Targeted Drug Delivery

Principal Investigator: Joo Ha Hwang, MD, PhD
Ultrasound-enhanced drug delivery for treatment of solid tumors:

The overall aim is to investigate the use of pulsed High-Intensity Focused Ultrasound (HIFU) for enhancing penetration of a chemotherapeutic agent into the interstitium of tumors. HIFU could be used in conjunction with novel drug delivery vehicles to deliver newer chemotherapeutic agents that overcome multi-drug resistance for more effective treatment of localized tumors while limiting systemic toxicity.

Background and Significance: In general, systemic administration of chemotherapeutic agents have some efficacy because the vascular endothelia of tumors are poorly developed resulting in “leaky” vessels allowing drugs to escape the systemic vascular space preferentially into the interstitium. The drug then must penetrate into the tumor via diffusion; however, penetration is typically limited due to the relatively high interstitial pressure of tumors. Overall, the effectiveness of chemotherapeutic agents is limited by their ability to selectively reach their target (tumor cells) without causing unwanted toxicities.

Packaging drugs in liposomes or nanoparticles is attractive, however barriers to delivery of ‘packaged’ chemotherapeutic agents include (1) the transport of drug/nanoparticle from the vascular space to the interstitial space, (2) penetration of the drug beyond the vascular barrier into the interstitial space and (3) intracellular delivery. Although most chemotherapeutic agents are small molecules that are actively transported through endothelial cells, the interstitial pressure is typically higher in tumors, limiting the passive diffusion of molecules into them. The transport of drugs once they enter the interstitial space is also limited to passive diffusion.  The concentration of doxorubican administered intravenously decreases exponentially with distance from tumor blood vessels, with drug concentration half its perivascular concentration at a distance of 40-50um, wereas teh mean distance from blood vessels to regions of hypoxia is 90-140 um [Primeau 2005]. 

The challenge of this project is to develop HIFU for targeted drug delivery enabling relatively larger drug molecules to selectively traverse the vascular barrier within a tumor and then penetrate the interstitial space of the tumor. Our studies suggest multi-faceted dependences on various ultrasound parameters, such as amplitude, frequency, pulse duration, pulse repetition frequency, and duty cycle. Figure 8 shows representative fluorescent images taken of the cross section of tissue samples. We used fluorescein isothiocyanate-dextran (FITC-dextran) nanoparticles (commonly used in drug delivery studies) for our studies. Increased fluorescence was seen at the edge of all samples in both control and HIFU treated groups. However, penetration was enhanced greatly with ultrasound. In some cases, consistent penetration was observed up to depths of 250 mm. The 20 kDa FITC-dextran appeared to penetrate to greater depths (up to 250 mm) compared to the 500 kDa FITC-dextran (up to 200 mm). However, the most intense fluorescence over the greatest depth was observed in specimens exposed to 500 kDa FITC-dextran. Although not shown here, in many samples penetration was observed to reach depths of approximately 500 mm compared to 50 mm in some of the controls, an order of magnitude enhancement.

Figure 8. Fluorescent images of cross sections of samples incubated with FITC-dextran (20 kDa or 500 kDa) and exposed to HIFU with a range of peak negative pressures. Scale bar represents 250 mm.

Qualitatively, depth of penetration and intensity increase with increasing acoustic intensity. A relationship between the pulse repetition frequency and enhanced penetration was also observed (data not shown). The tissue itself was not damaged (by cell lysis, data not shown) yet penetration depths of approximately 500 mm were observed. The penetrations achieved with ultrasound are thus significant.

The existing literature and our preliminary studies suggest that mechanistic contributions from cavitation, radiation force, microstreaming and temperature can be harnessed to impact a significant biologic response that improves the effectiveness of systemically administered chemotherapeutic agents. The studies here will leverage current studies being performed at UW. Those initial studies focused on inexpensive gels and ex vivo tissue. The next step is in vivo studies. Our Aims are to (1) Optimize vascular permeability in vivo using HIFU, and (2) Optimize tissue penetration with HIFU.

Vascular permeability: In vivo studies will be performed using a rat tumor model (Eker Tsc2 mutation) that spontaneously forms tumors with a native vascular supply (as opposed to an implanted subcutaneous tumor model).

Tumor vascularity will be characterized prior to and after ultrasound exposure by systemically injecting fluorescent nanoparticles of various sizes to assess those able to escape the vasculature. Penetration and distribution will be measured by measuring the distance the fluorescent nanoparticles are able to penetrate from the tumor blood vessels using an anti-CD31 (platelet/endothelial cell adhesion molecule 1) fluorescent monoclonal antibody to label tumor blood vessels. A composite fluorescent image will then be created to demonstrate the distribution and penetration of the fluorescent nanoparticles relative to the tumor blood vessels (Fig. 9).  The fluorescent intensity from the nanoparticles can then be quantified as a function of distance from the nearest blood vessel. The optimal parameters from these studies will then be used for subsequent in vivo penetration studies.

Figure 9. sample composite fluorescent image with doxorubicin (blue) and tumor vasculature (red). [from Primeau 2005].