Pulsed high intensity focused ultrasound mediated nanoparticle delivery: mechanisms and efficacy in murine muscle

Abstract

High intensity focused ultrasound (HIFU) is generally thought to interact with biological tissues in two ways: hyperthermia (heat) and acoustic cavitation. Pulsed mode HIFU has recently been demonstrated to increase the efficacy of a variety of drug therapies. Generally, it is presumed that the treatment acts to temporarily increase the permeability of the tissue to the therapeutic agent, however, the precise mechanism remains in dispute. In this article, we present evidence precluding hyperthermia as a principal mechanism for enhancing delivery, using a quantitative analysis of systemically administered fluorescent nanoparticles delivered to muscle in the calves of mice. Comparisons were carried out on the degree of enhancement between an equivalent heat treatment, delivered without ultrasound, and that of the pulsed-HIFU itself. In the murine calf muscle, Pulsed-HIFU treatment resulted in a significant increase in distribution of 200 nm particles (p < 0.016, n = 6), while the equivalent thermal dose showed no significant increase. Additional studies using this tissue/agent model also demonstrated that the pulsed HIFU enhancing effects persist for more than 24 h, which is longer than that of hyperthermia and acoustic cavitation, and offers the possibility of a novel third mechanism for mediating delivery.

Fig. 1

Measurement of temperature at the center of the ultrasonic focus by use of an interstitial thermocouple. The probe is positioned and exposures are given at six treatment points. (a) Temperature curves at six individual treatment points (x-axis is min.; y-axis is °C). (b) Schematic representation of the six treatment points and their relative position to the probe, whose coordinates are unknown. (c) Projected focal zone temperature at an individual treatment point using temperature increases due to heating at neighboring sites, which are fit to the bioheat equation. Note, the graphs (1 to 6) in ‘a’ correspond to the similarly positioned focal zones in ‘b’.

Fig. 2

(a) Temperature vs. time. “HT” shows the temperature of muscle measured during a “heat only” treatment using a heat lamp source; “HT Control” gives the simultaneous temperature measurement from the insulated control limb. Note that ambient limb temperature in air, as demonstrated by the initial temperature of the HT, is actually 34 °C, which is lower than the 37 °C bath temperature typically used for p-HIFU treatments (see above). “HIFU37” refers to p-HIFU treatment in a 37 °C bath; “HIFU34” to p-HIFU treatment in a 34 C bath. Both of these are simulated by extrapolating the measurements of Fig. 1 to predict the heating and cooling of a single point. “HIFU raster” refers to the temperature measured at a given location while a complete treatment is without a cool-down period between raster sites. (b) t43 equivalent thermal dose calculated for these temperature profiles. HT is greater than HIFU37 and HIFU raster in terms of both peak temperature and thermal dose. Note, the thermal dose for HT control and HIFU34 is approximately 0.

Fig. 3

Threshold processing of a typical fluorescent image. Large artifacts (vertical arrows) are recognized and removed from consideration, leaving the area fraction of fine particles (horizontal arrows) as the measure of delivery. Bar = 100 μm.

Fig. 4

Comparison of heat treatment (ambient limb temperature at 34 °C) to standard p-HIFU treatment with 37 °C bath, and low temperature pulsed HIFU treatment with 34 °C bath. Despite elevated absolute temperatures and thermal doses relative to the HIFU treatments, the heat-only treatment showed no significant increase in particle delivery. Bars indicate group medians and quartiles.

Fig. 5

Longevity of p-HIFU (HIFU37) effects out to 24 hrs. Particle delivery is not significantly affected by a 24 hr waiting period between p-HIFU treatment and tail vein injection. Bars indicate group medians and quartiles.

Fig. 6

Cross section of a p-HIFU treated region, showing muscle surface (arrows right, skin removed). An increase in fluorescent particles is observed near surface. Bar = 100 μm.

Fig. 7

(a) Spectra collected during treatments at 40 W and 20 W TAP. The spectra give the maximum value measured over 40 pulses, at a single raster point. The trailing edge at far right is outside of the pulse, and thus represents the level of system noise. The sweep runs from 0.1 to 5 MHz, and each y division represents 8.1 dB below the reference level of −4.1 dBm. (b) Representation of normalized (with respect to fundamental) spectra at 40 and 20 W TAP; 20 W H2O is the spectrum in degassed water for comparison.