Sequential Delivery and Cascade Targeting of Peptide

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Sequential Delivery and Cascade Targeting of Peptide Therapeutics for Triplexed Synergistic Therapy with RealTime Monitoring Shuttled by Magnetic Gold Nanostars Rong Wu, Qianhao Min, Jingjing Guo, Tingting Zheng, Li-Ping Jiang, and Jun-Jie Zhu Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b05877 • Publication Date (Web): 01 Mar 2019 Downloaded from http://pubs.acs.org on March 2, 2019

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Analytical Chemistry

Sequential Delivery and Cascade Targeting of Peptide Therapeutics for Triplexed Synergistic Therapy with Real-Time Monitoring Shuttled by Magnetic Gold Nanostars Rong Wu†, Qianhao Min†, Jingjing Guo†, Tingting Zheng*,†,‡, Liping Jiang*,†, and Jun-Jie Zhu† †State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210023, China ‡ Department of Chemistry, School of Chemistry and Molecular Engineering, East China Normal University, Dongchuan Road 500, Shanghai 200241, China. ABSTRACT: Due to the outstanding synergistic effects and low-toxicity, combination therapy exhibits more considerable potential in antitumor activity than monotherapy. Herein, a core-shell magnetic gold nanostar (Fe 3 O 4 @GNS, MGNS)-based system for co-delivery of a mitochondrial targeting amphipathic tail-anchoring peptide (ATAP) and a membrane-associated cytokine (tumornecrosis-factor-related apoptosis-inducing ligand (TRAIL) was constructed. The magnetic core can facilitate delivery of the drug vehicle by external magnetic field, which results in accurate accumulation and enhances tumor cellular uptake for preliminary targeting. TRAIL and ATAP could sequentially target and be released toward the plasma membrane and mitochondria, initiating the extrinsic and intrinsic apoptosis pathways, respectively. The gold shell of MGNS can cause local tumor hyperthermia due to broadband plasmon resonances in the near infrared region, which can act as a complement with the peptide drug to further enhance apoptosis. Both in vitro and in vivo experiments revealed that rationally integrating extrinsic apoptosis, intrinsic apoptosis and hyperthermia for triplexed synergistic therapy, enabled the smart drug vehicle with pinpoint peptide drug delivery capabilities, and minimized side effects, enhancing the antitumor efficiency.

INTRODUCTION Apoptosis, a cellular suicide program, plays a key role in human diseases.[1-3] Excessive proliferation is the result of evading apoptosis, which enable cancer cells survival even under hypoxic conditions and resist drugs.[4,5] Pro-apoptosis therapeutics as promising chemotherapeutics can have potent biological activities, which have garnered immense attention in recent years.[6,7] Since many pro-apoptosis therapeutics can induce cell apoptosis to achieve antitumor effect, induction and real-time monitoring of apoptosis in tumor cells would be essential in early assessment of antitumor efficiency.[8-11] Generally, apoptosis can be induced through intrinsic and extrinsic pathways. During extrinsic way, the recognition of death receptors and death ligands can activate caspase cascade, leading to proteolytic degradation of the cell architecture.[1,7] Especially, tumor nectosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), a membrane-mediated cytokine, specifically recognize its death receptor (DR4/DR5) for stimulation of rapid apoptosis.[12,13] The intrinsic pathway leads through mitochondrion damage, initiating the caspase cascade.[9] One particular example is a mitochondrial targeting amphipathic tail-anchoring peptide (ATAP), powerfully activating mitochondria-mediated apoptosis.[14] Activating both of internal and external apoptotic pathways can increase apoptosis efficiency, decreasing drug resistance and avoiding side effects over mono-apoptosis pathway.[15,16] Impressively, in addition to apoptosis inducing, ATAP and TRAIL can also respectively

achieve mitochondria and plasma membrane targeting for pinpoint therapy. Therefore, devising a co-delivery system with ATAP and TRAIL capable of cascade-targeting, and sequentially initiating the extrinsic and intrinsic apoptosis pathways is highly desirable. On the other hand, over the past decades, photothermal therapy (PTT) plays an increasingly important role due to its local antitumor capability.[17-19] PTT agents, such as gold,[20,21] carbon,[19] as well as other inorganic materials,[22-24] have obtained widespread application in cancer medicine. Among agents mentioned above, gold nanostars (GNSs) exhibit the superiority of PTT, which own high adsorption-to-scattering ratio and tunable plasmon properties.[17,25] Moreover, GNSs have served as ultra-efficient quenchers for chromophores through their nanosurface fluorescence resonance energy transfer (FRET) property in imaging application.[26] Interestingly, a recent report has verified that PTT can also enhance the pro-apoptotic capacity of peptide cytokine and mitochondrial membrane,[27] which can achieve complementary treatment with peptide therapeutics to synergistically enhance cancer cell apoptosis. Besides, lack of tumor-targeting capacity is detrimental to the therapy efficiency for GNSs-based therapeutic agents. [28,29] To ensure the PTT efficiency, preparing GNSs-based peptide agents that can target to tumor sites and integrate imaging functions during PTT, has been a tendency for accurate therapy. Herein, we constructed a core-shell Fe 3 O 4 @GNS–based

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Figure 1. Schematic illustration of drug vehicle for triplexed synergistic therapy with real-time monitoring. a) The synthesis of MGNSs, b) self-assembly process of MGNSs@ATAP/RGD-TRAIL/FAM-DEVD nanoconstructs and c) the antitumor mechanism of triplexed synergistic therapy. i) Internalization of vehicle following by target identification of TRAIL and RGD; ii) the release of ATAP in the cytoplasm; iii) the collapse of the mitochondrial structure triggered by ATAP resulting in intrinsic apoptosis; iv) imaging triggered by the cleavage of caspase-3. drug carrier that integrates multiple functions, including specific-cascade-targeting, high delivery efficiency, sequential apoptosis induction, and localized hyperthermia, forcefully enhancing cancer cell apoptosis efficiency. In Figure 1a and 1b, the designed nanosystem was composed of Fe 3 O 4 @GNS magnetic gold nanostars (MGNSs), polyethylenimine (PEI), polyethylene glycol (PEG) linker, arginine-glycine-aspartic

acid containing peptide-functionalized TRAIL (RGD-TRAIL), ATAP and caspase 3-responsive aspartic-glycine-valineaspartic acid (DEVD) peptide, which was obtained by multistep conjugations. For the antitumor process of triplexed synergistic therapy, as shown in Figure 1c, with a magnet, the Fe 3 O 4 core firstly served as a magnetic guiding unit for preliminary targeting, which allowed for the enhanced accumula-


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tion at the tumor sites.[30-33] PEI adsorbed on MGNSs (PEI@MGNSs) provided enough amino binding site for further RGD-TRAIL and ATAP conjugation, which were capable of further TRAIL and RGD-mediated plasma membrane dualtargeting (Process i, initiating the extrinsic apoptosis pathway) and ATAP-mediated mitochondria targeting (Process iii, initiating the intrinsic apoptosis pathways). N-succinimidyl 3-(2pyridyldithio) propionate (SPDP)[14,34] served as the heterobifunctional cross-linker between the thiol-terminated peptides and the amine-terminated PEI@MGNSs moieties to form redox-responsive (eg. glutathione) drug vehicles for free peptides release in the cytoplasm (Process ii),[35] which enhanced apoptosis. Furthermore, deriving from broad-band plasmon resonances in the NIR water window of gold shell, PTT therapy for local tumor hyperthermia was performed with NIR laser irradiation, which synergistically enhanced cancer cell apoptosis.[18,36,37] For apoptosis monitoring, a carboxyfluorescein (FAM)-conjugated DEVD (FAM-DEVD) peptide was designed to anchorage onto the surface of MGNSs to construct caspase 3-responsive MGNSs@FAM-DEVD nanocomplexes. Due to FRET, the green fluorescence of the FAM-DEVD peptide was completely quenched, which could be recovered through the FAM-DEVD peptide cleavage by apoptosisactivated caspase-3.[29,38] The “off-on” process exhibited in apoptotic cells, could in-time monitor the activity of caspase-3, and in turn self-evaluate the apoptosis efficiency (Process iv). We revealed that the smart vehicle, with rational multifunctionalities integration, is endowed with pinpoint peptide drug delivery and sequential targeting, and minimized side effects powerfully enhancing the antitumor efficiency.

MCL. Preparation of Magnetic Core-Shell Nanostars (MGNSs): Firstly, Fe 3 O 4 magnetic nanoparticles (Fe 3 O 4 NPs) were obtained through a typical coprecipitation method with some modification. Sodium citrate was used for coating Fe 3 O 4 NPs with mechanical stirring. Then, 10 mL of Fe 3 O 4 aqueous solution was injected into boiling HAuCl 4 (20 mL, 0.5 mM) solution for reducing Au3+ by sodium citrate to form the Au-shell coating (Fe 3 O 4 @Au Seed). The mixture was controlled in the state of boiling for another 10 min for seed growth. The dark purple seed was collected after stopping heating and cooling. Next, Fe 3 O 4 @Au seeds (7.5 mL), sodium citrate (0.5 mL, 1%) and hydroquinone solution (5 mL, 15mM) were added in order into HAuCl 4 solution (50 mL, 0.25 mM) under the mechanical stirring. The blue mixture was stirred continuously under room temperature for 1.5 h, and stabilized by PEG linker. Finally, MGNSs was obtained by centrifugation. Peptide Drug Loading: For PEI-coated MGNSs, the MGNSs (0.2 mg mL-1) and branched PEI (2 mg mL-1) were mixed overnight and then the mixture was purified by dialysis tubing (12000Da) for several days. Simultaneously, a heterobifunctional SH-PEG-COOH were boned to RGD-TRAIL, using EDC/NHS coupling reaction between amino and carbonyl groups and forming PEG-RGD-TRAIL. After that, a crosslinker (SPDP, 0.16mM) was used for linking ATAP and PEG-RGD-TRAIL to PEI-coated MGNSs, forming MGNSs@ATAP/RGD-TRAIL nanocomplexes and varying ratios. Finally, the MGNSs@ATAP/RGD-TRAIL nanocomplexes were mixed with the FAM-DEVD shaking overnight to form MGNSs@ATAP/RGD-TRAIL/FAM-DEVD nanocomplexes. The obtained MGNSs@ATAP/RGD-TRAIL/FAMDEVD nanonanocomplexes were then dispersed in D-PBS. Dark Field and Confocal Microscopy Imaging: 8 × 103 cells were seeded in each dish containing fresh DMEM medium. After 24 h incubation, designated nanoprobes were injected into each well for another incubation. Imaging was operated on a DFM and a two-photon CLSM excited by 488nm laser, respectively. For Magnetically Facilitated Delivery, 45 min incubation under magnetic exposure was performed. Measurement of Apoptosis and Mitochondrial Membrane Potential: When cells were going to be the early stage of apoptosis, phosphorus esters acyl serine (PS) could be outside of lipid membrane and recognized Annexin V. Apoptosis detection kit, containing Annexin V-FITC and PI, was used to distinguish apoptosis period. 4 × 105 cells were seeded in 6well plate and then cultured with designated nanoprobes for evaluating the apoptosis efficiency. Finally, the data were recorded immediately on a flow cytometer with 488 nm excitation. As for mitochondrial membrane potential, the changes can also be obtained by flow cytometry analysis using a JC-1 assay kit with 488 nm excitation. Pharmacokinetics and Biodistribution Measurements: HeLa tumor-bearing mice (tumor size = 50 mm3) were administered intravenously with 200 µL of MGNSs@ATAP/RGD-TRAIL (1 mg Au mL-1). After injection, at designated time point, major organs were peeled and blood were collected for ICP-MS analysis of Au element. In Vivo Antitumor Efficacy Assay: mice with 50 mm3 of tumor were divided into seven groups (n=3) randomly. From

EXPERIMENTAL Chemicals and Materials: The carboxyfluorescein-DEVDpeptide (FAM-DDDEVDGRRRRC, 1290.34 g/mol, 95% purity) and the amphipathic tail-anchoring peptide (ATAP, KFEPKSGWMTFLEVTGKICEMLSLLKQYC, 3411.14 g/mol, 95% purity) were purchased from GL Biochem. Ltd. (Shanghai, China). TRAIL and RGD-TRAIL were obtained from Jiangsu TargetPharma Laboratories Inc. (Changzhou, China). Gold(III) chloride hydrate (AuCl 3 ·HCl·4H 2 O) was provided by Shanghai Chemical Reagent Co. (Shanghai, China). Ethylene imine polymer (PEI, MW=10000) was obtained from Aladdin Industrial Corporation. N-Hydroxysuccinimide (NHS, 97%), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), 2-Mercaptoethanol (β-ME), Nsuccinimidyl 3-(2-pyridyldithio)propionate (SPDP, 95%) and α-thio-ω-carboxy poly(ethylene glycol)(MW=3500, SH-PEGCOOH) were provided by Sigma-Aldrich (St. Louis, USA). Caspase-3 cell activity detection kit, mitochondrial membrane potential assay kit and apoptosis detection kit were provided by Nanjing Keygen Biotech. Co. Ltd. (Jiangsu, China). Apparatus: UV−vis extinction spectra were obtained by a UV-3600 spectrophotometer. SEM and EDS analysis were measured on an S-4800 scanning electron microscope. TEM was operated on a JEOLJEM 200CX. Photoluminescence (PL) spectra were recorded on a RF-5301PC. DLS data were recorded on a Brookhaven BI-200SM instrument. MTT assay was performed on a Bio-Rad 680 microplate reader. CLSM experiments were operated on Leica TCS SP5 microscope. Flow cytometric analysis was performed on Cytomics FC 500



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were prepared by conventional coprecipitation method. Then, Fe 3 O 4 @Au core−shell particles were obtained through the reduction of HAuCl 4 onto citrate-stabilized Fe 3 O 4 NPs surface. These core-shell particles, which served as seeds, were subjected to a second reduction process of HAuCl 4 by hydroquinone to yield MGNSs. The size and surface roughness of MGNSs could be tuned by changing the hydroquinone/seeds ratio. The monodispersed Fe 3 O 4 NPs and Fe 3 O 4 @Au seeds were obtained with an average diameter of 4.7 ± 0.5 nm and 39.4 ± 8.4 nm (Figure S1), respectively. After the second reduction process to form MGNSs, the spiky morphology indeed gradually developed, and a uniform size of 100.8 ±12.1 nm was exhibited in Figure 2a and 2b. The scanning electron microscopy (SEM) image and corresponding elemental mapping results were shown in Figure 2c, indicating the homogeneous core-shell distribution of Fe and Au elements in the MGNSs. Energy dispersive spectroscopy (EDS) measurements showed that the content of Au changed from 25.67 wt% to 73.88 wt% during the second reduction process (Figure S2a). The lattice fringes of MGNSs were clearly observed in Figure 2d, which could be attributed to (111) and (200) planes of Au. X-ray diffraction (XRD) data fitted well to those of the Au and Fe 3 O 4 bulk phases (Figures 2e and S2b). The Fe 3 O 4 diffraction peaks could belong to the (220), (311), and (440) planes. After the second reduction process, diffraction peaks of Au distinguished from Fe 3 O 4 could be clearly obtained, which confirmed successful preparation of the polycrystalline MGNSs. The fabricated MGNSs showed long-time stability with spiky architectures and sizes in several physiological

day 1, the mice were intravenously injected via the tail vein with PBS (Group i), MGNSs@ATAP (Group ii), MGNSs@RGD-TRAIL (Group iii), MGNSs@ATAP/RGDTRAIL (Group iv). Mice in group ⅴ and ⅵ were intravenously injected with MGNSs (Group v) and MGNSs@ATAP/RGDTRAIL (Group vi), followed by NIR irradiation four times (1.5 W cm-2, 30 min) without a magnet and MGNSs@ATAP/RGD-TRAIL (Group vii) with NIR irradiation after each drug injection at 6h, 12h, 18h, 24h. A magnet (3291 Gs, 50*20*10 mm) was fixed on tumors with a piece of scotch tape. Then, the body weight and tumor size were recorded every 2 days. All animal operation was followed by the guidelines of the Institutional Animal Care and Use Committee. After treatment, the treated mice and the age-matched healthy mice without treatment were sacrificed by CO 2 asphyxiation for necropsy. Major organs were peeled and treated for HE and TUNEL staining, and examined by BX51 optical microscope. RESULTS AND DISCUSSION Preparation and Characterization of MGNSs@ATAP/RGD-TRAIL/FAM-DEVD Peptide Drug Vehicles. To substantiate our design, a self-assembly approach [39] was used to prepare MGNSs with multifunctional properties with some modifications. The approach was simple and gentle, avoiding the use of cytotoxic surfactant. As shown in Figure 1a, firstly, citrate-stabilized Fe 3 O 4 nanoparticles (NPs)

Figure 2. Characterization of MGNSs@ATAP/RGD-TRAIL/FAM-DEVD peptide drug vehicles. a) Scanning electron microscopy (SEM) image of MGNSs. b) Transmission electron microscopy (TEM) image of MGNSs. Inset: size distribution of MGNSs by DLS measurement. c) i) Bright field and corresponding EDS mapping images of ii) Fe, iii) Au elements, and iv) overlay chart. d) HRTEM image of MGNS particle. The inset is an entire MGNS particle. e) XRD patterns of i) Fe3O4 NPs, ii) MGNSs and the standard XRD patterns of iii) Fe3O4 and iv) Au NPs. f) Optical extinction spectra of colloidal i) Fe3O4 NPs, ii) Fe3O4@Au seed, iii) MGNSs and iv) MGNSs@ATAP/RGD-TRAIL/FAM-DEVD nanoconstruts. The vertical dash line indicates the wavelength of the NIR laser (808 nm). g) Temperature evolutions of colloidal suspensions of MGNS particles and solid Au NPs and of pure water upon exposure to 808 nm laser irradiation. The particle concentrations of MGNSs and solid Au NPs were both at 100 µg mL-1. h) The magnetic hysteresis loops of the MGNSs at 300 K. Inset: the photograph of magnetic separation process of the MGNSs from the solution.


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environments (Figure S3). As shown in Figure 2f, the colloidal MGNS particles showed a broad-band plasmon resonance covering NIR region and were promising for PTT. The temperature of MGNS colloids (100 µg mL−1) changed from ~ 24.2 °C to ~ 48.2 °C under 808 nm laser irradiation (1.5 W cm-2) for 20 min (Figure 2g). In contrast, only 1.0 °C and 1.4 °C increase was observed in pure water and solid sphere Au NPs respectively. In addition, photothermal efficiency of MGNSs didn’t degrade after multi-cycles tests, confirming the structural robustness. As shown in Figure 1b, FAM-DEVD peptides were bound to MGNSs through Au−S bond to form the MGNSs@FAMDEVD nanocomplexes and the green fluorescence of FAMDEVD peptide was completely quenched (Figure S4, this section was skipped during the process of exploring therapeutic efficiency). For peptide drug loading, branched PEI bond to MGNSs via electrostatic interactions, acting as a proton sponge and inducing endosomolysis of peptide vehicles within the cytosol instead of highly acidic endosomal environment in their most potent form.[40] Simultaneously, a heterobifunctional PEG linker with sulfydryl and carboxyl groups (SH-PEG-COOH) was used to couple with RGD-TRAIL to form PEG-RGD-TRAIL by the typical EDC/NHS coupling reaction between carboxyl and amino groups,[41] which provided a good dispersity and stability for RGD-TRAIL peptide due to the high hydrophilicity of PEG chain,[42] and increased the length of RGD-TRAIL peptide to expose targeting moieties. Finally, ATAP and PEG-RGD-TRAIL peptides were conjugated to PEI@MGNSs complexes through disulfide bonds (S-S) using the heterobifunctional cross-linker SPDP to form redox-responsive MGNSs@ATAP/RGD-TRAIL/FAMDEVD nanocomplexes. Microeletrophoresis measurements confirmed the stepwise assembly (Figure S5). The increased size (Figure S6) and slight red shift in UV-vis spectra (Figure 2f) also confirmed the stepwise assembly process. In addition, because of paramagnetic properties of the Fe 3 O 4 NP core of MGNSs, the peptide drug delivery vehicle exhibited highperformed magnetic properties without remanence or coercivity at 300 K (Figure 2h). Site-Specific-Cascade Targeting. The peptide drug carriers are endowed with the ability of delivering both peptide drugs and heat to targeted cancer cells and tumor, which could be separated from the reaction mixture conveniently (Figure 2h) with an efficient magnetic response. To validate the recognition between integrin and RGD[43,44] from MGNSs@ATAP/RGD-TRAIL nanocomplexes thus causing TRAIL bind onto the DR4/DR5-deficient plasma membrane more efficiently, HeLa cells that express sufficient integrin but deficient DR4/DR5 were used as a model to incubate with MGNSs, MGNSs@ATAP/TRAIL and MGNSs@ATAP/RGD-TRAIL nanocomplexes respectively for 2 h, followed by the dark field microscope (DFM) observation (Figure 3a i-iii). An evident difference in the distribution of nanocomplexes occurred in HeLa cells. After incubation with MGNSs@ATAP/RGD-TRAIL, the nanocomposites can bind cell membrane, while most of nanocomposites were observed within the cells that were cultured with MGNSs@ATAP/TRAIL, which exhibited that TRAIL from MGNSs@ATAP/RGD-TRAIL specifically recognized by the integrin could efficiently bind onto the cell membrane, and increase sojourn time on cell membrane for

promoting the recognition between TRAIL and death receptors, enhancing apoptosis. It made up for a loss of direct internalization of the conjugated TRAIL on MGNSs without RGD. Moreover, RGD modified TRAIL can enhance the facilitate targeting of cancer and RGD-TRAIL can enhance apoptosis in integrin-positive cancer cells[13]. After incubating HeLa cells with MGNSs@ATAP/RGD-TRAIL nanoconstructs for another 2 h, 4 h, and 10 h, the nanocomposites dispersed gradually inside the HeLa cells (Figure 3a iv-vi). In contrast, MGNSs nanocomposites without peptide modification did not attach to the cell surfaces at all even after cultured with HeLa cells for 24 h (Figure 3a i). Taken together, the DFM images well verified the site-specific-targeting capabilities of MGNSs@ATAP/RGD-TRAIL nanocomposites. Self-Evaluation of the Sequential Delivery and Apoptosis Efficiency. The ratio of RGD-TRAIL/ATAP co-immobilized on each MGNS particle is important and even can determine the overall apoptosis efficiency. Increasing the amount of RGD-TRAIL per MGNS would promote extrinsic apoptosis efficiency, while reducing ATAP would decrease intrinsic apoptosis efficiency. As shown in Figure S7, the maximum apoptosis efficiency was obtained at the RGD-TRAIL/ATAP ratio of 1/2 and it was found that 0.06 ng mL-1 RGD-TRAIL and 4 nM ATAP were present per 1 μg mL-1 MGNSs at this ratio (Experimental Section, Figure S8). Since caspase-3 could recover the quenched fluorescence signal of MGNSs@ATAP/RGD-TRAIL/FAM-DEVD nanoconstructs (Figure S4 In Supplementary Information), these nanoprobes could real-time monitor the cell apoptosis and self-evaluate the targeted delivery and apoptosis efficiency by flow cytometry and confocal laser scanning microscopy (CLSM). We chose HeLa cells, HL-60 cells, MDA-MB-231 cells and 293T cells as models to confirm sequential delivery and further verify cascade targeting, whose expression levels of integrin and DR4/DR5 were different (HeLa cells as models for high integrin and low DR4/DR5 expression cancer cells, HL-60 cells as high integrin and high DR4/DR5 expression cancer cells, MDA-MB-231 cells as low integrin and low DR4/DR5 expression cells, and HEK-293T cells as the integrin- and DR4/DR5-negative control[13,45]). After incubating HeLa cells with MGNSs@ATAP/RGD-TRAIL/FAM-DEVD nanocomplexes for 3 h, fluorescence signals recorded by CLSM were observed within the cells (Figure 3b ii) and strengthened with longer incubation time (Figures 3b i-v and S9). For comparison, stronger green fluorescence intensity was obtained in HL-60 cells after incubation with MGNSs@ATAP/RGD-TRAIL/FAM-DEVD nanocomplexes for only 12 h (Figures 3c i and S10-11). In contrast, weaker fluorescence signal was obtained in MDA-MB-231 cells after incubation with the nanocomplexes for 24 h (Figures 3c iii and S12), whereas 293T cells showed no fluorescence even after incubation for 36 h (Figure 3c iv). Besides of RGD-TRAIL and ATAP peptide-induced apoptosis, PTT further triggered the apoptosis in HeLa cells. After incubation of HeLa cells with MGNSs@ATAP/RGD-TRAIL/FAM-DEVD nanoprobes at 37 °C for 6 h, hyperthermia was performed by NIR laser (808 nm, 1.5 W cm-2) exposure for 20 min followed by another incubation reached to 24h. Much stronger fluorescence intensity was detected from NIR-illuminated group (Figure 3b vi and S9), which revealed the enhanced apoptosis with complementary PTT. Furthermore, according to the evaluation of An-



nexin V-FITC/PI apoptosis detection assay, optimal apoptotic efficacy was observed in MGNSs@ATAP/RGDTRAIL/FAM-DEVD nanocomplexes with NIR (Figure 3d).8,46 MGNSs@ATAP/RGD-TRAIL/FAM-DEVD nanocomplexes with NIR irradiation showed the lowest viability of 35.8%, with early apoptotic ratio of 37.7% and late apoptotic ratio of 26% (Figure 3d vi). For integrin- and DR4/DR5-sufficient HL-60 cells, MGNSs@ATAP/RGD-TRAIL/FAM-DEVD

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nanocomplexes with NIR irradiation showed enhanced apoptosis-inducing effect with early apoptotic ratio of 64.7% and late apoptotic ratio of 33.3% (Figure S13). In Vitro Cytotoxicity and Therapeutic Efficiency. The in vitro cytotoxicity of MGNSs@ATAP/RGD-TRAIL cultured with HeLa cells was tested through MTT assay. The biocompatibility of MGNS, PEI@MGNSs, and DEVD-FAM was firstly examined (Figure S14). Almost no significant cytotoxi-

Figure 3. In vitro imaging and therapeutic efficiency. a) Dark field microscope (DFM) images of i) MGNSs incubated with HeLa cells for 24 h, ii) MGNSs@ATAP/TRAIL incubated with HeLa cells for 2 h, and MGNSs@ATAP/RGD-TRAIL incubated with HeLa cells for iii) 2 h, iv) 4 h, v) 6 h, vi) 12 h. Scale bar indicates 10 μm. b) Time course of confocal fluorescence images of HeLa cells incubated with MGNSs@ATAP/RGD-TRAIL/FAM-DEVD nanoconstructs for i) 1 h, ii) 3 h, iii) 6 h, iv) 12 h, v) 24 h, and vi) 24h +NIR. Scale bar indicates 25 μm. c) Confocal fluorescence images of i) HL-60 cells incubated with MGNSs@ATAP/RGD-TRAIL/FAM-DEVD nanoconstructs for 12 h, ii) HeLa cells and iii) MDA-MB-231 cells incubated with MGNSs@ATAP/RGD-TRAIL/FAM-DEVD nanoconstructs for 24 h, and iv) 293T cells incubated with MGNSs@ATAP/RGDTRAIL/FAM-DEVD nanoconstructs for 36 h. Scale bar indicates 25 μm. d) Flow cytometry analysis of HeLa cells after staining with Annexin V-FITC and PI with different treatments. e) Flow cytometry-based JC-1 analysis of HeLa cells with different treatments. f) Viability of Hela cells with different treatments for 24h. g) Viability of different cells incubated with different concentrations of MGNSs@ATAP/RGD-TRAIL nanoconstructs with or without NIR laser illumination (808nm, 1.5 W cm-2, 10 min) for 48 h.


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city was exhibited even for MGNSs and PEI@MGNSs nanoconstructs at a high concentration of 20 µg mL−1 and DEVDFAM at 20 µM for 2 days, indicating good biocompatibility of MGNSs@FAM-DEVD as a peptide drug vehicle. Next, we tested the therapeutic efficiency of MGNSs@ATAP/RGDTRAIL nanoconstructs delivered to HeLa cells on 45 min magnetic field exposure (the optimized duration, Supplementary Information, Figure S15). As shown in Figure 3f, at equivalent peptides dosages and incubation times, the MGNSs@ATAP/RGD-TRAIL nanoconstructs showed a higher cytotoxicity than MGNSs@ATAP or MGNSs@RGDTRAIL nanoconstructs as the result of both RGD-TRAILmediated cell targeting and sequential apoptosis induction. Notably, treatment only with ATAP peptide, which lacks tumor-targeting capabilities, had negligible effect on cell viability (Figure S14d).28,29 Nevertheless, TRAIL can also act as “targeting ligand’’, which specifically bound with DR4/DR5 on the cell surface to activate extracellular apoptosis pathway, resulting in slight apoptosis effect (Figure S14e).[5] Treatment with only MGNSs with NIR did not show obvious decrease in the cell viability (Figure 3f). However, the combination of PTT with chemotherapeutic treatment led to greatly enhanced apoptosis effect. In Figure 3g, the half-maximal inhibitory concentration (IC 50 ) of MGNSs@ATAP/RGD-TRAIL nanoconstructs with NIR irradiation was 0.48ng mL-1 and 0.27 ng mL-1 (RGD-TRAIL concentration) for HeLa and HL-60 cells respectively, 1.5-fold and 1.14–fold lower than that of MGNSs@ATAP/RGD-TRAIL without NIR irradiation. In contrast, both MDA-MB-231 and 293T cells exhibited no outstanding cell apoptosis, and showed no obvious changes in cell viability with or without NIR illumination after incubation of MGNSs@ATAP/RGD-TRAIL nanoconstructs at 37 °C for 48 h. Hence, it suggests that while the loading of both RGDTRAIL and ATAP to the MGNSs increased its overall chemotherapeutic efficiency through extrinsic and intrinsic apoptosis pathways, the combination of PTT with chemotherapeutic treatment forming triplexed synergistic therapy led to the maximal apoptotic effect. Effect of MGNSs@ATAP Nanoconstructs on Mitochondrial Depolarization. Here, JC-1 assay was used to evaluate the change of mitochondrial membrane potential (MMP, ΔΨ m ), verifying that ATAP could cause mitochondrial dysfunction and induce apoptosis.[10,11] During early apoptosis, mitochondrial collapse results in mitochondrial depolarization. Due to its peculiarity of aggregation-mediated color change, JC-1 can translocate selectively to the mitochondria and distinguish the ΔΨ m . Generally, J-aggregates occur in healthy cells, exhibiting red fluorescence, whereas green monomers can be formed in apoptotic cells, which shows MMP depolarization. In Figure 3e, the control group shows intense red fluorescence of Jaggregates (99.4%). ATAP alone couldn’t cause mitochondrial collapse in HeLa cells, showing minimal green JC-1 monomers of 0.7% with red J-aggregates of 99.3%. After incubation with MGNSs@ATAP, obvious increase in the green fluorescence (13.4%) and decrease in red fluorescence both suggested increased mitochondrial depolarization, which confirmed the apoptosis. In a word, compared with unconjugated ATAP, the delivery of ATAP can cause significant mitochondrial dysfunction, eventually inducing cell apoptosis. Furthermore, cultured with MGNSs@ATAP under NIR irradiation, HeLa cells exhibited a further decrease in the red fluorescence,

which also verifyed the synergistic PTT effect on the mitochondrial-mediated apoptosis. In Vivo Antitumor Efficacy. For evaluating in vivo antitumor efficacy, HeLa tumor-bearing nude mice were employed as model. First, the biodistribution and pharmacokinetics of MGNSs@ATAP/RGD-TRAIL nanoconstructs were evaluated at multiple time points. The Au amount in blood, tissues, and organs were quantified by inductively coupled plasma mass spectrometry (ICP-MS). After drug injection, the Au concentration in blood exhibited progressive decrease, and dropped to 2 % at 48 h (Figure 4a). However, at tumor site, obvious accumulation was observed and 7 % was achieved at 12 h, which exhibited higher percentage than the group without a magnet (Figures 4b and S16). Besides, long-time accumulation of Au at tumor site from 24 h to 48 h indicated that the nanoconstructs could stay for an adequate time and enable continuous drug release for antitumor. The MGNSs@ATAP/RGD-TRAIL nanoconstructs showed slight accumulation in other organs, especially in liver and spleen (Figure 4c). Within the first 12 h after injection, the Au level in the organs continued to increase. The accumulation in liver and spleen may derive from the partial interception by the phagocytes.[47] Whereas, compared with liver and spleen, the accumulation of MGNSs@ATAP/RGD-TRAIL nanodrugs in kidney was lower, which further revealed the low renal toxicity. Then, six groups, 3 mice each group, were treated with different nanoconstructs with or without NIR illumination after the tumor size reached about 50 mm3 (Figure 4d). The control group was treated with PBS. Intravenous injection with nanodrugs (200 μL, 1 mg mL-1) was administered every two days. In Figure 4e, 4f and 4g, an obvious increase in weight and size was observed in control group (Group i). Treatment with MGNSs@ATAP (Group ii) or MGNSs@RGD-TRAIL (Group iii) exhibited inhibition, though the tumor was not completely eliminated. Nevertheless, tumors harvest from the mice treated with MGNSs@ATAP/RGD-TRAIL nanoconstructs (Group iv) exhibited a noticeable tumor size shrinking, further demonstrating the enhanced active targeting and sequential apoptosis-induction capability of MGNSs@ATAP/RGD-TRAIL nanoconstructs. Just acting as a PTT agent (Group v), though MGNSs couldn’t completely inhibit growth of the tumor, the tumor was suppressed through PTT by about 25%. As shown in Figure S17, PTT can enable the temperature of tumor site increase to ~ 45 °C, which is well-suited for hyperthermia and synergistically enhancing tumor cell apoptosis. Interestingly, with the treatment of MGNSs@ATAP/RGD-TRAIL nanoconstructs and hyperthermia (Group vii), the tumor growth was completely eliminated up to 14 days, indicating the outstanding triplexed synergistic effects. However, the therapeutic efficacy slightly decreased without a magnetic field, which confirmed that the magnetic targeting could lead to drug vehicle accumulation in tumor for enhancement therapy (Group vi). The antitumor efficacies with various treatments were also tested by the H&E and the TUNEL staining (Figure 4i).[37,48] Necrosis was obvio treatment with MGNSs@RGD-TRAIL nanoconstructs), Gro up iv (chemotherapeutic treatment with MGNSs@ATAP/RGD-TRAIL nanoconstructs), Group v (PT T using MGNSs nanoconstructs) and Group vi (chemotherapeutic treatment with MGNSs@ATAP/RGD-TRAIL nanocon-



structs without a magnet but coupled with NIR illumination), compared with those in the control group. Significantly, Group vii (chemotherapeutic treatment with MGNSs@ATAP/RGDTRAIL nanoconstructs under NIR illumination) exhibited more prominent necrosis in histological sections. Additionally, the highest level of the green fluorescence was observed in

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Group vii, confirming the outstanding tumor growth inhibition and enhanced apoptosis induced by combined chemo-PTT. The as-constructed MGNSs@ATAP/RGD-TRAIL peptide drug vehicles enhanced tumor accumulation and retention, and thus minimized side effects. During the therapy, all mice didn’t lose weight (Figure 4h) or die, which indicates that no

Figure 4. In vivo therapeutic efficacy. Au concentration in a) blood, b) tumor and c) different organs after treatment with MGNSs@ATAP/RGD-TRAIL nanoconstructs for pharmacokinetics and biodistribution measurements. Pictures of d) mice and e) tumors: i) control, ii) MGNS@ATAP, iii) MGNS@RGD-TRAIL, iv) MGNSs@ATAP/RGD-TRAIL，v) MGNSs + NIR irradiation, vi) MGNSs@ATAP/RGD-TRAIL + NIR irradiation without magnet (-MF) and vii) MGNSs@ATAP/RGD-TRAIL + NIR irradiation. f) Tumor weight at 15th day. g) Tumor volume and h) body weight. 3 mice each group. i) Histological observation of the tumor tissues obtained from mice after treatment. Hematoxylin and eosin (H&E) were used for staining tumor. For confirming apoptosis, the tumor sections were stained with Hoechst and fluorescein-dUTP (terminal deoxynucleotidyl transferase Dutp nick end labelling, TUNEL). A magnet was fixed on tumors for magnetic targeting. Scale bar indicates 100 μm.


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obvious side effects occurred. Moreover, organ damage wasn’t observed in all groups (Figure S18), which confirmed the minimal acute in vivo toxicity of the nanoconstructs. CONCLUSION In summary, we successfully developed a core-shell MGNS based nanosystem for sequential and site-specific delivery of RGD-TRAIL and ATAP. MGNS can forcefully deliver its cargos, RGD-TRAIL, ATAP and DEVD-FAM peptide to the tumor sites in a smart manner. MGNSs@ATAP/RGDTRAIL/FAM-DEVD nanocomplexes firstly accumulates at the tumor sites due to magnetic-mediate targeting and further accumulates at the tumor cell membrane by means of TRAILand RGD-mediated cell dual-targeting. RGD-TRAIL from MGNSs@ATAP/RGD-TRAIL/FAM-DEVD nanocomplexes specifically bind onto DR4/DR5 to activate the extrinsic apoptosis pathway. After endocytosis, with the redox-responsive cleavage, the free ATAP trigger potent mitochondriadependent intrinsic apoptosis pathway. In addition, the MGNS particles displayed a broad-band plasmon resonance in NIR region, which exhibited enhancement of therapeutic efficacy. In comparison to mono-chemotherapy or PTT, the rational combination therapy shows an outstanding synergistic effect on cancer cells. In vivo data also exhibited the effective tumor accumulation as well as outstanding tumor growth inhibitory ability. Moreover, the probe can monitor the cell apoptosis in real time for self-evaluation of the sequential delivery and apoptosis efficiency. It is worth pointing out that our strategy will open the view of antitumor exploration, which can synergistically utilize the extracellular and intracellular target and apoptosis. Besides, the co-delivery system can be imitated to deliver small-molecules, other pro-apoptotic protein and peptide for combination therapy to achieve a multi-synergetic antitumor efficacy.

REFERENCES (1) Denault, J.-B.; Salvesen, G. S. Caspases: Keys in the Ignition of Cell Death. Chem. Rev. 2002, 102, 4489–4500. (2) Fan, P.; Tyagi, A. K.; Agboke, F. A.; Mathur, R.; Pokharel, N.; Jordan, V. C. Modulation of nuclear factor-kappa B activation by the endoplasmic reticulum stress sensor PERK to mediate estrogeninduced apoptosis in breast cancer cells. Cell death Discov. 2018, 4, 15. (3) Juncadella, I. J.; Kadl, A.; Sharma, A. K.; Shim, Y. M.; Hochreiter-Hufford, A.; Borish, L.; Ravichandran, K. S. Apoptotic Cell Clearance by Bronchial Epithelial Cells Critically Influences Airway Inflammation. Nature 2013, 493, 547–551. (4) Delbridge, A. R.; Grabow, S.; Strasser, A.; Vaux, D. L. Thirty Years of BCL-2: Translating Cell Death Discoveries into Novel Cancer Therapies. Nat. Rev. Cancer 2016, 16, 99–109. (5) Scott, D. E.; Bayly, A. R.; Abell, C.; Skidmore, J. Small Molecules, Big Targets: Drug Discovery Faces the Protein–Protein Interaction Challenge, Nat. Rev. Drug Discov. 2016, 15, 533–550. (6) Chen, H. C.; Wang, Y. R.; Yao, Y. R.; Qiao, S. L.; Wang, H.; Tan, N. H. Sequential Delivery of Cyclopeptide RA-V and Doxorubicin for Combination Therapy on Resistant Tumor and In Situ Monitoring of Cytochrome c Release. Theranostics 2017, 7, 3781–3793. (7) Gray, B. P.; Brown, K. C. Combinatorial Peptide Libraries: Mining for Cell-Binding Peptides. Chem. Rev. 2014, 114, 1020–1081. (8) Hou, Z. Y.; Zhang, Y. X.; Deng, K. R.; Chen, Y. Y.; Li, X. J.; Deng, X. R.; Cheng, Z. Y.; Lian, H. Z.; Li, C. X.; Lin, J. UV-Emitting Upconversion-Based TiO 2 Photosensitizing Nanoplatform: NearInfrared Light Mediated in Vivo Photodynamic Therapy via Mitochondria-Involved Apoptosis Pathway. ACS Nano 2015, 9, 2584– 2599. (9) Holohan, C.; Schaeybroeck, S. V.; Longley, D. B.; Johnston, P. G. Cancer Drug Resistance: an Evolving Paradigm, Nat. Rev. Cancer 2013, 13, 714–726. (10) Saha, T.; Hossain, M. S.; Saha, D.; Lahiri, M.; Talukdar, P. Chloride-Mediated Apoptosis-Inducing Activity of Bis(Sulfonamide) Anionophores. J. Am. Chem. Soc. 2016, 138, 7558–7567. (11) Pai, J.; Hyun, S.; Hyun, J. Y.; Park, S. H.; Kim, W. J.; Bae, S. H.; Kim, N. K.; Yu, J.; Shin, I. Screening of Pre-MiRNA-155 Binding Peptides for Apoptosis Inducing Activity Using Peptide Microarrays. J. Am. Chem. Soc. 2016, 138, 857–867. (12) Jacob, N. T.; Lockner, J. W.; Kravchenko, V. V.; Janda, K. D. Pharmacophore Reassignment for Induction of the Immunosurveillance Cytokine TRAIL. Angew. Chem. Int. Ed. 2014, 53, 6628–6631. (13) Cao, L.; Du, P.; Jiang, S. H.; Jin, G. H.; Huang, Q. L.; Hua, Z. C. Enhancement of Antitumor Properties of TRAIL by Targeted Delivery to the Tumor Neovasculature. Mol. Cancer. Ther. 2008, 7, 851–861. (14) Shah, B. P.; Pasquale, N.; De, G. J.; Tan, T.; Ma, J. J.; Lee, K. B. Core–Shell Nanoparticle-Based Peptide Therapeutics and Combined Hyperthermia for Enhanced Cancer Cell Apoptosis. ACS Nano 2014, 8, 9379–9387. (15) Hu, Q. Y.; Sun, W. J.; Wang, C.; Gu, Z. Recent Advances of Cocktail Chemotherapy by Combination Drug Delivery Systems. Adv. Drug Deliv.Rev. 2016, 98, 19–34. (16) Sun, W.; Jiang, T.; Lu, Y.; Reiff, M.; Mo, R.; Gu, Z. CocoonLike Self-Degradable DNA Nanoclew for Anticancer Drug Delivery. J. Am. Chem. Soc. 2014, 136, 14722–14725. (17) Zhang, L.; Chen, Y.; Li, Z.; Li, L.; Saint-Cricq, P.; Li, C.; Lin, J.; Wang, C.; Su, Z.; Zink, J. I. Tailored Synthesis of Octopus-Type Janus Nanoparticles for Synergistic Actively-Targeted and ChemoPhotothermal Therapy. Angew. Chem. Int. Ed. 2016, 55, 2118–2121. (18) Zhang, Z.; Wang, J.; Chen, C. Near-Infrared Light-Mediated Nanoplatforms for Cancer Thermo-Chemotherapy and Optical Imaging Adv. Mater. 2013, 25, 3869–3880.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Additional Figures: SEM characterization, XRD characterization, fluorescence spectra, zeta potential values, CLSM images, Flow cytometry analysis, in vitro cytotoxicity evaluation, thermal images, and in vivo toxicity evaluation (HE) (pdf)

AUTHOR INFORMATION Corresponding Author *Email: [email protected] (T.T.Zheng) *Email: [email protected] (L.P.Jiang)

ACKNOWLEDGMENT We gratefully appreciate funding from the National Natural Science Foundation of China (21834004, 21475057 and 21605049), Shanghai Sailing Program (16YF1402700) and the International Cooperation Foundation from Ministry of Science and Technology (2016YFE0130100), We also thank Professor Zichun Hua from School of life science of Nanjing University for his help.



(19) De Volder, M. F. L.; Tawfick, S. H.; Baughman, R. H.; Hart, A. J. Carbon Nanotubes: Present and Future Commercial Application. Science 2013, 339, 535–539. (20) Zheng, T. T.; Grace Li, G. F.; Zhou, F.; Wu, R.; Zhu, J. J.; Wang, H. Gold‐Nanosponge‐Based Multistimuli‐Responsive Drug Vehicles for Targeted Chemo‐Photothermal Therapy. Adv. Mater. 2016, 28, 8218–8226. (21) Wang, S. J.; Tian, Y.; Tian, W.; Sun, J.; Zhao, S.; Liu, Y.; Wang, C. Y.; Tang, Y. X.; Ma, X. Q.; Teng, Z. G.; Lu, G. M. Selectively Sensitizing Malignant Cells to Photothermal Therapy Using a CD44-Targeting Heat Shock Protein 72 Depletion Nanosystem. ACS Nano 2016, 10, 8578–8590. (22) Tao, W.; Zhu, X. B.; Yu, X. H.; Zeng, X. W.; Xiao, Q. L.; Zhang, X. D.; Ji, X. Y.; Wang, X. S.; Shi, J. J.; Zhang, H.; Mei, L. Black Phosphorus Nanosheets as a Robust Delivery Platform for Cancer Theranostics. Adv. Mater. 2017, 29, 1603276. (23) Chen, W. S.; Ouyang, J.; Liu, H.; Chen, M.; Zeng, K.; Sheng, J. P.; Liu, Z. J.; Han, Y. J. L.; Wang, Q.; Li, J.; Deng, L.; Liu, Y. N.; Guo, S. J. Black Phosphorus Nanosheet-Based Drug Delivery System for Synergistic Photodynamic/Photothermal/Chemotherapy of Cancer. Adv. Mater. 2017, 29, 1603864. (24) Yang, Y.; Zhu, W. J.; Dong, Z. L.; Chao, Y.; Xu, L.; Chen, M. W.; Liu, Z. 1D Coordination Polymer Nanofibers for Low‐Temperature Photothermal Therapy. Adv. Mater. 2017, 29, 1703588. (25) Gao, Y.; Li, Y.; Chen, J.; Zhu, S.; Liu, X.; Zhou, L.; Shi, P.; Niu, D.; Gu, J.; Shi, J. Multifunctional gold nanostar-based nanocomposite: Synthesis and application for noninvasive MR-SERS imagingguided photothermal ablation. Biomaterials 2015, 60, 31–41. (26) Zdeněk, F.; Juřík, T.; Kovář, D.; Trnková, L.; Skládal, P. Nanoparticle-Based Immunochemical Biosensors and Assays: Recent Advances and Challenges. Chem. Rev. 2017, 117, 9973–10042. (27) Song, X.; Kim, S. Y.; Zhou, Z.; Lagasse, E.; Kwon, Y. T.; Lee, Y. J. Hyperthermia enhances mapatumumab-induced apoptotic death through ubiquitin-mediated degradation of cellular FLIP(long) in human colon cancer cells. Cell Death Dis. 2013, 4, e577. (28) Xu, X.; Wu, J.; Liu, Y.; Yu, M.; Zhao, L.; Zhu, X.; Bhasin, S.; Li, Q.; Ha, E.; Shi, J.; Farokhzad, O. C. Ultra-pH-Responsive and Tumor-penetrating Nanoplatform for Targeted SiRNA Delivery with Robust Anti-Cancer Efficacy, Angew. Chem. Int. Ed. 2016, 55, 7091– 7094. (29) Dam, D. H.; Culver, K. S.; Odom, T. W. Grafting Aptamers onto Gold Nanostars Increases in Vitro Efficacy in a Wide Range of Cancer Cell Types. Mol. Pharmaceutics 2014, 11, 580–587. (30) Ulbrich, K.; Hola, K.; Subr, V.; Bakandritsos, A.; Tucek, J.; Zboril, R. Targeted Drug Delivery with Polymers and Magnetic Nanoparticles: Covalent and Noncovalent Approaches, Release Control, and Clinical Studies. Chem. Rev. 2016, 116, 5338–5431. (31) Tian, Y.; Jiang, X.; Chen, X.; Shao, Z.; Yang, W. Doxorubicin-Loaded Magnetic Silk Fibroin Nanoparticles for Targeted Therapy of Multidrug-Resistant Cancer. Adv. Mater. 2014, 26, 7393–7398. (32) Wang, Y.; Wei, G. Q.; Zhang, X. B.; Huang, X. H.; Zhao, J. Y.; Guo, X.; Zhou, S. B. Multistage Targeting Strategy Using Magnetic Composite Nanoparticles for Synergism of Photothermal Therapy and Chemotherapy. Small 2017, 14, 1702994. (33) Tang, Z. M.; Li, D.; Sun, H. L.; Guo, X.; Chen, Y. P.; Zhou, S. B. Quantitative Control of Active Targeting of Nanocarriers to Tumor Cells Through Optimization of Folate Ligand Density. Biomaterials 2014, 35, 8015–8027. (34) Wu, M.; Meng, Q.; Chen, Y.; Zhang, L.; Li, M.; Cai, X.; Li, Y.; Yu, P.; Zhang, L.; Shi, J. Large Pore-sized Hollow Mesoporous Organosilica for Redox-Responsive Gene Delivery and Synergistic Cancer Chemotherapy. Adv. Mater. 2016, 28, 1963–1969. (35) Luo, Z.; Ding, X. W.; Hu, Y.; Wu, S. J.; Xiang, Y.; Zeng, Y. F.; Zhang, B. L.; Yan, H.; Zhang, H. C.; Zhu, L. L.; Liu, J. J.; Li, J. H.; Cai, K. Y.; Zhao, Y. L. Engineering a Hollow Nanocontainer Platform with Multifunctional Molecular Machines for Tumor-

Page 10 of 11

Targeted Therapy in Vitro and in Vivo. ACS Nano 2013, 7, 10271– 10284. (36) Lee, S. M.; Park, H.; Choi, J. W.; Park, Y. N.; Yun, C. O.; Yoo, K. H. Multifunctional Nanoparticles for Targeted Chemophotothermal Treatment of Cancer Cells. Angew. Chem. Int. Ed. 2011, 50, 7581–7586. (37) Zhong, Y. N.; Wang, C.; Cheng, L.; Meng, F. H.; Zhong, Z. Y.; Liu, Z. Gold Nanorod-Cored Biodegradable Micelles as a Robust and Remotely Controllable Doxorubicin Release System for Potent Inhibition of Drug-Sensitive and -Resistant Cancer Cells. Biomacromolecules 2013, 14, 2411–2419. (38) Min, Y. Z.; Li, J. M.; Liu, F.; Yeow, E. K. L.; Xing, B. G. Near-Infrared Light-Mediated Photoactivation of a Platinum Antitumor Prodrug and Simultaneous Cellular Apoptosis Imaging by Upconversion-Luminescent Nanoparticles. Angew. Chem. Int. Ed. 2014, 53, 1012–1016. (39) Perrault, S. D.; Chan, W. C. W. Synthesis and Surface Modification of Highly Monodispersed, Spherical Gold Nanoparticles of 50−200 nm. J. Am. Chem. Soc. 2009, 131, 17042–17043. (40) Pack, D. W.; Hoffman, A. S.; Pun, S.; Stayton, P. S. Design and Development of Polymers for Gene Delivery, Nat. Rev. Drug Discov. 2005, 4, 581–593. (41) Wróblewska, A.; Paluch, P.; Wielgus, E.; Bujacz, G.; Dudek, M. K.; Potrzebowski, M. J. Approach Toward the Understanding of Coupling Mechanism for EDC Reagent in Solvent-Free Mechanosynthesis. Org. Lett. 2017, 19, 5360–5363. (42) Jia, F.; Lu, X.; Tan, X.; Wang, D.; Cao, X.; Zhang, K. Effect of PEG Architecture on the Hybridization Thermodynamics and Protein Accessibility of PEGylated Oligonucleotides. Angew. Chem. Int. Ed. 2017, 56, 1239–1243 (43) Raj, M. H.; Yashaswini, B.; Ro¨ssler, J.; Salimath, B. P. Combinatorial Treatment with Anacardic Acid Followed by TRAIL Augments Induction of Apoptosis in TRAIL Resistant Cancer Cells by the Regulation of p53, MAPK and NFκβ Pathways. Apoptosis 2016, 21, 578–593. (44) Kim, K.; Lee, M.; Park, H.; Kim, J. H.; Kim, S.; Chung, H.; Choi, K.; Kim, I.; Seong, B. L.; Kwon, I. C. Cell-Permeable and Biocompatible Polymeric Nanoparticles for Apoptosis Imaging. J. Am. Chem. Soc. 2006, 128, 3490–3491. (45) Zheng, T. T.; Fu, J. J.; Hu, L. H.; Qiu, F.; Hu, M. J.; Zhu, J. J.; Hua, Z. C.; Wang, H. Nanoarchitectured Electrochemical Cytosensors for Selective Detection of Leukemia Cells and Quantitative Evaluation of Death Receptor Expression on Cell Surfaces. Anal. Chem. 2013, 85, 5609–5616. (46) Qian, C. G.; Yu, J. H.; Chen, Y. L.; Hu, Q. Y.; Xiao, X. Z.; Sun, W. J.; Wang, C.; Feng, P. J.; Shen, Q. D.; Gu, Z. Light‐Activated Hypoxia‐Responsive Nanocarriers for Enhanced Anticancer Therapy. Adv. Mater. 2016, 28, 3313–3320. (47) Dam, D. H. M.; Culver, K. S. B.; Kandela, I.; Lee, R. C.; Chandra, K.; Lee, H.; Mantis, C.; Ugolkov, A.; Mazar, A. P.; Odom, T. W. Biodistribution and in Vivo Toxicity of Aptamer-Loaded Gold Nanostars. Nanomed. Nanotechnol. Biol. Med. 2015, 11, 671–679. (48) Guo, X.; Wei, X.; Jing, Y. T.; Zhou, S. B. Size Changeable Nanocarriers with Nuclear Targeting for Effectively Overcoming Multidrug Resistance in Cancer Therapy. Adv. Mater. 2015, 27, 6450–6456.


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For TOC only


Sequential Delivery and Cascade Targeting of Peptide

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