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Nov 22, 2016 - Paclitaxel–Paclitaxel Prodrug Nanoassembly as a Versatile Nanoplatform for Combinational Cancer Therapy. Xiangfei Han§, Jinling Chen...
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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*,§ §

School of Pharmacy, Shenyang Pharmaceutical University, Shenyang 110016, China Key Laboratory of Structure-Based Drug Design and Discovery, Ministry of Education, Shenyang Pharmaceutical University, Shenyang 110016, China ⊥ School of Pharmacy, Shandong University, Jinan 250012, China ∥ School of Pharmacy, Guangdong Pharmaceutical University, Guangzhou 510006, China # School of Chinese Materia Medica, Guangzhou University of Chinese Medicine, Guangzhou 510405, China

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ABSTRACT: Recently, nanomedicine without drug carriers has attracted 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 selfassemble into uniform nanoparticles (NPs) with 1,1-dioctadecyl3,3,3,3-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 a mean diameter of about 150 nm and high stability in biological condition. A 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. KEYWORDS: disulfide, redox responsive, photothermal therapy, high drug loading, anticancer

1. INTRODUCTION

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 the 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) It is easy to scale up production and accelerate laboratory-to-industry translation.

Cancer is a prevailing threat to human beings because of 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 drug resistance. Over the past several years, many carrier-based drug-delivery strategies have been developed, including liposomes,2 polymeric nanoparticles,3 dendrimers, inorganic nanoparticles,4 protein analogous micelles, nanodiamonds, albumin-bound nanoparticles, and molecularly targeted nanoparticles. However, current carrier-based nanomedicines have serious disadvantages: (1) poor drug-loading capacity, (2) the burst release of the therapeutic drug from carriers, and (3) the difficulty of scale up owing to the complexity of multicomponent. Therefore, the development of nanomedicine which combines high drug payloads, targeted anticancer and controlled releasing activity is of prime interest for pharmacists. © 2016 American Chemical Society

Received: October 14, 2016 Accepted: November 22, 2016 Published: November 22, 2016 33506

DOI: 10.1021/acsami.6b13057 ACS Appl. Mater. Interfaces 2016, 8, 33506−33513

Research Article

ACS Applied Materials & Interfaces

further lead to significant cytotoxicity, which synergistically generated highly efficient thermo-chemotherapy with total tumor ablation. Although the 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.

Photothermal therapy (PTT) is a hyperthermia therapeutic approach that exploits photosensitive agents to kill cancer cells by heat generated from optical energy.7−9 Compared with traditional chemotherapy, PTT is noninvasive and does not damage normal tissues with localized laser exposure. There are many extraordinary inorganic photothermal agents as Au,10 MoS2,11,12 Bi2Se3,13,14 and TiO2.15 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 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, as a result of the uninhibited growth of tumor cells.20,21 The elevated GSH level in tumor cells was widely used for designing redox-sensitive nanosystem, and the disulfide bond was extensively utilized as a redox-responsive linkage to facilitate controlled release of anticancer drugs in tumor cells.22−26 Although many chemophotothermal combinational therapies were developed,27−30 there were few studies concentrating on redox-responsive selfassembly 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 paclitaxel−s−s−paclitaxel (PTX−s−s−PTX) conjugate is able to coassemble with DiR to form unanimous nanoparticles. The disulfide bond is used as a redox-responsive linkage to facilitate a rapid release of paclitaxel in tumor cells (Scheme 1) The NIR-light-riggered temperature increase could

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, 5-diphenyltetrazolium bromide (MTT) were obtained from Gibco (Beijing, China). Isoflurane was bought from RWD Life Science Co. Ltd. (Shenzhen, China). Fetal bovine serum (FBS) and calf serum (CS) were purchased from Hyclone (Beijing, China) and KeyGEN (Nanjing, China), respectively. Dimethyl sulfoxide (DMSO) was purchased from Kemeng (Tianjin, China). 1,1-Dioctadecyl-3, 3, 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 3 h at −30 °C under nitrogen atmosphere. The anhydrous THF solution of NMM (1.19 mmol) and PABA (1.19 mmol) were added dropwise to the reaction liquid, and the reaction was stirred at −30 °C for 4 h, and 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%). DTDPA-PABA (0.80 mmol) was dissolved in andryous THF, and 4.00 mmol of 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 −30 °C. 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%). DTDPA-NO2 (0.35 mmol) was dissolved in anhydrous dichloromethane; paclitaxel (0.70 mmol) and DMAP (8.10 mmol) were added to the dichloromethane solution. The reaction was allowed to cool 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%). 1 H NMR spectra were 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

Scheme 1. Redox-Sensitive Drug Release Mechanism of PTX−s−s−PTX Triggered by GSH (DTT)

33507

DOI: 10.1021/acsami.6b13057 ACS Appl. Mater. Interfaces 2016, 8, 33506−33513

Research Article

ACS Applied Materials & Interfaces pegylation. Finally, THF was evaporated. To prepare the DiR-loaded self-assembly nanoparticles, the prepared procedure was similar to that 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, DSPEPEG2000, 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 performed in triplicate were carried out via a Zetasizer (Nano ZS, Malvern. U.K.), 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 s and airdried. 2.4. Physical and Chemical Stability of DSNs. The changes of diameter and PDI of DSNs with or without being incubated in PBS (pH 7.4) containing 10% FBS with a time for 7 days were 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-buffered 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, and 24 h. At desired time intervals, 1 mL samples were 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−vis Detector on a reverse ODS Phenomenex -C18 column (250 mm × 4.6 mm, 5 μm) thermostated at T = 30 °C 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. 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−1 Å−2 constraint, and solvent was energy minimized for 2000 steps using the steepest descent (SD) method followed by a further 2000 steps using conjugate gradient algorithms. Subsequently, these initial harmonic restraints were gradually reduced to zero during energy minimizations. After that, the system was minimized by the SD method and switched to conjugate gradient every 3000 steps for a total of 6000 steps without harmonic restraints. Thereafter, the system was gently heated from 10K to 300 K, applying harmonic restraints with a force constant of 10 kcal mol−1 Å−2 on the solute atoms, and then equilibrated for 2000 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 longrange 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 structural representations. A simple study to explore the mechanism of the conjugate to selfassemble into nanoparticles was conducted by imaging the dynamics of crystal growth. A THF solution of paclitaxel and paclitaxel−

paclitaxel conjugate was placed onto a glass slide, respectively. After drying and desiccating, a drop of water was added to each sample. Pictures were taken after the slides were kept in a humidified chamber at room temperature for 4 h, 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 808 nm laser at 0.5 W cm −2. 2.7. Cell Culture. A549 lung cancer cells were cultured in RPMI Medium 1640 with 10% fetal bovine serum (FBS), 100 μg/mL streptomycin, and 100 units/mL penicillin in a humidified cell culture incubator at 37 °C, 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 °C, 5% CO2. The next day, the media was removed and then replaced with fresh media containing a 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, and DSNs were diluted by fresh media directly, with fresh media as the control group. When the cells were cultured for 48 h, 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 4 h. 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/DEAD Viability/ Cytotoxicity Kit to evaluate the PTT effects on cell viability. Cells were pretreated with all formulation for 4 h 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 570 nm 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)

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 nearinfrared 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 DISCUSSION 3.1. Synthesis of Paclitaxel−Paclitaxel Conjugate. First, 3,3′-dithiodipropionic acid (DTDPA) was reacted with p-aminobenzyl alcohol (PABA) with the aid of 4-methylmorpholine (NMM) and isobutyl chloroformate under N 2 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 33508

DOI: 10.1021/acsami.6b13057 ACS Appl. Mater. Interfaces 2016, 8, 33506−33513

Research Article

ACS Applied Materials & Interfaces

Figure 1. (A) Particle size and (B) TEM of DSNs. (C) Changes of diameter and PDI of DSNs at room temperature for 7 days. (D) Size changes of DSNs incubated in PBS (pH 7.4) containing 10% FBS for 7 days. (E) PTX−s−s−PTX chemical stability. (F) Cumulative release of PTX in pH 7.4 with or without DTT.

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, oPh1), 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, p-Ph2, oPh4), 7.19 (m, 2H, -SCH2CH2OCNH−), 7.06 (d, J = 8.1 Hz, 2H, 3′-NH), 6.27 (s, 2H, H-10), 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 = 11.5 Hz, 2H, 2′−OCO2CH2), 5.05 (d, J = 11.5 Hz, 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, Ha20), 4.18 (d, J = 8.3 Hz, 2H, Hb-20), 3.78(d, J = 6.9 Hz, 2H, H3), 3.02 (br.s, 4H, −CH2CH2SSCH2CH2-), 2.73 (br.s, 4H, −CH2CH2SSCH2CH2-), 2.55(m, 2H, Ha-6), 2.43 (s, 6H, 4OAc), 2.34 (m, 2H, Ha-14), 2.19 (s, 6H, 10-OAc), 2.08 (m, 2H, Hb-14), 1.88 (m, 2H, Hb-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). The 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 the 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. 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,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 7 days, 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,F, DSNs exhibit sustained release ( 0.05). Moreover, the cytotoxicity of all formulations upon 808 nm irritation observed was in the following order: PTX−s−s−PTX < PTX sol < DSNs. The IC50 of the DSNs group (28.98 nM) is lower than that of PTX−s−s−PTX (622.59 nM) and that of PTX sol (44.24 nM). A higher cytotoxicity was observed as the concentration of PTX was increased. The highest amount of cell death, where cell viability was 16.21%, was obtained by incubating the cells with DSNs with irradiation for 10 min. These results demonstrated that the combination of chemotherapy and PTT treatment caused a significant increase in cell death compared with treatment with chemotherapy alone or PTT alone, which clearly showed a synergistic effect of the two different therapeutic modalities. According to a number of reports, mild photothermal heating (e.g., to ≈ 43 °C) could accelerate the cellular uptake of chemotherapeutic drugs and thus enhanced the overall therapeutic efficacy in the combined therapy.7,33 In addition, Taxol has long been criticized for its serious cremophor EL-induced toxicities. In comparison with Taxol, DSNs do not have excipient-related toxicity. Moreover, DSNs could passively accumulate into the tumor site because of the enhanced permeability and retention (EPR) effects and had

DSNs and the accelerated dissociation of DSNs, confirming that DSNs had remarkable redox-responsive drug release. In addition, the intermediate PTX-SH was confirmed by LC-MS (Figure S5, S6). 3.4. Mechanism of the Conjugate Self−Assembling into Nanoparticles. It was commonly reported that hydrophobic drugs alone could not self-assemble into stable NPs, which required amphiphilic or ionic materials to balance the intermolecular force. This novel nanomaterial composed of hydrophobic prodrugs self-assembled unexpectedly into NPs by simple insertion of a disulfide bond between hydrophobic paclitaxel. To explore the self-assembly mechanism of the PTX−s−s−PTX, molecular dynamics (MD) simulations for the tetramer PTX−s−s−PTX were performed. As shown in Figure 2A, four molecules swiftly gathered to form a cluster of

Figure 2. (A) MD simulations of tetrameric PTX−s−s−PTX in water (yellow indicates s−s bond). (B) Imaging the dynamics of crystal growth. THF containing PTX and PTX−s−s−PTX was placed onto a glass slide. After drying and desiccating, a drop of water was added to each sample. The slides were placed into a humidified chamber at room temperature for 4 h, and pictures were taken. The left panel (I) shows PTX and the right panel (II) PTX−s−s−PTX. (C) The electrostatic potential map of PTX−s−s−PTX.

tetramers. After the self-assembly was completed, the conformation changed negligibly and the whole cluster moved together. The phenyl rings of PTX were curved inside the cluster without direct interactions between s−s bonds (yellow color). Considering the structure of the tetramer, we might infer that the main driving forces for the self-assembly of the PTX−s−s−PTX were noncovalent hydrophobic interactions and π−π stacking between PTX and PTX. It was known that crystal grew to form an ordered and repeating pattern of atoms or molecules, which precipitated drug from solution in favor of solvent−solvent interactions. For self-assembly of nanoparticles, such a thermodynamically favored process needed to be abrogated and assembled structures forced to interact in a direct method with the solution phase. Structural interactions observed in the MD simulations suggested that self-assembly of PTX−s−s−PTX might be supported by hampering crystallization, which was based on the disulfide bond. To further explore this concept, we examined the crystallization kinetics of PTX and PTX−s− s−PTX by optical imaging. Both PTX and PTX−s−s−PTX formed amorphous precipitate after THF evaporation. Four hours after hydration, crystals were detected for PTX (Figure 2B-I). In contrast, spherical particles (diameter