Cooperation therapy between anti-growth by photodynamic-AIEgens and anti-metastasis by small molecule inhibitors in ovarian cancer

Metastasis is one of the main causes of death and treatment failure in ovarian cancer. Some small molecule inhibitors can effectively inhibit the metastasis of primary tumors. However, they do not kill the primary tumor cells, which may lead to continuous proliferation. Herein, we have prepared a multifunctional nanoparticles named TPD@TB/KBU2046, which consisted of three functional moieties: (1) KBU2046 (small molecule inhibitor) that can inhibit the metastasis of the primary tumors, (2) TB (photodynamic-AIEgens) that may suppress the growth of the primary tumors, and (3) TPD, which contains TMTP1 (a targeting peptide, which specifically binds to highly metastatic tumor cells) that can enhance the TB/KBU2046 dosage in the tumor site. Methods: The TPD@TB/KBU2046 was prepared by nano-precipitation method. We linked the targeting peptide (TMTP1) to the nanoparticles via amidation reaction. TPD@TB/KBU2046 nanoparticles were characterized for encapsulation efficiency, particle size, absorption spectra, emission spectra and ROS production. The combinational efficacy in image-guided anti-metastasis and photodynamic therapy of TPD@TB/KBU2046 was explored both in vitro and in vivo. Results: The TPD@TB/KBU2046 showed an average hydrodynamic size of approximately 50 nm with good stability. In vitro, TPD@TB/KBU2046 not only inhibited the metastasis of the tumors, but also suppressed the growth of the tumors under AIEgens-mediated photodynamic therapy. In vivo, we confirmed that TPD@TB/KBU2046 has the therapeutic effects of anti-tumor growth and anti-metastasis through subcutaneous and orthotopic ovarian tumor models. Conclusion: Our findings provided an effective strategy to compensate for the congenital defects of some small molecule inhibitors and thus enhanced the therapeutic efficacy of ovarian cancer.


Introduction
The ovarian cancer is a common gynecological malignant tumor with the highest mortality rate [1][2]. As the early symptoms are not obvious and the diagnosis rate is low, most patients are clinically diagnosed as advanced stage [3][4]. At present, clinical treatment of ovarian cancer is still surgical resection combined with chemotherapy, but the 5-year survival rate is less than 40% [5]. Metastasis of ovarian cancer can occur via the transcoelomic, haematogeneous, or lymphatic route, and is responsible for the major Ivyspring International Publisher obstacle to the success of treatment [6][7]. Thus, inhibition of tumor metastasis is extremely important for ovarian cancer treatment.
Recently, many small molecule inhibitors, such as tea polyphenols, curcumin, KBU2046, BENC-511, ZD6474, et al, have been reported for inhibiting tumor metastasis [8][9][10][11][12]. Among them, KBU2046 is an innovative small molecule inhibitor, which has significant anti-metastasis effect and opens up a novel strategy for cancer treatment. Xu et al. demonstrated that KBU2046 can be inserted very precisely into the fissures of heat shock protein 90 and cell division cycle 37 heterocomplex, thereby affect the binding of the client kinase protein RAF1 to the heterocomplex, resulting in limited cell motility. Although KBU2046 can effectively inhibit the metastasis of tumor cells, it has no cytotoxic effect. Similarly, some small molecule inhibitors such as MPT0G211, DSHN, BDP5290 and BMS-777607 etc. also have good effect in inhibiting metastasis of tumors, but no effect on cell viability [13][14][15][16]. That is to say, even using these small molecule inhibitors, tumor cells can still proliferate in situ. The inherent properties of these small molecule inhibitors, including lack of cytotoxicity, make their biocompatibility excellent, but this is a defect in the treatment of tumors. Therefore, based on the defect of these small molecule inhibitors, it is necessary to introduce a therapeutic element to suppress the growth of the tumors.
Photodynamic therapy (PDT) is a burgeoning, noninvasive and safe therapeutic assay that utilized a photosensitizer, light of particular wavelength and oxygen to achieve its therapeutic effect [17][18][19][20]. Photosensitizer is able to generate reactive oxygen species (ROS) upon light irradiation which can induce tumor cell death [21][22][23][24]. As photosensitizer plays a critical role in undertaking the efficacy of PDT, the design and function of photosensitizer has been unremittingly improved over the decades [25][26][27]. Traditional photosensitizer easily aggregates in aqueous phase due to the π−π stacking and rigid planar structures, for instance, porphyrin and its derivatives. Due to the aggregation-caused quenching effect, fluorescence quenching and ROS production efficiency reduction, the application of traditional photosensitizers has been greatly affected [28][29][30]. Fortunately, Tang's group discovered the aggregation-induced emission luminogens (AIEgens), which provided a possibility to solve the issue of aggregation-caused quenching (ACQ) [31][32][33][34]. The AIEgens have been developed as specific nanomedicine for cancer theranostics, such as image-guided tumor resection, cell tracking, monitoring of drug delivery processes and detection of biological processes [35][36][37][38][39]. Importantly, some AIEgens with special structures can also be used as photosensitizers which possess high ROS production efficiency and strong fluorescence emission in aggregation state [40][41][42][43]. The integration of brightness and photosensitization within a single AIEgen exhibits great convenience for therapeutic strategy with simplicity and reproducibility.
Herein, we have successfully prepared a self-assembled nanoparticle (TPD@TB/KBU2046) containing small molecule inhibitor (KBU2046) and photodynamic-AIEgen (TB) (Scheme 1). In addition, TPD@TB/KBU2046 nanoparticles contain tumor targeting peptide (TMTP1), which specifically binds to highly metastatic tumor cells [44][45][46][47], thereby enhancing the accumulation of KBU2046 and TB in tumors. In vivo, our results showed that TPD@TB/ KBU2046 nanoparticles not only possess excellent tumor targeting, but also had cooperative effects of anti-metastasis and anti-growth, which provided a promising strategy for the treatment of ovarian cancer. into ultrapure water and stirred for 24 h. Then, the reaction product was added to water solution and purified by dialysis (MWCO 3500) in flowing ultra-pure water for 48 h. After lyophilization, the product TPD was obtained with a yield of 73.4%. fluoroisoflavanone (KBU2046, 0.8 mg) were simultaneously dissolved in tetrahydrofuran (THF) solution (1 mL). Sonicator with a microtip probe was used to stir the aforesaid solution for 5 min at 10 W. The mixed solution was added into the water, whereafter, the organic solvent THF in the mixed solution was blown dry with nitrogen. The solution was then filtered via a 0.2 μm filter membrane to get targeted nanoparticles. Nano-ZS ZEN3690 (Malvern Instruments) was used to detect the particle size at 25 °C. Particle size was measured in PBS buffer.

Animal model
The experimental animal scheme strictly complied with the requirements of the experimental animal ethics committee of Tongji Medical College and conformed to the principles of animal protection, animal welfare, and ethics. BALB/c nude mice were purchased from HFK Bioscience Co. (Beijing, China). They were fed normally and provided unlimited access to water. Briefly, 1 ×10 6 SKOV-3 or EGFP-SKOV-3 cells were subcutaneously inoculated into the right anterior side of female BALB/c nude mice. Tumor growth was measured using a vernier caliper. The tumor volume was calculated as: volume = 0.5× ((tumor length) × (tumor width) × (tumor width)).

Anti-tumor effects of nanoparticles were studied using subcutaneous tumor model
When the volume of SKOV-3 tumor reached around 20 mm 3 , SKOV-3 tumor-bearing mice were randomly split into five groups. Mice were injected with TPD@TB/KBU2046 (2.0 mg/ml, 200 μL)), PD@TB/KBU2046 (2.0 mg/ml, 200 μL), TPD@TB (2.0 mg/ml, 200 μL), TPD@KBU2046 (2.0 mg/ml, 200 μL) and TPD (2.0 mg/ml, 200 μL) via tail vein, respectively. After 12 h, the nanoparticles were distributed into the tumor and then the tumor area was exposed to white light (200 mW cm -2 ) for 20 min. The weight and tumor volume of the mice were daily recorded. The tumors and major organs were collected for histological observation by standard hematoxylin & eosin and immunohistochemical staining.
Anti-tumor effects of nanoparticles were studied using orthotopic ovarian cancer model

Fabrication and characterization of TPD@TB/KBU2046
According to our previous report, we synthesized the AIE-photosensitizer TPA-BDTO (TB) ( Figure S1) [48]. The fluorescence of TB increased with the increase of concentration ( Figure S2A). At the same time, with the decrease of the THF/water ratio, TB gradually aggregated and its fluorescence increased ( Figure S2B). These results indicated that TB was an AIEgen. The small molecule inhibitor KBU2046 was synthesized according to the literature ( Figure S3) [12]. PEG-DSPE (PD) was linked with TMTP1 by amidation reaction to yield the TMTP1-PEG-DSPE (TPD) ( Figure S4/5) [49][50]. UV-vis characteristic absorption spectra of TPD, TB and KBU2046 were shown in Figure 1A. KBU2046 and TB had two characteristic absorption peaks at 267 nm and 530 nm, respectively. The TPD@TB/KBU2046 was obtained via a modified nano-precipitation approach using TPD to form nanoparticles [51]. The loading contents of TB and KBU2046 in TPD@TB/KBU2046 determined by UV-vis spectrophotometer were 24.20% and 21.94%, respectively. The loading contents of TB and KBU2046 in PD@TB/ KBU2046 were 26.21% and 24.47%, respectively. The loading content of TB in TPD@TB was 30.40% and the loading content of KBU2046 in TPD@KBU2046 was 22.06%. UV-vis absorption spectra of TPD@TB/ KBU2046, PD@TB/KBU2046, TPD@TB, TPD@KBU 2046 and TPD nanoparticles were exhibited in Figure  1B. The TPD@TB/KBU2046 showed the main absorption peaks at 267 and 530 nm, which was in connection with KBU2046 and TB. The TPD@TB/ KBU2046, PD@TB/KBU2046 and TPD@TB possessed a strong emission at 530 nm, while TPD@KBU2046 and TPD almost displayed no fluorescence ( Figure 1C, Figure S7). Transmission electron microscopy (TEM) image confirmed the morphology and particle size of the TPD@TB/KBU2046 ( Figure 1D). Importantly, the TPD@TB/KBU2046 in PBS solution displayed great stability within 24 h at room temperature ( Figure 1E). The ROS indicator ABDA was used to confirm the ROS generation capability of TPD@TB/KBU2046, whose absorption decreased at 378 nm when ROS generated upon white light irradiation ( Figure 1F). The intersystem crossing process between the lowest singlet excited state (S1) and triplet excited state (T1) of TB induced the ROS production. Comparing with the commercial photosensitizer (Rose Bengal), TPD@TB/KBU2046 possessed almost as good photosensitivity as it ( Figure S8A/B). These results indicated that we have successfully prepared the TPD@TB/KB2046 nanoparticles, which had great stability and photosensitivity.

Nanoparticles-mediated PDT inhibited the proliferation of SKOV-3 cells
Five nanoparticles (TPD@TB/KBU2046, PD@TB /KBU2046, TPD@TB, TPD@KBU2046 and TPD) were respectively incubated with SKOV-3 cells for 4 h. Strong red fluorescence (TB) can be observed in the SKOV-3 cells by CLSM after cultured with TPD@TB/KBU2046, PD@TB/KBU2046 and TPD@TB (Figure 2A). After incubation with DCFH-DA and irradiation with white light, green fluorescence (DCF) was observed in the SKOV-3 cells in TPD@TB/KBU2046, PD@TB/KBU2046 and TPD@TB groups, which indicated that when TB was loaded into nanoparticles, it could enter SKOV-3 cells and produce excessive ROS under white light irradiation (Figure 2A). Furthermore, the viability of the SKOV-3 cells was evaluated using propidium iodide (PI) after cultured with TPD@TB/KBU2046, PD@TB/KBU2046, TPD@TB, TPD@KBU2046 and TPD upon light irradiation ( Figure 2B). The numbers of the death cells in TPD@TB/KBU2046, PD@TB/KBU2046 and TPD@ TB groups were more than that in TPD@KBU2046 and TPD groups, indicating that PDT effects were produced in TPD@TB/KBU2046, PD@TB/KBU2046 and TPD@TB groups. Meanwhile, the SKOV-3 cells viability was also evaluated by CCK-8 assay ( Figure  2C). With the increase of the nanoparticle concentration in culture medium, the cell viability in TPD@TB/KBU2046, PD@TB/KBU2046 and TPD@TB groups decreased, which was in contrast with that in TPD@KBU2046 and TPD groups. The above results demonstrated that the TB loaded into nanoparticles can be used for image-guided PDT. To investigate the cytotoxic mechanism, we carried out western blotting to reveal the expression of proliferation factor proliferating cell nuclear antigen (PCNA) during the PDT process [52,53]. As shown in Figure 2D, the expression of PCNA in SKOV-3 cells decreased in TPD@TB/KBU2046, PD@TB/KBU2046 and TPD@TB groups, while it was strong in TPD@KBU2046 and TPD groups, which indicated that treating SKOV-3 cells with TB-loading nanoparticles could effectively reduce the expression of PCNA and inhibit cell proliferation under white light irradiation.

Anti-metastasis efficiency of the nanoparticles in vitro
The effects of different nanoparticles on the migration capability of SKOV-3 cells were examined by cell scratch test. As pointed in Figure 3A/B, the width of the scratches in these five groups was basically the same at the beginning of the scratch test. However, 36 h after incubation, the scratch width of TPD@TB/KBU2046, PD@TB/KBU2046 and TPD@ KBU2046 groups was significantly wider than that of the other two groups ( Figure 3A/C). Moreover, the metastatic potential of the SKOV-3 cells was assessed by transwell assay after incubated with different nanoparticles, which represented that the numbers of the metastatic cells in TPD@TB/KBU2046, PD@TB/ KBU2046 and TPD@KBU2046 were significantly less than that in the other two groups ( Figure 3D/E). The scratch test and transwell assay confirmed that SKOV-3 cells treated with nanoparticles containing small molecule inhibitor KBU2046 had lower migration and metastasis capability. Vimentin was related to the metastasis of the tumor cells, and the higher its expression, the stronger its metastasis capability [54]. Expression of Vimentin in SKOV-3 cells treated with TPD@TB/KBU2046, PD@TB/ KBU2046 and TPD@KBU2046 nanoparticles was lower than that in other groups ( Figure 3F). This result revealed that the small molecule inhibitor KBU2046-entrapped nanoparticles can inhibit tumor cell invasion and metastasis via Vimentin pathway.

Biodistribution and tumor targeting of nanoparticles in vivo
It was known that only the materials with hemolysis rate less than 5% can be applied to intravenous injection [55]. Hemolysis test was used to assess the hemocompatibility of nanoparticles ( Figure  4A). Negligible hemolysis happened in the five experimental groups as well as the phosphate buffered saline (negative control), however, lots of hemoglobin was observed in the ultrapure water (positive control). The hemolysis ratio of the five experimental groups was lower than 5%, which indicated that the nanoparticles were appropriate for intravenous injection. In vivo, the pharmacokinetics showed that TPD@TB/KBU2046 remained in the circulation for a relatively long time without non-specific scavenging, which was conducive to the enrichment of nanoparticles in tumors ( Figure 4B). In order to confirm the targeting effect of nanoparticles in vivo, the SKOV-3 cell line expressing green fluorescent protein (EGFP-SKOV-3) was used to establish a tumor-bearing mice model, and then the tumors were localized according to the spontaneous fluorescence ( Figure 4C To further demonstrate the tumor targeting of nanoparticles, EGFP-SKOV-3 tumor-bearing mice were treated with TPD@TB/KBU2046, PD@TB/ KBU2046, TPD@TB, TPD@KBU2046 and TPD, respectively. The biodistribution of EGFP and TB in the tumors and organs were detected by IVIS and was shown in Figure 4H/I. Due to the presence of TB in the nanoparticles, fluorescence was observed in the tumors and organs in TPD@TB/KBU2046, PD@TB/ KBU2046 and TPD@TB groups, but not in TPD@ KBU2046 and TPD groups. Further analysis showed that strong fluorescence signals were observed in tumors of TPD@TB/KBU2046 and TPD@TB groups, but weakness in the PD@TB/KBU2046 group (without TMTP1 peptide). These results suggested that nanoparticles with TMTP1 peptide exhibited excellent targeting efficacy in cancer therapy.

Cooperation therapy between anti-growth and anti-metastasis to inhibit tumor growth and metastasis in subcutaneous tumor model
In order to investigate the anti-tumor efficacy of the nanoparticles in vivo, we divided SKOV-3 tumor-bearing mice with tumor volume of approximately 20 mm 3 into five groups: (1) TPD@TB/KBU2046; (2) PD@TB/KBU2046; (3) TPD@ TB; (4) TPD@KBU2046; and (5) TPD. The mice in each group were exposed to white light. As shown in Figure 5A, the growth of the tumors in TPD@TB/ KBU2046, PD@TB/KBU2046 and TPD@TB groups was both suppressed compared with TPD group. The inhibition rate of TPD@TB/KBU2046 group was 72.3%, that of TPD@TB group was 65.09%, and that of PD@TB/KBU2046 group was 30.81%. These results suggested that tumor growth can be significantly inhibited when TB and TMTP1 were simultaneously contained in nanoparticles. H&E staining revealed that tumor tissues in TPD, PD@TB/KBU2046 and TPD@KBU2046 groups were compact, whereas tumors in TPD@TB/KBU2046 and TPD@TB groups were sparse, and a large number of necrotic tumor cells could be found ( Figure 5B). TUNEL staining exhibited abundant apoptotic tumor cells in TPD@TB/KBU2046 and TPD@TB groups, few apoptotic cells in PD@TB/KB2046 group, and almost no apoptosis in TPD and TPD@KBU2046 groups ( Figure 5C). These results indicated that nanoparticles containing TMTP1 and TB developed superb PDT efficacy, thereby causing increased apoptosis/ necrosis of tumor cells.
The anti-metastasis effect of nanoparticles was evaluated according to the tumor boundary and lymph node metastasis. The black arrow in Figure 5D showed a clear tumor boundary, suggesting that tumor cell in TPD@TB/KBU2046 and TPD@KBU2046 groups had a weaker invasive capability. Meanwhile, metastatic tumors (white arrow) appeared in the lymph nodes in the PD@TB/KBU2046, TPD@TB, and TPD groups, but not in the TPD@TB/KBU2046 and TPD@KBU2046 groups ( Figure 5E). These results consistently implied that the nanoparticles contained both TMTP1 and KBU2046 inhibited tumor invasion and metastasis.
Ki-67 was a marker that reflected the proliferative capability of tumor cells [56]. The numbers of Ki-67 positive cells in TPD@TB/KBU2046 and TPD@TB groups were significantly less than the other groups, indicating that targeted PDT significantly suppressed tumor cell proliferation ( Figure 5F). Vimentin expression in tumors was significantly correlated with invasion and metastases. Vimentin was highly expressed in tumor in PD@TB/KBU2046, TPD@TB, and TPD groups, but lowly expressed in TPD@TB/KBU2046 and TPD@KBU2046 groups ( Figure 5G). In conclusion, our results suggested that when the nanoparticles contained TMTP1, TB and KBU2046, can they achieve both anti-metastasis and anti-growth effects in vivo.
Low toxicity or even no toxicity of biological materials is a prerequisite for the further application [57]. The weight of mice did not decrease significantly, and there was no significant difference among the groups ( Figure 6A). Meanwhile, the weight of the organs such as heart, liver, spleen, lung and kidney of mice in different treatment groups had no significant difference ( Figure 6B). H&E staining of the organs in the different groups were also performed. As displayed in Figure 6C, there were no pathological changes in heart, liver, spleen, lung and kidney in each group after injected with different nanoparticles [58,59]. In conclusion, the above results showed that the nanoparticles had good biocompatibility in living mice and can be used for biomedical applications in vivo.

Cooperation therapy between anti-growth and anti-metastasis to reduce recurrence and metastasis after surgery in orthotopic ovarian tumor model
In order to test the universality of this strategy and further improve the therapeutic effect of PDT, we used an orthotopic ovarian tumor model, combined with surgical treatment to indirectly increase the penetration depth of light, thereby improving the curative effect of PDT. Before treatment, we found that TPD@TB/KBU2046 also had a good targeting effect on orthotopic ovarian tumor ( Figure 7B). 15 mice were divided into five groups: (1) TPD@TB/ KBU2046; (2) PD@TB/KBU2046; (3) TPD@TB; (4) TPD@KBU2046; and (5) TPD. All the mice were treated according to the procedure shown in Figure  7A. After treatment, all mice were executed and the distribution of EGFP-SKOV-3 tumors in the abdominal cavity was detected by IVIS. Recurrence and metastasis of ovarian cancer were shown by the yellow arrow ( Figure 7C). Recurrence and metastatic ovarian cancer can produce cancerous ascites, which reflect the condition of ovarian cancer to a certain extent [60]. Our statistical results showed that ascites formation was almost not detected in TPD@TB/ KBU2046 group (0.12 ±0.03 mL), but a large number of ascites were formed in PD@TB/KBU2046 (0.33 ±0.08 mL), TPD@TB (0.29 ±0.14 mL), TPD@KBU2046 (0.35 ±0.11 mL) and TPD groups (0.94 ± 0.19 mL), which meant that TPD@TB/KBU2046 group had the best therapeutic effect ( Figure 7D). The relative fluorescence intensity of recurrent and metastatic tumors in TPD@TB/KBU2046, PD@TB/KBU2046, TPD@TB, TPD@KBU2046 and TPD groups were 0.04 ±0.01, 0.5 ± 0.11, 0.49 ± 0.46, 0.48 ± 0.24, and 1.52 ±0.46, respectively ( Figure 7E). In conclusion, these results suggested that cooperative therapy between anti-metastasis by small molecule inhibitors and anti-growth by photodynamic-AIEgens could effectively inhibit the recurrence and metastasis of ovarian cancer after surgery. Irradiation time: 20 min. Data were presented as the mean ± SD (n =5). After treated with different nanoparticles, (B) the weight of heart, liver, spleen, lung and kidney was measured, and (C) the histological changes of organs were observed by H&E staining. Data were presented as the mean ± SD (n =5). Data were presented as the mean ± SD (n =3) and analyzed by two-sided Student's t-test. n.s. not significant, * P <0.05, ** P <0.01 *** P <0.001.

Conclusions
In conclusion, we proposed an effective selfassembled TMTP1-targeted nanoparticle entrapping AIEgen and small molecule inhibitor for imageguided PDT and anti-metastasis therapy. The targeting effect of TPD@TB/KBU2046 nanoparticles based on TMTP1 can increase the concentration of AIEgen (TB) and small molecule inhibitor (KBU2046) in the tumor site and reduce toxic effects toward normal tissues. The nanoparticles were demonstrated to exhibit superb ROS generation efficiency as well as excellent anti-metastasis capability. TPD@TB/ KBU2046 can achieve cooperative treatment of antimetastasis therapy and image-guided anti-growth for ovarian cancer both in vitro and in vivo. All in all, these findings implied that using photodynamic-AIEgens to compensate for the congenital defects of small molecule inhibitors may be a simple and efficient strategy.