1.

Introduction

Photodynamic therapy (PDT) is an attractive treatment strategy that is minimally invasive with little to no systemic toxicity.^1,2 PDT utilizes photosensitizers (PSs) to harvest the power of light and transform it into reactive oxygen species (ROS), a powerful cancer-killing grenade. The ROS are short-lived, limiting the effect of PDT to the irradiated area only. Currently, several drugs have been approved across the globe for PDT treatment. Most notably, Photofrin^® was approved back in 1993 in Canada for bladder cancer treatment and later for other cancers and diseases by FDA in the US and other regions in the world.³ A major drawback of common PSs is their lack of specific uptake. Even though PDT toxicity is limited in the region exposed to light irradiation only, normal cells in the same region still suffer from unnecessary PDT damage. This limitation can be overcome by adding targeting ligand to the PS design. With the introduction of a targeting moiety, PDT benefits from two advantages: (1) specific targeting can lead to significant higher uptake in malignant cells, which leads to more efficient treatment outcome with fewer damage to innocent cells and (2) lower dose of PSs would be needed for similar therapeutic efficacy, which further lowers side effects of the treatment. Previously, our lab developed PDT agents that specifically target type-2 cannabinoid receptor (${\mathrm{CB}}_2\mathrm{R}$)^4,5 or translocator protein^6,7 with successful anticancer therapy outcome in murine tumor models.

In addition to targeting functionality, a new advance of PDT agent design is to include ROS-cleavable linker for on-demand drug release. We developed a heterobifunctional thioketal linker for such a purpose.⁸ The You laboratory recently reported several prodrugs that can be activated by ROS produced from PS and encouraging combinatory therapeutic effects have been observed.^9–12 For example, they synthesized a light-activatable paclitaxel prodrug for combined PDT and chemotherapy.¹³ As compared to the non-ROS-cleavable analog, this light-activatable paclitaxel prodrug showed greatly improved therapeutic outcome in ovarian cancer cells. However, this prodrug has aggregation issues in aqueous solution and is lack of targeting functionality. In a more recent study, polyethylene glycolylated folic acid molecules with different sizes of polyethylene glycol (PEG) were attached to the light-activatable paclitaxel prodrug for improved water solubility and targeted delivery.¹⁴ Unfortunately, these targeted prodrugs still suffer from aggregation issue and the structures involve many components, including a phthalocyanine PS, folic acid, PEG, ROS-cleavable linker and paclitaxel. It is more desirable to develop a water soluble, targeted, and light activatable prodrug with a relatively simply design.

In this study, we synthesized a light-activatable cannabinoid prodrug, IR700DX-TK-mbc94, for combined and target-specific PDT and cannabinoid therapy. This prodrug consists of only three components: a highly hydrophilic phthalocyanine PS, ROS-sensitive thioketal linker, and cannabinoid. Cannabinoids have attracted great attention as promising antitumor drugs as they can produce ROS, inhibit pro-metastatic Id1 gene expression, and upregulate autophagy-mediated cell death.¹⁵ The cannabinoid molecule we chose targets ${\mathrm{CB}}_2\mathrm{R}$ overexpressed in various types of cancers, such as glioma, lymphoma, skin, prostate, lung, and breast cancer.^16,17 Upon light irradiation, the PS produces cytotoxic ROS, which simultaneously cleaves the ROS-sensitive linker and subsequently releases cannabinoid drug. Therefore, this prodrug offers targeted and on-demand combinatory PDT and cannabinoid therapy with a simple design. We found that treatment with IR700DX-TK-mbc94 caused remarkably improved therapeutic outcome as compared to nonactivatable IR700DX-mbc94.

2.

Materials and Methods

2.3.

Synthesis of mbc94-SH and mbc94-TTPA

3-(Tritylthio)propionic acid 5.9 mg ($17\mu \mathrm{mol}$), HBTU (8 mg, $21\mu \mathrm{mol}$), and DIEA (3.9 mg, $30\mu \mathrm{mol}$) were dissolved in anhydrous DMF (0.6 mL) under argon atmosphere at 0°C with stirring. Mbc94 (12 mg, $17\mu \mathrm{mol}$) dissolved in anhydrous DMF (0.6 mL) was added dropwise to the mixture after which the reaction mixture was slowly warmed to room temperature and stirred for additional 16 h. The resulting mixture was dried and separated by column chromatography using a gradient of 2% to 5% methanol in dichloromethane. The desired product (mbc94-TTPA) was yielded as an oil (10.7 mg). The trityl protection was subsequently removed by dissolving the product in dichloromethane (1.0 mL) and trifluoroacetic acid (0.3 mL) at $-20°\mathrm{C}$ followed by addition of triethylsilane ($50\mu \mathrm{L}$). After removal of solvent, the mixture was separated by column chromatography using a mixture of dichloromethane/methanol/ammonia = 100:5:0.5 to yield the final compound (mbc94-SH) as an oil (1 mg, 7% after two steps).

${}^1\mathrm{H}$ NMR of mbc94-TTPA (400 MHz, ${\mathrm{CDCl}}_3$) $\delta$ 7.44-7.36 (m, 7 H), 7.36-7.30 (m, 2H), 7.29-7.17 (m, 7H), 7.17-7.10 (m, 2H), 7.07-6.91 (m, 4H), 6.81 (s, 1H), 5.62 (t, $J=8.1\mathrm{Hz}$, 1H), 5.29 (s, 2H), 3.94 (s, 1H), 3.80 (dd, $J=12.7$, 1.7 Hz, 1H), 3.08 (q, $J=9.1\mathrm{Hz}$, 2H), 3.00-2.86 (m, 4H), 2.76 (t, $J=9.5\mathrm{Hz}$, 2H), 2.50-2.42 (m, 2H), 3.28 (s, 1H), 2.22 (t, $J=9.4\mathrm{Hz}$, 1H), 2.05 (t, $J=9.4\mathrm{Hz}$, 1H), 1.82-1.18 (m, 17H), 1.16 (s, 3H), 1.10 (s, 3H), 0.85 (s, 3H).

Fig. 1

Structures of IR700DX-TK-mbc94 and IR700DX-mbc94.

${}^1\mathrm{H}$ NMR of mbc94-SH (400 MHz, ${\mathrm{CDCl}}_3$) $\delta$ 7.33 (t, $J=10.0\mathrm{Hz}$, 3H), 7.19-6.87 (m, 6H), 6.79 (s, 1H), 5.62 (t, $J=8.1\mathrm{Hz}$, 1H), 6.66-6.45 (br s, 1H), 5.25 (s, 2H), 3.88 (s, 2H), 3.80 (dd, $J=13.1$, 1.9 Hz, 1H), 3.20 (q, $J=9.2\mathrm{Hz}$, 2H), 3.00 (t, $J=8.9\mathrm{Hz}$, 2H), 2.75 (t, $J=9.4\mathrm{Hz}$, 2H), 2.56 (t, $J=8.7\mathrm{Hz}$, 2H), 2.33 (s, 1H), 1.84-1.92 (m, 17H), 1.25 (s, 3H), 1.17 (s, 3H), 1.10 (s, 3H).

3.

Results

Previously, we described the synthesis of compound 1,⁸ a heterobifunctional ROS-responsive thioketal linker, where the two distinct functional groups provide readily assessable point for conventional amide coupling. This unique feature renders compound 1 an excellent ROS-activatable linker connecting two functionalities, a phthalocyanine PS, IR700DX and a cannabinoid, mbc94 (Fig. 2). Intermediate 2a was prepared by coupling mbc94 and 1 via amide coupling using HBTU. The trifluoroacetamide protection was then removed using base hydrolysis to give 2b. Finally, 2b was mixed with IR700DX-NHS in dimethyl sulfoxide to yield compound IR700DX-TK-mbc94 (3). To study the therapeutic effect of the activated prodrug, we also synthesized mbc94-SH by attaching 3-(tritylthio)propionic acid to mbc94 through amide coupling, followed by deprotection of the thiol group in an acidic condition (Fig. 3). We found that IR700DX-TK-mbc94 and IR700DX-mbc94 efficiently displaced the high-affinity radioligand $[{}^3\mathrm{H}]\mathrm{CP}\text{-}55940$ ($K_d=0.39\mathrm{nM}$) from human ${\mathrm{CB}}_2\mathrm{R}$ protein with similar nanomolar affinities ($K_i=2.67\pm 1.78\mathrm{nM}$ for IR700DX-TK-mbc94 versus $1.86\pm 1.38\mathrm{nM}$ for IR700DX-mbc94), which are roughly 20-fold higher than mbc94-SH ($K_i=47.7\pm 16\mathrm{nM}$).

Fig. 2

Synthesis of IR700DX-TK-mbc94.

Fig. 3

Synthesis of mbc94-SH.

To validate the hypothesis that the thioketal linker is cleavable by the in situ generation of ROS, we quantified the amount of thiol generation upon light irradiation. We originally attempted to use the classic Ellman’s assay for thiol quantitation.^22,23 Unfortunately, this method is not suitable as the Ellman’s reagent is prone to artifacts from redox reaction, which is inevitable due to the ROS generation. We therefore used the 7-diethylamino-3-(4’-maleimidylphenyl)-4-methylcoumarin (CPM) assay^24,25 to determine the ROS-induced thioketal cleavage. CPM is a weakly fluorescent compound but its fluorescent can be activated upon reaction with thiol via maleimide-thiol coupling reaction.^26,27

Upon light irradiation, IR700DX generates ROS, which can cleave the nearby thioketal linker and subsequently produce two thiol fragments and one acetone. As expected, over the 30-min light irradiation period, we observed steady increase of fluorescence from CPM mixed with IR700DX-TK-mbc94 (Fig. 4). Light irradiation of IR700DX-mbc94 and IR700DX caused similarly low level of CPM fluorescence and the vehicle group showed negligible signal, suggesting that ROS production alone may also lead to CPM fluorescence activation. Nevertheless, CPM fluorescence caused by light activation of IR700DX-TK-mbc94 is much higher than the other groups ($p<0.0001$). To investigate whether the increased CPM fluorescence may be caused by higher level of ROS production, we further compared type I and type II ROS produced from IR700DX-TK-mbc94 and IR700DX-mbc94 (Fig. 5). Under the same dose of light irradiation, IR700DX-TK-mbc94 produced almost the same amount of type I (APF indicating free radical, RFU APF: 7731 for IR700DX-TK-mbc94 versus 7760 for IR700DX-mbc94 after 30-min irradiation), and less amount of type II (SOSG indicating singlet oxygen, RFU SOSG: 8901 for IR700DX-TK-mbc94 versus 11412 for IR700DX-mbc94 after 30-min irradiation, 22% less) ROS than IR700DX-mbc94, indicating that ROS production did not contribute to the increased CPM fluorescence in the IR700DX-TK-mbc94 group. These combined data suggest that thiol production from IR700DX-TK-mbc94 dominated the CPM fluorescence activation.

Fig. 4

Fluorescence intensity of CPM over 30 min of light irradiation at 690 nm is compared among four groups, including IR700DX-TK-mbc94, IR700DX-mbc94, IR700DX, and vehicle.

Fig. 5

ROS production of IR700DX-TK-mbc94 and IR700DX-mbc94 analyzed by (a) AFP (type I ROS) and (b) SOSG (type II ROS) assays.

The in vitro therapeutic results are summarized in Fig. 6. To demonstrate the advantages of the light-activatable combination therapy strategy, all therapeutic results are compared between IR700DX-TK-mbc94 and the nonactivatable IR700DX-mbc94 analog. Experimental setup is demonstrated in Fig. 7. To ensure uniformed light irradiation, light dose was determined using an optical power meter and each group of cells ($n=4$) was positioned in the center of the irradiated region. We first treated ${\mathrm{CB}}_2\mathrm{R}+$ mouse delayed brain tumor ${\mathrm{CB}}_2$-mid DBT cells with two concentrations (0.5 and $1\mu \mathrm{M}$) of IR700DX-TK-mbc94 or IR700DX-mbc94, washed unbound PSs, and applied light irradiation [Figs. 6(a) and 6(b)]. We found that right after light irradiation, 59% death was observed in cells treated with $1\mu \mathrm{M}$ of IR700DX-TK-mbc94, whereas no significant cell death was seen in the IR700DX-mbc94 treatment group ($p<0.0001$). After these cells were incubated for additional 24 h, the IR700DX-TK-mbc94 treatment group showed increased cell death to 97% and the IR700DX-mbc94 group showed 74% cell death ($p=0.001$), suggesting that programmatic apoptotic cell death was involved in both groups, but necrosis was only involved in the IR700DX-TK-mbc94 group. At the lower PS concentration ($0.5\mu \mathrm{M}$), no significant cell death was observed in either group right after light irradiation; however, after additional 24 h incubation, the IR700DX-TK-mbc94 group showed 78% cell death as compared to insignificant cell death in the IR700DX-mbc94 group ($p<0.0001$). In addition, we found that the cellular uptake of IR700DX-TK-mbc94 group can be significantly blocked by the ${\mathrm{CB}}_2\mathrm{R}$ ligand SR144528 [44% blocking effect, $p=0.02$, Fig. 6(c)], supporting target-specific binding of IR700DX-TK-mbc94. We also studied light dose-dependent therapy using three light doses (18, 36, and $54\mathrm{J}/{\mathrm{cm}}^2$) and two concentrations (1 and $2\mu \mathrm{M}$) [Fig. 6(d)]. It is obvious that higher light dose or concentration led to greater cell death. Interestingly, mbc94-SH alone did not cause significant cell death (Fig. 8). We also found that although light treatment using IR700DX-TK-mbc94 caused almost complete death of ${\mathrm{CB}}_2$-mid DBT cells, the same treatment appears to be safe to HEK-293 normal cell control (Fig. 9). These data suggest that IR700DX-TK-mbc94 is remarkably effective in treating cancer cells in a target-specific manner.

Fig. 6

PDT treatment using IR700DX-TK-mbc94 led to increased cancer cell death as compared to PDT with IR700DX-mbc94. (a,b) Concentration-dependent and progressive cell death caused by PDT using (a) 0.5 or (b) $1\mu \mathrm{M}$ IR700DX-TK-mbc94 or IR700DX-mbc94. Progressive cell death was observed after PDT treatment (24 h post-PDT versus right after PDT treatment). (c) Cellular uptake of IR700DX-TK-mbc94 can be blocked by a ${\mathrm{CB}}_2\mathrm{R}$ ligand, SR144528. (d) Light dose-dependent cell death caused by PDT using IR700DX-TK-mbc94. Cells were treated with 18, 36, or $54\mathrm{J}/{\mathrm{cm}}^2$ 690-nm light after incubation with 1 or $2\mu \mathrm{M}$ IR700DX-TK-mbc94.

Fig. 7

In vitro PDT study using 96-well plates. (a) LED light was used to irradiate cells treated with IR700DX-TK-mbc94 or IR700DX-mbc94. (b) To ensure uniformed light irradiation, light dose was determined using an optical power meter and each group of cells ($n=4$) was positioned in the center of the irradiated region. Other groups of cells were positioned outside of the irradiated region.

Fig. 8

mbc94-SH alone is not efficient in cancer cell treatment.

Fig. 9

Normal cell control (HEK-293) showed negligible death as compared to ${\mathrm{CB}}_2\mathrm{R}$-expressing DBT-2D4 cells after treatment with PDT using $2\mu \mathrm{M}$ IR700DX-TK-mbc94 and $54\mathrm{J}/{\mathrm{cm}}^2$ light dose.

4.

Discussion

The combination of multiple therapeutics for cancer treatment has gained growing interest. Such a practice usually leads to enhanced outcome. In some cases, more favorable synergistic effects are achieved, offering remarkable therapeutic outcome and fewer side effects. Recently, we demonstrated the synergistic outcome by combing two PDT agents that bind to distinct cellular localization.⁶ Although combination therapy can be easily realized by administration of multiple therapeutics individually, a more attractive approach is targeted delivery of therapeutics to the tumor site in one package, where they can be activated or released on demand. This approach is likely to further improve the therapeutic outcome and safety. Often, a cleavable linker is included in the drug delivery system design that is sensitive to conditions associated with tumor cells or microenvironment, such as protease, acidic pH, and oxidative stress.²⁸ Although dozens of cleavable linkers have been developed, the cleavage is usually controlled only spatially, but not temporally. In addition, these drug delivery systems are typically difficult to develop, involving multifunctional nanosystems or small molecules with many components. Here, we aimed to develop a multifunctional prodrug, which has a simple design but allows for targeted combinatory therapy and on-demand activation both spatially and temporally. This was achieved by utilizing a unique ROS-activatable heterobifunctional linker, a ${\mathrm{CB}}_2\mathrm{R}$-targeted cannabinoid prodrug, and a highly hydrophilic PS with six sulfonate groups.

One interesting finding is that both IR700DX-TK-mbc94 and IR700DX-mbc94 bind to ${\mathrm{CB}}_2\mathrm{R}$ with a much higher ($\sim 20$ folds) affinity than mbc94-SH. This is surprising because typically attachment of a large dye molecule to a small targeting ligand compromises the binding, as we have observed in our previous studies.^19,29 Although the exact binding mechanism is yet to be investigated, it is possible that IR700DX facilitated the interaction by binding to additional sites on ${\mathrm{CB}}_2\mathrm{R}$. It has been previously reported that phthalocyanine dyes can bind to certain proteins.³⁰ It is noteworthy that the binding affinity of IR700DX-mbc94 to ${\mathrm{CB}}_2\mathrm{R}$ has been previously reported by us determined by live cell fluorescence binding study ($K_d=42\mathrm{nM}$), which is considerably lower than the binding affinity reported in this study ($K_i=1.86\mathrm{nM}$). However, such a significant difference is not surprising, because the previous binding study used live murine cells but human ${\mathrm{CB}}_2\mathrm{R}$ protein was used in this binding study. Our previous study has shown that largely different binding affinities were determined when protein versus intact cells were used.³¹ In addition, human and mouse ${\mathrm{CB}}_2\mathrm{Rs}$ only share 82% amino acid identity.³²

IR700DX-TK-mbc94 and IR700DX-mbc94 share the same PS and therefore, the amount of ROS produced from each agent is presumed to be the same. However, based on the SOSG studies, the singlet oxygen produced from IR700DX-TK-mbc94 appears to be less than that from IR700DX-mbc94. This is in fact expected because some singlet oxygen produced from IR700DX-TK-mbc94 was consumed to cleave the ROS-activatable thioketal linker and therefore not able to be detected by SOSG. Because singlet oxygen is the main phototoxicity source from IR700DX,⁴ such loss of singlet oxygen would reduce the therapeutic outcome of IR700DX-TK-mbc94 as compared to IR700DX-mbc94. In contrast, we found that IR700DX-TK-mbc94 produced much higher therapeutic outcome than IR700DX-mbc94, suggesting that the ROS-activated prodrug, mbc94-SH, greatly contributed to the treatment. Interestingly, mbc94-SH alone, even with a concentration of $5\mu \mathrm{M}$, did not show significant therapeutic effect, whereas IR700DX-TK-mbc94 treatment with much lower concentrations caused dramatic cell death. We speculate that IR700DX-TK-mbc94, which has a higher binding affinity to ${\mathrm{CB}}_2\mathrm{R}$ than mbc94-SH, facilitated delivery of mbc94-SH to the target site. As a result, mbc94-SH cleaved from IR700DX-TK-mbc94 is more effective than if it is directly used to treat ${\mathrm{CB}}_2\mathrm{R}+$ cells. It is also possible that the remarkable therapeutic outcome from IR700DX-TK-mbc94 is attributed to the synergism between PDT and cannabinoid therapy.

5.

Conclusions

In conclusion, we developed a far red light-activatable prodrug, IR700DX-TK-mbc94, which binds to ${\mathrm{CB}}_2\mathrm{R}$ with a nanomolar affinity and synergizes PDT and cannabinoid therapy. IR700DX-TK-mbc94 showed superior efficacy than IR700DX-mbc94 in the therapeutic studies. With many favorable features including targeting specificity, on-demand activation, combinatory therapy, high hydrophilicity and safety, IR700DX-TK-mbc94 has great potential in treating ${\mathrm{CB}}_2\mathrm{R}$-positive tumors.