The use of ultrasound to enhance the extravasation of drugs and nanoparticles has been widely reported; however, reports of significant enhancements have been accompanied by reports of failed protocols. Alternatively, reports of enhanced accumulation by radiofrequency ablation or by mild hyperthermia have led to consistent, well-characterized protocols. Here, by labeling liposomes and albumin with a positron emission tomography or fluorescent tracer, individualized protocols for ultrasound treatment of epithelial and EMT tumors have been developed.
Without ultrasound, we found that the accumulation of liposomes was ~1.5 times greater in epithelial, as compared with EMT, tumors. When ultrasound was applied within 1 hour of particle injection, accumulation was enhanced in the insonified, as compared with the contralateral, tumor with a concomitant increase in both the rate and volume of accumulation. Across both epithelial and EMT tumors, maximum and mean accumulation in the tumor interstitium were increased in the insonified tumors. The effect of ultrasound differed with both thermal and mechanical ultrasound indices. Increasing the CEM43 from 1.6 to 4.5 increased the accumulation of particles in EMT tumors to a maximum of 9.2±3.0%ID/g for a CEM43 of 4.5, while accumulation peaked in epithelial tumors at 14.7%±3.0ID/g. Given the narrow therapeutic window for current chemotherapeutics, this increase in tumor accumulation could significantly improve efficacy.
Enhanced delivery to the EMT phenotype is of particular interest as such tumors are aggressive and frequently difficult to treat (39
). Here, a significant enhancement of accumulation was observed in both the EMT and epithelial models, yet the parameters required to maximize the effect differed with the tumor biology. Accumulation increased in both EMT and epithelial tumors with a PNP of 2.4 MPa; however, a greater thermal dose decreased accumulation in epithelial tumors. The mean vascular volume in epithelial tumors was more than double that in the EMT phenotype. We hypothesize that the decreased accumulation in epithelial tumors with a higher thermal dose results from vascular stasis in the large vessels that are prevalent in the epithelial (but not EMT) phenotype (12
). On the contrary, accumulation in the EMT phenotype was greatest at the highest thermal dose evaluated here, where small regions of heat-mediated necrosis were evident. For EMT tumors, the proportional increase in transport with increasing CEM43 likely results from decreased interstitial pressure, increased apparent permeability and cell damage (40
Mechanical effects of ultrasound stem from multiple sources, including cavitation, radiation pressure, and expansion and contraction of space between lipid membrane bilayers (41
). Potential benefits of leaflet stretching include stimulation of mechano-sensitive membrane proteins, rearrangement of cytoskeletal elements, membrane perforation increasing drug uptake, and increased tissue permeability (42
). As demonstrated in , accumulation was enhanced in epithelial tumors at a PNP of 1.1 and 2.4 MPa; however, accumulation was not maximized in EMT tumors until a PNP of 2.4 MPa was applied. Following the transformation of Met-1 cells from an epithelial to an EMT phenotype, we observed downregulation of E-cadherin, cytokeratin 8/18 (CK8/18), and CK19 expression, but upregulation of vimentin. E-cadherin expression is important for maintaining cell-cell contacts and tissue organization (43
). Vimentin has a number of critical functions within the cell that involve attachment, migration, and cell signaling (45
). In addition, vimentin modulates the negative effects of mechanical and thermal stresses (46
). Our results suggest that imposing a mechanical stress by ultrasound (in addition to heat) enhances accumulation in the EMT phenotype, presumably due to weakened cell-cell adhesion and increased vimentin expression.
The mechanisms for the effect of ultrasound on apparent permeability and particle accumulation were also investigated. First, techniques for the estimation of vascular volume, clearance rate and water content were validated by measurements for the quadriceps muscle (internal control), where values were similar to those previously reported for C57BL/6 mice (5
). Ultrasound reduced the intratumoral pressure and increased Pap
in our tumor model without significant alterations to vascular volume and tissue water content. The measured changes in these two parameters likely contributed to the enhanced accumulation of particles within the tumor. Interestingly, ultrasound reduced intratumoral pressure even after euthanasia; therefore, the effect cannot be solely attributed to changes in blood flow, protein expression or macrophage recruitment. We hypothesize that this reduction is likely due to the effect of insonation and hyperthermia on the extracellular matrix and will evaluate this effect further in future studies. Thermal absorption and mechanical agitation can influence the extracellular matrix, particularly collagen integrity (48
). Also, collagen content is known to influence extracellular matrix hydraulic conductivity (49
PET provided a convenient method to assess the accumulation and apparent vascular permeability over time and across the tumor volume; such measurements can be directly compared with classical estimates of vascular clearance. PET-based pharmacokinetic modeling has previously been developed for small molecule therapeutics, but nuclear medicine-driven techniques to evaluate the pharmacokinetics of nanoparticles are currently unavailable. While small molecules distribute in seconds to minutes, nanoparticles extravasate from the blood pool in hours to tens of hours, and therefore nanoparticle distribution at the time of injection provides a direct measurement of organ and tumor vascular volume. With PET, the circulating radioactivity can be measured based on cardiac chamber activity. With estimates of circulating particle concentration and tumor vascular volume, PET can be applied to estimate the rate of nanoparticle extravasation for each acquisition.
In our supplemental information
, we detail the relationship between the PAP
parameter derived through the image-guided model with the classical parameters of solute transport coefficient, clearance, and 30 minute albumin clearance (Cl30
are related as described by SI Eq6,
is the tumor volume, r
is the mean radius of the blood vessels within the tumor, and η
is the plasma volume fraction in the tumor. In our study, both the apparent permeability (as determined by PET) and the clearance (as determined by fluorescent albumin tracers) were increased as a result of ultrasound. Not surprisingly, due to the difference in diameter, the ultrasound-induced increase in apparent permeability for liposomes was greater than the induced increase in clearance for albumin (~30% increase in Cl30
for albumin and ~70% increase in PAP
for liposomes as assessed by PET, averaged across epithelial and EMT tumors). One limitation of these studies is that we were not able to determine whether the net flux of solute into the tumor interstitium was due to increased extravasation or a decrease in the net clearance from the tumor. Regardless of the mechanism, both albumin clearance and PET imaging of liposomes assess net flux of solute between the tumor vasculature and interstitium, and demonstrated the effectiveness of ultrasound to enhance accumulation. A further limitation is that we selected one peak treatment temperature to study (42°C) and the optimal protocol is expected to vary with changes in this treatment temperature.
In summary, we applied ultrasound to increase nanoparticle-based drug extravasation and accumulation in both epithelial and EMT tumors. Using both the thermal and mechanical properties of ultrasound, effective treatment times were shorter than times typically used in mild hyperthermia treatments (5-18 minutes compared to durations of 1 or more hours). In addition, we observed a decrease in intratumoral pressure after only 5 minutes as compared to the reported 6 hours required for IFP reduction with whole body hyperthermia (50
). Our study clearly demonstrates that differences in tumor phenotype must be recognized in treatment planning and suggests that imaging techniques, such as those applied here, may provide the information required to optimize treatment for individual characteristics. Such individual differences can be viewed as an opportunity or obstacle for such treatments. For our ultrasound treatment parameters and a target temperature of 42°C, thermal dose must be limited when treating highly vascular epithelial tumors; whereas delivery can be increased in the poorly vascularized EMT phenotype with a higher mechanical and thermal dose.