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Volume 6 Number 3
21 January 2018 Pages 341-528
This is an Accepted Manuscript, which has been through the Royal Society of Chemistry peer review process and has been

Journal of
Materials Chemistry B
Materials for biology and medicine

ISSN 2050-750X

Wei Wei, Guanghui Ma et al.
Macrophage responses to the physical burden of cell-sized particles
accepted for publication.
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DOI: 10.1039/D0TB01692F

Tumor pH-Triggered “Charge Conversion” Nanocarriers with On-Demand Drug Release for Precise Cancer Therapy

Bo-Ai Ma †, ‡, Chun-Yang Sun †,*

† Department of Radiology and Tianjin Key Laboratory of Functional Imaging, Tianjin Medical University General Hospital, Tianjin 300052, P.R. China
‡ School of Food and Biological Engineering, Hefei University of Technology, Hefei, Anhui 230009, P.R. China
E-mail: [email protected] (C. Y. Sun)

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ABSTRACT DOI: 10.1039/D0TB01692F

Combined X-ray-induced photodynamic therapy (X-PDT) and chemotherapy are of great interest for tumor treatment, but their outcome is still hindered by insufficient drug delivery without tumor specificity and the difficulty of switching to chemotherapy during the X-PDT process. Herein, we report an efficient strategy for preparing a nanocarrier, DANPVP&DOX, with slight-acidity-induced charge conversion and hypoxia- motivated doxorubicin (DOX) release to achieve a more precise and synchronous therapeutic effect. Upon a change in the extracellular pH (pHe) in the tumor matrix, the surface charge of DANPVP&DOX converted from negative to positive via dimethyl maleate degradation. Following the increased internalization by tumoral cells, exposure of verteporfin (VP) in DANPVP&DOX to low-dose X-ray radiation resulted in O2 consumption in the cytoplasm to produce cytotoxic reactive oxygen species (ROS), which caused cell killing. Moreover, the hypoxic conditions formed in the tumor area
specifically promoted DANPVP&DOX dissociation and on-demand DOX release. Consequently, DANPVP&DOX significantly increased the therapeutic efficacy through X- PDT and cascade chemotherapy. More importantly, this strategy could potentially be extended to various therapeutic agents other than anticancer drugs for precise drug delivery and cancer treatment.

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1.INTRODUCTION DOI: 10.1039/D0TB01692F

Currently, chemotherapy remains the most common treatment, second to surgery, in clinical cancer therapy.1 In addition, photodynamic therapy (PDT), which generates cytotoxic reactive oxygen species (ROS) to cause irreversible cellular damage, has garnered great attention in terms of its noninvasiveness, safety, controlled phototoxicity and negligible drug resistance.2, 3 Because the penetration distance of visible light or near infrared light in biological tissues is short, PDT can only be used for treatment of superficial lesions in clinic. 4, 5 To further enhance PDT’s potency, some PDT agents were found to be effectively activated by deeply penetrating X-ray radiation, allowing X-PDT to overcome the tissue depth limitation of the conventional light-triggered PDT (∼1 cm).6-8 More importantly, the combination of chemotherapy and X-PDT would further improve the anticancer efficacy via unique therapeutic mechanisms.9, 10
Entering tumoral cells is necessary for both photosensitizers (PSs) and chemotherapeutics to function at the sites of action.11-13 Despite great efforts, co- delivery of PSs and chemotherapeutics using traditional delivery systems with anionic or neutral charge suffers from insufficient agent uptake, resulting in low therapeutic efficacy.14, 15 In contrast, it is well known that the positively charged surface on nanoparticles facilitates cellular internalization through enhanced interaction with the cell membrane. However, their applications are bottlenecked by tight binding to serum proteins and accelerated clearance from blood circulation in vivo.16, 17 Charge conversion systems may simultaneously enable delayed blood clearance and improved tumoral cell uptake in combination therapy.18-21 These systems could avoid nonspecific

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absorption to negative surfaces in the bloodstream, and promote DOI:intracellular10.1039/D0TB01692F endocytosis with tumor cell selectivity by positive surface charges stimulated by
specific physiological microenvironments in tumors.22-24 Among the various stimuli- responsive compounds, 2,3-dimethylmaleic anhydride has been widely used to design charge conversion systems because of the superior sensitivity of dimethyl maleate to the stable extracellular acidity (pHe ~6.8) in the tumor matrix.25, 26
Unlike X-PDT, which allows the PSs to work inside the nanoparticle, liberating the chemotherapeutics from the nanocarriers efficiently after entering the cells is essential for their interaction with targeted biosubstances.27-29 Moreover, increasing evidence has demonstrated that chemotherapeutic agent release should be coordinated with the X- PDT process to maximize the combined therapeutic effect. However, for delivery systems designed based on pH-, reduction-, or enzyme-stimulated release mechanisms, the time at which to initiate X-PDT is difficult to determine owing to the difficulty in real-time monitoring of peaks in the intracellular drug concentration.30-35 Accordingly, “on-demand” release triggered by X-PDT itself is believed to be the most feasible method to realize synchronous X-PDT and chemotherapy.36-40 Generally, the X-ray irradiation of PSs could lead to the consumption of high levels of O2 to generate ROS, creating a local hypoxic environment.41, 42 Fortunately, systems that respond to either abundant ROS or hypoxia have been proposed, with their development involving the exploitation of thioketal, phenylboronic ester or nitroimidazole (NI) moieties.39, 43, 44 Considering the advantages of the X-ray radiation, such as high repeatability, deep penetrability and spatiotemporal controllability, using hypoxia-activated nanocarriers

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in the combination of X-PDT and chemotherapy is highly potential for DOI:locoregional10.1039/D0TB01692F and efficient cancer cell killing.45, 46
In light of the abovementioned findings, a delivery system integrated with both charge conversion and hypoxia-responsive drug release could be promising for more precise and advanced anticancer therapy. In this study, we report nanocarriers capable of pHe-facilitated tumoral cell internalization and hypoxia-facilitated drug release. The micellar nanocarriers were self-assembled from diblock poly(ethylene glycol)- polyphosphoesters (PEG-b-P(AEP-g-DA/NI)) containing both 3,4-dimethoxy-N- methylamphetamine (DMMA) and NI moieties in the side group, photosensitizer VP and chemotherapeutic DOX (Scheme 1), hereinafter referred to as DANPVP&DOX. DANPVP&DOX, with a negative charge, was stable in the blood circulation and passively accumulated in tumor tissues through the enhanced permeability and retention (EPR) effect. Following extravasation of DANPVP&DOX from tumor vessels, slight localized acidity could cause increased tumoral cell uptake via acidity-sensitive DMMA degradation and subsequent charge switching. Finally, X-ray radiation to VP not only produced ROS to induce cell killing but also generated hypoxic conditions.47, 48 Under acute hypoxic conditions, hydrophobic NI was selectively converted into a hydrophilic aminoimidazole (AI) moiety with the help of NADPH, thus leading to the disassembly of DANPVP&DOX for cytoplasmic release of the DOX payload. Therefore, tumor cell apoptosis can be realized by cascade-amplifying therapeutic effects. Excitingly, the effectiveness of DANPVP&DOX plus X-ray radiation was verified in both in vitro cellular- level and in vivo animal-level studies.

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DOI: 10.1039/D0TB01692F

Scheme 1. Schematic illustration of DANPVP&DOX for precise drug delivery by cascade pHe-facilitated cellular internalization and hypoxia-activated drug release. The negative surface charge of DANPVP&DOX prolonged its blood circulation after systemic injection, and the charge switching by extracellular acidity lead to increased tumoral cell uptake. Next, the X-ray radiation of VP efficiently produced ROS and hypoxic conditions to dissociate DANPVP&DOX for cytoplasmic DOX release.


2.1Animals. BALB/c nude mice (female, 6 weeks old) and BALB/c mice (female, 6 weeks old) were purchased from Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). All in vivo experiments were performed according to Tianjin

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Medical University Guidelines for Animal Research, and were approved byDOI:Tianjin10.1039/D0TB01692F Medical University Animal Care and Use Committee.
2.2Synthesis of diblock PEG-b-P(AEP-g-DMA/NI). The diblock copolymer of PEG-b-PAEP was synthesized by ring-opening polymerization via previously reported method. Then, PEG-b-PAEP grafted with cysteamine hydrochloride (Cya) and (2-nitro- 1H-imidazol-1-yl) methanethiol (NI-SH) (denoted as PEG-b-P(AEP-g-Cya/NI)) was obtained by thoil-ene “click” reaction. The procedure was conducted as follow: in a quartz flask, NI-SH (71.6 mg, 0.45 mmol), cysteamine hydrochloride (51.1 mg, 0.44 mmol), 2,2-dimethoxy-2-phenyl acetophenone (DMPA, 7.1 mg) were mixed in PEG- b-PAEP solution (143.5 mg, in 8 mL THF), followed by purging with N2 for 15 min. Then the mixture was stirred at 25 °C under ultraviolet light (365 nm) irradiation. After 1 h, the reaction was stopped and the solution was further stirred overnight. The product was obtained by dialysis (MWCO 3500 Da) against ddH2O at 4 °C and lyophilization.
PEG-b-P(AEP-g-Cya/NI) (0.22 g), triethylamine (40 μL), pyridine (40 μL) and 2,3- dimethylmaleic anhydride (35 mg) were mixed in 5 mL of MDSO under N2 atmosphere. The mixture was kept at 25 °C for 36 h and the product PEG-b-P(AEP-g-DMA/NI) was obtained followed by precipitation in diethyl ether, filtration and lyophilization. To prepare a control polymer, succinic anhydride was grafted to PEG-b-P(AEP-g-Cya/NI) by a similar procedure (denoted as PEG-b-P(AEP-g-SA/NI)).
2.3Preparation of Drug-Loaded Nanoparticles. The VP&DOX loaded- nanoparticles were fabricated using a nanoprecipitation method. Briefly, PEG-b- P(AEP-g-DMA/NI) (10.0 mg), DOX(1.0 mg) and VP (1.0 mg) were dissolved in 2 mL

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of DMSO, and then dropwisely added into 10 mL of ddH2O under stirring. AfterDOI:further10.1039/D0TB01692F stirring for 30 min, the solution was dialyzed against ddH2O to remove DSMO dialysis
tubing (MWCO 3500 Da). The free VP and DOX after encapsulation were removed by centrifugation at 1500 rpm. The obtained nanoparticles were denoted as DANPVP&DOX, and the VP and DOX were loaded into PEG-b-P(AEP-g-SA/NI)-based nanoparticle to prepare the control group, which was denoted as SANPVP&DOX.
2.4ROS Generation in Vitro. 1O2 generation of VP-loaded nanoparticles was measured by singlet oxygen green sensor (SOSG) based on the reference.8 200 μL of free VP, DANPVP&DOX or SANPVP&DOX was mixed with SOSG (final concentration is 5 μM), respectively. Following the X-ray radiation, the SOSG emission fluorescence at 525 nm were recorded by H-7000 fluorescence spectrophotometer (Hitachi, Japan, Ex = 488 nm) to reflect 1O2 production.
2.5DOX Release in Vitro under X-Ray Activation. To measure the DOX release after X-ray irradiation, SANPVP&DOX or DANPVP&DOX was suspended in phosphate buffer (PB, 20 mM, pH 7.4). The solution was irradiated with X-ray at different dose and then transferred into the dialysis tubing (MWCO 14000 Da), which was immersed in PB at 37 °C with gentle shaking (60 rpm). At predetermined intervals, the external PB buffer was collected and replaced by the fresh buffer. The DOX content in collected PB was analyzed by high performance liquid chromatography (HPLC).
2.6Cellular Uptake of Nanoparticles. MDA-MB-231 cells were seeded in 24-well plates at a density of 5 × 104 cells per well and incubated for 12 h. Then, cells were treated with DMEM medium containing DANPVP&DOX or SANPVP&DOX, which was

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pretreated at pH 6.8 or 7.4 for 2 h, respectively. The cells were further incubatedDOI: for 410.1039/D0TB01692F h, and then washed twice with cold PBS. The cellular internalization of DANPVP&DOX or SANPVP&DOX was measured by flow cytometry and HPLC according to previously
reported method.

For the confocal laser scanning microscope (CLSM) observation, MDA-MB-231 cells were seeded onto 12 mm coverslips at 20000 cell density and incubated with DMEM medium containing SANPVP&DOX or DANPVP&DOX, which was pretreated at pH 6.8 or 7.4 for 2 h, respectively. Following another 4 h of incubation, the cells were washed with PBS and fixed with 4% paraformaldehyde, then stained with Alexa Fluor 488 phalloidin (Invitrogen, Carlsbad, CA) and 4ʹ,6-diamidino-2-phenylindole (DAPI) sequentially following the standard protocol before imaging on Zeiss LSM 810 microscope.
2.7In Vitro Cytotoxicity. MDA-MB-231 cells were seeded into 96-well plates (5000 cell density) and incubated overnight at 37 °C. Next, the medium was replaced with fresh DMEM medium containing DANPVP&DOX or SANPVP&DOX at different DOX concentrations, which was pretreated at pH 6.8 or 7.4 for 2 h. After further incubation for 4 h, the cells were washed with PBS and exposed with X-ray (4 Gy) for 10 min. Thereafter, the cells were further incubated for 48 h at 37 °C, and the cell viabilities was measured by a standard 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay using a Bio-Rad 680 microplate reader.
MDA-MB-231 cells were seeded in the 24-well plates (50000 cell density) and incubated with DOX, DANPVP&DOX or SANPVP&DOX ([DOX] = 2 μg/mL), which was

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pretreated at pH 6.8 or 7.4 for 2 h. After further incubation for 4 h, the cells wereDOI:washed10.1039/D0TB01692F with PBS and exposed with X-ray (4 Gy) for 10 min. The cells were further incubated
for 48 h, collected and analyzed using an Annexin V-FITC apoptosis detection kit I (BD Biosciences) according to the manufacture’s procedure.
2.8Pharmacokinetic and Biodistribution In Vivo. Female BALB/c mice were divided into 3 groups (n = 4) and received i.v. injection with DOX, DANPVP&DOX or SANPVP&DOX ([DOX] = 10 mg/kg), respectively. After 0.25, 0.5, 1, 2, 4, 8, 12, 24 or 48 h, 100 μL of blood samples were collected from the retroorbital plexus of the mouse eye and the concentration of DOX in plasma was analyzed followed by a published method.23
To evaluate the biodistribution of DOX-loaded nanoparticles, BALB/c nude mice bearing MDA-MB-231 xenografts were received i.v. injection with DOX, DANPVP&DOX or SANPVP&DOX ([DOX] = 10 mg/kg), respectively. At 12, 24 and 48 h post-injection, the mice were sacrificed, and the solid tumors and major organs were harvested, washed with PBS. The DOX concentration in tumors and other organs were detected using HPLC.
2.9Tumor Growth Inhibition in Vivo. The mice bearing MDA-MB-231 xenograft were divided randomly into six groups (n = 5) and received an intravenous injection every 5 days of PBS, VP&DOX ([DOX] = 2.5 mg/kg), DANPVP&DOX or SANPVP&DOX ([DOX] = 2.5 mg/kg) when the tumor volume was about 100 mm3. After 24 h post- administration, the tumor region was irradiated with X-ray (4 Gy) for 10 min. The Tumor size was monitored and calculated by following formula: tumor volume = 0.5 ×

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length × width2. After the last day, mice were euthanized and tumor tissuesDOI: were10.1039/D0TB01692F collected for the terminal transferase dUTP nick-end labeling (TUNEL) analysis.
2.10Biosafety Evaluation. BALB/c mice were divided into six groups (n = 5) and treated with various formulation every day. On the 4th day, the blood sample were collected for enzyme-linked immunosorbent assay and hematologic toxicity analysis according to the manufacturer’s instructions. After euthanizing the mice, major organs were fixed in 4% paraformaldehyde overnight and embedded in paraffin. The paraffin- embedded organ slices were stained with H&E and observed by Nikon TE2000U optical microscope.
3.Results and Discussion

3.1Preparation and Characterization of TKHCENPDOX.

In our design, both pHe-triggered charge switching and hypoxia-triggered on- demand release could be achieved by functional ligands located on PEG-b-P(AEP-g- DA/NI) pendant groups. The synthesis of PEG-b-P(AEP-g-DA/NI) is shown in Figure S1. First, the diblock copolymer PEG-b-PAEP was obtained by ring-opening polymerization using 1.5.7-triazabicyclo-[4.4.0]dec-5-ene as the organic catalyst. According to the 1H NMR spectrum in Figure S2, the chemical structure of PEG-b- PAEP was verified, and the degree of polymerization of AEP was determined to be 57. Next, NI-SH and cysteamine hydrochloride were attached to the allyl groups of PAEP to obtain PEG-b-P(AEP-g-Cya/NI) under 365 nm irradiation through thiol-ene “click” chemistry. In the 1H NMR spectrum, the resonance peak corresponding to the allyl groups disappeared completely, and the modification efficacy of Cya was calculated as

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54.4% based on the ratio of the integrated area of peak h at 2.79 ppm (h) and DOI:peak e at10.1039/D0TB01692F 1.94 ppm (e, Figure S3). Finally, some of the amino groups on PEG-b-P(AEP-g-Cya/NI)
were converted to carboxyl groups via 2,3-dimethylmaleic anhydride conjugation. To ensure a negative surface potential of DANP, nearly 60% of the amino groups were consumed during the reaction, as determined by 1H NMR analysis (Figure S4). On the other hand, the non-pHe-responsive succinic anhydride was used to prepare insensitive PEG-b-P(AEP-g-SA/NI) as the control copolymer (Figure S5). Conjugation of hydrophobic NI groups to hydrophilic PEG-b-PAEP resulted in PEG-b-P(AEP-g- DA/NI) or PEG-b-P(AEP-g-SA/NI), which were amphiphilic. The critical micelle concentrations of both PEG-b-P(AEP-g-DA/NI) and PEG-b-P(AEP-g-SA/NI) were determined to be 0.66×10-3 mg/mL and 1.29×10-3 mg/mL, respectively (Figure S6).
Thereafter, the amphiphilic PEG-b-P(AEP-g-SA/NI) and PEG-b-P(AEP-g-DA/NI) formed drug-loaded nanoparticles in aqueous solution via nanoprecipitation and were denoted as SANPVP&DOX and DANPVP&DOX, respectively. As shown in Figure 1A and 1B, the diameter of both micellar nanoparticles measured by dynamic light scattering (DLS) and transmission electron microscope (TEM) was ~120 nm (detailed value was shown in Table S1). Due to the PEG and polyphosphoesters outside the shell, SANPVP&DOX and DANPVP&DOX maintained their sizes for over 168 h (Figure 1C) in phosphate-buffered saline (PBS). Furthermore, the UV-vis absorption spectra of DANPVP&DOX and SANPVP&DOX in Figure 1D exhibited characteristic absorption bands at approximately 690 nm and 470 nm, which correspond to those of free VP and DOX, respectively. According to the UV-vis spectra, the DOX and VP drug loading contents (DLCs) of

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DANPVP&DOX were calculated as 3.71% and 4.29%, respectively, whichDOI: were10.1039/D0TB01692F

comparable to those of SANPVP&DOX (Table S1).

Figure 1. Diameter and morphology of DANPVP&DOX (A) and SANPVP&DOX (B). The scale bar is 200 nm. (C) Change of hydrodynamic size in PBS solution (pH 7.4). (D) The UV-Vis absorption spectra of free VP, free DOX, DANPVP&DOX and SANPVP&DOX.
Because dimethyl maleate has been shown to gradually hydrolyze in response to slight acidity, DANPVP&DOX was expected to undergo negative to positive charge conversion in the extracellular tumor environment. From the results shown in Figure 2A, a dramatic change in the zeta potential of DANPVP&DOX from -16.7 mV to +8.5 mV in 40 min could be detected at pH 6.8. Although the zeta potential of DANPVP&DOX slowly increased to –10.4 mV when incubated at pH 7.4, it remained negative for 120 min. Meanwhile, the zeta potential of SANPVP&DOX was stably maintained at approximately -22.7 mV, indicating that tumor pH stimulates the degradation of only

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DANPVP&DOX. In protein absorption experiments (Figure 2B), we found thatDOI:DANP10.1039/D0TB01692F incubated at pH 6.8 significantly interacted with bovine serum albumin and fibrinogen. Conversely, both DANP and SANP showed minimal protein adsorption under physiological pH conditions. These results are in good agreement with the pH-induced
charge conversion of DANP and indicate that the blood clearance of DANP and SANP would be decreased when their zeta potentials are negative.

Figure 2. (A) Zeta potential change of DANP and SANP after incubation at pH 7.4 or 6.8 for different time periods. (B) Fibrinogen (FBG) and bovine serum albumin (BSA) dsorption on the nanoparticles at 37 °C, pH 7.4 or pH 6.8. (C) DOX release profiles from DANPVP&DOX following X-ray exposure at different doses. (D) X-ray-stimulated pulsed release of DOX from DANPVP&DOX. The samples were exposed with X-ray for 5 min at different time points indicated by the arrows.

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DOI: 10.1039/D0TB01692F

As expected, DANPVP&DOX would undergo PDT effect with X-ray irradiation, and the generated hypoxic environment would reduce NI to AI to facilitate nanoparticle dissociation and subsequent DOX release. To examine the X-ray-triggered 1O2 production of VP-loaded nanoparticles, SOSG was used as an indicator and its enhancement of fluorescence intensity was monitored. As shown in Figure S7, the emission fluorescence at 525 nm of SOSG in the SANPVP&DOX and DANPVP&DOX solutions increased by 9.98- and 9.60-folds, respectively, within 2 min. Moreover, the SOSG fluorescence elevation nearly disappeared after adding vitamin C as an ROS scavenger, demonstrating that 1O2 was indeed produced by encapsulated VP.
Next, in vitro oxygen consumption and hypoxia generation mediated by VP combined with X-ray radiation were evaluated using an O2-sensitive phosphorescent probe. Notably, both DANPVP&DOX and SANPVP&DOX consumed O2 faster with the combined treatment, accompanied by an increase in radiation time (Figure S8). Furthermore, the hypoxia-motivated conversion of the hydrophobic NI to hydrophilic AI moiety on the side groups of the PAEP segment was investigated by UV-vis spectroscopy. As shown in Figure S9, the absorption peak of DANP moved from 332 nm to 268 nm under hypoxic conditions, suggesting the transition of NI to AI via six electron shift with the assistance of NADPH.39, 49-51 Since the amphiphilic copolymers were converted to hydrophilic, the hydrodynamic diameter of DANPVP&DOX and SANPVP&DOX decreased to ~20 nm after X-ray exposure (Figure S10), indicating hypoxia-stimulated nanoparticle disassembly.

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Moreover, the profile of DOX release from DANPVP&DOX and SANPVP&DOXDOI:under10.1039/D0TB01692F irradiation or in the dark was analyzed using HPLC. As shown in Figure 2C, DANPVP&DOX exhibited rapid DOX release within the first 4 h, and a power density- dependent release behavior over 24 h was observed. The X-ray irradiation at 1.0, 2.0
and 4.0 Gy resulted in 21.65±2.76, 50.41±2.12 and 72.39±2.05% loaded DOX release, respectively, which was significantly higher than that from DANPVP&DOX in the dark. In addition, the DOX liberated from DANPVP&DOX presented a pulsatile and controlled pattern following the X-ray on/off cycle (Figure 2D). Meanwhile, SANPVP&DOX exhibited comparable DOX release profiles under the same conditions (Figure S11), suggesting that neither DMMA nor SA modification would hinder drug release. Collectively, these results demonstrated that external X-ray irradiation can accelerate drug release by generating hypoxia and triggering NI group reduction.
3.2Cellular Internalization of DANPVP&DOX in Vitro

To study whether the charge reversal of DANPVP&DOX can increase its cellular uptake in a weakly acidic environment, MDA-MB-231 cells were incubated for 4 h with DANPVP&DOX or SANPVP&DOX pretreated at pH 7.4 or 6.8, respectively. After incubation, the intracellular DOX content was determined by flow cytometry and HPLC, respectively. As displayed in Figure 3A, DANPVP&DOX or SANPVP&DOX pretreated at pH 7.4 showed comparable cellular uptake since their surface charge remained negative under neutral conditions. However, cell uptake of DANPVP&DOX pretreated at pH 6.8 was significantly more efficient than that of SANPVP&DOX pretreated at pH 6.8, indicating that the positive zeta potential overcomes the original limitation on the interaction

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between cells and DANPVP&DOX. Moreover, DANPVP&DOX or SANPVP&DOX was DOI:pretreated10.1039/D0TB01692F at pH 6.8 or 7.4 for 2 h and then co-incubated with MDA-MB-231 cells. After a 4 h incubation, the intracellular VP content in MDA-MB-231 cells quantitatively determined by HPLC was 3.92±0.34 μg per mg protein for DANPVP&DOX at pH 6.8,
which was ~2.11 and ~2.25 times higher than those of DANPVP&DOX and SANPVP&DOX at pH 7.4 (Figure 3B).

Figure 3. (A) The intracellular DOX fluorescence in MDA-MB-231 cells after incubation with SANPVP&DOX or DANPVP&DOX at pH 6.8 or 7.4. (B) Quantitative analyses of the VP content in MDA-MB-231 cells by HPLC after incubation with VP, SANPVP&DOX or DANPVP&DOX for 4 h. *p<0.05.

On the other hand, the enhanced cellular internalization at pH 6.8 was further visualized by CLSM observation. As shown in Figure 4, the cellular uptake of SANPVP&DOX was unaffected by the pH, while much stronger red fluorescence was clearly detected in the cytoplasm of cells treated with DANPVP&DOX at pH 6.8 than in cells treated with DANPVP&DOX at pH 7.4. These results are in good agreement with previous reports, suggesting that weak acidity substantially promotes the cellular

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uptake of DANPVP&DOX by changing its surface potential.52, 53 DOI: 10.1039/D0TB01692F

Figure 4. Cellular uptake and subcellular distribution of SANPVP&DOX or DANPVP&DOX under different pH conditions. DAPI (blue) and Alexa Fluor 488 phalloidin (green) were used to stain cell nuclei and F-actin, respectively. The scale bar is 50 μm.
3.3Intracellular DOX Release and Cytotoxicity of DANPVP&DOX In Vitro.

We next investigated the intracellular DOX release triggered by PDT-induced hypoxic environment. Using hypoxia detection probes with X-ray irradiation, the formed local hypoxic microenvironment during PDT process in MDA-MB-231 cells was shown in Figure 5A. In comparison with other groups, the treatment of

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DANPVP&DOX+X-ray at pH 6.8 led to the strongest fluorescence signal of DOI:hypoxia10.1039/D0TB01692F generation, which could be explained by charge conversion-facilitated VP internalization within tumor cells. Subsequently, MDA-MB-231 cells were incubated
with DANPVP&DOX or SANPVP&DOX pretreated at pH 7.4 or 6.8 for 2 h, and then irradiated with X-ray (4 Gy) for 10 min. The cells were further incubated with fresh DMEM medium for 2 h, followed by the observation via CLSM (Figure 5B). Owning to most conspicuous hypoxia formation by DANPVP&DOX+X-ray at pHe, the DANPVP&DOX underwent rapid dissociation and subsequently released a large amount of DOX that specifically transferred from cytoplasm into the nuclei.

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DOI: 10.1039/D0TB01692F

Figure 5. (A) Confocal images of MDA-MB-231 cells with hypoxia detection probes with X-ray radiation. The scale bar is 20 μm. (B) Intracellular delivery of SANPVP&DOX and DANPVP&DOX on MDA-MB-231 cells with X-ray radiation. The scale bar is 20 μm. (C) Relative MDA-MB-231 cell viabilities after incubation with DANPVP&DOX or SANPVP&DOX with or without X-ray radiation. The DANPVP&DOX or SANPVP&DOX were pre-cultured at different pH for 2 h. *p < 0.05. (D) MDA-MB-231 cell apoptosis induced by DANPVP&DOX or SANPVP&DOX at pH 6.8 or 7.4 ([DOX] = 2.0 μg/mL).

Because DOX works with DNA major groove intracellularly, it is reasonable that

effective entry into cells and hypoxia-triggered DOX release into the cytoplasm of

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DANPVP&DOX were subsequently accompanied by improved tumor cell DOI:proliferation10.1039/D0TB01692F

inhibition. After verifying the good biocompatibility of both SANP and DANP on MDA- MB-231 cells (Figure S12), the cells were incubated with DANPVP&DOX or SANPVP&DOX pretreated at different pH conditions, and then treated with 4 Gy radiation. Furthermore, the cells were cultured for 48 h for the MTT assay. In comparison to both formulations without irradiation, the cells treated with DANPVP&DOX+X-ray or SANPVP&DOX+X-ray exhibited enhanced anticancer activity at pH 7.4 owing to the combined effect of X- PDT cytotoxicity and hypoxia-activated chemotherapy (Figure 5C). More importantly, the pH 6.8-induced charge conversion of DANPVP&DOX led to the highest anticancer activity regardless of X-ray irradiation. The cell viability of DANPVP&DOX+X-ray at pH 6.8 was significantly reduced to 37.15 ± 5.13% when the DOX concentration was 2.0 μg/mL, whereas SANPVP&DOX+L treatment at pH 6.8 resulted in 56.29 ± 3.50% cell viability.
Furthermore, cell apoptosis after treatment, as mentioned above, was evaluated by Annexin-V/propidium iodide (PI) staining. As displayed in Figure 5D, DANPVP&DOX+X- ray and SANPVP&DOX+X-ray induced apoptosis in 26.85% and 25.74% of MDA-MB- 231 cells at pH 7.4, respectively. Meanwhile, the percentage of apoptotic cells (25.84%) was similar when the cells were treated with SANPVP&DOX+X-ray at pH 6.8. Notably, treatment with DANPVP&DOX+X-ray at pH 6.8 induced the highest apoptotic ratio (55.53%) among these treatments. These results were consistent with the uptake and DOX release observations, verifying that enhanced cytotoxicity is a result of pHe- induced charge conversion and hypoxia-triggered chemotherapy.

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3.5Pharmacokinetics and Biodistribution of DANPVP&DOX In Vivo. DOI: 10.1039/D0TB01692F

In protein adsorption experiments, we demonstrated that both DANP and SANP, with a negative charge, can effectively avoid protein adsorption and thus potentially delay blood clearance. Their pharmacokinetic profiles were then evaluated by HPLC following intravenous injection. As shown in Figure 6A, DANPVP&DOX and SANPVP&DOX provided blood circulation of DOX that was comparable and longer than that of free DOX. Moreover, the pharmacokinetic parameters were calculated using a noncompartment model. As an important parameter of drug retention in the bloodstream, the area under the curve (AUC0-t) of SANPVP&DOX and DANPVP&DOX was 12.68-fold and 10.23-fold greater, respectively, than that of free DOX (76.403 μg/mL*h). Meanwhile, the elimination half-life time (t1/2z) of SANPVP&DOX and DANPVP&DOX extended to 20.214 and 20.517 h, respectively. The accelerated clearance of DANPVP&DOX after 6 h was likely due to the degradation of a small amount of DMMA groups at pH 7.4. The pharmacokinetic data illustrated that the negative charge of DANPVP&DOX was relatively stable in blood and effectively decreased the nonspecific interactions between DANPVP&DOX and other components.

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DOI: 10.1039/D0TB01692F

Figure 6. (A) Plasma DOX concentration versus time following systemic injection of DANPVP&DOX, SANPVP&DOX or free DOX (n = 4). (B) DOX concentration in tumor tissues at different time intervals. ∗p < 0.05 versus free DOX. Quantitative analysis of DOX content in major organs at 24 h (C) and 48 h (D) after i.v. injection of DANPVP&DOX, SANPVP&DOX or free DOX.

The tumor accumulation and biodistribution of DANPVP&DOX were further examined in mice bearing MDA-MB-231 xenografts. The mice received an i.v. injection of free DOX, DANPVP&DOX or SANPVP&DOX, and the DOX content in tumor tissues and other organs was quantitatively measured using HPLC. As shown in Figure 6B, treatment with DANPVP&DOX or SANPVP&DOX significantly increased DOX retention in tumors at 24 h from 0.75 ± 0.26 to 3.31± 0.28 and 2.15± 0.31 %ID/g tumor, respectively. Considering that SANPVP&DOX has more opportunities to enter tumor tissues via blood

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circulation and the EPR effect, the higher tumor accumulation of DANPVP&DOX DOI:could be10.1039/D0TB01692F attributed to its enhanced cellular internalization and the continual elimination of SANVP&DOX, which was not taken up by tumoral cells. Additionally, the DOX distribution in other organs at 24 h and 48 h was also determined. As displayed in Figure
6C and 6D, due to their comparable pharmacokinetic profiles and physicochemical properties, both DANPVP&DOX and SANPVP&DOX yielded similar DOX contents in major organs. Because the liver and spleen belong to the reticuloendothelial system (RES), DANPVP&DOX and SANPVP&DOX resulted in obvious accumulation in both organs, which is in agreement with previous reports.54
3.6Tumor Growth Inhibition and Biosafety of DANPVP&DOX In Vivo. Considering that DANP could deliver both VP and DOX into tumoral cells more
efficiently than SANP, the antitumor activity of DANPVP&DOX in vivo was evaluated in mice bearing MDA-MB-231 xenografts. The mice were randomly divided into six groups (n = 5) and received different formulations via i.v. injection every five days during the treatment. With the tumor accumulation data as a guide, the tumor tissues were exposed to X-ray (4 Gy) for 10 min at 24 h post injection. In comparison with the negative control group (PBS group, Figure 7A), the mice treated with free VP&DOX plus X-ray radiation moderately inhibited MDA-MB-231 tumor growth, with a tumor inhibition rate (TIR) of ~47.1%. In the absence of X-ray exposure and DOX liberation, the inefficient intracellular DOX release from both DANPVP&DOX and SANPVP&DOX resulted in a TIR less than ~30%. Although DANPVP&DOX could deliver more DOX to tumor cells via pHe-induced charge conversion, there was no significant difference

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between the two nanocarriers. Moreover, the SANPVP&DOX+X-ray group DOI:exhibited10.1039/D0TB01692F remarkable tumor growth inhibition, which could be attributed to the X-PDT effect and
boosted DOX release. In contrast, tumor growth was almost completely suppressed in the group treated with DANPVP&DOX plus X-ray irradiation (TIR = 88.1%), validating the efficiency of the combination of acidity-facilitated drug uptake and hypoxia- activated DOX release. As shown in Figure 7B, the tumor weight of the DANPVP&DOX+X-ray group was the smallest among several groups after the treatment. Furthermore, widespread regions of apoptotic cells (TUNEL-positive) were visualized in tumor slices after treatment with DANPVP&DOX+X-ray by immunohistochemical staining, suggesting its highest anticancer effect (Figure 7C). To demonstrate the therapeutic advantage of X-PDT for deep tumors, the mice bearing MDA-MB-231 xenografts were received i.v. injection of DANPVP&DOX and the irradiation of 4.0 Gy X- ray with the pork tissues of 1.5 cm thickness lain on top. As shown in Figure S13, the tumor growth of DANPVP&DOX+X-ray group was partially slowed with a decreased TIR of ~74.2%. Although its treatment efficacy was impeded by tissue thickness, the impact was much less severe than that of traditional PDT, which exhibit almost no efficacy beyond 1 cm thickness.6

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DOI: 10.1039/D0TB01692F

Figure 7. (A) Tumor growth on mice bearing MDA-MB-231 tumor xenograft with different treatments (n = 5). The injections were performed on days 0, 5 and 10 with an equivalent [DOX] = 2.5 mg/kg. *p < 0.05. (B) The weight of the MDA-MB-231 xenograft tumors excised on day 15. *p < 0.05. (C) H&E analysis of tumor tissue slices after the treatments. The scale bar is 200 μm. (D)TUNEL analysis of tumor tissue slices. The scale bar is 100 μm.

According to our design, the VP and DOX molecules were protected by DANP in the circulating blood and showed minimal toxicity to normal organs without X-ray exposure. Except for free VP&DOX treatment, no noticeable mouse body weight change was observed when the mice received other formulations during the whole therapeutic period (Figure S14). As shown in Figure S15, the hematological assessment

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revealed no significant differences following the various treatments. DOI:Moreover, the10.1039/D0TB01692F enzyme-linked immunosorbent assay (ELISA) measurements of ALT, AST and BUN
(Figure S16) further demonstrated that DANPVP&DOX+L did not induce notable liver or kidney damage.

In summary, we have developed a novel drug-delivery platform that combines X- PDT and cascade chemotherapy for improved cancer therapy. Under physiological conditions, the obtained nanocarrier DANPVP&DOX, with a negative charge, and PEGylation could efficiently protect encapsulated VP and DOX, minimizing the side effects on normal tissues. After accumulating into the tumor interstitium, pHe is able to trigger a change in the surface charge of DANPVP&DOX to positive to enhance cellular internalization by tumoral cells. Under X-ray irradiation, DANPVP&DOX not only converts O2 to cytotoxic 1O2 to induce cancer cell apoptosis but also facilitates DOX release via intracellular hypoxia-activated disassembly of itself. Because of the specificity of local tumor acidity and the controllability of an external X-ray radiation, the promoted combination effect occurred precisely within tumor tissues. Therefore, this strategy provides new avenues to design stimuli-responsive drug delivery systems with precise and advanced therapeutic efficiency in cancer treatment.

Conflicts of interest

There are no conflicts to declare. Acknowledgements
This work was supported by the National Natural Science Foundation of China

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(51603150), Tianjin Municipal Science and Technology DOI:Commission10.1039/D0TB01692F

(17JCQNJC02200) and Tianjin Medical University General Hospital (ZYYFY2016041). The authors have declared that no competing interests exist.
Notes and references

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DOI: 10.1039/D0TB01692F

The pHe-triggered “charge conversion” nanocarriers were developed for combined X- ray-induced photodynamic therapy (X-PDT) and hypoxia-activated chemotherapy.