Pulsed-high intensity focused ultrasound and low temperature-sensitive liposomes for enhanced targeted drug delivery and antitumor effect

Abstract

Purpose: To determine if pulsed-high intensity focused ultrasound (HIFU) could effectively serve as a source of hyperthermia with thermosensitive liposomes to enhance delivery and efficacy of doxorubicin in tumors.

Experimental design: Comparisons in vitro and in vivo were carried out between non-thermosensitive liposomes (NTSL) and low temperature-sensitive liposomes (LTSL). Liposomes were incubated in vitro over a range of temperatures and durations, and the amount of doxorubicin released was measured. For in vivo experiments, liposomes and free doxorubicin were injected i.v. in mice followed by pulsed-HIFU exposures in s.c. murine adenocarcinoma tumors at 0 and 24 h after administration. Combinations of the exposures and drug formulations were evaluated for doxorubicin concentration and growth inhibition in the tumors.

Results: In vitro incubations simulating the pulsed-HIFU thermal dose (42 degrees C for 2 min) triggered release of 50% of doxorubicin from the LTSLs; however, no detectable release from the NTSLs was observed. Similarly, in vivo experiments showed that pulsed-HIFU exposures combined with the LTSLs resulted in more rapid delivery of doxorubicin as well as significantly higher i.t. concentration when compared with LTSLs alone or NTSLs, with or without exposures. Combining the exposures with the LTSLs also significantly reduced tumor growth compared with all other groups.

Conclusions: Combining low-temperature heat-sensitive liposomes with noninvasive and nondestructive pulsed-HIFU exposures enhanced the delivery of doxorubicin and, consequently, its antitumor effects. This combination therapy could potentially produce viable clinical strategies for improved targeting and delivery of drugs for treatment of cancer and other diseases.

Fig. 1

Fraction of doxorubicin (DOX) release in vitro as a function of temperature (T) and time (t): (A) t = 2 min and T = 20°C to 42°C and (B) T = 42°C and t = 1to 12 min. NTSLs did not release a detectable amount of doxorubicin even at the peak temperature of 42°C and the maximum incubation period of 12 min. For 2-min incubations, LTSLs started releasing doxorubicin at a temperature of 39°C, reaching almost 50% at 42°C. At a temperature of 42°C, release of doxorubicin at 2 min in LTSLs was ~ 50% and nearly 100% by 12 min. Points, mean (n = 4); bars, SE.

Fig. 2

Local drug delivery in murine adenocarcinoma tumors using free doxorubicin, NTSLs, or LTSLs, with or without pulsed-HIFU exposures. Liposomes or free doxorubicin (2 mg/kg) were first injected i.v. followed by exposures in the tumors (400 mm³) at 0 and 24 h after administration. Immediately after the exposures, animals were sacrificed, and tumors were assayed for doxorubicin content. Significant differences were not found between exposed and unexposed tumors in mice receiving NTSLs at either exposure time point. The same occurred for free doxorubicin. Although accumulated doxorubicin was greatest in unexposed tumors receiving NTSLs (at 24 h), the highest mean concentration of doxorubicin was found in tumors receiving LTSLs and pulsed-HIFU exposures. Differing lowercase letters between mean doxorubicin concentrations indicate a significant difference of at least P = 0.05. Columns, mean (n = 5); bars, SE.

Fig. 3

Local drug delivery in murine adenocarcinoma tumors using only LTSLs, with or without pulsed-HIFU exposures. Lag time between i.v. liposome injection and exposures in tumors (400 mm³) was varied. Immediately afterwards, animals were sacrificed, and tumors were assayed for doxorubicin content. Whereas doxorubicin in unexposed tumors continued to accumulate with time, lag time between injection and exposures in tumors did not affect content of doxorubicin. Columns, mean (n = 5); bars, SE.

Fig. 4

Left, growth curves for mice receiving saline, NTSLs, or LTSLs, with or without pulsed-HIFU exposures. Tumors were treated (doxorubicin, 5 mg/kg) when they reached a size of 200 mm³, and the mice were sacrificed when the tumors reached a size of at least 500 mm³. Right, number of days posttreatment for tumors to reach 500 mm³. Tumors receiving LTSLs and pulsed-HIFU exposures grew significantly slower than tumors in all other groups (P < 0.05). Points/columns, mean (n = 6); bars, SE.