PaclitaxelâPaclitaxel Prodrug Nanoassembly as a Versatile

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Paclitaxel-paclitaxel Prodrug Nanoassembly as a Versatile Nanoplatform for Combinational Cancer Therapy Xiangfei Han, Jinling Chen, Mengjuan Jiang, Na Zhang, Kexin Na, Cong Luo, Ruoshi Zhang, Mengchi Sun, Guimei Lin, Rong Zhang, Yan Ma, Dan Liu, and Yongjun Wang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b13057 • Publication Date (Web): 22 Nov 2016 Downloaded from http://pubs.acs.org on November 22, 2016

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ACS Applied Materials & Interfaces

Paclitaxel-paclitaxel Prodrug Nanoassembly as a Versatile Nanoplatform for Combinational Cancer Therapy

Xiangfei Han§, Jinling Chen‡, Mengjuan Jiang§, Na Zhang‡, Kexin Na§, Cong Luo§, Ruoshi Zhang§, Mengchi Sun§, Guimei Lin⊥, Rong Zhang‖, Yan Ma#, Dan Liu*, ‡ and Yongjun Wang*, § §

School of Pharmacy, Shenyang Pharmaceutical University, 103 Wenhua Road, Shenyang,

110016, China. ‡

Key Laboratory of Structure-Based Drug Design and Discovery, Ministry of Education,

Shenyang Pharmaceutical University, China. ⊥

School of Pharmacy, Shandong University, Jinan 250012, China.

‖

School of Pharmacy, Guangdong Pharmaceutical University, Guangzhou, China.

#

School of Chinese Materia Medica, Guangzhou University of Chinese Medicine, Guangzhou

510405, China KEYWORDS: disulfide, redox responsive, photothermal therapy, high drug loading, anticancer.

ACS Paragon Plus Environment

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ABSTRACT: Recently, nanomedicine without drug carriers attracts many pharmacists’ attention. A novel paclitaxel-s-s-paclitaxel (PTX-s-s-PTX) conjugate with high drug loading (~78%, w/w) was synthesized by conjugating paclitaxel to paclitaxel by using disulfide linkage. The conjugate could self-assemble into uniform nanoparticles (NPs) with 1, 1-dioctadecyl-3, 3, 3, 3-tetramethylindotricarbocyanine iodide (DiR) encapsulated within the core of PTX-s-s-PTX NPs for photothermal therapy (PTT). The DiR-loaded self-assembled nanoparticles (DSNs) had mean diameter of about 150 nm and high stability in biological condition. Disulfide bond is utilized as a redox-responsive linkage to facilitate a rapid release of paclitaxel in tumor cells. DSNs indicated significant cytotoxicity as a result of the synergetic chemo-thermal therapy. DSNs were featured with excellent advantages, including high drug loading, redox−responsive releasing behavior of paclitaxel, capability of loading with photothermal agents and combinational therapy with PTT. In such a potent nanosystem, prodrug and photothermal strategy are integrated into one system to facilitate the therapy efficiency.

1. INTRODUCTION Cancer is a prevailing threat to human beings owing to its high incidence, severe mortality and low survival rate.1 In spite of the discovery of several potent new therapeutic molecules, the clinical use and efficacy of conventional anticancer agents is still hampered by nonspecific biodistribution, rapid metabolism and clearance and multitude drug resistance. Over the past several years, many carrier-based drug delivery strategies have been developed, including liposomes,2 polymeric nanoparticles,3 dendrimers, inorganic nanoparticles4, protein analogous micelles, nanodiamonds, albumin-bound nanoparticles and molecularly targeted nanoparticles. However, current carrier-based nanomedicines have serious disadvantages: (1) poor drug loading


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capacity, (2) the burst release of the therapeutic drug from carriers, (3) the difficulty of scale up owing to the complexity of multicomponent. Therefore, the development of nanomedicine which combines high drug payloads, targeted anti-cancer and controlled releasing activity is of prime interest for pharmacists. Nowadays much attention has been paid to one-component nanomedicine. One-component nanomedicine (OCN) with one single chemical substance allows for precise control and optimization of the drug loading capacity and physicochemical properties.5,6 Compared with traditional carrier-based nanomedicine, OCN does not require any additional carriers and can itself possess desired physicochemical features for preferential accumulation at target sites. In general, OCN has the following unique properties: (1) OCN allows for precise control over the drug loading. High drug loading capacity can also be obtained; (2) The physicochemical features of the OCN can be tuned by simply optimizing the molecular structure, which affects the drug loading, circulation time in vivo and drug release; (3) easy scale-up production and accelerate laboratory-to-industry translation. Photothermal therapy (PTT) is a hyperthermia therapeutic approach that exploits photosensitive agents to kill cancer cells by heat generated from optical energy.7,8,9 Compared with traditional chemotherapy, PTT is noninvasive and does not damage normal tissues with localized laser exposure. There were such many extraordinary inorganic photothermal agents as Au,10, MoS2,11,12 Bi2Se313,14 and TiO215. However, unlike organic materials, inorganic NPs are often rapidly sequestered from the blood and accumulate in reticuloendothelial system (RES) organs (liver, spleen, etc.) in spite of pegylation. A majority of pegylated NPs end up in the liver and spleen after circulation, resulting in elevated potential long-term toxicity.16 In addition, inorganic materials were difficult to degrade in biological condition which might cause


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unpredictable side effect, hindering their clinical application.17 Furthermore, the therapeutic efficiency of phototherapy was based on both the power density of the NIR light and irradiation time together. It is imperative to balance laser power and irradiation time, because long therapeutic time might reduce the compliance of patients and hinder the clinic application. DiR is a lipophilic near-infrared (NIR) fluorescent dye with strong light-absorbing capability, which is commonly used for in vivo imaging. Moreover, DiR has a maximal emission wavelength of 780 nm with high fluorescence quantum yield, which makes it a promising candidate for PTT application. However, the lipophilic DiR tends to aggregate in water, leading to low fluorescence quantum yield and fluorescent intensity. The high fluorescent intensity and photostability are obtained by encapsulating DiR into nanoparticles. Recently, DiR has been used for in vivo optical imaging and cell tracking in combination with polymeric nanoparticles.18,19 However, its potential photothermal effect was scarcely investigated. The cytosolic GSH concentration in tumor cells is several times higher than that in normal cells, owing to the crazy growth of tumor cells.20,21 The elevated GSH level in tumor cells was widely used for designing redox-sensitive nanosystem, and disulfide bond was extensively utilized as a redox-responsive linkage to facilitate controlled release of anticancer drugs in tumor cells.22,23,24,25,26 Although

many

chemo-photothermal

combinational

therapies

were

developed,27,28,29,30 there were few literatures concentrating on redox-responsive self-assembly prodrugs as OCN encapsulating photothermal agents. In this context, we described herein a new concept of nanomedicine with high drug loading, redox−responsive releasing properties and loading with imaging or photothermal agent. The PTX-s-s-PTX conjugate is able to co-assemble with DiR to form unanimous nanoparticles.


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Disulfide bond is used as a redox-responsive linkage to facilitate a rapid release of paclitaxel in tumor cells. (Scheme1) The NIR-light triggered temperature increase could further lead to significant cytotoxicity, which synergistically generated highly efficient thermo-chemotherapy with total tumor ablation. Although disulfide bond has been widely used as redox-sensitive linkage for designing anticancer conjugates, to our knowledge, this is the first attempt to develop DiR-loaded prodrug-nanoplatform based on disulfide bond-bridged hydrophobic conjugates of PTX and PTX for thermo-chemo combinational therapy. 2. EXPERIMENTAL SECTION 2.1 Materials and Instruments. Paclitaxel (PTX) was purchased from Beijing Maisuo Chemical Technology Co Ltd (Beijing, China). 4-methylmorpholine (NMM), p-aminobenzyl alcohol (PABA), isobutyl chloroformate (IBCF), pyridine, 4-nitrophenyl chloroformate, 3, 3’-dithiodipropionic acid (DTDPA) and 4-Dimethylaminopyridine (DMAP) were bought from Aladdin Industrial Corporation (Shanghai, China). 2-distearoyl-snglycero-3-phosphoethanolamine-N-[methoxy (polyethylene glycol)-2000] (DSPE-PEG2000) was obtained from Shanghai Advanced Vehicle. The human lung cancer cells (A549) were purchased from the cell bank of Chinese Academy of Sciences (Beiijng, China). Roswell Park Memorial Institute (RPMI-1640), Dulbecco’s Modified Eagle Medium (DMEM, high glucose), trypsin and 3-(4, 5-dimethylthiazol-2-yl)-2, 5diphenyltetrazoliumbromide (MTT) were obtained from Gibco (Beijing, China). Isoflurane was bought from RWD life science Co. Ltd. (Shenzhen, China).Fetal bovine serum (FBS) and CS were purchased from Hyclone (Beijing, China) and KeyGEN (Nanjing, China), respectively. Dimethylsulfoxide (DMSO) was purchased from Kemeng (Tianjin, China). 1, 1-dioctadecyl-3, 3,


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3, 3-tetramethylindotricarbocyanine iodide (DiR) was brought from AAT Bioquest (Beiijng, China). All other reagents and solvents used in this article were of analytical grade. 2.2 Synthesis of Paclitaxel-Paclitaxel Conjugate. 3, 3’-dithiodipropionic acid (DTDPA, 0.48 mmol), NMM (1.05 mmol) and isobutyl chloroformate (IBCF, 1.05 mmol) were dissolved in anhydrous THF. The mixture was stirred for 3h at -30 oC under nitrogen atmosphere. The anhydrous THF solution of NMM (1.19 mmol) and PABA (1.19 mmol) was added dropwise to the reaction liquid and the reaction was stirred at 30oC for 4 h, then the solution was allowed to room temperature for another 12 h. White solid was obtained through recrystallization from dichloromethane/menthol (DTDPA-PABA, 27.5%). 0.80 mmol DTDPA-PABA was dissolved in andryous THF and the 4.00 mmol pyridine was added. The THF solution of 4-nitrophenyl chloroformate (4.80 mmol) was added dropwise to the mixture. The reaction lasted 4 h under nitrogen protection at -30oC. The solution was stored at room temperature overnight. At the end of the reaction, the solvent was evaporated and washed by dichloromethane. The insoluble substance was remaining DTDPA-PABA. Finally, the organic layer was washed with saturated NH4Cl and NaCl solution for three times. Then it was purified by silica gel column chromatography (HEX/EA= 3:1) to give a white solid (DTDPA NO2, 30.2%). 0.35 mmol DTDPA-NO2 was dissolved in anhydrous dichloromethane，paclitaxel（0.70 mmol) and DMAP (8.10mmol) were added to the dichloromethane solution. The reaction was allowed to room temperature overnight. The organic layer was washed with saturated NH4Cl solution. Lastly, it was purified by silica gel column chromatography (PET/Acetone = 1:1) to give a white solid (PTX-s-s-PTX, 57.2%).


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1

H-NMR spectra was recorded on a Bruker ARX-400 NMR spectrometer operating at 400

MHz and using deuterated chloroform (CDCl3) as solvent. The chemical shifts were corrected against residual solvent signals. The accurate mass of sample was performed using a Bruker micrOTOF-Q time of flight mass spectrometer, mass range of m/z was 50 ~ 3000, ion type is [M + Na] 2+. 2.3 Preparation and Characterization of DSNs. PTX-s-s-PTX

conjugate

could

self-assemble

into

nanoparticles

with

previous

nanoprecipitation method. The THF solution of PTX-s-s-PTX was added into water with vigorous stirring, the self-assembly nanoparticles were formed and DSPE - PEG2000 was then added to the solution for pegylation. Finally, THF was evaporated. To prepare the DiR-loaded self-assembly nanoparticles, the prepared procedure was similar to mentioned above. The difference was that DiR and PTX-s-s-PTX were first dissolved in THF, and then added to water with vigorous stirring. For DSNs, the ratio of paclitaxel-paclitaxel conjugate, DSPE-PEG2000 and DiR was 5:1:2.5 (w/w). The concentration of DiR was quantified using its absorbance at 762 nm with a mass extinction coefficient of 196.3 mL mg

−1

cm−1. The size and zeta potential of

DSNs were measured by DLS. The measurements that performed in triplicate were carried out via a Zetasizer (Nano ZS, Malvern. UK) and the results were evaluated by mean ± standard deviation (SD). The morphology of DSNs was observed through TEM (JEM2100, JEOL. Japan) operated at an accelerating voltage of 200 kV. The samples were prepared by dropping 10 µL of nanoparticle solution on a carbon-coated copper grid for 1 min and then dried by filter paper. Finally, 1 % phosphotungstic acid (5 µL) was dropped for 30 seconds and air dried. 2.4 Physical and Chemical Stability of DSNs.


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The changes of diameter and PDI of DSNs with or without being incubated in PBS (pH 7.4) containing 10% FBS with time for 7d measured by DLS. The release of paclitaxel from DSNs and the degradation of paclitaxel-paclitaxel conjugate were studied under shaking (100 rpm) at 37 °C in the phosphate buffer saline (PBS, pH 7.4) containing 30% ethanol with or without 10 mM Dithiothreitol (DTT), respectively. Typically, 1 mL of DSNs dispersion (0.5 mg/mL) was added to 30 mL of release media. The release studies were conducted at different time intervals: 0, 0.5,1, 2, 4, 6, 8, 12, 24h. At desired time intervals, 1 mL samples was withdrawn and determined. The content of paclitaxel and paclitaxel-paclitaxel conjugate was determined by high-performance liquid chromatography (HPLC) with Waters e2695 Separations Module and Waters 2489 UV/Visible Detector on a reverse ODS Phenomenex -C18 column (250 mm×4.6 mm, 5 µm) thermostated at T = 30 ℃ with UV detection at 227 nm using a mixture of acetonitrile/water (75:25, v/v) as a mobile phase with a flow rate of 1 mL/min. Each experiment was repeated in triplicate and the results were expressed by mean ± SD. The accurate mass of intermediate PTX-SH was confirmed using HPLC and Bruker microTOF-Q time of flight mass spectrometer, mass range of m/z was 50 ~ 3000, ion type is [M + Na] +. 2.5 The Mechanism of the Conjugate Self-assembling into Nanoparticles. A tetramer structure of PTX-s-s-PTX for molecular dynamics (MD) simulations was generated by GaussView 5.0.8 (Gaussian, Inc., Wallingford, CT). Antechamber was used to generate parameters of the PTX-S-S-PTX for the AMBER and associated GAFF force fields, and the AM1-bcc model was used to generate the atomic charges. The MD simulations, including the energy minimization, were performed by using AMBER 11 software package. The system was solvated with the TIP3P water model in a truncated octahedron box with a 10 Å distance around the solute using xLEAP. The PTX-S-S-PTX molecules were fixed with a 50 kcal mol-1Å-


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2constrain, and solvent was energy minimized for 2,000 steps using the steepest descent (SD) method followed by a further 2,000 steps using conjugate gradient algorithms. Subsequently, these initial harmonic restraints were gradually reduced to zero during energy minimizations. After that, the system was minimizedby the SD method and switched to conjugate gradient every 3,000 steps for a total of 6,000 steps without harmonic restraints. Thereafter, the system was gently heated from 10K to 300K, applyingharmonic restraints with a force constant of 10 kcal mol-1Å-2 on the solute atoms, and then equilibrated for 2,000 ps. Finally, production MD 7 simulation was carried out for 10 ns to check the self-assembly process. The particle mesh Ewald (PME) summation method was applied to treat the long range electrostatic interactions with a periodic boundary condition. All bonds involving hydrogen atoms were restricted by the SHAKE algorithm. The time step in all MD simulations was 2 fs. PyMOL and VMD software were used to visualize the trajectories and to depict structuralrepresentations. A simple study to explore the mechanism of the conjugate to self-assemble into nanoparticles was conducted by imaging the dynamics of crystal growth. THF solution of paclitaxel and paclitaxel-paclitaxel conjugate was placed onto a glass slide, respectively. After drying and desiccating, add a drop of water to each sample. Pictures were taken after the slides were kept in a humidified chamber at room temperature for 4h and pictures were taken. 2.6 Photothermally Induced Temperature Changing of DSNs. The photothermal conversion abilities of PTX-s-s-PTX NPs, DiR solution and DSNs (500 µg mL −1, in terms of DiR, respectively) was evaluated by recording the temperature of them under the irradiation of a 808nm laser at 0.5W cm −2. 2.7 Cell Culture.


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A549 lung cancer cells were cultured in RPMI Medium 1640, respectively with 10% fetal bovine serum (FBS), 100 µg/mL streptomycin and 100 units/mL penicillin in a humidified cell culture incubator at 37℃, 5% CO2. The cells were subcultured by 0.25% trypsin without EDTA when grown up to 80%. 2.8 Cell Viability Assay. A549 cells were seeded in a 96-well plate (5000 cells/100 µL/well) and cultured at 37 ℃, 5% CO2. Next day, the media was removed and then replaced with fresh media containing series of concentrations of formulations. Paclitaxel were dissolved by DMSO and then diluted by fresh media. The final concentration of DMSO was 0.1%. PTX-s-s-PTX, DiR solution, DSNs were diluted by fresh media directly, with fresh media as the control group. When the cells were cultured for 48h, the media was displaced by another 100 µL fresh media, then, 15 µL of MTT reagent (5 mg/mL) was added to the media and the cells were incubated continuously for 4h. Then the media was replaced with 100 µL DMSO and vibrated for 10 min. The photothermal cytotoxicity of DSNs in the A549 cells was determined by LIVE/DEADViability/Cytotoxicity Kit to evaluate the PTT effects on cell viability. Cells were pretreated with all formulation for 4h, and then exposed to the 808 nm laser at a power density of 0.5 W cm

−2

for 5 min. By contrast,

cells in PTX solution without NIR irradiation were performed as control. The effects of these formulations on the cytotoxicity were evaluated using the standard MTT assay. The absorbance at 570nm of each well was measured on a microplate reader. Eq. (1) was employed to calculate the cell inhibition rate: Cell inhibition rate (%) = 1-Ae/Ac

(1)


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In Eq. (1), Ae is the mean absorbance of the experimental group and Ac is the mean absorbance of the control group. The IC50 values were calculated by SPSS software. 2.9 In Vivo Targeting Effect. The tumor biodistribution of DSNs was assessed by tumor-bearing A549 mice (Laboratory Animal Center of Shenyang Pharmaceutical University, Shenyang, Liaoning, China) using the IVIS in vivo imaging system (PerkinElmer). The near infrared fluorescence dye DiR was loaded in DSNs for near-infrared (NIR) fluorescence imaging. Tumor-bearing mice were established by injecting a suspension of 1 × 106 A549 cells in PBS into the right axillary flank of female kunming mice. Mice with subcutaneous tumors of approximate 150 mm3 were subjected to treatment. DSNs was injected intravenously via the tail vein into the tumor-bearing mice to trace profiles of the tumor accumulation and biodistribution of DSNs. Mice were sacrificed at 24 h and the ex vivo biodistribution imaging signals of DiR in each organ and tumor were immediately detected. 2.10

Statistical Analysis. All the results were represented as mean ± SD. The statistical significance was determined

using SPSS software. P < 0.05 was considered significant, and P < 0.01 was considered highly significant. 3. RESULTS AND DISCUSSIONS 3.1 Synthesis of Paclitaxel-Paclitaxel Conjugate. Firstly, 3, 3'-dithiodipropionic acid (DTDPA) was reacted with p-aminobenzyl alcohol (PABA) with the aid of 4-methylmorpholine (NMM) and isobutyl chloroformate under N2


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atmosphere. Then DTDPA-PABA was activated by 4-nitrophenyl chloroformate (PNPCF) to achieve the carbonate derivative. Finally, DTDPA-NO2 was reacted with paclitaxel to gain the target compound PTX-s-s-PTX. (Figure S1) The accurate m/z

(2+)

1112.3583 ions was

determined as a double sodium adduct ([C116H122N4O34S2Na2]2+ ions) for PTX-s-s-PTX. (Figure S2) The predicted molecular formula is matched with actual molecular formula. The chemical structure of PTX-s-s-PTX was confirmed by 1H-NMR (Figure S3). The 1H NMR spectra of PTX-s-s-PTX conjugate (400 MHz, CDCl3) was analyzed as following: δ 8.12 (d, J = 7.4 Hz, 4H, o-Ph1), 7.71 (d, J = 6.7 Hz, 4H, o-Ph3), 7.61 (t, J = 7.0 Hz, 2H, p-Ph1), 7.52-7.50 (m, 6H, m-Ph1, p-Ph3), 7.48-7.46 (m, 8H, o-Ph2, m-Ph2), 7.40-7.36 (m, 14H, m-Ph3, m-Ph4, pPh2, o-Ph4), 7.19 (m, 2H, -SCH2CH2OCNH-), 7.06 (d, J = 8.1 Hz, 2H, 3’-NH), 6.27 (s, 2H, H10), 6.20 (t, J = 8.4 Hz, 2H, H-13), 5.96 (d, J = 8.3 Hz, 2H, H-3’), 5.67 (d, J = 7.0 Hz, 2H, H-2), 5.45 (br.s, 2H, H-2’), 5.14 (d, J = 11.5Hz, 2H, 2’-OCO2CH2-), 5.05 (d, J = 11.5Hz, 2H, 2’OCO2CH2-), 4.96 (d, J = 9.3 Hz, 2H, H-5), 4.40 (q, 1H, H-7), 4.29 (d, J = 8.3 Hz, 2H, H-20), 4.18 (d, J = 8.3 Hz, 2H, H-20), 3.78(d, J = 6.9 Hz, 2H, H-3), 3.02 (br.s, 4H, CH2CH2SSCH2CH2-), 2.73 (br.s, 4H, -CH2CH2SSCH2CH2-), 2.55(m, 2H, H-6), 2.43 (s, 6H, 4-OAc), 2.34 (m, 2H, H-14), 2.19 (s, 6H, 10-OAc), 2.08 (m, 2H, H-14), 1.88 (m, 2H, H-6), 1.79 (s, 6H, Me-18), 1.67 (s, 6H, Me-19), 1.20 (s, 6H, Me-17), 1.13 (s, 6H, Me-16). 1H-NMR spectrum displayed new peaks at δ 5.14/5.05 and δ 3.02/2.73, which can be attributed to the methylene protons of PABA and the disulfide linker, respectively. Furthermore, the chemical shift of H2’ in PTX moved to the low field to 5.45, indicating that successful conjugation at 2’OH of paclitaxel. The results above indicated that paclitaxel-s-s-paclitaxel conjugate was successfully obtained by conjugating paclitaxel with paclitaxel via disulfide bond. 3.2 Characterization of DSNs.


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The morphology and size distribution of DSNs were characterized by transmission electron microscopy (TEM) and dynamic light scattering (DLS). The mean diameter and zeta potential of DSNs measured by DLS were 151.8±2.597 nm and -33.9±0.777 mV, respectively. TEM image demonstrates that the DSNs possess spherical morphology with a size of about 150 nm (Figure 1A and B). For DSNs, the drug loading of PTX was 58.82% while that of DiR was 29.41%. However, the drug loading of PTX is 78.33% for PTX-S-S-PTX NPs. In addition, some other hydrophobic drugs were also encapsulated in such nanoparticles. (Figure S4) 3.3 Physical and Chemical Stability of DSNs. The diameter and the polydispersity index (PDI) of DSNs measured by DLS remained unchanged within 7d, indicating good stability of DSNs in aqueous solution and in PBS containing 10% FBS. (Figure 1C, D) Dithiothreitol (DTT), which was prevalent GSH simulatant, was used to investigate the reduction behavior of DSNs. As shown in Figure 1E and F, DSNs exhibit sustained release (

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