Tumor-Microenvironment-Responsive Nanoconjugate for Synergistic

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Tumor Microenvironment-Responsive Nano-Conjugate for Synergistic Anti-Vascular Activity and Phototherapy Pingping Liang, Xiaoyu Huang, Ya Wang, Dapeng Chen, Changjin Ou, Qi Zhang, Jinjun Shao, Wei Huang, and Xiaochen Dong ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.8b06478 • Publication Date (Web): 16 Oct 2018 Downloaded from http://pubs.acs.org on October 17, 2018

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Tumor Microenvironment-Responsive Nano-Conjugate for Synergistic Anti-Vascular Activity and Phototherapy Pingping Liang,† Xiaoyu Huang,† Ya Wang,† Dapeng Chen,† Changjin Ou,† Qi Zhang,‡ Jinjun Shao,†* Wei Huang,§ Xiaochen Dong†* †Key

Laboratory of Flexible Electronics (KLOFE) & Institute of Advanced Materials (IAM),

Nanjing Tech University (NanjingTech), 30 South Puzhu Road, Nanjing 211800, China. E-mail: [email protected]; [email protected] ‡School

of Pharmaceutical Sciences, Nanjing Tech University (NanjingTech), 30 South Puzhu

Road, Nanjing 211800, China. Shaanxi Institute of Flexible Electronics (SIFE), Northwestern Polytechnical University (NPU),

§

127 West Youyi Road, Xi'an 710072, China

ACS Paragon Plus Environment

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ABSTRACT Insufficient oxygen supply (hypoxia), short half-life (< 40 ns) of singlet oxygen and upregulation of the heat shock proteins expression in solid tumor impede the photodynamic and photothermal therapeutic efficacy. Herein, a near-infrared carrier-free nano-conjugate DAA with synergistic anti-vascular activity and pH-responsive photodynamic/photothermal behavior was designed and synthesized to improve cancer treatment efficacy. Obtained by self-assembly approach, the biocompatible DAA nanoparticles (NPs) displayed amplifying pH-responsive photodynamic/photothermal performance in acidic tumor microenvironment (TME) due to the protonation of diethylaminophenyl (DEAP) units. Most important, the anti-vascular agent 5, 6dimethylxanthenone-4-acetic acid (DMXAA), targeting to the vascular endothelial growth factor (VEGF), can be smartly released from the pro-drug DAA via the ester bonds hydrolysis at the sub-acid endocytosis organelles in the endothelial cells, which can effectively destroy the vascular to prevent tumor proliferation and metastasis. Hence, DAA NPs can specifically target to vascular endothelial cells and the tumorous lysosomes with desired cellular damage property in vitro. Therefore, the tumors can be ablated completely with no recurrence and side effects in vivo, which implies that DAA NPs provide a promising approach for cancer treatment via synergistic anti-vascular activity and photodynamic/photothermal therapy. KEYWORDS: tumor microenvironment, targeted, anti-vascular activity, photodynamic therapy, photothermal therapy


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Compared with traditional cancer treatment methods, such as radiotherapy and chemotherapy, cancer photodynamic therapy (PDT) and photothermal therapy (PTT) exhibit great attention because of the advantages of high selectivity, non-invasiveness and no-resistance.1-4 However, some critical issues, such as insufficient oxygen supply (hypoxia),5-7 short half-life (< 40 ns) of singlet oxygen (1O2) with a quite low diffusion radius (< 20 nm), largely limit PDT efficacy.8,9 In addition, up-regulation of the heat shock proteins expression severely reduce the therapy efficiency and impedes PTT in clinical application.10-12 Thereby, other therapeutic modalities should be combined with PDT/PTT to optimize the therapeutic approaches and improve the anticancer efficiency. As known to all, the microenvironment characteristic of tumorous angiogenesis formation is a recognized hallmark of tumor proliferation or metastasis, and the maximum diameter of tumor can only grow to 1 mm in an avascular environment.13-15 Moreover, recently, passive tumor-targeted drug delivery system has developed as a promising method to selectively accumulate antineoplastic agent through enhanced permeability and retention (EPR) effect at tumor site.16-18 Although conceptually impressive, from intravenous injection site to the intra-tumor, the drug accumulation encounters biological barriers of huge diffusional hindrance from extracellular matrix (caused by interstitial fluid pressure), deep penetration into the tumor site and fast circulation in the blood stream.19-21 To overcome these obstacles, active-targeted drug-delivery approaches have been widely employed to transport macromolecules or drugs to cancer vasculature or cancer cells. The active-targeted anti-vascular strategy was appeared as a potential method for cancer treatment recent years.22-25 However, most anti-vascular agents exhibit a poor lipid solubility and membrane permeability, they should be conjugated with central structure via lipophilic group (such as ester or amide) for permeation into tumor tissue


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and hydrolytic release at tumor site to specifically kill the vascular endothelial cells. In this way, tumor proliferation and metastasis behaviour could be effectively inhibited.26-29 In addition, the characteristic hypoxia of tumor microenvironment (TME) can further stimulate the up-regulation of glycolysis, resulting tumorous acidity (pH 5.0-6.8),32-34 which is different from normal tissues (pH 7.4) and make pH-responsive materials present great potential to enhance cancer therapeutic efficiency.35-37 Based on the tumorous acidity of TME, recently, many synergistic nano-systems are developed by surfactant modification the therapeutic agents to improve the water solubility; however, these processes are always complicated.[13] Accordingly, self-assembly approach has attracted great attention to form carrier-free and TMEresponsive nanoparticles with narrow size distribution, superb bio-imaging and therapeutic efficacy.38-41 Diketopyrrolopyrrole (DPP) derivatives, near-infrared (NIR) absorbing dyes, have proved as hopeful photosensitizers (PSs) with excellent photo-stability, high molar extinction coefficient and

brilliant

reactive

oxygen

species

(ROS)

generation.38,42

For

example,

Furyl

diketopyrrolopyrrole (FDPP) exhibits a lower energy gap (caused by the intramolecular charge transfer and co-planarity), leading to a red-shift absorbance and a high fluorescence quantum yield compared with traditional phenyl-diketopyrrolopyrrole.43,44 Additionally, the flanked Nalkyl long side chains on the FDPP core can achieve suitable lipo-hydro partition coefficients (logP).45 Furthermore, it is reported that diethylaminophenyl (DEAP) unit could exhibit great promise as a pH-responsive group to amplify the PDT/PTT efficiency in the acidic TME (extracellular matrix pH 6.5-6.8, endosomes and lysosomes: pH 4.5-6.0).46 Moreover, 5, 6dimethylxanthenone-4-acetic acid (DMXAA), appeared as an active-targeting anti-vascular agent, could effectively destroy the vascular to prevent tumor proliferation and metastasis.


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However, DMXAA shows poor anti-tumor efficiency due to undesirable lipid solubility and membrane permeability when used alone. To successfully accumulate into tumor site and kill the vascular endothelial cells with deep drug-penetration and abundant hydrolytic release, DMXAA should be conjugated with some PSs to realize TME-responsive synergistic therapy. Herein, we rationally designed and synthesized a carrier-free nano-conjugate DAA for synergistic anti-vascular activity and pH-responsive PDT/PTT. Photophysical measurements indicated that the self-assembled DAA NPs (diameter 55 ± 2 nm) present a high molar extinction coefficient, pH-responsive intersystem crossing ability and non-radiative decay. With the stimulus of TME, the DMXAA and photosensitizer DPP-4 NPs could be released in situ from DAA NPs via the hydrolysis of ester bond. Then they could specifically target to vascular endothelial cells and sub-cellular lysosome in vitro. Accordingly, the anti-vascular activity of DMXAA as well as phototherapy ability of DPP-4 make the nano-conjugate DAA presents dramatically enhanced cancer treatment efficiency to ablate the tumor completely in vivo (Scheme 1).


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Scheme 1. Representation of DAA NPs formation along with the acidic hydrolysis of DMXAA. And the anti-vascular and pH-responsive cancer PDT/PTT of DAA NPs at tumor site.

RESULTS AND DISCUSSION Synthesis and characterization The synthesis route of nano-conjugate DAA is shown in Figure S1. First, DPP-1 reacted with 1,6-dibromohexane to afford DPP-2 under base condition, which was then brominated in the presence of N-bromosuccinimide (NBS) to obtain DPP-3. And DPP-4 was synthesized via the reaction of Pd-catalyzed Suzuki coupling between DPP-3 and N, N-diethyl-4-(tetramethyl-1,3,2dioxaborolan-2-yl)aniline. Finally, DAA was successfully obtained via reacting DPP-4 with


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DMXAA (5, 6-dimethylxantheonone-4-acetic acid) in 70% yield. 1H NMR,

13C

NMR, and

MALDI-TOF Mass spectroscopy were applied to characterize the structure and composition of all the intermediates and DAA (Figure S2-S7). Furthermore, the single crystal of DAA molecule was obtained via slow evaporation method from dichloromethane-ethanol solution. As indicated in Scheme 1, the conjugated backbone of DAA exhibited nearly coplanar conformation with the torsion angles of only +2.82o and +11.33o, respectively. DPP-2, DPP-3, DPP-4 and DAA are easily dissolved in organic solvents, such as DMF, DCM and THF. A photograph of their THF solution with a color change from yellow to blue is presented in Figure 1a. In addition, the aim product DAA showed almost the same absorption profiles with DPP-4 ranging from 500 nm to 700 nm. While the two typical characteristic absorbance peaks of DMXAA at 278 nm and 339 nm in the ultra-violet region further confirm the successful conjugation of DMXAA onto DPP-4 (Figure S8a). In order to prepare the hydrophilic DAA NPs for bio-application, the simple self-assembly approach was adopted.38 Figure 1b shows that the obtained DAA NPs can homogeneously disperse in phosphate buffer saline (PBS) compared with that of DAA (in PBS). According to the transmission electron microscope (TEM) image, the DAA NPs display a well-defined spherical morphology. And the average diameter measurement by dynamic light scattering (DLS) indicates that the DAA NPs exhibit an average value of 55 ± 2 nm with a narrow size distribution (Figure 1c). The small sized nano-conjugate can improve retention via targeting system into the tissue and result long-term blood circulation and high extravasation tendency through tumor vascular.24 Moreover, DAA NPs display a broad absorbance band (λmax: 670 nm), existing a 27 nm red-shift compared with DAA (Figure 1d), which owing to the conjugated backbone planarization as well as strong intramolecular and intermolecular π-π stacking of nanostructured


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DAA. In addition, the maximum emission of DAA NPs peaks at ~700 nm, the large spectral overlap leads to obvious self-inner filter effect, which will quench singlet and triplet excitons and increase photothermal conversion efficiency in certain degree. All these results demonstrate that the NIR-absorbing DAA NPs was an ideal candidate for targeted cancer phototherapy. pH-responsive behaviour and DMXAA release performance Under weakly acidic environment, H+ could bond to pH-responsive DEAP groups of DAA and made its HOMO energy level lower than that of the central chromophore, which could induce the fluorescence reduction as well as photodynamic and photothermal efficiency increase,47,

48

in

which, DAA can be gradually protonated to form DAA1H and DAA2H by continual addition of CF3COOH (TFA) (Figure 1g). As shown in Figure 1e, DAA exhibited a maximum characteristic absorbance peak at 642 nm. With the addition of TFA, the intensity of the characteristic peak decreased greatly and the change was not obvious between 390 nm to 500 nm. The change in intensity of purple colour absorption (400-460 nm) and red colour absorption (650-760 nm) makes the blue DAA solution (in THF) gradual turning into bluish purple and purple (Figure S9a) with two new peaks appeared at around 1000 and 1102 nm. These phenomena indicate DAA possess obvious pH response, which is advantage for the TME responsiveness. Moreover, Figure S9b also displays the degenerative fluorescence changes during the whole process under the 365 nm excitation, proving the sequential protonation progress of DAA as well. Furthermore, Figure S8b shows that the fluorescence (674 nm) of DAA in THF is significantly decreased with the addition of TFA, which further demonstrates the pH-responsive behaviour of DAA. Figure 1f shows that the absorbance of DAA NPs at pH 5.0 is weaker than that at pH 7.4 at the same concentration, which is in consistence with the result of the DAA upon acidification.


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Hence, according to Beer-Lambert law A = εcL, the molar extinction coefﬁcient (ε) of DAA NPs at 670 nm was calculated to be 1.91 × 104 M-1 cm-1 (Figure S8d-e), demonstrating its excellent light-absorbing ability. In addition, the fluorescence (698 nm) of DAA NPs at pH 7.4 is stronger than that at pH 5.0, confirming a pH-responsive fluorescent characteristic with declining radiation transition (Figure S8c).

Figure 1. (a, b) Photographs of DPP-2, DPP-3, DPP-4, DAA in THF, and DAA, DAA NPs in PBS. (c) TEM image of DAA NPs (PBS, pH 7.4). Inset: DLS size distribution of DAA NPs. (d) Normalized absorbance of DAA in THF and DAA NPs in PBS. (e) Absorbance of DAA in THF with adding different concentrations of TFA. (f) Absorbance spectra of DAA NPs in PBS (pH 7.4 and 5.0). (g) Proposed protonation mechanism of DAA caused by H+.


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To determine the 1O2 generation ability of DAA at different pH value, the absorbance intensity change of 3,4-diphenylisobenzofuran (DPBF, 1O2 probe) at 416 nm within 64 seconds was investigate systematically.49 As shown in Figure S8f-8h, it suggests that the 1O2 generation ability of DAA exhibits an obvious acidity dependence, and the efficiency for singlet oxygen generation was calculated to be 39.7% (12.5 mM TFA), which is higher than some literatures, such as aluminum phthalocyanine tetrasulfonate (38%) and DTDPPBr2 (16.2%).50,51 The 1O2 generation ability of DAA NPs (in PBS) was also measured via Singlet Oxygen Sensor Green (SOSG) indicator.52 As shown in Figure 2a-2c, the fluorescence intensity (531 nm) of SOSG mixed with DAA NPs at pH 5.0 presents a stronger 1O2 generation ability compared with that at pH 7.4, further indicating the acidity activated 1O2 generation of DAA NPs. Figure 2d displays the temperature changes of DAA NPs (in PBS, pH 7.4) under 660-nm laser (0.8 W/cm2) with its concentration increase (0, 10, 20, 40, 60, 80 and 100 μg/mL). It can be observed that the respective temperature increases is about 4, 9.5, 15, 17.5, 19, 22.6 and 27.4 °C, displaying an excellent photothermal effect and obvious concentration dependence of DAA NPs. Meanwhile, we observe that the temperature increase (24.9 °C, 10 min, 660-nm laser irradiation) of DAA NPs at pH 5.0 is higher than that pH 7.4 (22.6 °C), demonstrating an acidity enhanced photothermal effect of DAA NPs (Figure 2e). In addition, the temperature change of DAA NPs was similar with that of DPP-4 in same condition (Figure S8i), indicating the conjugation of DMXAA with DPP-4 almost no larger improvement on its photothermal conversion efficiency (PTC). And the PTC of DAA NPs (pH 7.4) was calculated to be 48.6% (Figure 2f-2g), which is significantly better than some reported photothermal agents.49, 53 Good light-stability of the photosensitizer is required for its biomedical application. After laser illumination, the UV-vis-NIR absorbance, TEM and DLS measurements don’t display obvious


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changes (Figure S9c-9d), indicating the superb light-stability of DAA NPs. Furthermore, after five alternate laser irradiation cycles (on: 5 min; off: 5 min), the temperature elevation of DAA NPs PBS dispersion does not exhibit obvious decrease, which further shows an excellent photothermal stability (Figure 2h). This superb photothermal effect and stability of DAA NPs make it can be a promising cancer phototherapeutic agent. In addition, the in situ release of DMXAA from DAA NPs in vitro is estimated by dialysis system at 37 °C.23,28 As shown in Figure 2i, the release of DMXAA from DAA NPs is promoted by the low pH value, which indicates the weak acidic conditions is favourable for the release of DMXAA due to the ester bonds hydrolysis. It also can be concluded that the acidic TME of endocytosis organelles is useful for triggering the release of DMXAA for synergistic antivascular activity and phototherapy in vivo.


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Figure 2. (a, b) Fluorescence spectra (at 531 nm) of SOSG (1.5 μg/mL) mixed with DAA NPs (80 μg/mL) at different pH (7.4 and 5.0) excited at 480 nm wavelength. (c) Fluorescence intensity increase curves of SOSG from (a) and (b). (d, e) Photothermal curves of DAA NPs (in PBS) at different concentrations and pH values (control: pH 7.4 and 5.0 PBS). (f) Photothermal curve of DAA NPs aqueous dispersion (2 mL, 80 μg/mL) during laser on and off (0.8 W/cm2, control: DI water). (g) Linear cooling time vs -ln(θ) acquired from (f). (h) Heating and cooling curves of DAA NPs in PBS (80 μg/mL) for five cycles (660-nm, 0.8 W/cm2). (i) In vitro DMXAA release kinetics from DAA NPs in PBS with different pH value.

In vitro examination Inspired by the superb synergistic PDT/PTT efficiency of DAA NPs, the toxicity was estimated by a MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay with HeLa cells in vitro. As demonstrated in Figure 3a, the cell viability significantly declined with dosedependence and caused a half maximal inhibitory concentration (IC50) of 9 μM under laser irradiation (660-nm, 0.8 W/cm2). Compared to the HeLa cells threated with DAA NPs without laser irradiation, the cell viability was approximate 94% even at a high concentration (18 μM), suggesting almost no dark cytotoxicity of DAA NPs. The superb biocompatibility, high phototoxicity and low dark-toxicity of DAA NPs make it a promising photosensitizer for cancer phototherapy.

Furthermore,

the

flow-cytometric

assay

with

Annexin

V-fluorescein

isothiocyanate (Annexin V-FITC) and Propidium Iodide (PI) staining was obtained to quantitatively determine the apoptosis caused by DAA NPs at different stages. As shown in Figure 3b, the cell apoptosis also showed a dose-dependence with considerable percentage of


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4.0, 28.7, 54.6 and 88.7%, suggesting the excellent phototherapeutic efficiency of DAA NPs for combined therapy. To further visualize the live (green) and dead (red) cells killed by DAA NPs under 660-nm laser irradiation, the HeLa cell was co-stained by calcein-AM/PI.54, 55 As a result, more apoptotic cells (Figure 3d, with DAA NPs) were observed compared with the control group (Figure 3c, without DAA NPs), implying DAA NPs having excellent photo-ablation ability to cancer cells. In addition, encouraged by the anti-vascular activity of DMXAA and its considerable release from DAA NPs, the destruction of existing vessels assay in vitro was carried out with Human Umbilical Vein Endothelial Cells (HUVECs).56,

57

As shown in Figure 3e, interestingly, with

DAA NPs incubating of the capillary-like tubes from HUVECs on BD Matrigel, the vascular length, tightness and intersections showed decreased tendency (right panel) compared with no DAA NPs group (middle panel), further confirming the abundant release of DMXAA from DAA NPs with desired anti-vascular activity.

Figure 3. (a, b) MTT assay and Flow cytometry analysis of HeLa cells (660-nm laser, 0.8


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W/cm2). (c, d) Fluorescence images (200×) of calcein-AM (green)/PI (red) co-staining HeLa cells incubated without and with DAA NPs (18 μM) for 24 h (37 °C, 0.8 W/cm2, 10 min). (e) Existing vessels destruction assay in vitro, left panel: representative vessel pictures (Bright-field) of HUVECs treated without DAA NPs. middle panel: calcein-AM staining of the existing vessels from the left panel; right panel: calcein-AM staining of the existing vessels treated with DAA NPs (pH 5.0, 18 μM, 10 h) (Scale bar: 100 μm).

Fluorescent living cell imaging for cellular uptake behaviour was captured by confocal microscope. Figure 4a shows that the HeLa cells treated with DAA NPs (pH 7.4) displayed stronger cytoplasmic fluorescence than that in pH 5.0, indicating the weakly acidic TME could decrease the radiation decay for the improvement of photodynamic and photothermal effect. Hence, using DCFH-DA (2',7'-Dichlorodihydrofluorescein diacetate) as ROS probe, the fluorescence intensity of DCF (oxidation product of DCFH-DA) in cytoplasm (treated with DAA NPs) at pH 5.0 is brighter than that of pH 7.4 (Figure 4b), demonstrating that DAA NPs possess the pH-responsive photodynamic performance in vitro. As exhibited in Figure 4c, the subcellular localization behaviour of DAA NPs was detected by Green Lyso-Tracker, ER-Tracker and MitoTracker.58 And the yellow colour, overlapping of red (DAA NPs) and green (Lyso-Tracker) fluorescence, could be clearly observed in the lysosomes, suggesting DAA NPs were mainly pumped across the lysosomal membrane from cytosol (pH 7.2) to lysosomes (pH 4.5-5.0). This phenomenon further confirms that the weak acidity in lysosomes can activate the endocytic DAA NPs to amplify the synergetic phototherapy efficiency.


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Figure 4. (a, b) Confocal fluorescence images of HeLa cells incubated with DAA NPs (9 μM) at pH 7.4 and 5.0 (DAA NPs and DCFH-DA excited at 633 nm and 488 nm, respectively). (c) Subcellular localization of DAA NPs at pH 7.4 in HeLa cells (Scale bar: 10 μm, trackers excited at 488 nm).

In vivo examination Inspired by the excellent in vitro performance of DAA NPs, a series of pre-experiments were carried out before the synergistic anti-vascular activity and phototherapy in vivo. Firstly, the scratch-wound healing assay were performed to detect the invasive ability of HeLa cells (incubation with DAA NPs of different concentrations).59 As presented in Figure 5a, the wound nearly healed after 24 h when the concentration of DAA NPs was 0 μM. Compared to the incubated groups (4.5, 9 and 18 μM), the cell migration ability significantly decreased with a dose-dependence, verifying the outstanding cancer cells ablation effect and the reduced crisis of cancer metastasis ability of DAA NPs. In addition, the fluorescence and photothermal imaging of DAA NPs were recorded on the HeLa tumor-bearing nude mice, respectively. Figure 5b shows


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that the fluorescence intensity increased gradually and reached the maximum at 12 h after the intravenous injection, then decreased due to the metabolism of DAA NPs, which offered the time window guidance for phototherapy in vivo. The fluorescence images of tumor and organs (heart, liver, spleen, lung and kidney) showed that the tumor presented the strongest fluorescence signal compared with other organs, which further indicates that the DAA NPs mainly accumulated into the tumor site through the EPR effect (Figure 5c). And the phenomenon of weak hepatic fluorescence comes from the fact that liver is the most important detoxifying organ, proving the perfect targeting and ideally metabolizable performance of DAA NPs in vivo. To further confirm the targeting performance, the photothermal imaging of the tumor by 660-nm laser (0.8 W/cm2) irradiation was carried out at 12 h post-injection. The tumor temperature steadily increased to 57.0 °C (ΔT = 20.8 °C) within 8 min (Figure 5d). On the contrary, little temperature change (ΔT = 3.7 °C) was detected in the control group in the same condition, revealing the excellent targeting and photothermal imaging to guide the cancer phototherapy of DAA NPs.


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Figure 5. (a) Scratch-wound healing assay: HeLa cells migration at different concentrations of DAA NPs. (b) Tumor fluorescence image of mice with DAA NPs i.v. injection. (c) Fluorescence images of organs from the sacrificed mice. (d) Thermal images of tumor-bearing mice with or without DAA NPs injection (100 μg/mL, 100 μL).

At last, the synergistic anti-vascular activity and pH-responsive PTT/PDT efficiency of DAA NPs was investigated systematically with Xenograft HeLa tumor-bearing nude mice. 24 mice


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were randomly separated into 4 groups (n = 6): (I) saline with laser irradiation as control group. (II) DAA NPs without laser irradiation. (III) DPP-4 NPs with laser irradiation. (IV) DAA NPs with laser irradiation as treatment group. Compared with group II (with anti-vascular activity of DMXAA released from DAA) and group III (with pH-responsive phototherapy of DPP-4), all tumors in IV group were completely ablated at third treatment (intravenous injection for every two days, 4 min laser irradiation). Then the scars fell off and there was no recurrence after one month. However, the tumor volumes of the mice in control group steadily expanded to ~3500 mm3 within 18 days (Figure 6a). The visually therapeutic results in different group was displayed in Figure 6c, suggesting the amplified synergistic cancer therapy ability of DAA NPs in vivo. The tumors from all groups were operated by CD34-immunohistochemical staining as well as H&E (Hematoxylin and Eosin) staining to explore the different treatment effects.47, 60 As shown in Figure 6d and 6e, the cells in group I arranged closely with no vessel damage and necrosis, proving good biocompatibility, low dark toxicity and side effects of DAA NPs in vivo. Compared with group II (slightly serious vessel destruction and little apoptosis) and group III (minor vessel damage and mild necrosis), the vessel length in group IV was shortest and obviously cell apoptosis or necrosis were observed, indicating the significant cancer ablation of DAA NPs caused by synergistic anti-vascular activity of DMXAA and phototherapy of photosensitizer DPP-4. In addition, no abnormalities in body weight during treatment (Figure 6b) and no inflammation of organ (heart, liver, spleen, lung and kidney) H&E staining were observed after treatment (Figure S10), further implying no side effects of DAA NPs.


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Figure 6. (a, b) Tumor volume and body weight recording (**P< 0.01). (c) Photographs of four group mice after treatment. (d) CD34 and (e) H&E tumor tissue staining photographs after the treatment from the four groups (200×, tumor from group IV is obtained after second treatment).

CONCLUSION In summary, a promising therapeutic agent DAA has been successfully synthesized by conjugating DEAP and DMXAA groups onto the furyl-diketopyrrolopyrrole (FDPP) core. And self-assembly approach was adopted to obtain the carrier-free DAA nanoparticles. In which, the


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pH-responsive DEAP moiety at sub-cellular lysosomes (pH 4.5-5.0) can be protonated in acidic tumor microenvironment to improve its photodynamic and photothermal efficiency. More importantly, DMXAA groups can be released via ester bonds hydrolysis at the sub-acid endocytosis organelles in the endothelial cells (pH 5.0-6.0) with deep drug accumulation to achieve anti-vascular activity, accordingly, inhibiting tumor proliferation and metastasis. In vitro, DAA NPs, with excellent biocompatibility, little dark toxicity but strong photo-toxicity, can induce a lysosomes and endothelial cells localization as well as high cell apoptosis percentage. Therefore, in vivo, the tumors can be ablated completely with no recurrence. Conclusively, this rationally designed nano-conjugate DAA NPs present a promising approach for precise and effective tumor treatment in clinic.

EXPERIMENTAL SECTION Synthesis of DPP-1 Potassium tert-butoxide (16.00 g), tert-amyl alcohol (100 mL) and dimethyl succinate (5.84 g,) were added in 4-cyanofuran (10.00 g) and tert-amyl alcohol (20 mL) at 110 °C under N2 atmosphere. Afterward, methanol (5 mL) was extracted from the mixture and the reaction was cooled to 65 °C. After two hours, methanol (150 mL) was added to terminate the reaction. Then, acetic acid (10 mL) was injected and stirred overnight at 65 oC. The crude product was washed with methyl alcohol and water to obtain DPP-1 (7.10 g, yield: 27%). 1H NMR (500 MHz, DMSO-d6) δ (ppm) 8.62 (d, J = 4.3 Hz, 2H), 8.58 (d, J = 4.2 Hz, 2H), 8.0 (t, J = 7.6 Hz, 2H), 7.24 (t, J = 7.2 Hz, 2H).

13C

NMR (126 MHz, DMSO-d6) δ 161.12, 146.76, 143.74, 131.24,

116.68, 113.56, 112.83, 107.57, 41.03, 37.82.


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Synthesis of DPP-2 DPP-1 (5.36 g), 1, 6-dibromide hexane (12.2 g) and potassium hydroxide (2.80 g) were dissolved in N, N-dimethyl formamide (150 mL) and stirred at 25 °C for 24 hours. Then solvent was removed, and crude product was purified by chromatography on silica column (PE/DCM, V/V = 1:6) to obtain DPP-2 (5.87 g, yield: 50%). 1H NMR (500 MHz, CDCl3) δ8.36 (d, J = 4.3 Hz, 2H), 7.71 (d, J = 1.8 Hz, 2H), 6.75 (d, J = 7.2 Hz, 2H), 4.46-3.95 (m, 4H), 3.45 (t, J = 7.1 Hz, 4H), 1.57 - 1.47 (m, 16H).

13C

NMR (126 MHz, CDCl3) δ 160.85, 144.92, 133.61, 120.21,

113.51, 106.44, 77.48, 75.95, 42.18, 33.73, 32.63, 29.98, 27.83, 25.97. Synthesis of DPP-3 DPP-2 (1.01 g) was dissolved in acetic acid (0.5 mL) and chloroform (50 mL). NBromosuccinimide (NBS) (0.66 g) was added under dark at 25 °C for 4 hours. Next, the solvent was removed and the residue was purified on silica column (PE/DCM, V/V = 1:5) to afford DPP-3 (1.11 g, yield: 87%). 1H NMR (500 MHz, CDCl3) δ 8.30 (d, J = 4.3 Hz, 2H), 6.69 (d, J = 4.2 Hz, 2H), 4.17 (t, J = 7.6 Hz, 4H), 3.45 (t, J = 7.2 Hz, 4H), 1.96-0.91 (m, 16H).

13C

NMR

(126 MHz, CDCl3) δ 160.51, 146.15, 132.49, 126.40, 122.23, 115.62, 106.32, 77.73, 42.30, 33.56, 32.84, 32.52, 32.04, 29.82, 27.78, 27.30, 26.01, 25.29. Synthesis of DPP-4 DPP-3 (0.80 g), N, N-diethyl,4-(tetramethyl-1,3,2-dioxaborolan-2-yl) aniline (0.65 g), tris(dibenzylideneacetone)dipalladium(0)

(48.7

mg),

tri-o-tolyl

phosphine

(32.4

mg),

tetrabutylammonium bromide (34.3 mg) and potassium carbonate (1.47 g) were dissolved in toluene (20 mL) and DI water (3 mL) at 90 °C under N2 atmosphere for 24 hours. Then solvent was removed, and product was purified on silica column (PE/DCM, V/V = 1:5) to achieve DPP4 (0.41 g, yield: 43%). 1H NMR (400 MHz, CDCl3) δ 8.38 (s, 2H), 7.56 (d, J = 8.7 Hz, 4H), 6.70


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(d, J = 8.9 Hz, 6H), 4.22 (t, J = 7.3 Hz, 4H), 3.44 - 3.36 (m, 12H), 1.89 - 1.82 (m, 8H), 1.51 (s, 8H), 1.21 (t, J = 7.0 Hz, 12H) (Figure S2).

13C

NMR (101 MHz, CDCl3) δ 148.18, 142.69,

131.73, 126.13, 123.08, 116.56, 111.54, 106.186, 44.496, 42.422, 33.87, 32.81, 30.202, 28.04, 26.330, 12.653 (Figure S3). MALDI-TOF MS: calculated for C46H56Br2N4O4 886.267; found: 887.602 (M+) (Figure S4). Synthesis of DAA DPP-4 (0.44 g), 5, 6-Dimethylxantheonone-4-acetic acid (0.31 g) and potassium carbonate (0.14 g) were dissolved in tetrahydrofuran (THF, 10 mL) and N, N-Dimethylformamide (DMF, 10 mL) at 90 oC for 24 hours. Solvent was removed, and product was purified by chromatography on silica column (DCM/EA, V/V = 1:5) and recrystallization to acquire DAA (0.45 g, yield: 70%). 1H

NMR (400 MHz, CDCl3) δ 8.35 (s, 2H), 8.21 (dd, J = 8.0, 1.5 Hz, 2H), 8.02 (d, J = 8.1 Hz,

2H), 7.58 - 7.55 (m, 2H), 7.51 (d, J = 8.7 Hz, 4H), 7.27 (d, J = 6.1 Hz, 1H), 7.24 (s, 1H), 7.12 (d, J = 8.1 Hz, 2H), 6.66 (d, J = 8.8 Hz, 6H), 4.12 (m, 8H), 3.92 (s, 4H), 3.37 (d, J = 6.8 Hz, 8H), 2.35 (d, J = 2.3 Hz, 12H), 1.75 (m, 4H), 1.64 (m, 3H), 1.60 (s, 1H), 1.50 - 1.42 (m, 4H), 1.41 1.34 (m, 4H), 1.18 (t, J = 7.0 Hz, 12H) (Figure S5). 13C NMR (101 MHz, CDCl3) δ 177.42, 170.73, 154.12, 153.95, 148.16, 144.48, 135.92, 131.635, 126.00, 125.09, 123.79, 123.35, 121.47, 119.53, 116.403, 111.46, 106.084, 65.36, 44.45, 42.29, 35.94, 30.28, 28.49, 26.81, 26.18, 20.66, 12.62, 11.51 (Figure S6). MALDI-TOF MS: calculated for C80H82N4O12 1290.593; found: 1289.674 (M+) (Figure S7). Preparation of DAA NPs DAA nanoparticles were obtained by self-assembly method. 1 mg DAA (in 500 μL THF) was added drop-wise into 5 mL PBS (pH 7.4, 6.5 or 5.0) at room temperature. During this procedure,


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the DAA self-assembled into NPs. After 5 min stirring, THF was removed by continuous nitrogen bubbling. Characterization and Morphology Analysis The morphology of DAA NPs was characterized by TEM. The dimensions of the NPs were determined by DLS measurement on Malvern Zetasizer (Nano-ZS, Malvern Instruments, Ltd., UK) instrument. The UV-Vis-NIR and fluorescence spectrum data were recorded by quartz cuvettes with optical path-length of 1 cm in wavelength range of 200-1300 nm and 520-900 nm. Singlet Oxygen Generation The singlet oxygen generations of DAA and DAA NPs were detected through monitoring oxidation of diphenylisobenzofuran (DPBF, 2 × 10-5 M, 64 s) and Singlet Oxygen Sensor Green (SOSG, 1.5 μg/mL, 20 min) at different pH. The absorbance of DPBF at 416 nm and fluorescence intensity of SOSG at 531 nm was recorded by UV-Vis-NIR and Fluorescence spectrophotometer, respectively. Measurement of Photothermal Performance Photothermal performance of DAA NPs was recorded by IR thermal camera and BMIR software to monitor temperature and thermal images as literatures.[61-63] PBS dispersion of DAA NPs was exposed to the laser (660-nm, 0.8 W/cm2) at different pH (7.4 and 5.0) and different concentrations (0, 10, 20, 40, 60, 80 and 100 μg/mL) for 10 min. Photo-stability of DAA NPs For evaluating photo-stability, UV-vis-NIR spectra and morphology of DAA NPs before and after five heating (laser on: 5 min) and cooling (laser off: 5 min) cycles under 660-nm laser irradiation were recorded. Photothermal Conversion (PTC) Efficiency


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DAA NPs aqueous dispersion irradiated with 660-nm laser (0.8 W/cm2) for 10 min. Then the laser was removed when temperature reached a plateau. And the temperature was monitored by IR thermal camera per 20 s for 20 min. DI water of the same volume was also tested as contrast. The follow equation was used to calculate the PTC efficiency: η = [hS(Tmax-Tsurr) - Q0]/[I(1 - 10-A660)]

(1)

(h represents the heat transfer coefficient; S represents the sample container surface area. Tmax represents the steady-state maximum temperature. Tsurr represents the ambient room temperature. Q0 represents the energy input by the same solvent without NPs in the same quartz cuvette after laser irradiation). θ was introduced to calculate the hS, which was defined as follow: θ = (T-Tsurr)/(Tmax-Tsurr)

(2)

hence, hS was calculated by the follow equation: τs = cdmd/hS

(3)

τs represents the characteristic thermal time constant; and the heat capacity cd of deionized water is about 4.2 J g-1 k-1, md represents the mass of the dispersion. Q0 was calculated using the following equation: Q0 = hS(Tmax - Tsurr)

(4)

In vitro Drug Release Firstly, DAA NPs (2 mL, 80 μg/mL) was transferred into a dialysis tube (MWCO = 500), they were incubated in PBS (60 mL, pH 7.4, 6.5 and 5.0) at 37 °C, respectively. Then ambient buffer solution (2 mL) was collected and replaced with fresh PBS (2 mL). The released percentage of DMXAA was determined by absorbance recording using UV-vis-NIR spectrophotometer at predetermined time intervals.


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Cell Culture and Incubation Conditions HeLa cancer cells were cultured in fresh DMEM (Dulbecco’s Modified Eagle’s Medium), containing 1% double resistant (streptomycin and penicillin) and 10% inactivated FBS at 37 °C under a humidified 5% CO2 and 95% air atmosphere. The cancer cells were split about every 3 days before they reached 90% confluence. In vitro Cell Cytotoxicity HeLa cells were seeded into two 96-well plates at a density of about 1 × 105 cells per well (the number of the replication well is five) and incubated in a humidified atmosphere for 24 hours. Then, the medium was replaced by the fresh complement medium with different concentrations of DAA NPs (0, 4.5, 9, 14, and 18 μM) for another 24 hours. Hereafter, one of the two plates was still kept in dark and the other was irradiated with a 660-nm laser (0.8 W /cm2). After 12 hours, the MTT solution (5 mg/mL, 20 µL) was added to each well for 4 hours. To dissolve the purple formazan, DMSO (150 μL per well) was added. And absorbance intensity was detected with a microplate reader at the O.D (optical densities) of 492 nm. Cell viability values were calculated by formula: Cell viability (%) = absorbance of experimental group/ absorbance of control group × 100%. Apoptosis Study by Flow Cytometry HeLa cells were seeded into a 12-well plate and divided into 4 groups. Control group (without DAA NPs) and three treatment groups (4.5, 9 and 18 μM DAA NPs) were all irradiated with 660-nm laser (0.8 W /cm2, 4 min). After incubation for 12 hours, the cells were stained with annexin VFITC and propidium iodide, measured by flow cytometer.


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AM/PI Staining AM/PI staining to trace the living/dead cells, the DAA NPs incubated HeLa cells in two groups (0 and 18 μM) were irradiated with 660-nm laser (0.8 W /cm2) and stained with mixture of AM/PI (AM: 2 μM, PI: 8 μM). Then fluorescence images were obtained by an inverted fluorescence microscope (Nikon, Japan) to observe the dead and living cells. (PI excited at 533 nm, AM excited at 465 nm). Tube Disruption Assay Thawy Matrigel Matrix (10 µL per well) was added into an angiogenesis slide on ice and incubated at 5% CO2, 37 °C to form gel. Human Umbilical Vein Endothelial Cells (HUVECs, 2 ×104 cells per well) were seeded on the gel in Endothelial Cell Growth Medium to generate complete network. Thereby, the medium was replaced with fresh medium containing 0 and 18 μM of DAA NPs PBS dispersion (pH 5.0) and incubating for another 4 hours in dark. After the PBS three times rewashing, Calcein-AM solution (50 µL, 6.25 µg mL-1) was used to dye in dark (30 min). Then micro-tube structure (10×) was photographed by an inverted fluorescence microscope (Nikon, Japan) with 465 nm excitation. In vitro Cellular Uptake of DAA NPs For cell imaging, HeLa cells were seeded into two confocal culture plates and incubated in 2 mL culture media for 24 hours. Hereafter, the medium was replaced by the fresh complement medium of 9 μM DAA NPs PBS dispersion (pH 7.4) for 24 hours in dark (name this method as A). Then the cells were incubated with paraformaldehyde (4%) for 20 min and rinsed with PBS. After staining with DAPI (4', 6-diamidino-2-phenylindole) for 3 min, the two plates were rinsed thrice with PBS (pH 7.4) then refilled with 1 mL PBS with pH 7.4 and 5.0. The fluorescence


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images (excited at 633 nm) were recorded by laser scanning up-conversion luminescence microscope equipped (Olympus IX 70). Intracellular ROS Assay Incubate HeLa cells as above method (A). After this, the cells were incubated in the DCFH-DA for 25 min, paraformaldehyde (4%) for 20 min and DAPI for 3 min in dark. After rinsing thrice with PBS (pH 7.4) and refilling with 1 mL PBS with pH 7.4 and 5.0, respectively, the two plate cells were radiated by 660-nm laser (0.8 W /cm2, 4 min). At last, cell fluorescence images were performed with the laser scanning up-conversion luminescence microscope equipped, too (DAA NPs excited at 633 nm, DCF excited at 488 nm). Sub-Cellular Localization Incubate HeLa cells as above method (A). Afterwards, Green Mito-Tracker, Lyso-Tracker and ER-Tracker dyeing liquid were added into the three culture plates, respectively. The cell images were also performed with the laser scanning up-conversion luminescence microscope equipped. (DAA NPs, Green Mito-Tracker, Lyso-Tracker and ER-Tracker were excited at 633 nm, 488nm, 488 nm and 488nm, respectively). The Scratch-wound Healing Assay HeLa Cells were seeded into a 6-well plate and adhered for 80% confluence with healing plugins to create denuded area. After cell attachment, the healing plug-ins was taken out and cells were washed with PBS then further incubated with different concentrations of DAA NPs for 100% confluence (except the denuded area). Subsequently, pictures were taken as initial contrast (0 h). Then, the cells were irradiated by 660-nm laser (0.8 W/cm2) and were taken by microscope. Animals and Tumor Model


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All mice (five weeks aged, 18-20 g weight) were obtained from Comparative Medicine Centre of Yangzhou University. Research Center for Laboratory Animals of Yangzhou University of Traditional Chinese Medicine (Yangzhou, China) approved all procedures. In addition, the experiment was performed in strict accordance with National Institutes of Health (NIH) guidelines for the care and use of laboratory animals (NIH Publication no. 85-23 Rev. 1985). To protect the human subjects, the investigators adhered to the policies of applicable law. In Vivo Fluorescence Imaging The mice were anaesthetized with 2 L/min oxygen flow with 2% isoflurane during intravenous injection of DAA NPs and imaging. And the contrast data at different time points (0, 2, 4, 6, 12 and 24 h) were obtained using the fluorescence imaging system with 660-nm excitation wavelength. After this, the mice were killed by cervical dislocation. Next, organs (tumor, heart, liver, kidneys, lung and spleen) were obtained and imaged on the same imaging system. In Vivo Antitumor Assay The HeLa tumor models were created by subcutaneous cells injection (4 × 106 cells in 200 µL PBS). All tumor-bearing nude mice were carefully arranged with free access to water and food in ventilated cages and randomly divided into four groups (n = 6, including Saline, DAA, DPP4+Laser, DAA+Laser) when the tumor volumes reached about 100-110 mm3. Antitumor assay was performed every two days. The tumor volumes (V = 0.5 ab2, a; b: the largest and smallest superficial diameter) and body weights were measured by caliper and digital scale. Histology Sample Preparation After treatment of 18 days, tumor-bearing nude mice were sacrificed to obtain the tumors and r organs (heart, liver, spleen, lung and kidney) for histological analysis by H&E (hematoxylin and


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eosin) staining. In addition, the density of tumor vascular was evaluated by anti-CD34 antibody to count CD34+ microvessel. Finally, the slices were observed with an optical microscopy.

ASSOCIATED CONTENT Supporting Information available: materials and characterization. This material is available free of charge via the Internet at http://pubs.acs.org. ACKNOWLEDGMENTS The work was supported by the National Natural Science Foundation of China (Xiaochen Dong, program no. 61525402, 61775095), Natural Science Foundation of Jiangsu Province (Jinjun Shao, program no. BK20161012), Jiangsu Provincial Graduated Training Innovation Project (Pingping Liang, program no. KYCX18_1120).

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For Table of Contents Only

A carrier-free nano-conjugate DAA for synergistic anti-vascular activity and pH-responsive cancer photodynamic/photothermal therapy was synthesized for dramatically enhanced anticancer performance. The biocompatible DAA nanoparticles display superb pH-responsive photodynamic/photothermal efficiency in acidic tumor microenvironment due to the protonation of diethylaminophenyl (DEAP) units. Most important, the anti-vascular agent 5, 6dimethylxanthenone-4-acetic acid (DMXAA), targeting to the vascular endothelial growth factor (VEGF), can be smartly released from DAA in the endothelial cells, which can effectively destroy the vascular to prevent tumor proliferation and metastasis in vivo.


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Tumor-Microenvironment-Responsive Nanoconjugate for Synergistic

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