Ultrasound Molecular Imaging for the Guidance of Ultrasound-Triggered Release of Liposomal Doxorubicin and Its Treatment Monitoring in an Orthotopic Prostatic Tumor Model in Rat

Abstract

Liposome encapsulation of drugs is an interesting approach in cancer therapy to specifically release the encapsulated drug at the desired treatment site. In addition to thermo-, pH-, light-, enzyme- or redox-responsive liposomes, which have had promising results in (pre-) clinical studies, ultrasound-triggered sonosensitive liposomes represent an exciting alternative to locally trigger the release from these cargos. Localized drug release requires precise tumor visualization to produce a targeted and ultrasound stimulus. We used ultrasound molecular imaging (USMI) with BR55, a vascular endothelial growth factor receptor 2 (VEGFR2)-targeted ultrasound contrast agent, to guide ultrasound-triggered release of sonosensitive liposomes encapsulating doxorubicin (L-DXR) in an orthotopic prostatic rodent tumor model. Forty-eight hours after L-DXR injection, local release of doxorubicin was triggered with a confocal ultrasound device with two focused transducers, 1.1-MHz center frequency, and peak positive and negative pressures of 20.5 and 13 MPa at focus. Tumor size decreased by 20% in 2 wk with L-DXR alone (n = 9) and by 70% after treatment with L-DXR and confocal ultrasound (n = 7) (p < 0.01). The effect of doxorubicin on perfusion/vascularity and VEGFR2 expression was evaluated by USMI and immunohistochemistry of CD31 and VEGFR2 and did not reveal differences in perfusion or VEGFR2 expression in the absence or after the triggered release of liposomes. USMI can provide precise guidance for ultrasound-triggered release of liposomal doxorubicin mediated by a confocal ultrasound device; moreover, the combination of B-mode imaging and USMI can help to follow the response of the tumor to the therapy.

Introduction

Liposome-encapsulated drugs have been a fast-growing field of research since approval of the PEGylated unilamellar liposome-encapsulated doxorubicin Doxil in 1995 for the treatment of Kaposi's sarcoma (James 1995). Doxil was subsequently approved for metastatic breast cancer, ovarian cancer and multiple myeloma (Goncalves et al. 2020). To date, several formulations of liposome-based products are in clinical trials or approved for different purposes, including analgesics, photodynamic therapy, fungal diseases, viral vaccines and cancer therapy (Bulbake et al. 2017; Goncalves et al. 2020). The advantages of liposomal formulations compared with free drugs include an extended circulation time in blood, reduced side effects (Horowitz et al. 1992; Thorn et al. 2011) and intratumor accumulation through the so-called enhanced permeability and retention effect (EPR) described in the 1980s (Matsumura and Maeda 1986). Despite these advantages, one of the main limitations of liposomal doxorubicin formulations remain the relatively small number of injected liposomes that can reach and finally accumulate within the tumor space (Golombek et al. 2018). This weak accumulation, combined with the slow release of the drug from the liposome, prevents the therapeutic window from being reached and therefore may limit the efficacy.

Although the EPR effect has been largely described in several animal models, its occurrence in patients remains debated (Moghimi and Farhangrazi 2014; Nichols and Bae 2014; Golombek et al. 2018; Swetha and Roy 2018; Zhou et al. 2020), and several strategies to improve the EPR effect have been investigated as a way to increase the concentration of liposomes at the target site, including alteration of the tumor micro-environment and endothelial cell lining, aided by external sources such as radiation, hyperthermia, photodynamic therapy and ultrasound (Dhaliwal and Zheng 2019; Park et al. 2019).

Another approach to potentiate liposomal drug-based treatment is to locally trigger the release of the drug from the liposome to increase its bioavailability in a short period and reach a therapeutic window. With respect to the latter approach, formulations of thermo-, pH-, light-, enzyme- or redox-responsive liposomes have shown promising results in preclinical and clinical studies (Lee and Thompson 2017; Abri Aghdam et al. 2019). In a phase 1 clinical trial using ultrasound-induced hyperthermia to trigger targeted drug delivery of doxorubicin from thermosensitive liposomes (TARDOX study), the use of ultrasound-induced hyperthermia resulted in an enhanced delivery of doxorubicin in solid liver tumors (Lyon et al. 2018; Gray et al. 2019). Formulations of liposomes that can release their cargo in response to the mechanical ultrasound stimulus of cavitation, so-called sonosensitive liposomes, have also been investigated. In this context, Evjen et al. (2011) have described a sonosensitive liposomal doxorubicin (L-DXR) formulation based on the unsaturated phospholipid 1,2-dierucoyl-sn-glycero-3-phosphocholine.

By use of a dedicated confocal ultrasound device to induce mechanical stress on the liposomes through acoustic cavitation, precise release of encapsulated drug could be triggered on demand (Somaglino et al. 2011; Evjen et al. 2013). Previous studies have found in vitro that the degree of release from these liposomes is proportional to the cavitation dose applied, and that under cavitational stress, more than twice the amount of DXR is released compared with the clinical formulation Caelyx (Mestas et al. 2014). This cavitational procedure was successfully tested in vivo in a subcutaneous rat prostatic carcinoma model (AT2 phenotype of the Dunning R3327) in which the activation of L-DXR 48 h postinjection limited tumor growth (Fowler et al. 2013; Mestas et al. 2014). Such inertial cavitation treatment does not alter the structure and cytotoxicity of doxorubicin, is safe and did not promote cancer cell dissemination in a highly metastatic 4T1 breast tumor model in mice (Lafond et al. 2016). Moreover, pulsed cavitational ultrasound was successfully tested to treat calcified bioprosthetic valve stenosis (Villemain et al. 2017) and a First-in-Man clinical trial has been conducted to remotely treat aortic stenosis (NCT03779620).

An important constraint to locally trigger the release of doxorubicin from liposomes is the need for treatment guidance on precise application of the cavitation stimulus. This need was highlighted when tested in an orthotopic pancreatic murine model (Camus et al. 2019), in which the use of a very high frequency (40 MHz) preclinical ultrasound scanner was required to localize the tumor and guide the treatment.

For the clinical treatment of tumors in deep organs such as the prostate and pancreas, where such high-frequency imaging is not possible, the use of gas microbubbles as intravascular ultrasound contrast agents is an alternative imaging option (Wang et al. 2020; Klibanov 2021). Thus, contrast-enhanced ultrasound (CEUS) would be an asset as it offers real-time imaging with high spatial resolution to delineate the lesion's extent and can also provide valuable information on perfusion (Frinking et al. 2020). Although 3-D matrices are available, and the feasibility of 3-D CEUS imaging in humans has been reported (Xiang et al. 2013; Sridharan et al. 2015; El Kaffas et al. 2017), as has its ability to identify responders to different cancer therapies based on monitoring of perfusion changes (El Kaffas et al. 2020), 3-D CEUS imaging is currently the standard in clinics, which limits its attractivity for treatment guidance.

Ultrasound molecular imaging (USMI) could provide an even more specific alternative, as tumor visualization and delineation based on the detection of specific tumor endothelial characteristics could be achieved (Wang et al. 2020). USMI has been validated in preclinical models using cationic microbubbles (Diakova et al. 2020), targeted microbubbles against von Willebrand factor (Shim et al. 2015), α_v-integrins (Leong-Poi et al. 2003) or tumoral biomarkers, including B7 H3 (Bachawal et al. 2020), Netrin 1 (Wischhusen et al. 2018) and vascular endothelial growth factor receptor 2 (VEGFR2) with BR55 (Pochon et al. 2010). BR55 was tested in an orthotopic prostatic G-Dunning model, where the feasibility of tumor visualization in the prostate was determined (Tardy et al. 2010). In a chemo-induced rat mammary tumor model, it was found that USMI can provide a volumetric tumoral delineation and that the therapeutic follow-up of tumoral response to an anti-angiogenic treatment is feasible at the anatomical, functional and molecular levels in a single exam (Helbert et al. 2020). Recently, BR55 has been successfully translated in the clinic for the detection of angiogenesis in breast and ovarian tumors in women (Willmann et al. 2017) and prostate cancer in men (Smeenge et al. 2017).

In the present study, we used USMI with BR55 to guide ultrasound-triggered release of liposomal doxorubicin in a preclinical prostate tumor model. Sonosensitive L-DXR were injected into rats bearing orthotopic G-Dunning rat prostate tumors. USMI was used to visualize and delineate tumors within the prostate to specifically select ultrasound treatment locations. Finally, USMI was used to evaluate tumor response after ultrasound-induced doxorubicin release from liposomes.