(512b) Ultrasonic and Liposomal Drug Delivery to Tumors

Our previous research has developed ultrasonically-activated micellar drug carriers that can control delivery in space and time by focusing ultrasound (US) non-invasively on the specific tissue to be treated. Recently we have developed a liposomal drug carrier that is even more effective than our micellar system in reducing and even eliminating colon tumors in a rat model. This drug-containing carrier is injected systemically into the blood stream, and ultrasound is focused only on the tumor. As the blood carries the delivery device through the ultrasonic field in the tumor, the drug is released at the site where US is focused.

Methods and Materials

Doxorubicin (Dox) was sequestered in liposomes made from phosphatidyl choline, cholesterol, and distearoyl-sn-glycero-3-phospho-ethanolamine-PEG-2000. The lipids were deposited onto a glass test tube and rehydrated with ammonium sulfate buffer (pH~4.5). The liposomes were formed by vortexing followed by repeated extrusion through a 0.2 um membrane. Light scattering measurements showed that the liposomes were about 250 um in diameter (number-average diameter). Dox was loaded by a pH gradient method. The drug formulation was stored at 4°C until use within 4 weeks.

About 40 four-wk-old BDIX rats were inoculated with DHD cancer cells on each hind leg to grow subcutaneous tumors. Therapy began when tumors were at least 1 mm in diameter, usually after about 3 weeks of growth. Dox in stealth liposomes was injected (4.4 mg/kg or 2.5 mg/kg) via tail vein. US at 20 kHz was applied for 15 minutes and 1 W/cm2 (temporal average) to only one tumor, while the other tumor acted as an untreated control. This treatment was repeated weekly for 4 to 6 weeks. Some tumors were insonated before drug infusion, while others were insonated after infusion. Both tumor sizes were measured every wk following 15 minutes of insonation. Because the volume of each tumor was slightly different on the first day of measurement, the volumes were normalized by dividing them by the volume on day 1.

Results and Discussion

Our results showed that in those rats receiving 4.4 mg/kg Dox, the insonated tumors retreated rapidly and actually disappeared in more than 75% of the rats. The control side tumors, receiving liposomal Dox, but no ultrasound, slowed in growth rate, but regressed in less than 25% of the rats, and disappeared in only a few rats. Statistically, the application of ultrasound caused a slower or negative growth rate compared to the control tumors (p < 0.001). In rats receiving 2.5 mg/kg, the results were not as impressive, indicating that the higher dose is more effective in causing tumor regression. But both data sets showed that the insonated tumors quickly regressed in size, usually much more than the control side.

Our previous results on tumor growth rate indicate that application of US after systemic injection of micelle-encapsulated (not the liposomes used herein) Dox significantly suppressed tumor growth, even though pharmacokinetics studies showed that there were not tremendous increases in Dox concentration in tissues (only ~15%) in the insonated tumor compared to the bilateral non-insonated tumor. Is ultrasound also acting on the cell membranes as well as on the carrier? If so, then it would not matter what carrier is used, as long as the drug is present during insonation. To this end we previously completed a series of tumor growth studies using free Dox (non-encapsulated) in the same rat model described above. A statistical examination of the data of treated vs non-treated growth rate showed that rats receiving free Dox did not have any change in the tumor growth on the insonated tumor compared to the control (p=0.92). Thus the use of micelles as a carrier had a significant impact compared to free Dox. This was the main finding that led us to examine carefully the nature of the drug carriers. The recent results of delivery from our stealth liposomes solidified our thesis that the nature of the carrier is significant, as is the drug dosage. Changing the carrier from a micelle to a stealth liposome produced a tremendous positive difference in tumor treatment.

Conclusion

Colorectal model tumor growth in a rat model is very dependent upon the application of ultrasound and the carrier by which the Dox is delivered. It is also dependent upon the concentration of Dox applied in the treatment. Ultrasound combined with Dox delivered in our stealth liposomes is much more effective in reducing tumor growth than Dox delivered from our micelles. Furthermore, US combined with free Dox does not change growth rate compared to free Dox without US. More research will reveal the mechanisms by which ultrasound and liposomes effectively produce tumor regression.

Acknowledgments

The authors gratefully acknowledge Brigham Young University for funding.