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Radiotherapy-Controllable Chemotherapy from ROSResponsive Polymeric Nanoparticles for Effective Local Dual Modality Treatment of Malignant Tumors Te-I Liu, Ying-Chieh Yang, Wen-Hsuan Chiang, Chun-Kai Hung, YuanChung Tsai, Chi-Shiun Chiang, Chun-Liang Lo, and Hsin-Cheng Chiu Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.8b00942 • Publication Date (Web): 25 Jul 2018 Downloaded from http://pubs.acs.org on July 25, 2018

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Radiotherapy-Controllable Chemotherapy from ROS-Responsive Polymeric Nanoparticles for Effective Local Dual Modality Treatment of Malignant Tumors Te-I Liu1§, Ying-Chieh Yang2§, Wen-Hsuan Chiang3, Chun-Kai Hung1, Yuan-Chung Tsai1, ChiShiun Chiang1, Chun-Liang Lo4, and Hsin-Cheng Chiu1*

1

Department of Biomedical Engineering and Environmental Sciences, National Tsing Hua

University, Hsinchu 30013, Taiwan 2

Department or Radiology, National Taiwan University Hospital Hsin-Chu Branch, Hsinchu

30013, Taiwan 3

Department of Chemical Engineering, National Chung Hsing University, Taichung 40227,

Taiwan 4

Department of Biomedical Engineering, National Yang-Ming University, Taipei 11221,

Taiwan

*Corresponding Author: Email: [email protected] §

These authors contributed equally to this work

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ABSTRACT: Radiotherapy is one of the general approaches to deal with malignant solid tumors in clinical treatment. To improve therapeutic efficacy, chemotherapy is frequently adopted as the adjuvant treatment in combination with radiotherapy. In this work, a ROSresponsive nanoparticle (NP) drug delivery system was developed to synergistically enhance the antitumor efficacy of radiotherapy by local ROS-activated chemotherapy, taking advantages of the enhanced concentration of reactive oxygen species (ROS) in tumor during X-ray irradiation and/or reoxygenation after X-ray irradiation. The ROS-responsive polymers, poly(thiodiethylene adipate) (PSDEA) and PEG-PSDEA-PEG, were synthesized and employed as the major components assembling in aqueous phase into polymer NPs in which an anticancer camptothecin analogue, SN38, was encapsulated. The drug-loaded NPs underwent structural change including swelling and partial dissociation in response to the ROS activation by virtue of the oxidation of the nonpolar sulfide residues in NPs into the polar sulfoxide units, thus leading to significant drug unloading. The in vitro performance of the chemotherapy from the X-ray irradiation pre-activated NPs against BNL 1MEA.7R.1 murine carcinoma cells showed comparable cytotoxicity to free drug and appreciably enhanced effect on killing cancer cells while the X-ray irradiation being incorporated into the treatment. The in vivo tumor growth was fully inhibited with the mice receiving the local dual modality treatment of X-ray irradiation together with SN38-loaded NPs administered by intratumoral injection. The comparable efficacy of the local combinational treatment of X-ray irradiation with SN38-


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loaded NPs to free SN38/irradiation dual treatment corroborated the effectiveness of ROSmediated drug release from the irradiated NPs at tumor site. The IHC examination of tumor tissues confirmed the significant reduction of VEGFA and CD31 expression with the tumor receiving the local dual treatment developed in this work, thus accounting for the absence of tumor regrowth compared to other single modality treatment. KEYWORDS: Radiotherapy, Irradiation controlled chemotherapy, Drug delivery system, ROS-responsive polymer

1. INTRODUCTION Radiotherapy, by adopting high-energy ionizing radiation to suppress tumor growth and kill cancer cells, has been one of the general approaches for decades to deal with malignant solid tumors in clinical cancer treatment.1 With the high-energy ionizing irradiation, not only the direct breakage of DNA takes place, but also a high level of reactive oxygen species (ROS), such as hydroxyl radicals, singlet oxygen, hydrogen peroxide etc., are generated.2,3 These ROS are usually highly chemically reactive, capable to attack the surrounding tissues by oxidation reaction, causing the damage of cell structure, the breakage of DNA strands (particularly the damage of double-stranded DNAs which are more difficult to repair) and cells death.4,5



To achieve the best therapeutic efficacy with effective tumor growth inhibition, chemodrug is frequently used before, during and after radiotherapy as the adjuvant therapy for synergistic treatment of susceptible cancers.6 Chemotherapy when being adopted in combination with radiotherapy for synergistic responses, not only simply serves as an anticancer agent that induces cell apoptosis and reduces tumor size for better radiotherapeutic effect, but also as a sensitizer that enhances the radiosensitivity of cancer cells.7,8 For instance, cisplatin and camptothecin analogues are anticancer drugs that inhibit DNA replication and repairing in the S phase of cell cycle, thus exacerbating the apoptosis of the cancer cells receiving irradiation treatment.9,10 However, the poor lesion selectivity of chemodrugs usually leads to severe adverse effects, limits drug dosage application and thus reduces therapeutic efficacy.11 To address this issue, numerous strategies for effective drug delivery have been developed in the last two decades.12,13 Adopting functionalized nanoparticles (NPs) as a delivery system of chemotherapy has been considered a prominent strategy to improve drug delivery and accumulation at tumor sites and enhancing drug stability under physiological conditions.14,15 Encapsulation of hydrophobic anticancer agents into NP formulations largely overcomes the difficulty from their inborn water-insoluble property which severely limits their clinical usage.16 In addition, the development of smart drug delivery systems also enables the anticancer therapy to be more selective toward


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targeted sites, for instance, taking advantages of the naturally preferred NP accumulation in solid tumor via angiogenesis and the adoption of targeting ligands capable to complementarily bind targeted cell receptors.17-19 Drug release from the NP carrier systems in a spatially/temporally controllable manner is also crucial to the success of drug delivery for effective anticancer treatment. 20,21 Approaches involving the uses of internal stimuli, such as tumor acidic pH, the high level of GSH and oxidative stress, have been utilized in controlling NP morphology and drug liberation. 22-24 On the other hand, adopting external stimuli to activate drug release and/or therapeutic action from NP therapy systems provides even a wider spectrum (including photo, magnetic, temperature, acoustic wave, and mechanical stress etc.) alongside more precise controls at both locations and timing.25-28 For dual modality treatment in combination with chemotherapy, photodynamic therapy (PDT) and X-ray irradiation therapy both emphasizing on local treatment rely in large measure on the therapeutic action of ROS. Owing to the high oxidative reactivity, the ROS is obviously a good candidate to serve as a stimulus for anticancer drug release from NP delivery systems. Taking advantages of the facile activation process, the photodynamic mediated release of anticancer chemotherapy from NP carrier systems has been previously reported.29,30 Nevertheless, the restricted tissue penetration depth of visible light is one of the major limitations in its applications.31 It has been well recognized that a high level of ROS are produced at



treated tissues during X-ray irradiation and reoxygenation after irradiation without the concern of tissue penetration limitation.32,33 Nevertheless, it was rarely reported to utilize the X-ray induced ROS as a trigger for drug liberation from the ROS-responsive NP carrier systems. In this work, ROS-responsive polymers, poly(thiodiethylene adipate) (PSDEA) and PEG-PSDEA-PEG, were synthesized and employed for the preparation of polymeric NPs encapsulating a nonpolar camptothecin analogue, SN38, for synergistic treatment with X-ray irradiation against malignant solid tumors. The ROS-mediated oxidation reaction of the hydrophobic sulfide residues of PSDEA and PEG-PSDEA-PEG acting as the major components of the polymeric NP carriers into the hydrophilic sulfoxide moieties induces significant structural change of the NPs and drug release. The structural transformation in response to the ROS treatment was characterized by 1HNMR, DLS and TEM. The in vitro cytotoxicity of chemotherapy either alone upon ROS pre-activation of the sulfide-containing NPs or dual modality therapy with X-ray irradiation against cancer cells was evaluated. The validation of the radiotherapycontrollable drug release from the ROS-responsive NP therapy system in vivo was conducted by evaluating the growth inhibition of tumor receiving the NPs intratumorally with X-ray activation in comparison with the combinational treatment of free drug (SN38) with X-ray irradiation. The schematic of the preparation of ROS responsive NPs


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and of the strategy for the synergistic treatment of combining X-ray irradiation with radiotherapy-controlled chemotherapy is illustrated in Scheme 1.

Scheme 1. Schematic illustration of the fabrication and oxidized mechanism of ROSresponsive nanotherapeutics for assisting the radiotherapy by controlled SN38 release.

2. EXPERIMENTAL SECTION 2.1. Materials Monomethoxy-poly(ethylene glycol) (mPEG, Mw 5000 g/mol) and rose bengal were obtained from Tokyo Chemical Industry (Tokyo, Japan). 7-Ethyl-10-hydroxycamptothecin (SN38) was purchased from SINOVA (Bethesda, MD). Dimethyl adipate, 2,2’-thiodiethanol,



titanium tetrabutoxide, 1,6-hexanediol, chloroform-d, lipase isolated from candida antarctica, 2’,7’-dichlorofluorescin diacetate and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma-Aldrich (St Louis, MO). Dulbecco's modified Eagle medium (DMEM), Hoechst 33342, Singlet Oxygen Sensor Green S36002 (SOSG), Alexa Fluor® 488, anti-mouse CD31 antibody, and anti-VEGFA antibody were acquired from Invitrogen (USA). Organic solvents were obtained from Alfa Aesar (UK). Deionized water was produced from Milli-Q Synthesis (18 MΩ, Millipore). All other chemicals were reagent grade and used as received. Mouse liver carcinoma cell line BNL 1MEA.7R.1 was obtained from Food Industry Research and Development Institute (Taiwan). 2.2. Synthesis and characterization of ROS-responsive polymers ROS-responsive polymer, PSDEA, was synthesized by bulk polymerization. In brief, dimethyl adipate and 2,2’-thiodiethanol were mixed at a molar ratio 1:0.9 and the mixture was heated to 150 oC under nitrogen atmosphere and stirring for 3 h to remove water in the reaction mixture. Titanium tetrabutoxide (0.5 wt%) was added and the reaction carried out at 205 oC for 8 h. The resulting solution was cooled to the ambient temperature under reduced pressure. The crude product was dissolved in chloroform and purified by precipitation from methanol three times. PSDEA was collected by filtration. Poly(1,6-hexamethylene adipate) (PHMA), an ROSinsensitive polymer used as a control, was prepared similarly, yet with 2,2’-thiodiethanol being replaced by 1,6-hexanediol. The structures and the average molecular weights of the polymers


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were determined, respectively, by 1H-NMR (500 MHz, Bruker Avance 500 NMR) in DMSOd6 at ambient temperature and gel permeation chromatography (GPC; Agilent 1100 series equipped with PLgel 5 mm, 7.5 mm x 300 mm, Agilent 79911GP-503) using THF as the eluent at a flow rate of 1.0 mL/min under RI detection. The PEG standards (Polymer Laboratories, Varian, Inc) were employed for the molecular weight calibration in GPC analyses. The in vitro biodegradation study of PSDEA was carried out by incubation of the polymer (3.0 mg/mL) in PBS containing lipase (1000 Units/mL) at 37 oC for prescribed time intervals, followed by lyophilization and GPC analysis using THF as the eluent. The structural response of PSDEA (3.0 mg/mL) to the ROS treatment was carried out by incubation of the polymer in aqueous H2O2 solution (1.0 M) for prescribed time intervals at 37 oC. The structure of the sample collected by lyophilization was characterized by 1H-NMR in DMSO-d6 at ambient temperature and GPC under the same conditions as aforementioned. Preparation of PEG-PSDEA-PEG was conducted in a one-pot reaction. It was carried out first by the bulk polymerization of dimethyl adipate and 2,2’-thiodiethanol for PSDEA synthesis as described previously. It was followed by transesterification of PSDEA with mPEG, by which the polymerization reaction was also stopped. The mixture of dimethyl adipate (1.9 g) and 2,2’-thiodiethanol (1.2 g) was heated to 150 oC under nitrogen for 3 h. After the addition of titanium tetrabutoxide, the reaction was carried out at 205 oC for 8 h. mPEG (5000 g/mol, 6 g) was added and the reaction further proceeded for 96 h under the identical conditions. The



crude product was dissolved in water and purified by tangential flow filtration (MWCO 10KD, mPES hollow fiber, Spectrum, mounted with a MAP-TFF System, LEF Science). The product was collected by lyophilization. The triblock copolymer, PEG-PHMA-PEG, was also prepared using 1,6-hexandiol instead of 2,2’-thiodiethanol. The structures and the averages molecular weights of the triblock copolymers were determined by 1H-NMR and GPC as aforementioned. 2.3. Preparation and characterization of SN38-loaded ROS-responsive NPs SN38-loaded ROS-responsive NPs were prepared by the nanoprecipitation approach. In brief, PEG-PSDEA-PEG (5 mg), PSDEA (0.75 mg) and SN38 (1.0 mg) were dissolved in DMSO (0.2 mL) and the solution was added dropwise into phosphate buffer (pH 7.4, 1.8 mL) under sonication (bath type) for 10 min. The NPs were purified by dialysis (MWCO 12000~14000) against phosphate buffer (pH 7.4, 0.01 M) for 3 days. The aqueous NP dispersion was filtered (1.2 μm) and stored at 4 oC before use. The SN38-loaded ROSresponsive NPs were referred to hereinafter as SS-NPs. The drug-loaded ROS-insensitive NPs prepared from the assembling of SN38, PHMA and PEG-PHMA-PEG as a control sample were also attained in a similar manner and are referred to as SH-NPs in this work. The drug encapsulation efficiency was determined by the measurement of the drug absorbance at 388 nm (Hitachi Double Beam Spectrophotometer U-2900, Japan) in DMSO. The drug loading efficiency and content were calculated as follows. Drug loading efficiency (LE) % = (weight of drug loaded) / (weight of drug in feed) × 100 %


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Drug loading content (LC) % = (weight of drug loaded) / (weight of nanoparticles) × 100 % The drug-free NPs, both ROS-responsive and non-responsive, were prepared similarly, yet in the absence of SN38 and abbreviated herein as S-NPs and H-NPs, respectively. The measurements of the hydrodynamic diameter (Dh) and zeta potential of the NPs in phosphate buffer (pH 7.4, 0.1 M) were conducted on a Malvern Zetasizer Nano ZS90 (USA) at 25 °C. The colloidal stability of the NPs was evaluated in terms of the size variation with time by dynamic light scattering (DLS) measurement in either PBS or DMEM containing 10% FBS over 7 days. To assess the NP morphology, the mean hydrodynamic radii (Rh) of the NPs were also determined by DLS on a Brookhaven BI-200SM goniometer equipped with a BI-9000 AT digital correlator using a solid-state laser (30 mW, λ = 637 nm) at 90° while the radii of gyration (Rg) were estimated by the static light scattering (SLS) measurements in the angle range 60140o from Brookhaven BI-200SM goniometer. The NP morphology was also examined by transmission electronic microscopy (TEM, JEOL JEM-2100F) after phosphotungstic acid staining. 2.4. Morphologic responses of NPs to ROS treatment The ROS production in this study for the NP characterization was induced either by photodynamic reaction or X-ray irradiation of prescribed dosages in gray (Gy). The photodynamic reaction was carried out using rose bengal (5.0 M) as the sensitizer under laser light irradiation (0.5 W/cm2) at 525 nm for prescribed durations. The X-ray irradiation was



produced by the linear accelerator (Elekta SLI-5951, Sweden) at the National Taiwan University Hospital Hsin-Chu Branch. To compare the level of ROS generation between X-ray irradiation and PDT treatment, SOSG was employed as a fluorescence probe of ROS in this study. The aqueous SOSG solution (10.0 M) was added into the 96-well plate (200 L/well) and subjected to either X-ray irradiation (0, 5, 10, 15 Gy) or PDT treatment (5.0 M rose bengal, 0.5 W/cm2 at 525 nm). The fluorescence intensity of SOSG was determined using a SpectraMax M5 microplate reader (excitation/emission ~480/540 nm). Both SS-NPs and SH-NPs in phosphate buffer (pH 7.4, 0.1 M) immediately after the ROS treatment induced by the photodynamic reaction for various prescribed durations (0, 5, 10 and 20 min) were characterized by DLS and TEM. The time-evolved particle size variation of the NPs in phosphate buffer after the ROS treatment was examined by DLS at 25 oC. The ROS generation was induced by either photodynamic reaction of rose bengal (5.0 M) under photoirradiation (0.5 W/cm2 at 525 nm) for 5 min or X-ray irradiation (15 Gy). The irradiation dosage of 15 Gy was also selected in the in vivo experiments as reported elsewhere.34,35 2.5. ROS-activated SN38 release The release of SN38 from the ROS-pretreated SN38-loaded NPs at an SN38 concentration of 90.0 μM in PBS at 37 °C was evaluated in terms of the change of the drug fluorescence intensity with time.36 The excitation was conducted at 388 nm on a Hitachi F-7000 fluorescence spectrophotometer and the fluorescence intensity at 530 nm was recorded within a time period


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of 168 h. The ROS activation of the NPs was carried out by either photodynamic reaction of rose bengal (5.0 M) under photo-irradiation at 525 nm for prescribed durations (5 and 10 min) or X-ray irradiation (15 Gy). The fluorescence intensity of free SN38 (90.0 M) was adopted to represent the fluorescence emission upon the complete release of SN38 from the NPs. The aqueous solubility of SN38 is ca 38 g/mL (ca 97 M) as reported elsewhere.37 2.6. In vitro cellular uptake To examine the cellular uptake of SN38, BNL 1MEA.7R.1 cells (2.5 x 105 cells/well) were seeded onto 22 mm round glass coverslips, placed in a 6-well plate, and cultured for 24 h at 37oC under 5% CO2 conditions. The cells were then co-incubated at 37 oC for 12 h, respectively, with free SN38 (20 μM), SS-NPs and SH-NPs of the equivalent SN38 dose with and without ROS pretreatment. The ROS pretreatment of the NPs was carried out by either the photodynamic reaction of rose bengal for 5 min or X-ray irradiation (15 Gy). After being washed three times with PBS, the cells were fixed with 4% formaldehyde. To identify cell morphology and intracellular SN38 location, cells were stained with F-actin Staining KITGreen Fluorescence-Cytopainter (Abcam) for 30 min and rinsed with PBS three times. Cellular uptake of SN38 was directly visualized by laser scanning confocal microscopy (LSCM, Zeiss LSM 780, Germany) with the fluorescence channels for SN38 (ochest channel) and F-actin marker (FITC channel). For quantitative expression of the cell uptake, the mean SN38 fluorescence intensity per cell was obtained by normalization of the total SN38 fluorescence



intensity with the number of cells (140-150 cells) (n = 3). 2.7. In vitro chemotherapy of SN38-loaded NPs BNL 1MEA.7R.1 cells (8 × 103 cells/well) were seeded in a 96-well plate and incubated in DMEM containing 10% FBS and 1% penicillin at 37 oC for 24 h. Cells were then coincubated, respectively, with free SN38, SS-NPs and SH-NPs (with and without ROS pretreatment) of various SN38 concentrations for additional 12 h. The aqueous solution of free SN38 was prepared by dissolution of SN38 in DMSO to a concentration of 25 mM, followed by dilution with the aqueous medium to the desired concentrations. The DMSO content in the aqueous medium was less than 1% (v/v). The ROS pretreatment was induced by either photodynamic reaction of rose bengal for 5 min or X-ray irradiation (10 and 15 Gy) as described previously. The cell culture medium was then replaced with fresh DMEM and the cells were further incubated for 24 h. MTT assay was used to determine the cell viability. In brief, MTT (0.25 mg/mL, 200 μL) was added to each well, followed by incubation at 37 oC for 4 h. After discarding the culture medium, DMSO (200 μL) was added to dissolve the precipitates and the resulting solution was measured for absorbance at 570 nm using a SpectraMax M5 microplate reader. The IC50 values (the 50% inhibitory concentration) were attained from the dose-vs-toxicity curves using Sigmaplot. Drug-free NPs, S-NPs and H-NPs were also characterized for their cytotoxicity in a similar manner. 2.8. In vitro combinational radio/chemotherapy


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To evaluate the cytotoxic effect of combinational radio/chemotherapy, the BNL 1MEA.7R.1 cells (1 × 103 cells/well) were seeded in a 96-well plate and incubated in DMEM containing 10% FBS and 1% penicillin at 37oC for 24 h. Cells were then co-incubated, respectively, with free SN38, SS-NPs and SH-NPs at an SN38 concentration of 0.312 μM, followed by X-ray irradiation (0, 10 and 15 Gy) treatment. The aqueous solution of SN38 in culture medium was prepared as aforementioned. After 18-h co-incubation, the medium was replaced with fresh DMEM and incubation further proceeded for additional 30 h. MTT assay was used to determine the cell viability. 2.9. Tumor growth inhibition Male BALB/cByJNarl mice (4~5 weeks old), purchased from National Laboratory Animal Center (Taiwan), were cared in accordance with the Guidance Suggestions for the Care and Use of Laboratory Animals, approved by the Administrative Committee on Animal Research at the National Tsing Hua University (Taiwan). To establish tumor model, 8 x 105 BNL 1MEA.7R.1 cells (100 L) were subcutaneously injected into the right thigh of mice. With tumor reaching ca 200 mm3 in size, mice were divided into 8 groups with 5 mice in each group (n = 5) and treated, respectively, with PBS, free SN38 (containing less than 1% DMSO), SS-NPs and SH-NPs (in 20.0 μL PBS) by intratumoral injection at a single SN38 dose of 2.0 mg/kg with and without X-ray irradiation. PBS was employed as a blank control. The tumor bearing mice were subjected to X-ray irradiation (15 Gy) at the tumor region at 2 h post-



injection. Tumor size and body weight were evaluated daily. Tumor volume (V) was determined as follows: V = L × W2/2, where W is the tumor measurement at the widest point and L the tumor dimension at the longest point. The mice were euthanized and sacrificed at day 17 post-injection and the tumors were harvested, weighed and embedded in OCT compound (Sakura Finetek, USA) and then stored at -80oC before use. Tumor sections were H&E stained and histologically examined by an Olympus IX70 inverted microscope (Japan). 2.10. Immunohistochemical (IHC) identifications Rat anti-mouse CD31, anti-VEGFA antibodies with Alexa Fluor 488® goat anti-rat as the secondary antibody were used for the IHC identifications of tumor angiogenesis in the tissue cryosections. The cell nuclei were stained with propidium iodide (PI). All stained tumor sections were examined by LSCM with the fluorescence channels for Alexa Fluor 488 and PI. The CD31-identified areas were analyzed with Image-Pro Plus. The integrated optical density (IOD) of CD31 (also termed as the CD31 density) was determined based on the area and intensity of red pixels in each selected tumor field. Twenty randomly selected fields were imaged and the mean IOD of CD31 was attained. The IOD value of VEGFA was estimated similarly from twenty randomly selected fields. 2.11. Tumor reoxygenation after X-ray irradiation The reoxygenation in tumor tissues after X-ray irradiation treatment was examined in terms of the ROS generation by the staining of tumor tissue with DCFH-DA.38 The OCT on


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the tissue slides was removed carefully and the tumor tissue region circled with ImmEdge Pen (H-4000, Vector, USA). The tumor tissue in the circle was stained with DCFH-DA (10 μM) at 37 oC for 30 min. After being washed three times with PBS, the tissue was stained with Hoechst 33258 for the cell nucleus identification. The stained tumor sections were examined by LSCM. 2.12. Statistical Analysis. All data were presented as mean value ± SD. Statistical analysis was performed with Prism 5.0 software (GraphPad Software) by an unpaired t-test and one-way ANOVA with Bonferroni multiple comparisons. Statistical significance is indicated as (n.s.) P > 0.05, (*) P < 0.05, (**) P < 0.005 and (***) P< 0.001.

3. RESULTS AND DISCUSSION 3.1. Synthesis and characterization of the ROS-responsive (co)polymers. To promote the therapeutic efficacy of radiotherapy against malignant solid tumors by virtue of combining effective chemotherapy at the tumor sites, a radiotherapy-controllable NP drug delivery system comprising the anticancer agent, SN38, and the ROS-responsive polymers, PSDEA and PEG-PSDEA-PEG, in polymer NP structure was developed in this work. The synthesis of PSDEA was performed by bulk polymerization through transesterification reaction of dimethyl adipate with 2,2’-thiodiethanol. The synthetic route and chemical structure of PSDEA is illustrated in Figure 1a. The chemical structure was confirmed by 1H-NMR



(Figure 1a), showing the feature signals of ethylene protons from thiodiethylene at δ 4.22 and 2.75 ppm and of butylene protons from adipate at δ 2.34 and 1.65 ppm with the ratio of the overall signal integrals of ca 1.0. The weight-average molecular weight is ca 1.52 x 104 g/mol and the polydispersity 1.96 as estimated by GPC (Table S1). The tri-block copolymer, PEGPSDEA-PEG, was prepared in a one-pot reaction manner first by stepwise polymerization of dimethyl adipate with 2,2’-thiodiethanol as aforementioned. It was followed by transesterification of the PSDEA chain segments with mPEG under the identical conditions, by which the polymerization reaction was also concomitantly terminated. The 1H-NMR spectrum of the purified product (Figure 1b) illustrates the feature signal of ethylene protons from mPEG at ca δ 3.64 ppm in addition to the proton signals from the PSDEA blocks identical to those as aforementioned in Figure 1a. With the average molecular weight of mPEG being ca 5000 g/mol, the average molecular weight of the triblock copolymer was estimated to be 1.2 x 104 g/mol while the PSDEA block was ca 2000 g/mol, based upon the integral ratio of the pertinent proton signals. The result is in agreement with the datum from GPC measurement (Table S1). To prepare ROS-insensitive NPs as a negative control sample, the polymer, PHMA, and the triblock copolymer, PEG-PHMA-PEG were synthesized and characterized in a similar manner. The results are shown in Figure S1 and Table S1. In addition to the structural and molecular weight characterization, the structural response of PSDEA to enzymatic cleavage and ROS-induced oxidation were also examined. The


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biodegradability of PSDEA by lipase was confirmed by monitoring changes of the polymer elution profiles from the GPC measurements after co-incubation with lipase at 37 oC with time. As shown in Figure S2, the intensity of polymer peak in terms of either peak height or peak area decreased in a proportional manner to the reaction time with lipase, meanwhile the peak intensity ascribed to low molecular weight products was significantly enhanced. Obviously, the enzymatic action of lipase on PSDEA occurred by the cleavage of ester linkage, leading to the reduction of molecular weight and the production of small fragments in the reaction mixture. The capability of PSDEA to undergo ROS-induced oxidation was performed by co-incubation of the polymer with H2O2 as a ROS generation source in aqueous phase and the structural change was monitored by 1H-NMR as a function of incubation time. The results are shown in Figure S3. In agreement with the 1H-NMR measurements reported previously,39,40 downfield shifts of the feature signals of the methylene protons right adjacent to the sulfide groups from δ 2.77 to 2.97~3.10 ppm and of the next methylene protons from 4.21 to 4.46-4.53 ppm were clearly observed, indicating the oxidation of the sulfide to sulfoxide units. Such a change from sulfide to sulfoxide residues plays a vital role in activating drug release owing to the resulting structural change of the polymeric NPs upon the transformation of hydrophobic cores into hydrophilic polymeric chain segments. In contrast, the chemical shift of the proton signals from PHMA remained unchanged over 24-h incubation in aqueous H2O2 solution, indicating the high resistance of the polymer to ROS-induced oxidation (Figure S3). Nevertheless, the GPC



analysis showed the elution time of PSDEA after the oxidation reaction remained almost unchanged, indicating a negligible variation in chain length of the polymer after H2O2 treatment (Figure S4).


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Figure 1. Synthetic routes, chemical structures and 1H-NMR (500 MHz) spectra (in CDCl3 at 25 oC) of (a) PSDEA and (b) PEG-PSDEA-PEG.



3.2. The characterization of the ROS-responsive NPs. The ROS-responsive SN38-loaded nanoparticles (SS-NPs) were prepared by the nanoprecipitation technique. The mean particle sizes of SS-NPs in pH 7.4 phosphate buffer determined by DLS and in dried state examined by TEM are ca 109 nm and 110 nm in diameter (Figures 2 and S5). The DLS size distribution profiles of SS-NPs, SH-NPs and drug-free HNPs and S-NPs in phosphate buffer (pH 7.4, 25 oC) are illustrated in Figure S5. The Rg and Rh of SS-NPs were determined by SLS and DLS (both on a Brookhaven BI-200SM goniometer), respectively, and the Rg/Rh ratio was employed to evaluate the structure of SS-NPs.41-43 The results show that, with the Rg of SS-NPs being 42.9 ± 3.9 nm and Rh 51.7 ± 4.4 nm, their ratio (0.829) strongly suggests that the NPs are in the spherical micelle architecture, in consistence with the TEM observation (Figure 2). The TEM images also revealed that these NPs were well dispersed and spherical, indicating the strong amphiphilic property of PEG-PSDEA-PEG capable to prevent the NPs from aggregation in aqueous phase. The particle size of ca 100 nm is appropriate for promoting tumor accumulation of nanocarriers via enhanced permeability and retention (EPR) effect.44 Owing to its additional role as a radiosensitizer, SN38 was selected as the chemodrug loaded into polymeric NPs. The drug loading efficiency and capacity of SS-NPs are ca 71.8% and 11.1 wt%, respectively, as determined by fluorescence measurements in DMSO. As a control sample, the ROS-insensitive NPs (SH-NPs) from the assembling of SN38, PHMA and PEG-PHMA-PEG were also obtained similarly to SS-NPs


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and their characterizations with respect to particle size, size distribution and drug loading are also shown in Figure S5 and Table 1. In addition to the Dh value, the values of polydispersity index (PDI) and light scattering intensity of the NPs are also included in Table 1. Apparently, either the presence of the sulfide residues or the incorporation of SN38 can appreciably increase the light scattering intensity and decrease the PDI by increasing the hydrophobicity of the solid cores and the amphiphilic property of NPs in aqueous phase. Table 1. DLS Data and Drug Loading Characteristics of NPs.a,b

Sample

SN38-loaded S-NPs (SS-NPs) SN38-loaded H-NPs (SH-NPs) Drug-free S-NPs (S-NPs) Drug-free H-NPs (H-NPs)

Size (Dh, nm)

PDI

Count rate (kcps)

SN38 Loading Efficiency (%)

Capacity (wt%)

109.4 ± 9.8 0.16 ± 0.03 5361.2 ± 380.2

71.8 ± 1.1

11.1 ± 0.5

104.6 ± 9.0 0.19 ± 0.01 4456.0 ± 490.9

73.8 ± 1.2

12.5 ± 0.1

89.4 ± 9.1 0.21 ± 0.09 1441.0 ± 543.7

-

-

81.9 ± 7.8 0.31 ± 0.02

-

-

421.0 ± 17.8

a

DLS measurement was performed in phosphate buffer (pH 7.4, I = 0.1 M) at 25 oC. bError bars represent mean ± s.d. (n ≥ 10).

The excellent colloidal stability of SS-NPs and SH-NPs in PBS (pH 7.4) and cell culture DMEM (10% FBS) was observed by the essentially unchanged particle sizes over a time period of 7 days (Figure S6). It is also important to note that the size variation of the SS-NPs in aqueous solutions before and after lyophilization was rather insignificant (Figure S6), indicating its prominent colloidal stability in lyophilized form for long-term storage.



3.3. ROS-induced morphological variation and drug release The responses of the sulfide-containing NPs (SS-NPs) in morphologic change and drug release to ROS treatment was examined. The ROS treatment of the NPs for various durations was induced primarily by the photodynamic reaction of rose bengal under laser light irradiation at 525 nm and the morphology of the NPs was examined immediately after the treatment. The photodynamic reaction was employed instead of direct X-ray irradiation because of the facile photo-activation process, the easy accessibility and the flexibility to immediately undergo morphologic examination. Both significant increases in Dh and decreases in light scattering intensity were clearly observed owing to the increased swelling and decreased structural compactness of SS-NPs treated with the photodynamic reaction (Figures 2b and c). The TEM images also illustrate the significant increases of particle size with increasing the treatment duration (Figure 2c). In addition to the enhanced swelling, the NPs, from the TEM examination, became partially disintegrated when being subjected 20-min photodynamic reaction and large debris were thus produced. This is because of the ROS-induced oxidation of the hydrophobic sulfide residues of PSDEA and PEG-PSDEA-PEG in polymeric NPs to the hydrophilic sulfoxide groups. By contrast, the SH-NPs comprising PHMA and PEG-PHMA-PEG is more resistant to the oxidation, leading to the well preservation of their spherical structure.


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Figure 2. (a) Particle size distribution profiles of SS-NPs before and right after ROS treatments of various durations. (b) Light scattering intensity (kcps) of SS-NPs as a function of the duration of ROS treatment. Error bars represent mean ± s.d. (n = 5). (c) TEM images of SSNPs receiving ROS treatments of various durations. Scale bars are 200 nm. (d) TEM images of SH-NPs before and after ROS treatment (20 min). ROS generation was induced by photoirradiation (525 nm, 0.5 w/cm2) of rose bengal (5.0 μM) in phosphate buffer (pH 7.4).

Figure 3 shows the change of Dh of SS-NPs and SH-NPs with time after receiving the ROS treatment induced either by 5-min photodynamic reaction (rose bengal) or X-ray



irradiation (15 Gy). The particle size of the SH-NPs remained essentially unchanged even at 24 h after the ROS treatment, indicating the high tolerance of the NPs against ROS oxidation. By contrast, the SS-NPs kept enlarged in size over 16 h after the oxidation reactions, indicating the time requirement for the full change in NP structure after the oxidation of the sulfide groups into the sulfoxide residues of the ROS-sensitive polymers. The ROS levels by the X-ray irradiation (15.0 Gy) and the photodynamic activation of rose bengal (5.0 M; power density 0.5 w/cm2 at 525 nm for 5 min) in terms of the fluorescence intensity of SOSG as an ROS probe are rather similar as illustrated in Figure S7. Therefore, comparable changes in size of SS-NPs occurring after the ROS treatments induced by these two approaches under the conditions employed were observed.

Figure 3. (a) Time-evolved mean particle sizes (Dh) of SS-NPs and SH-NPs after ROS treatments induced by either photodynamic reaction or X-ray irradiation. (b) Relative particle


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size changes of SS-NPs and SH-NPs over 24 h after ROS treatment. The irradiation dose was 15 Gy. The photodynamic ROS generation was induced by photo-irradiation (525 nm, 0.5 w/cm2) of rose bengal (5.0 μM) for 5 min in phosphate buffer (pH 7.4). Samples were analyzed with one-way ANOVA, followed by Bonferroni correction for multiple comparisons. ***P < 0.001 and n.s. P > 0.05. Error bars represent mean ± s.d. (n = 5).

The capability of the drug-loaded NPs to undergo ROS-triggered drug release is crucial to the success of combining radiotherapy with locally triggered chemotherapy at tumor site. The controllable drug release in response to the ROS pretreatment was examined in terms of the increase of the SN38 fluorescence intensity at 530 nm with time by virtue of the release of SN38 originally in the quenched state when being buried within the hydrophobic cores of NPs. Owing to the hydrophobic nature of SN38 resulting in severe adherence of the drug species to the inner surface of dialysis membrane, the dialysis technique was not adopted herein for the in vitro drug release measurements. The effect of the entrapment and aggregation of SN38 within hydrophobic cores of polymer NPs on the intensity of fluorescence emission was observed for the SS-NPs in the absence of ROS treatment in Figure 4a. The quench effect is attributed to the drug aggregation via hydrophobic interaction or π-π stacking.45 Figure 4a shows the time-evolved fluorescence spectra of SS-NPs with the excitation at 388 nm after receiving the 15-Gy irradiation treatment. The fluorescence emission spectrum of free SN38 at



an equivalent dose was adopted to represent the full release of the SN38 species from the NPs. Appreciable increases of the fluorescence intensity of SN38 with time were observed upon the X-ray irradiation of the SS-NPs. Assuming a linear relationship between the increment of the SN38 fluorescence intensity (at 530 nm) and the amount of drug release, the relative drug release index (%) defined below was employed as a measure of the SN38 release from the NPs.

Relative Drug Release Index (%) =

FL Intensity530 nm, time point − FL Intensity530 nm, 0 h × 100% FL Intensity530 nm, Free SN38 − FL Intensity530 nm, 0 h

where the fluorescence intensity of free SN38 at the same concentration (90.0 M) in PBS was employed to represent the complete drug release. The fluorescence emission spectra of SS-NPs (the SN38 concentration of 90.0 M) in the H2O/DMSO solutions as a function of the DMSO content (Figure S8) also show a comparable fluorescence intensity for complete drug release upon the dissociation of the NP structure with the DMSO content being equal to or greater than 30% v/v. The DMSO-induced disruption of the NP structure was corroborated by the dramatic reduction in light scattering intensity (from 9550 to 770 kcps) by the DLS measurements.


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Figure 4. (a) Fluorescence emission spectra of SS-NPs in PBS with time after the ROS pretreatment induced by X-ray irradiation (15 Gy). Free SN38 at an equivalent dose was adopted as a positive control. (b) Relative drug release index of SN38 from SS-NPs and SHNPs with time after the ROS pretreatments. ***P < 0.001 and n.s. P > 0.05 at last time point. P-values were determined by one-way ANOVA with Bonferroni correction for multiple comparisons. Error bars represent mean ± s.d. (n = 5).

Figure 4b shows the relative drug release indices of SS-NPs and SH-NPs over a time period of 180 h after ROS pretreatment induced either by photodynamic reaction or X-ray irradiation. In agreement with the insignificant structural change of SH-NPs in response to ROS pretreatment (Figure 2), the SH-NPs were found rather insensitive in drug release to the ROS treatment, except for the initial drug burst release. The burst release at the initial stage was attributed to the drug species originally adsorbed onto the surface areas of the hydrophobic micellar cores.46-48 By contrast, the drug release from the SS-NPs was significantly enhanced with the NPs receiving the photodynamic or X-ray irradiation activation. As expected, the prolonged duration of the photodynamic treatment from 5 to 10 min inevitably increased the ROS generation (Figure S7), thus leading to the substantially increased drug release from SSNPs. Comparable drug release profiles of SS-NPs pretreated respectively with the 15-Gy Xray irradiation and the 5-min photodynamic reaction at 525 nm using rose bengal as the



photosensitizer were observed (Figure 4b), implying that the ROS-induced oxidative effects by the two approaches on the NP structure are similar as aforementioned (Figure 3). 3.4. In vitro cytotoxicity of the ROS-responsive nanoparticles. The cytotoxicity of drug-free NPs (S-NPs and H-NPs) with or without the ROS pretreatment against murine BNL 1MEA.7R.1 hepatocellular carcinoma cells was determined by MTT assay. The results are shown in Figure S9. Irrespective of the ROS pretreatment induced by the photodynamic reaction, both S-NPs and H-NPs exhibited negligible cytotoxicity against the cancer cells in the concentration range employed. The data demonstrate that the drug-free NPs and their oxidized products particularly from S-NPs are essentially nontoxic toward the cancer cells. It is noteworthy that the cytotoxicity of rose bengal employed herein as a photodynamic sensitizer with or without the pretreatment of photo-irradiation was negligible (Figure S10). The cellular uptake of SN38 from SS-NPs and SH-NPs, respectively, before and after the ROS treatment by the BNL 1MEA.7R.1 cancer cells was examined by LSCM. Free SN38 was also employed for comparison. The LSCM images are illustrated in Figure 5a. The fluorescence signals of SN38 within the cancer cells after co-incubation with ROS-insensitive SH-NPs were rather weak, irrespective of the ROS pretreatment. The fluorescence intensity was slightly enhanced for the SS-NP sample without the ROS treatment. By contrast, the intracellular fluorescence signals were highly increased for the SS-NP sample pretreated with


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either photodynamic reaction or X-ray irradiation. Apparently, in the absence of the structural change of NPs induced by the ROS oxidation, the drug release from the NPs (SH-NPs and HHNPs) occurring either extracellularly or intracellularly is rather limited. The slight increase of the intracellular signal intensity of SN38 from the ROS-responsive sample without the pretreatment is ascribed partly to the oxidative stress in cancer cells. It is well known that the entry of free drug into cells relies mainly on passive diffusion through cell membranes while the endocytic pathway is usually adopted for the uptake of NPs into cells. Since the disruption of the SS-NPs is a time-requiring processes after the ROS pretreatment (Figure 3), the drug release may occur from the pretreated SS-NPs intra- or extracellularly. This implies that the drug uptake by the cancer cells may occur via either the passive diffusion of the SN38 species released extracellularly or endocytosis while still being entrapped within the NPs. Figure 5a, however, demonstrates the strong dependence of the intracellular drug accumulation upon the ROS activation of SS-NPs induced by either photodynamic reaction or X-ray irradiation. The in vitro chemotherapeutic effect of SS-NPs receiving the ROS pretreatment on cancer cells was studied. SH-NPs and free drug were also employed as the controls. As shown in Figure 5b, the viability of the cancer cells incubated with various NPs and free drug was reduced proportionally to the SN38 dose. The cytotoxicity of the ROS pre-treated SS-NPs against the cancer cells was most profound among all the NP formulations and comparable to free SN38. The values of the 50% cellular growth inhibition (IC50) are also summarized in



Table S2. The data revealed that the SH-NPs and SS-NPs alone in the absence of ROS activation exhibited ineffective cytotoxic effect, with the IC50 values as high as 15.59 and 15.32 μM, respectively. By contrast, the IC50 value of the SS-NPs pre-activated by the 15-Gy X-ray irradiation was reduced to 2.07 μM, comparable to free drug (2.28 μM) and ca 7-fold lower than that without activation. Considering the comparable anticancer efficacy between free SN38 and X-ray pre-irradiated SS-NPs against BNL 1MEA.7R.1 cancer cells, it is highly plausible that SN38 was released from the SS-NPs to a substantial level extracellularly and thus the cell entry was virtually governed by the rapid passive diffusion.49 The X-ray irradiation dose also plays an important role influencing the drug release rate and thus the cytotoxicity. With an increase in the irradiation dose from 10 to 15 Gy, the IC50 value of the SS-NPs against the cancer cells was reduced ca 2-fold. In addition to the evaluation of the in vitro anticancer effect of the ROS-mediated chemotherapy from the ROS-responsive NP system, the combinational therapeutic effect from X-ray irradiation together with radiation-induced chemotherapy on the BNL 1MEA.7R.1 cells was also examined. The X-ray irradiation doses of 0, 10 and 15 Gy were employed because, in the irradiation dose range, the additional chemotherapeutic performance on cell apoptosis can still be easily identified. The results are shown in Figure 5c. Without the chemotherapy, the cell viability was reduced proportionally to the X-ray irradiation dose. A significant difference in the chemotherapeutic efficacy between SS-NPs and SH-NPs at an irradiation dose of 10 Gy was observed, indicating the prominent


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X-ray effect at this dose to trigger drug release from SS-NPs. By contrast, the chemotherapy effect of SS-NPs without the X-ray irradiation was found similar to the SH-NPs. In spite of a slow process, the drug release from both SS-NPs and SH-NPs might also be induced via the enzymatic cleavage of polymers by lipase inside the cancer cells. With the irradiation dose being increased to 15 Gy, the chemotherapeutic effect of SS-NPs was enhanced as expected, substantially higher than that of the SH-NP counterpart and comparable to that of free SN38. In contrast to the SH-NPs remaining insensitive to the irradiation, the results clearly demonstrate the effective response of the SS-NPs in mediating drug release to the X-ray stimulus at the doses of both 10 and 15 Gy.



Figure 5. (a) LSCM images of the cellular uptake of SN38 from SH-NPs and SS-NPs by BNL 1MEA.7R.1 hepatic carcinoma cells. The ROS pretreatment was induced either by photodynamic reaction (525 nm, 0.5 w/cm2) of rose bengal (5.0 μM) for 5 min or X-ray irradiation (15 Gy). Free SN38 was used as positive control. F-actin was stained in green. Scale


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bars are 20 μm. The mean SN38 fluorescence intensity per cell was obtained by normalization of the total SN38 fluorescence intensity with the number of cells (140-150 cells) (n = 3). (b) Viability of BNL 1MEA.7R.1 cells treated with SS-NPs and SH-NPs of different SN38 concentrations with or without the ROS pre-activation (n = 5). (c) Viability of BNL 1MEA.7R.1 cells after combinational treatment of radiotherapy (0, 10 and 15 Gy) with chemotherapy (n = 5). The concentrations of free SN38 and SN38 within SS-NPs and SH-NPs were 0.312 μM. Samples were analyzed with one-way ANOVA, followed by Bonferroni correction for multiple comparisons. ***P < 0.001 and n.s. P > 0.05. Error bars represent mean ± s.d.

3.5. In vivo therapeutic efficacy and reoxygenation of irradiated tumor tissues. To validate this therapeutic strategy and investigate the in vivo X-ray irradiation mediated drug release from the ROS-responsive drug delivery system in terms of tumor growth inhibition, the BALB/c mice bearing subcutaneous BNL 1MEA.7R.1 tumors were treated, respectively, with free drugs and NPs by intratumoral injection, followed by X-ray irradiation (15 Gy). The tumor size and body weight of tumor-bearing mice were evaluated daily over 17 days postinjection. As presented in Figure 6a, the tumor volume from the mice treated only with SHNPs and SS-NPs reached ca 2200 mm3, indicating similar, yet insufficient chemotherapeutic effects of SS-NPs and SH-NPs in the absence of pertinent external stimulus. The results are in



agreement with the in vitro anticancer effects of these two NPs (Figure 5), the enhanced level of ROS in tumor cells and the presence of intracellular lipase might be important in mediating the drug release. Although the sound tumor growth inhibition was observed with the X-ray irradiation (15 Gy) alone, the combinational treatment of radiotherapy with ROS-mediated chemotherapy from SS-NPs showed an essentially full inhibition of tumor growth, similar to the radiotherapy/free SN38 group. These data manifest that the combinational radio/chemotherapy exhibited more prominent antitumor efficacy than the single modality treatment. More importantly, this in vivo study validated the radiotherapy-activated chemotherapy from the SS-NP system in combination with the X-ray irradiation for effective local treatment of malignant solid tumors. In comparison with photodynamic therapy which has also been employed as a trigger of chemotherapy from the ROS-responsive NP delivery systems,31,50 X-ray irradiation endows the superior light tissue penetration and therefore the more effective activation for chemotherapy. In conjunction with the data of the in vivo tumor growth inhibition, the tumors harvested from the sacrificed mice receiving SSNPs/radiotherapy were obviously smaller than those from both the SH-NPs and PBS groups as illustrated in Figure 6b and c. The variation of the body weights of mice receiving various treatments is shown in Figure 6d. No apparent change in body weight was observed in all experimental groups, implying the absence of severe acute toxicity from the treatments. Severe tumor tissue damage was observed by the H&E examination in both the groups receiving the


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free drug/radiotherapy and SS-NPs/radiotherapy treatment (Figure 6e). In agreement with the reduction in tumor mass as aforementioned, such damages signify the prominent antitumor effect from the combined SS-NPs/radiotherapy approach by virtue of its chemotherapeutic efficacy comparable to free drug. Again, this demonstrates radiotherapy-mediated chemotherapy from the SS-NP system developed in this work in combination with local X-ray irradiation for superior antitumor efficacy.



Figure 6. (a) Tumor inhibitory effect of various treatments (PBS, free SN38, SH-NPs and SHNPs) with or without 15 Gy X-ray irradiation (+/- irradiation) on BNL 1MEA.7R.1 tumorbearing mice in the subcutaneous model (n = 5 each group) and the area under the curves (AUCs) attained from the tumor growth profiles. A single dose of chemotherapy (SN38 2.0


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mg/kg) was administered by intratumoral injection at day 0. Samples were analyzed with oneway ANOVA (**P < 0.01 and n.s. P > 0.05). (b) Morphology of the tumors in each group harvested from the sacrificed mice. (c) Tumor mass harvested at day 17 post treatment (the end point). Samples were analyzed with one-way ANOVA (*P < 0.05). (d) Body weights of the tumor-bearing mice post treatment. (e) H&E examination of tumor sections harvested from the tumor-bearing mice at the end point of the treatments. Bonferroni correction was applied for multiple comparisons. Scale bars are 200 μm. Error bars represent mean ± s.d. (n = 5).

Although the tumor growth was appreciably inhibited from the mice treated with X-ray irradiation alone or together with SH-NPs, the tumor regrowth was observed at day 11 after the treatment (Figure S11). By contrast, the regrowth was efficiently inhibited by the dual modality treatment of either SS-NPs/radiotherapy or SN38/radiotherapy. As reported previously, tumor reoxygenation usually occurs after radiotherapy treatment, resulting in the increased ROS level and HIF1-α stabilization in tumor.38,51,52 The latter further upregulates the expression of vascular endothelial growth factor A (VEGFA).38 VEGFA is a crucial cytokine to tumor angiogenesis required for tumor regrowth and cancer cell proliferation.53-55 Thus, the IHC staining of VEGFA and a tumor angiogenesis marker, CD31, in tumor tissue was conducted. The signal distributions of VEGFA and CD31 in tumor tissues are illustrated in Figure 7a. The fluorescence signal intensity of both VEGFA and CD31 in tumors treated,



respectively, with SS-NPs/radiotherapy and SN38/radiotherapy is significantly reduced compared to the groups receiving PBS, radiotherapy and SH-NPs/radiotherapy treatments. The integrated optical density (IOD) values of VEGFA and CD31 signals in the tumor tissue of each experimental group were attained by normalizing the mean IOD of the images against the mean IOD in the PBS group.56,57 The IOD of CD31 signal is also known as the CD31 density or the mean vessel density. The reduction of the IOD values of both VEGFA and CD31 signals in the SS-NPs/radiotherapy group (also the free SN38/radiotherapy group as a positive control) compared to other treated groups, strongly implies the significant decrease of angiogenesis and tumor regrowth (Figure 7b and c). The enhanced ROS generation resulting from tumor reoxygenation after X-ray irradiation was also examined by DCFH-DA staining.58,59 As shown in Figure S12, the DCFH-DA signal in the irradiated tumor was substantially increased compared to that without irradiation. The time-evolved DCFH-DA signal intensity was also observed. Owing to the increase in ROS in the tumor receiving the X-ray irradiation, the ROSmediated chemotherapy from the SS-NPs became more persisted compared to the SH-NP group, leading to a prominent reduction in angiogenesis and tumor regrowth via apoptosis of tumor cells.


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Figure 7. (a) Fluorescence images of tumor sections harvested from the mice bearing BNL 1MEA.7R.1 tumor at day 17 after treatments. Cell nuclei, VEGFA and CD31 in tumor sections were stained and identified. Scale bars are 200 μm. (b,c) IOD of VEGFA and CD31 in tumors receiving various treatments. Samples were analyzed with one-way ANOVA, followed by Bonferroni correction for multiple comparisons. *P < 0.05, **P



< 0.01, ***P < 0.001, and n.s. P > 0.05. Error bars represent mean ± s.d. (n = 20).

4. CONCLUSIONS A dual modality treatment strategy combining radiotherapy with radiotherapymediated chemotherapy from the ROS-responsive SS-NP system was developed in this work for effective local treatment of malignant solid tumors. The SS-NPs comprised PSDEA and PEG-PSDEA-PEG as the major component of polymeric NPs in which SN38 was entrapped by hydrophobic association. The ROS-mediated oxidation of the hydrophobic sulfide residues in polymer main chains into the hydrophilic sulfoxide groups led to the structural change of polymer NPs and the release of chemodrug. The in vitro cytotoxicity study against BNL 1MEA.7R.1 hepatic cancer cells demonstrated a comparable chemotherapeutic effect of the X-ray pre-activated SS-NPs to free drug. A significant difference in therapeutic efficacy between the combined treatments of ROSresponsive SS-NPs/radiotherapy and ROS-insensitive SH-NPs/radiotherapy in vitro was also observed. The evaluation of the in vivo therapeutic performance of the combined SS-NPs/radiotherapy treatment in which the SS-NPs were administered by intratumoral injection against hepatic carcinoma in the subcutaneous model showed essentially full inhibition of tumor growth similar to the free SN38/radiotherapy approach, strongly indicating the effective drug release upon the activation of SS-NPs


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by X-ray irradiation. The IHC examination of tumor tissues has also revealed that, with the tumor being treated with X-ray irradiation, the reoxygenation-induced ROS increase in tumor further facilitated the persistent chemotherapeutic effect from SS-NPs on suprression in angiogenesis and tumor regrowth. Future studies will focus on the combined local treatment of radiotherapy with the SS-NP system via intravenous administration against the hepatic solid tumor in the orthotopic tumor-bearing animal model for improved therapeutic effect and reduced adverse side effects.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: XXXXXXXXX. Chemical structures, synthetic routes and 1H-NMR spectra of PHMA and PEG-PHMA-PEG (Figure S1), the biodegradability of PSDEA (Figure S2), the 1H-NMR spectra of oxidized PSDEA and PHMA (Figure S3), GPC elution profiles of PSDEA before and after ROSoxidation (Figure S4), DLS size distribution profiles of different NPs (Figure S5), the colloidal and storage stability of SS-NPs and SH-NPs (Figure S6), evaluation of ROS generation by SOSG (Figure S7), fluorescence emission spectra of SS-NPs in H2O/DMSO solutions (Figure S8), cytotoxicity of drug-free S-NPs and H-NPs (Figure



S9), cytotoxicity of rose bengal against cancer cells (Figure S10), tumor regrowth profiles after 15-Gy X-ray irradiation alone and combined irradiation/SH-NP treatments (Figure S11), and DCFH-DA staining of ROS in irradiated tumor (Figure S12). Molecular weight characterization of polymers (Table S1) and IC50 of SN38 in different formulations against BNL 1MEA.7R.1 hepatic carcinoma cells (Table S2).

AUTHOR IMFORMATION Corresponding Authors *E-mail: [email protected] Author Contributions §

These authors contributed equally to this work

Notes The authors declare no competing financial interest.

ACKNOWLEDGEMENTS This work is supported by the Ministry of Science and Technology, Taiwan (MOST 1042627-M-007-009, MOST 105-2627-M-007-007 and MOST 106-2627-M-007-002) and National Tsing Hua University, Taiwan (104N2742E1).


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Radiotherapy-Controllable Chemotherapy from ROS-Responsive Polymeric Nanoparticles for Effective Local Dual Modality Treatment of Malignant Tumors

Te-I Liu1§, Ying-Chieh Yang2§, Wen-Hsuan Chiang3, Chun-Kai Hung1, Yuan-Chung Tsai1, Chi-Shiun Chiang1, Chun-Liang Lo4, and Hsin-Cheng Chiu1*


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Radiotherapy-Controllable Chemotherapy from ... - ACS Publications

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