Rationale and Objectives
A doxorubicin-loaded microbubble has been developed that can be destroyed with focused ultrasound resulting in fragments, or “nanoshards” capable of escaping through the leaky tumor vasculature, promoting accumulation within the interstitium. This study uses a rat liver cancer model to examine the biodistribution and tumoral delivery of this microbubble platform compared with de novo drug-loaded polymer nanoparticles and free doxorubicin.
Materials and Methods
Microbubbles (1.8 μm) and 217-nm nanoparticles were prepared containing 14-C labeled doxorubicin. Microbubbles, nanoparticles, a combination of the two, or free doxorubicin were administered intravenously in rats bearing hepatomas, concomitant with tumor insonation. Doxorubicin levels in plasma, organs, and tumors were quantified after 4 hours and 7 and 14 days. Tumors were measured on sacrifice and evaluated with autoradiography and histology.
Results
Animals treated with microbubbles had significantly lower plasma doxorubicin concentrations (0.466 ± 0.068%/mL) compared with free doxorubicin (3.033 ± 0.612%/mL, P = .0019). Drug levels in the myocardium were significantly lower in animals treated with microbubbles compared to free doxorubicin (0.168%/g tissue vs. 0.320%/g, P = .0088). Tumors treated with microbubbles showed significantly higher drug levels than tumors treated with free doxorubicin (2.491 ± 0.501 %/g vs. 0.373 ± 0.087 %/g, P = .0472). These tumors showed significantly less growth than tumors treated with free doxorubicin ( P = .0390).
Conclusions
Doxorubicin loaded microbubbles triggered with ultrasound provided enhanced, sustained drug delivery to tumors, reduced plasma and myocardium doxorubicin levels, and arresting tumor growth. The results suggest that in situ generation of nano particles provides a superior treatment over injection of free drug and also de novo synthesized nanoparticles.
The hyperpermeable vasculature of a tumor created during angiogenesis is characterized by pore sizes ranging from 380 to 780 nm, allowing nanoparticles (NP) to extravasate into the interstitium . Additionally, the tumor architecture generally lacks adequate lymphatic drainage, allowing these particles to accumulate over time .
Ultrasound contrast agents (UCA) are small (generally 1–6 μm) gas bubbles encapsulated within a stabilizing shell. When the agent is exposed to ultrasound, the gas core of the agent will expand and contract with a wall velocity on the order of hundreds of meters per second . These agents are generally restricted to circulation within the vascular system because of their size, but are small enough to penetrate into angiogenic vessels . When UCA are exposed to sufficient ultrasound intensity, the agents can cavitate and create enough shear force to rupture cell membranes and increase the permeability of the capillary wall, allowing particles to escape the vessel and penetrate tens of microns into the tumor interstitium . Vascular permeability can be enhanced through the use of UCA combined with targeted ultrasound .
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Open full size image
Figure 1
Schematic of ultrasound triggered drug delivery. Doxorubicin loaded UCA pass freely through the vasculature until exposed to ultrasound where they experience 1) acoustic radiation forces that push the microbubbles to the vessel wall. The oscillating pressure wave will also lead to 2) microbubble cavitation as the gas core expands and contracts in response to changes in pressure. When exposed to sufficiently strong ultrasound pulses, the microbubble will undergo 3) inertial cavitation resulting in the destruction of the polymer shell, creating drug loaded polymer fragments less than 400 nm. The energy released in the process of microbubble destruction is sufficient to 4) enhance the permeability of the vessel wall. The fragments can then begin to 5) accumulate within the tumor interstitium, potentially, through acoustic radiation force and with time, circulating fragments will accumulate though the enhanced permeability and retention effect. Lodged polymer fragments can 6) slowly degrade providing a sustained localized release of the chemotherapeutic agent.
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Materials and methods
Materials
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Microbubble Fabrication
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Nanoparticle Fabrication
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Cell Culture
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Animals
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Tumor Implantation
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Treatments
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Plasma Doxorubicin Quantification
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Organ Doxorubicin Quantification
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Tumor Growth Measurements
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Histology and Autoradiography
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Statistical Analysis
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Results
Plasma Doxorubicin Levels
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Organ and Tumor Doxorubicin Levels
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Tumor Growth
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Autoradiography and Histology
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Discussion
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Table 1
Actual Drug Levels Generated by Ultrasound Treatment of Drug-loaded Contrast Agent
Time Myocardium (ng/mg Tissue) Spleen (ng/mg Tissue) Left Liver (ng/mg Tissue) Right Liver (ng/mg Tissue) Lungs (ng/mg Tissue) Tumor (ng/mg Tissue) 4 hours 0.554 ± 0.035 22.272 ± 1.482 9.637 ± 1.297 8.577 ± 1.179 2.525 ± 0.832 4.174 ± 0.840 7 days 0.314 ± 0.052 4.815 ± 1.250 4.552 ± 1.213 4.718 ± 1.248 0.630 ± 0.081 2.340 ± 0.829 14 days 0.281 ± 0.028 8.099 ± 1.679 3.99 ± 0.767 4.061 ± 0.699 0.688 ± 0.199 3.478 ± 1.809
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Conclusion
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Acknowledgments
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