulation in the tumor tissue 24 hours after injection.
The blood half-life of the nanoparticles was determined to be approximately 10 hours. Ex vivo biodistribution measurements at 24 hours post-injection, were consistent with the in vivo PET/CT measurements. Nanoparticle accumulation was then investigated on a single-cell level with immunostaining techniques.
Flow cytometry indicated that nanoparticles were found to preferentially accumulate in tumor-assisted macrophages. Immunoflurescence imaging of tumor samples also indicated specific targeting of endothelial cells by the nanoparticles. This technique can help researchers identify promising nanomedicines for clinical translation.
Once mastered, this technique can be done in three days if it is performed properly. When radiolabeling nanomedicines, it's important to apply high standards of quality control. Following this procedure, other nanomedicines, such as nanoemulsions, polymeric nanoparticles, micelles, antibodies, and antibody fragments, can be labeled in order to measure their in vivo performance in tumor-bearing mice.
