Tumor mitochondria-targeted photodynamic therapy with a translocator protein (TSPO)-specific photosensitizer

doi:10.1016/j.actbio.2015.09.033

Acta Biomaterialia

Volume 28, December 2015, Pages 160-170

https://doi.org/10.1016/j.actbio.2015.09.033 Get rights and content

Abstract

Photodynamic therapy (PDT) has been proven to be a minimally invasive and effective therapeutic strategy for cancer treatment. It can be used alone or as a complement to conventional cancer treatments, such as surgical debulking and chemotherapy. The mitochondrion is an attractive target for developing novel PDT agents, as it produces energy for cells and regulates apoptosis. Current strategy of mitochondria targeting is mainly focused on utilizing cationic photosensitizers that bind to the negatively charged mitochondria membrane. However, such an approach is lack of selectivity of tumor cells. To minimize the damage on healthy tissues and improve therapeutic efficacy, an alternative targeting strategy with high tumor specificity is in critical need. Herein, we report a tumor mitochondria-specific PDT agent, IR700DX-6T, which targets the 18 kDa mitochondrial translocator protein (TSPO). IR700DX-6T induced apoptotic cell death in TSPO-positive breast cancer cells (MDA-MB-231) but not TSPO-negative breast cancer cells (MCF-7). In vivo PDT study suggested that IR700DX-6T-mediated PDT significantly inhibited the growth of MDA-MB-231 tumors in a target-specific manner. These combined data suggest that this new TSPO-targeted photosensitizer has great potential in cancer treatment.

Statement of Significance

Photodynamic therapy (PDT) is an effective and minimally invasive therapeutic technique for treating cancers. Mitochondrion is an attractive target for developing novel PDT agents, as it produces energy to cells and regulates apoptosis. Current mitochondriatargeted photosensitizers (PSs) are based on cationic molecules, which interact with the negatively charged mitochondria membrane. However, such PSs are not specific for cancerous cells, which may result in unwanted side effects. In this study, we developed a tumor mitochondria-targeted PS, IR700DX-6T, which binds to translocator protein (TSPO). This agent effectively induced apoptosis in TSPO-positive cancer cells and significantly inhibited tumor growth in TSPO-positive tumor-bearing mice. These combined data suggest that IR700DX-6T could become a powerful tool in the treatment of multiple cancers that upregulate TSPO.

Graphical abstract

Introduction

Photodynamic therapy (PDT) is a clinically approved, minimally invasive, and highly controllable therapeutic procedure, which has become popular as an alternative or additional approach to conventional cancer treatments, such as chemotherapy and surgery [1], [2]. A regime of PDT requires three key components: photosensitizer (PS), light irradiation, and oxygen [3]. In the presence of oxygen, a PS is activated by irradiation at a specific wavelength to produce reactive oxygen species (ROS), such as singlet oxygen and free radicals, which consequently lead to cell death [4]. Undoubtedly, development of effective PSs is essential to the advance of PDT. In recent years, a number of PSs have been developed for research as well as clinical use, such as porphyrin derivatives, chlorins, and phthalocyanines [5], [6].

The efficacy of PDT largely depends on the tumor-selectivity and subcellular localization of PSs. Upon administration, different PSs may locate to distinct cell organelles, such as mitochondria, lysosomes, and plasma membranes, depending on their physicochemical and binding properties, such as lipophilicity, charge, and chemical structure [7]. In fact, the subcellular distribution of PSs often correlates with specific type of cell death [8]. For example, antibody-PS conjugates used in photoimmunotherapy bind to plasma membrane and often lead to necrotic cell death [9].

Among the various subcellular targets, the mitochondrion holds particularly great promise as a PDT target, as it plays an essential role in supplying energy for cells and regulating cell apoptosis [10], [11], [12]. For this reason, great effort has been focused on developing new mitochondria-specific PDT agents [13], [14]. Current mitochondria-targeting PSs are mostly based on cationic molecules, because the negative charges on mitochondria membranes allow for ionic interaction with these PSs. However, given that mitochondrion is a universal cell organelle, such PSs can also damage mitochondria in healthy cells, leading to unwanted side effects. To address this limitation, we aimed to develop a PS that specifically binds to tumor mitochondria.

In the present study, we chose the 18 kDa translocator protein (TSPO), previously termed the peripheral benzodiazepine receptor (PBR), as the target for PDT. TSPO is a protein mainly found on the outer mitochondrial membrane and associated with a number of cellular processes, such as cholesterol transport, steroidogenesis, cell proliferation, porphyrin transport and apoptosis [15]. Although normal tissues and organs express TSPO at various levels, significantly increased expression level of TSPO has been found in multiple cancers including breast [16], [17], colorectal [18], prostate [19], and brain cancer [20]. In addition, higher TSPO expression levels correlate with increased tumor aggressiveness and metastasis as well as with a poorer prognosis [18]. Furthermore, deregulation of TSPO expression or function has been reported to contribute to cell apoptosis. Consequently, TSPO is a promising target for improved cancer treatment efficacy.

Since TSPO regulates porphyrin transport, much effort has been invested into TSPO-targeted PDT using endogenous and exogenous porphyrin molecules, as summarized in a recent review [21]. Unfortunately, most of these TSPO-PDT studies resulted in limited efficacy and selectivity. To improve the TSPO targeting effect, Chen et al. recently developed a TSPO targeted PS for bi-functional positron emission tomography (PET) imaging and PDT by conjugating ¹²⁴I-labeled TSPO ligand, PK11195, with a well known porphyrin derivative, HPPH, as the PS [22]. The resulting agent showed significantly improved PDT efficacy over the HPPH PS in a breast cancer mouse model, although lengthy synthetic process and skin damage was involved.

In this study, we report a new TSPO-targeted PS, IR700DX-6T, which consists of a phthalocyanine PS (IR700DX), a six-carbon linker, and a TSPO ligand (DAA1106) with a high binding affinity to TSPO. This construct allows for effective PDT with only low-power LED light irradiation, thus avoiding potential skin phototoxicity issues. The photo therapeutic effect of IR700DX-6T was examined using both TSPO-positive and TSPO-negative human breast cancer cells. Furthermore, we evaluated the in vivo PDT efficacy using a breast cancer xenograft animal model.

Section snippets

General

The solvents used are of ACS grade or HPLC grade. The PS, IR700DX-NHS ester, was purchased from LI-COR Bioscience (Lincoln, NE). ¹H NMR spectra were recorded on a Bruker Avance III 400 MHz system. Mass spectra were recorded on a Waters LCT Premier mass spectrometer. UV/Vis absorption spectra were recorded on a Cary 100 Bio UV–vis Spectrophotometer (Agilent Technologies, Santa Clara, CA). Fluorescence emission spectra were recorded on a Cary Eclipse fluorescence spectrophotometer (Agilent

Synthesis, spectroscopic, stability, and binding properties of IR700DX-6T

In our previous study, we developed a DAA1106 analog with a six-carbon linker and terminal amino group, 6-TSPOmbb732 (Scheme 1), which was subsequently conjugated to fluorescent dyes for TSPO-targeted imaging [23], [28]. Building upon this success, we attached this functional DAA1106 analog to a phthalocyanine dye, IR700DX-NHS, via a simple acylation reaction (Scheme 1). The resulting IR700DX-6T exhibits maximum absorption and emission at 690 nm and 699 nm in water, respectively (Fig. 1A).

The

Discussion

We see considerable opportunity in targeting the TSPO for PDT treatment. In addition to the elevated expression levels in various types of cancers, TSPO plays an important role in the regulation of apoptosis. Specifically, through interaction with several proteins in the outer and inner mitochondrial membrane, TSPO regulates the mitochondrial permeability transition pore (MPTP), which controls mitochondrial membrane integrity by maintaining mitochondrial transmembrane potential [30], [31].

Conclusions

In conclusion, we developed a novel TSPO-targeted PS, IR700DX-6T. In vitro study indicated that IR700DX-6T induced apoptotic cell death in TSPO-positive but not TSPO-negative breast cancer cells. Remarkably, in vivo PDT study suggested that IR700DX-6T-mediated PDT significantly inhibited the growth of MDA-MB-231 tumors in a target-specific manner. These results suggest that IR700DX-6T-PDT may have great potential in treating TSPO-positive tumors.

Conflict of interest

No potential conflicts of interest relevant to this article are reported.

Acknowledgement

We thank Dr. Carolyn J. Anderson at the University of Pittsburgh for providing MDA-MB-231 and MCF-7 cells. We appreciate Kathryn Day and Joseph Latoche for maintaining the animal imaging facility. This work was supported by the NIH (United States) Grant #R21CA174541 (PI: Bai) and the startup fund provided by the Department of Radiology, University of Pittsburgh, United States. This project used the UPCI imaging facilities supported, in part, by award P30CA047904.

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¹: These authors contributed equally to this work.

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