Ultrasound activatable antiangiogenic sonosensitizer for VEGFR associated glioblastoma tumor models

Angiogenic signaling pathway is a major contributing factor in cancer recurrence and progression, which can cause significantly reduced treatment outcomes, especially in the oxygen‐dependent photo‐ and sonodynamic therapies. VEGF and its receptor (VEGFR) play a crucial role in angiogenesis progression; precisely, upregulated VEGF signaling is mainly associated with angiogenesis progression in many types of cancers. Herein, we report a sunitinib‐conjugated sonosensitizer (TK‐RB: tyrosine kinase‐rose bengal) to enhance the anticancer efficacy through VEGF inhibition‐mediated antiangiogenesis in conjunction with cellular/tumor damage by ROS generated under ultrasound irradiation. TK‐RB reveals good selectivity and cytotoxicity toward VEGFR‐positive cells (U87MG) over VEGFR‐negative cells (MCF‐7). The fluorescent imaging analysis in vivo/ex vivo and the tumor growth investigation in nude mice with U87MG glioblastoma tumor xenografts demonstrate that rose bengal having tyrosine kinase inhibitor (TK‐RB) provides an enhanced antitumor effect. The current strategy will make a great contribution to optimizing anticancer performance by utilizing sonodynamic therapy together with antiangiogenics in several different malignancies.


INTRODUCTION
Generation of reactive oxygen species (ROS), such as superoxide radical, hydroxyl radical, singlet oxygen, etc. from excited sensitizers have gained much attention due to its tremendous cytotoxic effects with a noninvasive and spatiotemporal manner. [1] Photo-irradiation of sensitizer is one strategy (photodynamic therapy, PDT) to excite the molecule to generate such cytotoxic ROS. [2,3] As PDT gets importance in cancer treatment because of its less side effects, many researchers have focused on penetration depth to overcome its shallow tissue penetration from laser's innate limitation. [4] In spite of numerous efforts such as two-photon (TP) [5][6][7] and near infrared (NIR) [8,9] light irradiation to increase the penetration in deeply seated tumors, those strategies have not guaranteed the deep tissue penetration yet. [10] Recently, multifarious trials have been reported to surmount the difficulty of penetration with various energy delivery methods such as ultrasound (US) (sonodynamic therapy), [11][12][13] X-ray (X-ray-induced photodynamic therapy), [14,15] chemiluminescence (chemiluminescence-induced photodynamic therapy), [16,17] and alternative magnetic field (AMF) (magnetic hyperthermia). [18,19] Among assorted irradiation sources, US can be a promising tool to activate sensitizers for cancer treatment due to its high tissue penetration at depths exceeding 10 cm, [20] noninvasiveness, cost-effectiveness, and safe clinical modality. [21] Since US (>20 kHz) was used as a diagnostic tool in various fields, several efforts have been contributed to apply US usage for therapeutic purposes. High-intensity focused ultrasound (HIFU) is one of the most widely used therapeutic techniques with low frequency and high power among various US applications. [22] To emphasize the therapeutic efficacy of US-mediated cancer treatment, Umemura utilized US to activate the hematoporphyrin (Hp), which is called sonodynamic therapy (SDT). [23] Mode of action of SDT is still controversial with several hypotheses such as sonoluminescence (SL) from acoustic cavitation and direct ROS generation from pyrolysis, etc. [24,25] The most convincing hypothesis is that under US irradiation, cavitating bubbles are generated and are collapsed. By implosion of these bubbles, the energy is emitted in the form of SL, which is transferred to sonosensitizer to excite it from its ground state (S 0 ) to excited state (S 1 ). Further, through intersystem crossing (ISC) and energy or electron transfer to tissue oxygen, ROS are generated to diminish the cancer cells. [11,24] Vascular endothelial growth factor (VEGF), a homodimeric glycoprotein, is a member of the platelet-derived growth factor's (PDGF) family. [26] VEGF and its receptor (VEGFR) are the central considerable fundamental regulators in cancer proliferation and play a crucial role in angiogenesis progression. [27][28][29] Consumption of molecular oxygen is one critical defect in cancer. Especially, a low level of oxygen concentration in cells causes hypoxia in cancer, which promotes angiogenesis to supply more oxygen and nutrients from blood vessel and leads to more malignant and aggressive cancer. [30] Upregulated VEGF signaling is mainly associated with angiogenesis progression in many types of cancers including glioblastoma, one of the brain tumors bearing highly severe and malignant properties. [31] Targeting the VEGF underlying mechanism can directly impact the proliferation, metabolism, and morphology of glioma as well as many types of cancer cells due to inhibition of angiogenic signaling. [32][33][34][35][36][37] Sunitinib, a potent VEGFR tyrosine kinase inhibitor with high inhibitory performance (IC 50 : 4 ∼ 55 nM), is approved by the U.S. Food and Drug Administration (FDA). [38] In recent years, specific receptor/enzyme inhibitors conjugated with chemotherapeutics [39] or with photosensitizers [3] have gained much attention due to their multiple pathways targeting various kinds of cancers. Moreover, inhibition of the angiogenic signaling pathway in conjunction with PDT results in suppressing tumor growth. [40] Alternatively, in this context, we envisage that the inhibition of VEGFR signaling in combination with SDT is expected to offer a synergistic effect on malignant tumor suppression.
In this investigation, we report a sunitinib conjugated sonosensitizer (TK-RB: tyrosine kinase-rose bengal) to enhance the anticancer performance through antiangiogenesis in conjunction with cellular damage by ROS. Rose bengal (RB) of TK-RB serves as a sonosensitizer to generate ROS by US irradiation. [41,42] RB and its derivatives have long been considered as efficient photo-and sonosensitizers because of its high efficiency in ROS generation to destruct tumors. [43][44][45] RB exerts a large extent of cytotoxic effects with other combinations to demolish the tumor models, extensively. [46,47] Besides, the fluorescence property of RB helps to trace the cellular uptake and biodistribution of TK-RB in malignant cells and tumors. [48] Thus, the combination of RB with VEGFR tyrosine kinase inhibitor is expected to show a superior antitumor effect by angiogenesis signaling inhibition-mediated sonodynamic therapy toward glioblastoma.

RESULTS AND DISCUSSION
Synthesis of TK-RB is shown in Figure 1A. Briefly, the reaction of 5-fluoro-2,3-indoledione with 5-formyl-2,4-dimethyl-1H-pyrrole-3-carboxylic acid in piperidine/EtOH gave com-pound 1. Next, compound 1 was treated with 2 [39] in the presence of HATU and DIPEA in DMF to afford 3. Compound 4 was prepared from the reaction of RB with the propargyl bromide in acetonitrile (ACN). Finally, the TK-RB was obtained by a copper-catalyzed alkyne-azide click reaction of 3 with 4. All the synthesized compounds were characterized by analytical techniques, such as nuclear magnetic resonance ( 1 H and 13 C NMR) and mass spectrometry (FAB-MS) ( Figures  S7-S12).
First, the photophysical properties (UV-Vis absorption and FL emission spectrum) of TK-RB in DMSO and ACN solutions were investigated, which were well matched with those of RB ( Figure 1B,C). TK-RB showed a strong absorption band at 573 and 568 nm and an emission peak at 598 and 591 nm in DMSO and ACN, respectively, which are suitable for biological applications. We next implemented ROS generation experiments to confirm the US-triggered sonodynamic effect of TK-RB. The well-known singlet oxygen sensor, 2,2,6,6-tetramethylpiperidine (TEMP) [49] for electron spin resonance (ESR) was used to confirm the singlet oxygen generation. ESR analysis indicated that the TEMP itself showed no significant singlet oxygen generation regardless of the US irradiation. On the other hand, the signal of TEMPO was significantly enhanced by US irradiation with TK-RB ( Figure 1D), indicative of singlet oxygen generation from RB of TK-RB under US exposure. These results confirmed that sonosensitizer was an important resource to generate singlet oxygen by US.
As discussed above, TK-RB consists of RB and VEGFR inhibitor, which targets upregulated VEGFR tumor types. To assess it, first, we examined the VEGFR protein expression in different cancer cell lines (human glioblastoma [U87MG] and human breast cancer [MCF-7]) using Western blot analysis. The Western blot results indicated that the U87MG cells exhibited elevated VEGFR expression levels, whereas the MCF-7 cells had almost negligible VEGFR protein expression ( Figure S1). Next, to see the selectivity of TK-RB toward VEGF-positive cells over VEGF-negative cells, we performed cellular uptake experiments of TK-RB and RB in U87MG and MCF-7 cells using confocal microscopy. As seen in Figure 2A, U87MG cells uptake TK-RB seven-fold greater than MCF-7 does, which proves the targetability of sunitinib toward VEGFR-positive U87MG cells. By contrast, RB showed no significant cell-penetrating behavior toward U87MG and MCF-7 cells ( Figure S2). In further investigation of cytotoxicity experiments, we tested the dark toxicity of TK-RB in both U87MG and MCF-7 cell lines without US irradiation. Negligible cytotoxicity was observed up to 10 µM ( Figure S3). Moreover, we performed the cell viability experiments of TK-RB in various conditions with a function of US laser power (0.5 and 1.0 W/cm 2 ) and irradiation time (30 and 60 s). As indicated in Figure 2B, even low laser power with a short irradiation time showed susceptible cytoxicity in VEGFR-positive U87MG cells, whereas negligible effects in VEGFR-negative MCF-7 cells ( Figure S4).
It is well known that VEGF and its receptor (VEGFR) are the crucial fundamental regulators in angiogenesis progression. [27] The VEGF signals promote the angiogenesis through phosphorylation of serine/threonine kinase ERK (pERK). [50] The blockage of VEGFR can lead to the progress of antiangiogenic signaling pathway, decreasing the pERK signals. [51]  To investigate the antiangiogenic role of sunitinib in TK-RB, Western blotting experiments were also implemented. Upon increasing the dosage of TK-RB, p-ERK expression levels were dramatically decreased ( Figure 2C) because of the VEGF inhibition-mediated antiangiogenic property of TK-RB. Further, the ROS generation capability of TK-RB was investigated using DCFH-DA staining in vitro. Strong fluorescence images were observed in US-treated U87MG cells with TK-RB, compared to other control cells ( Figure 2D and Figure S5), indicating that the RB conjugated with sunitinib is capable of generating ROS to a great extent. Next, we performed cell death assay with calcein-AM (green, live cell) and PI (red, dead cell) staining. Intense red fluorescent signals of the PI channel were observed in TK-RB with US-treated U87MG cells. Whereas, no apparent cell death was seen in MCF-7 cells regardless of US treatment and other control treatments of U87MG ( Figure 2E). Taken together, the TK-RB penetrates VEGFR-positive cells (U87MG) much better than VEGFR-negative cells (MCF-7) and exhibits significant and selective cytotoxicity to U87MG cells by ROS generated under US irradiation. Moreover, VEGFR inhibition property of sunitinib in TK-RB provides an antiangiogenic signaling pathway, so-called synergistic effect of TK-RB on U87MG cells.
To gain insight into the in vivo tumor targetability of TK-RB, nude mice bearing U87MG glioblastoma tumor xenografts were subjected to time-dependent fluorescent images analysis upon intravenous injections of RB and TK-RB. As seen in Figure 3A,B, TK-RB-treated groups showed strong fluorescence images in the tumor region. A significant fluorescence enhancement of the images was observed up to 8 h; beyond that it showed a gradual decrease in fluorescent intensity. During this period, no notable signals were detected from the RB-treated groups. Moreover, to trace the biodistribution of these sonosensitizers (RB and TK-RB), each organ and tumor was harvested upon the sacrificing mice and their fluorescence intensities were examined ( Figure 3C,D). In the case of TK-RB-treated group, significant fluorescence intensity was observed in the tumor region, representing the enhanced tumor accumulation of TK-RB in solid tumors. The minimum fluorescence intensity was perceived in the tumor region processed with RB. Both sonosensitizers (RB and TK-RB) showed accumulation in the liver, which might be due to the predominant clearance through the hepatobiliary system. Collectively, these data confirmed the promising tumor targetability of TK-RB attributable to the sunitinib moiety.
We further assessed the US-mediated therapeutic efficacy of TK-RB in nude mice bearing U87MG glioblastoma tumor xenografts. In TK-RB-treated group, significant tumor regression was observed in the tumor region under US irradiation ( Figure 4A-C and Figure S6). Noticeably, no tumor suppression was observed with other control groups (control, RB w/ or w/o US, TK-RB w/o US). During this treatment period, such a remarkable bodyweight change was . Statistical significance was determined by two-way ANOVA test with a post hoc Bonferroni test. Different letters (e.g., a-b) signify statistically distinct datasets (p < 0.05) not observed in all the treated groups ( Figure 4D). Taken together, all these observations supported the conclusion that the combination of RB with a tyrosine kinase inhibitor (TK-RB) provides an enhanced antitumor effect.

CONCLUSION
In conclusion, we have developed a sunitinib-conjugated RB sonosensitizer (TK-RB) to enhance the anticancer effect by VEGFR inhibition-mediated antiangiogenesis with cellular/tumor damage by ROS generated under US irradiation. The in vitro results revealed that the conjugation of RB with a tyrosine kinase inhibitor (sunitinib) could increase the RB uptake toward VEGFR-positive cells (U87MG), followed by marked ROS generation under US irradiation, exhibiting remarkable cytotoxic effect to the U87MG cancer cells. Moreover, VEGFR inhibition property of sunitinib in TK-RB offers an antiangiogenic signaling pathway to increase the cytotoxic effect of TK-RB on U87MG cells. Fluorescent image analysis of in vivo/ex vivo and tumor growth studies in nude mice bearing U87MG glioblastoma xenografts strongly support the conclusion that RB-conjugated tyrosine kinase inhibitor (TK-RB) provides enhanced antitumor efficacy. Thus, the current strategy can lead to new vital developments toward cancer cells/tumors for their enhanced anticancer performance.