Microbubble-enhanced ultrasound to deliver an antisense oligodeoxynucleotide targeting the human androgen receptor into prostate tumours

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Abstract

We have shown recently that downregulation of the androgen receptor (AR), one of the key players in prostate tumor cells, with short antisense oligodeoxynucleotides (ODNs) results in inhibition of prostate tumor growth. Particularly with regard to an application of these antisense drugs in vivo, we now investigated the usefulness of microbubble-enhanced ultrasound to deliver these ODNs into prostate cancer cells.

Our short antisense AR ODNs were loaded onto the lipid surface of cationic gas-filled microbubbles by ion charge binding, and delivered into the cells by bursting the loaded microbubbles with ultrasound. In vitro experiments were initially performed to show that this kind of delivery system works in principle. In fact, transfection of prostate tumor cells with antisense AR ODNs using microbubble-enhanced ultrasound resulted in 49% transfected cells, associated with a decrease in AR expression compared to untreated controls. In vivo, uptake of a digoxigenin-labelled ODN was found in prostate tumour xenografts in nude mice following intratumoral or intravenous injection of loaded microbubbles and subsequent exposure of the tumour to ultrasound, respectively. Our results show that ultrasound seems to be the driving force of this delivery system. Uptake of the ODN was also observed in tumors after treatment with ultrasound alone, with only minor differences compared to the combined use of microbubbles and ultrasound.

Introduction

We have shown recently that the inhibition of AR expression with an antisense androgen receptor (AR) oligodeoxynucleotide (ODNasAR) results in prostate tumour growth retardation in vitro [1] and in vivo [2]. Thus, eliminating the expression of AR in prostate tumour cells with antisense ODNs may be an innovative strategy to treat prostate cancer. One of the major limitations of using antisense ODNs in the clinic, however, is their safe and efficient delivery to the prostate. Therefore, a number of different delivery techniques have been proposed in the past to improve gene delivery into the prostate [3]. We investigated the usefulness of microbubble contrast agents in combination with ultrasound to transfer an antisense ODN into prostate tumours. The principle behind this technique is the use of contrast agent microbubbles as transport vehicles, which carry the ODN to the tumour tissue upon intravenous injection. There, the loaded microbubbles are burst by ultrasound resulting in delivery of the ODN into the adjacent tissue by a mechanism called sonoporation [4], [5]. One main advantage of this technique is that microbubble-enhanced ultrasound is already routinely used in patients for improved diagnostic imaging and is therefore known as safe and non-toxic. Moreover, the use of focused ultrasound treatment gives the possibility to release the drug specifically at the tumour site.

The development of novel therapies for prostate cancer is an important issue due to the limited selection of efficient treatment modalities. Whereas organ-confined prostate cancer can be treated by surgery, androgen ablation is the main choice of treatment for patients with advanced prostate cancer [6], [7]. However, although the majority of patients respond to this treatment, most tumours relapse and progress during therapy despite castrate levels of testosterone. One of the key molecules contributing to prostate cancer growth is the AR which is expressed in all stages of prostate cancer, including those which are resistant to androgen ablation treatment [8], [9], [10]. Since, increased AR expression and promiscuous inappropriate transcriptional AR activation is associated with hormone-refractory prostate cancer [11], [12], [13], [14], [15], [16], [17], [18], the AR represents an interesting target for a novel therapy. The present study was therefore undertaken to investigate if microbubble-enhanced ultrasound can be used to deliver the ODNasAR into prostate tumours. We demonstrate here that the ODNasAR can be delivered into prostate tumor xenografts in nude mice through microbubble-enhanced ultrasound resulting in a significant uptake of the ODN in the tumour and tumour-associated stromal compartments as compared with untreated controls. Ultrasound is considered as the driving force of this system since there is also significant uptake of the ODN in tumors after delivery with ultrasound alone.

Section snippets

Antisense oligonucleotides

A 15-nucleotide short antisense phosphorothioate ODN was directed against the CAG polyglutamine region of the human AR gene which was tested for its activity previously [1] (5′-CTGCTGCTGCTGCTG-3′, ODNasAR, GenXpress, Vienna, Austria). To investigate tissue distribution in vivo, we used a digoxigenin-labelled ODN (5′-CTGCTGCTGCTGCTG-3′-digoxigenin, Eurogentec S.A., Belgium). A fluorescein-labelled ODN (5′-CTGCTGCTGCTGCTG-3′-fluorescein, GenXpress) was used to demonstrate charge-coupling to

Binding of ODNasAR to cationic microbubbles

Initially, we analyzed whether our 15 nucleotides-short ODNasAR can be loaded onto the lipid surface of cationic microbubbles by ion charge binding. The principle of this process is schematically depicted in Fig. 1. Using spectrophotometry we found that by incubating 1 μM of the ODNasAR with 2 × 107 microbubbles 42% of the ODNasAR were bound to the lipid surface whereas the residual amount of the ODN was not bound to the microbubbles and recovered from the aqueous phase. Loading of the ODN to

Discussion

Previous studies in our lab demonstrated that downregulation of the AR with short antisense ODNs is a powerful strategy to inhibit prostate cancer cell growth in vitro as well as in vivo [1], [2]. Although in vitro, these ODNs can be easily and efficiently delivered into prostate cancer cells either by electroporation or by the use of lipid-based transfection reagents, their delivery into prostate tumours in vivo still represents one of the major limiting factors for a clinical use.

Several

Acknowledgements

We want to thank Reinhold Ramoner for help with flow cytometry, Hannes Steiner and Andreas P. Berger for their technical assistance with animal experiments. Moreover, we want to thank Radu Rogojanu for his help with TissueQuest software. This work was supported by the Austrian Research Foundation (FWF P16882-B13) and the EU FP6 program (FP6-504587).

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