Synthesis and characterisation of ultrasound imageable heat-sensitive liposomes for HIFU therapy

Abstract

Background/objective: Novel approaches allowing efficient, readily translatable image-guided drug delivery (IGDD) against solid tumours is needed. The objectives of this study were to: 1) develop echogenic low temperature sensitive liposomes (E-LTSLs) loaded with an ultrasound (US) contrast agent (perfluoropentane, PFP), 2) determine the in vitro and in vivo stability of contrast agent encapsulation, 3) co-encapsulate and characterise doxorubicin (Dox) E-LTSL, and cellular uptake and cytotoxicity in combination with high intensity focused ultrasound (HIFU).

Method: E-LTSLs were loaded passively with PFP and actively with Dox. PFP encapsulation in E-LTSL was determined by transmission electron microscopy (TEM), and US imageability was determined in tissue-mimicking phantoms and mouse tumour model. Dox release from E-LTSL in physiological buffer was quantified by fluorescence spectroscopy. Cellular uptake and cytotoxicity of E-LTSL in the presence of HIFU-induced mild hyperthermia (∼40-42 °C) was determined in a 3D tumour spheroid model.

Results: TEM and US confirmed that the PFP emulsion was contained within LTSLs. Phantom and animal studies showed that the E-LTSLs were echogenic. Temperature versus size increase and Dox release kinetics of E-LTSLs demonstrated no difference compared to LTSL alone. Dox release was <5% within 1 h at baseline (25 °C) and body (37 °C) temperatures, and was >99% under hyperthermia. E-LTSL plus HIFU achieved significantly greater Dox uptake in spheroids and cytotoxicity compared to body temperature.

Conclusion: A stable US-imageable liposome co-loaded with Dox and PFP for in vivo IGDD was developed. Data suggest that HIFU can induce cellular uptake and toxicity with E-LTSLs.

Keywords: Echogenic heat-sensitive liposome; HIFU; Image Guided Drug Delivery; Perfluoropentane; Spheroid; Tumor.

Figure 1

Determination of 1,3-PD in E-LTSL by GC-MS. (A) Derivatized 1,3-PD (MW: 162), (B) Confirmation of presence of derivatized 1,3-PD following two rounds of column purification.

Figure 2

TEM images. (A) LTSL, (B) spherical E-LTSL encapsulating PFP-1,3-PD emulsion at 37 °C, (C) E-LTSL encapsulating PFP-PD emulsion with membrane deformation at 42 °C caused by hyperthermic relaxation of liposome membrane.

Figure 3

Ultrasound images of LTSL and E-LTSL from the phantom study. (A) Column purified PFP mixture containing PFP or 1,3-PD plus PFP showing no echogenicity (dark round black circle, top panel), whereas column purified E-LTSL sample showing similar echogenicity like unpurified PBS mixture containing PFP (white circular regions, bottom panel), (B) US intensity of LTSL and E-LTSL from 25–42 °C. A substantial increase in US intensity is noted at higher temperatures.

Figure 4

Tumour vascular contrast enhancement following intravenous injection of E-LTSL in a mouse model. A sustained increase in contrast compared to preinjection control was noted at (A) 0 min, (B) 5 min, (C) 10 min, (D) 15 min.

Figure 5

(A) Size to temperature analysis of LTSL and E-LTSL demonstrating stable hydrodynamic diameter (~150 nm) throughout the 25–42 °C range, (B) E-LTSL vial following 48 h storage at 4 °C with no visual evidence of particle accumulation.

Figure 6

Thermoscan assay in physiological buffer between 25–42 °C. (A) Release of Dox from E-LTSL was relatively greater in serum than in PBS. (B) Release profile of LTSL demonstrates a similar profile to E-LTSL (A).

Figure 7

Dox release kinetics. (A, B) Release from E-LTSL and LTSL in serum as a function of time (30 min) at constant temperature. (C, D) Release from E-LTSL and LTSL in PBS as a function of time (30 min) at constant temperature.

Figure 8

(A) Dox release as a function of sonication time (0–5 min). (B) Dox release in the presence of continuous-wave focused US (3–12 MHz).

Figure 9

Mild hyperthermia-induced drug release. (A) Significantly greater release in heated sample (LTSL & ELTSL, 42°C) relative to unheated control at 37°C was noted in cell supernatant. (B) Greater intracellular accumulation of the released drug was noted in 3D tumour spheroid upon HIFU plus E-LTSL and LTSL mild hyperthermia treatment compared to untreated samples (*p<0.05).

Figure 10

Cytotoxicity of LTSL and E-LTSL at body temperature and HIFU-induced mild hyperthermia in (A) C-26 cells, (B) A549 cells. All data were normalised to untreated control. Significant toxicity of released drug was noted at 42 °C compared to body temperature (*p<0.05).