Elsevier

Ultrasonics

Volume 48, Issue 4, August 2008, Pages 260-270
Ultrasonics

Drug-loaded nano/microbubbles for combining ultrasonography and targeted chemotherapy

https://doi.org/10.1016/j.ultras.2007.11.002Get rights and content

Abstract

A new class of multifunctional nanoparticles that combine properties of polymeric drug carriers, ultrasound imaging contrast agents, and enhancers of ultrasound-mediated drug delivery has been developed. At room temperature, the developed systems comprise perfluorocarbon nanodroplets stabilized by the walls made of biodegradable block copolymers. Upon heating to physiological temperatures, the nanodroplets convert into nano/microbubbles. The phase state of the systems and bubble size may be controlled by the copolymer/perfluorocarbon volume ratio. Upon intravenous injections, a long-lasting, strong and selective ultrasound contrast is observed in the tumor volume indicating nanobubble extravasation through the defective tumor microvasculature, suggesting their coalescence into larger, highly echogenic microbubbles in the tumor tissue. Under the action of tumor-directed ultrasound, microbubbles cavitate and collapse resulting in a release of the encapsulated drug and dramatically enhanced intracellular drug uptake by the tumor cells. This effect is tumor-selective; no accumulation of echogenic microbubbles is observed in other organs. Effective chemotherapy of the MDA MB231 breast cancer tumors has been achieved using this technique.

Introduction

All energy-based tumor treatment modalities, including thermal treatments, require prior imaging. The field of thermal tumor therapy has advanced rapidly, particularly for high intensity focused ultrasound (HIFU), which causes tumor ablation by coagulative necrosis of tumor tissue. Some other clinical applications of thermal therapy such as adjuvant hyperthermia with radiation therapy, drug and gene delivery, or coagulation of tumor blood vessels can be envisioned [1].

For most systems, MRI remains the imaging modality of choice due to the possibility of combining tumor imaging with MRI thermometry, which would eventually allow development of feedback treatment control systems; MRI imaging is used for example, in the ExAblate® 2000 (InSightec, Israel) system for the treatment of uterine fibroids. However, the possibility for combining diagnostic and therapeutic ultrasound for tumor imaging and treatment appears more attractive, for reasons of simplicity, time and cost-effectiveness. Dual-modality ultrasound imaging-treatment systems have recently entered the market (e.g. Ablatherm (EDAP, Lyon, France); and Sonablate (Focus Surgery, Indianapolis, Indiana) for treating prostate cancer; HIFU JC Tumor Therapy System (Chongqing HIFU Technology, Chongqing, China) for treating a vide variety of tumors); a miniature ultrasound-based dual-modality device for image guided soft tissue ablation (Guided Therapy Systems, Mesa, Arizona) and some other instruments are in the development stage.

However, ablative techniques present a number of problems related to the precise control of heat deposition. Patient motion and breathing during the treatment are also problematic. Currently, long treatment times, incomplete treatment of large targets, a non-uniform thermal dose distribution, and unintended normal tissue damage continue to impede broad penetration of HIFU therapies into clinical practice.

Here, we describe a therapeutic technique that utilizes non-thermal mechanisms of ultrasound interaction for targeted drug delivery and tumor imaging. This technique may rely on the same instruments that are used for HIFU, but driven at substantially lower ultrasound energies. In addition, thermal treatment and ultrasound-mediated drug delivery may complement each other because drug delivery works well in the highly perfused regions of a tumor (generally the periphery), while HIFU can destroy a poorly perfused tumor core. Again, tumor imaging prior to treatment is a crucial element of the proposed technique.

Contrast-enhanced ultrasonic tumor imaging is still in its infancy. Development of ultrasound contrast agents and the concomitant use of harmonic ultrasound imaging has made possible imaging of liver [2], breast [3], and prostate tumors [4]. Recently, ultrasound-stimulated acoustic emission of microbubbles has been used for color Doppler imaging of liver metastases [5]. Polymeric ultrasound contrast agents with targeting potential have been explored by the Wheatley group [6], [7], [8], [9]. Molecularly targeted microbubbles have been successfully used for the ultrasonic imaging of angiogenesis [10], [11], [12], [13], [14], [15], [16]. Bubble targeting using ultrasound radiation force has also been developed [17], [18], [19], [20].

Combining contrast-enhanced ultrasound tumor imaging with targeted drug delivery is a challenging task; drug loading onto lipid-coated microbubbles has been investigated for this purpose [21], [22], [23]. We have recently developed novel ultrasound-sensitive multifunctional nanoparticles composed of nanoscale polymeric micelles that function as drug carriers and nano- or microscale echogenic bubbles that combine the properties of drug carriers, enhancers of the ultrasound-mediated drug delivery, and long-lasting ultrasound contrast agents [24], [25]. Drug carrying, tumor-targeting, and retention in the tumor volume are functions of the micelles and/or nanobubbles; ultrasound contrast properties are provided by the microbubbles formed in a tumor volume by the coalescence of nanobubbles. The structure and properties of these systems are discussed below.

Section snippets

Block copolymers

Biodegradable diblock copolymers poly(ethylene oxide) -block-poly(l-lactide) (PEG-PLLA) and poly(ethylene oxide)- block-poly(caprolactone) (PEG-PCL) in which the molecular weight of either block was 2000 Da were bought from JCS Biopolytech Inc., Toronto, Ontario, Canada.

Nanoemulsion preparation

Perfluoropentane (PFP) nanoemulsions dispersed in a solution of polymeric micelles were produced by introducing an aliquot of a sterilized PFP into a micellar solution of a copolymer in sterilized plastic test tubes that were

Phase diagrams of the micelle/microbubble systems

The phase state of the designed systems at room temperature was evaluated for two biodegradable micelle-forming copolymers, PEG-PLLA and PEG-PCL, for various copolymer/PFP concentrations and concentration ratios. The phase diagram is presented schematically in Fig. 1. The three zones in the phase diagram along the direction of increasing PFP concentration correspond to: zone (1) – micelles only (no nanodroplets); PFP is dissolved in the micelle cores; zone (2) – micelles coexist with nano- or

Discussion

The compositions developed allowed contrast-enhanced tumor imaging and targeted drug delivery to be provided by the same multifunctional drug carrier system. Drug carrying, tumor-targeting, and retention in the tumor volume were provided by the nanobubbles; ultrasound contrast agent properties were provided by the microbubbles of micron size formed by coalescence of the nanobubbles in tumor tissue. The microbubbles also served as enhancers of ultrasound-mediated drug delivery.

The microbubbles

Conclusions

The polymeric copolymer-stabilized perfluoropentane nanoemulsion systems undergo nanodroplet/microbubble conversion in vivo, accumulate locally in tumor tissue and coalesce into larger, highly echogenic microbubbles, which provide for long-lasting ultrasound contrast in the tumor, and allows on-demand release of the encapsulated drug under the action of therapeutic ultrasound.

Acknowledgement

This work was supported by the NIH R01 EB1033 Grant to N.R.

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