Nitric Oxide Stimulated Programmable Drug Release of Nanosystem

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Nitric Oxide Stimulated Programmable Drug Release of Nanosystem for Multidrug Resistance Cancer Therapy Li Wang, Yun Chang, Yanlin Feng, Xi Li, Yan Cheng, Hui Jian, Xiaomin Ma, Runxiao Zheng, Xiaqing Wu, Keqiang Xu, and Haiyuan Zhang Nano Lett., Just Accepted Manuscript • DOI: 10.1021/acs.nanolett.9b01869 • Publication Date (Web): 30 Aug 2019 Downloaded from pubs.acs.org on August 30, 2019

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

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Nitric

Oxide

Stimulated

Programmable

Drug

Release of Nanosystem for Multidrug Resistance Cancer Therapy Li Wang,1,2 Yun Chang,2,3 Yanlin Feng,2,4 Xi Li,*1 Yan Cheng,2 Hui Jian,2 Xiaomin Ma,1 Runxiao Zheng,2, 4 Xiaqing Wu,2, 4 Keqiang Xu,2, 4 Haiyuan Zhang*2, 3, 4 1School

of Chemistry and Life Science, Changchun University of Technology, Changchun

130012 (P.R. China). 2Laboratory

of Chemical Biology, Changchun Institute of Applied Chemistry, Chinese Academy

of Sciences, Changchun 130022 (P.R. China). 3University

of Chinese Academy of Sciences, Beijing 100049 (China).

4University

of Science and Technology of China, Hefei, Anhui 230026 (China).

*Corresponding author: Haiyuan Zhang, Laboratory of Chemical Biology, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022 (P.R. China). 2University of Science and Technology of China, Hefei, Anhui 230026 (P.R. China); University of Chinese Academy of Sciences, Beijing 100049 (China); Tel: 86-431-85262136; Fax: 86-431-85262582Email: [email protected]; Xi Li, School of Chemistry and Life Science, Changchun University of Technology, Changchun 130012 (P.R. China). Email: [email protected]

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ABSTRACT: Nitric oxide (NO) molecular messenger can reverse the multidrug resistance (MDR) effect of cancer cells through reducing P-glycoprotein (P-gp) expression, beneficial for creating a favorable microenvironment for treatment of doxorubicin (Dox)-resistant cancer cells. Development of sophisticated nanosystems to programmably release NO and Dox becomes an efficient strategy to overcome the MDR obstacles and achieve promising therapeutic effects in Dox-resistant cancer. Herein, a NO stimulated nanosystem was designed to engineer a significant time gap between NO and Dox release, promoting MDR cancer therapy. A o-phenylenediamine– containing lipid that can hydrolyze in response to NO was embedded in the phospholipid bilayer structure of liposome to form NO-responsive liposome, which could further encapsulate Larginine (L-Arg)/Dox-loaded gold@copper sulfide yolk-shell nanoparticls (ADAu@CuS YSNPs) to form

ADLAu@CuS

YSNPs. Under 808 nm laser irradiation, the unique resonant energy

transfer (RET) process and reactive oxygen species (ROS) generation in the confined space of ADLAu@CuS

YSNPs could effectively convert L-Arg into NO, regionally destabilizing the

phospholipid bilayer structure, as a result of NO release. However, at this early stage, Dox could not be released from YSNPs due to the molecular scaffold limit. As the NO release progressed, the NO-responsive liposome layer was deteriorated more severely, allowing Dox to escape. This NO and Dox sequential release of

ADLAu@CuS

YSNPs could significantly inhibit P-gp

expression and enhance Dox accumulation in Dox-resistant MCF-7/ADR cells, leading to promising in vitro and in vivo therapeutic effects and presenting their great potential for MDR cancer therapy.

KEYWORDS: Copper sulfide, Nanosystem, Nitric oxide, Doxorubicin, Programmable release.


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Gas therapy is emerging as a promising therapeutic modality for cancer treatment due to its “green” approach with negligible side effect.1,

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Various therapeutic gaseous molecular

messengers have been applied in gas therapy,3, 4 of which nitric oxide (NO) exhibits a prominent physiological and pathological role in various cellular life activities.5-7 Recently, NO molecular messenger has been reported to enable the reversal of the multidrug resistance (MDR) effect of cancer cells through reducing P-glycoprotein (P-gp) expression level,8 which prompts to develop novel NO-delivery nanosystems for MDR cancer therapy. However, most of current NOdelivery nanosystems are mainly focusing on NO production/releasing nanosystems9-11 and shed less light on NO stimulated nanosystem,12 which dramatically limits the development of sophisticated nanosystems to spatiotemporally control the NO release in combination with chemotherapy for cancer. o-Phenylenediamine group can react with NO to produce an amide-derived benzotriazole moiety,13 which can undergo spontaneous hydrolysis.12,

14

This means o-phenylenediamine

groups potentially can be incorporated into the NO-delivery nanosystem to regulate the structure or performance of nanosystems, exhibiting the NO-responsive behavior. Moreover, considerable effects have currently been contributed to generate biocompatible NO donors, however, the majority of them (such as N-diazeniumdiolate (NONOate)-based and S-nitrosothiols (RSNO)based compounds)15, 16 suffer from side effects and rapid clearance in vivo, remaining obstacles to their clinical application. Fortunately, L-arginine (L-Arg) as a natural amino acid is found to be able to produce NO with the help of inducible NO synthase,17, 18 or in the presence of reactive oxygen species (ROS: including H2O2 and 1O2).19-21 Recently, it was found the incident energy (such as ultrasound or near infrared light) can boost this ROS-mediated L-Arg conversion,22 suggesting the intense energy transformation process can play a critical role in NO production.


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Resonance energy transfer (RET) has been proposed as an electromagnetic field mediated plasma energy transfer process. After the combination of plasma metal and semiconductor, the RET process can directly excite the semiconductor by relaxation of the local surface plasmon dipole and effectively generate electron-hole pairs in the semiconductor.23 RET process from plasmonic metal to semiconductor presents an intense energy transformation process and has been applied in gold (Au) nanorod (NR) embedded copper sulfide (CuS) yolk-shell (YS) nanoparticles (NPs) (Au@CuS YSNPs) to enhance the photoactivity upon NIR laser activation.24, 25 Recently, faceted Au NRs were reported to exhibit more active surface reactivity than conventional Au NRs because of the existence of high index facets that potentially can generate abundant ROS,26,

27

while inheriting the excellent plasmonic property. This means

Au@CuS YSNPs using faceted Au NRs as core probably can efficiently convert the loaded LArg into NO due to the intense energy transformation and potent ROS generation in the confined space. In the present study, NO-responsive liposomal Au@CuS YSNPs were designed to sequentially release NO and Dox for MDR cancer therapy (Figure 1). A series of faceted Au NRs with similar localized surface plasmon resonance (LSPR) absorbance maximum at 808 nm were embedded in CuS hollow NPs (HNPs) to form various Au@CuS YSNPs, aiming at screening out the most efficient ROS-generating NPs under NIR laser irradiation. NO-responsive liposome was designed through embedding hydrophobic o-phenylenediamine–containing lipid in the phospholipid bilayer structure of liposome, which can be regionally destabilized by NO due

to the hydrophobicity-to-hydrophilicity conversion of o-phenylenediamine–containing lipid in response to NO. After being loaded with both L-Arg and doxorubicin (Dox), Au@CuS YSNPs were encapsulated in NO-responsive liposome to form ADLAu@CuS YSNPs. Upon 808 nm laser


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phospholipid bilayer structure of NO-responsive liposome layer, as a result of NO release. However, at this early stage, Dox could not be efficiently released out of ADLAu@CuS YSNPs together with NO because of its larger molecular scaffold compared with that of NO. With the NO release process proceeding, the liposome layer was deteriorated more severely, allowing Dox to go through the membrane gradually, as a result of Dox release. This NO and Dox sequential release behavior of

ADLAu@CuS

YSNPs is beneficial for treatment of Dox-resistant human

breast cancer (MCF-7/ADR), where the early-stage NO release can inhibit P-gp expression and create favorable microenvironment for the latte-stage Dox accumulation and therapy in MCF7/ADR cells. Moreover, NIR-irradiated

ADLAu@CuS

YSNPs could exhibit additional

photothermal and photodynamic therapeutic effect for cancer in combination with chemotherapy of Dox. RESULTS AND DISCUSSION Characterization of Au@CuS YSNPs. A series of faceted Au NRs were prepared by using conventional Au NRs (Au1 NRs; 96.8 ± 2.2 nm in length; 21.5 ± 1.6 nm in diameter) as a template,24 where the end geometries of Au NRs were evolved through regulating the molar ratio of Cu2+/hexadecyltrimethylammonium bromide (CTAB) to form Au2, Au3, and Au4 NRs. Transmission electron microscope images (TEM) (Figure 2A) showed that these conventional and faceted Au NRs have similar lengths (92.8 ± 3.5 to 100.5 ± 2.6 nm) and diameters (20.2 ± 1.8 to 23.6 ± 1.2 nm) (Figure S1A) but different end geometries. High resolution-TEM (HRTEM) revealed Au1-Au4 NRs had different {220}, {111}, {221}, and {110} facets (Figure S1B) at the ends. The UV-Vis-NIR absorption spectra showed that A1-Au4 NRs had similar absorption maximum peaks at 808 nm (Figure S2). Based on these Au NRs, a series of Au@CuS YSNPs were prepared using Au@Cu2O core-shell NPs (CSNPs) as a template.28 The


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surface of Au@Cu2O CSNPs was further converted into a CuS shell by sodium sulfide (Na2S) through the Kirkendell effect, leading to the formation of Au@CuS YSNPs.28 TEM image of Au1-4@CuS YSNPs revealed their clear yolk-shell structure with diameters of 121.5 ± 3.4 nm to 128.6 ± 6.2 nm (Figure 2B). All these NPs could be well dispersed in water and their hydrodynamic sizes ranged from 190.3 ± 6.5 to 201.7 ± 5.7 (Figure S3). After dispersed in cell culture medium, these YSNPs showed weakly increased hydrodynamic sizes (Figure S4). X-ray diffraction (XRD) data revealed the cubic phase of Au (JCPDS No. 04-0784) and the covellite phase of CuS (JCPDS No. 06-0464) of Au1-4@CuS YSNPs (Figure S5). All Au1-4@CuS YSNPs showed the similar crystalline structure. UV-Vis-NIR spectroscopy of Au1-4@CuS YSNPs was carried out to study their optical properties (Figure S6). Due to the overlap of LSPR of Au and CuS, the LSPR absorption of Au1-4@CuS YSNPs in the NIR region was significantly enhanced. Brunauer-Emmett-Teller (BET) measurement on Au2@CuS YSNPs indicated their surface area of 338 m2 mg-1 and pore size of 2.9 nm (Figure S7), respectively, which will facilitate the drug loading and release of Au2@CuS YSNPs. Photodynamic and photothermal properties. When the wavelength of the incident NIR laser overlaps with the LSPR peak of the Au core, the RET process from Au core to CuS shell gets very potent, significantly promoting the ROS formation of Au@CuS YSNPs, especially using faceted Au NRs as core. The NIR laser-activated ROS formation of Au1-4@CuS YSNPs was evaluated through 2',7'-dichlorofluorescein (DCF) assays. Without 808 nm laser irradiation, all these YSNPs could not generate increased DCF fluorescence intensity, suggesting no ROS generation on these particles (Figure S8). However, upon 808 nm laser irradiation (0.5 W cm-2, 10 min), a significant increase in DCF fluorescence intensity was detected in Au 1-4 @CuS


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YSNPs, meaning that abundant ROS generation in these particles (Figure 2C). Among them, Au2@CuS YSNPs could generate the largest amount of ROS according to enhanced DCF fluorescence intensity, followed by Au3@CuS, Au4@CuS, and Au1@CuS YSNPs. The different ROS production ability of Au1-4@CuS YSNPs was ascribed to the different lattice activity of Au NRs because upon 808 nm laser irradiation Au2 NRs could generate the highest level of ROS, followed by Au3, Au4, and Au1 NRs (Figure S9). In order to highlight the key role of the yolkshell structure in photodynamic property, Au2 NRs and CuS HNPs were physically mixed to explore their ROS production properties. Figure S10 shows the ROS generation in the physical mixture was much weaker than that of Au2@CuS YSNPs, demonstrating the RET process significantly promotes the photon-to-hot electron energy conversion. The NIR-activated photothermal property of Au@CuS YSNPs was investigated through monitoring the temperature elevation of their aqueous suspension using thermometer and thermal infrared imaging. Figure 2D shows that upon 808 nm laser irradiation (0.5 W cm-2), the temperature of all these Au1-4@CuS YSNPs was rapidly elevated within 10 min, exhibiting the similar photothermal processes, and their photothermal transduction efficiencies were further calculated to be 40~44% (Figure S11A-S11H and Table S1) based on Roper and colleagues’ methods.29 The similar photothermal profiles of Au1-4@CuS YSNPs are dramatically distinct from their photodynamic profiles showing a different ROS generation trend, because the photothermal property is ascribed to RET process-mediated photon-heat conversion while the photodynamic property originates from RET process and the active crystalline plane of Au NRs,30, 31 where the LSPR-induced RET energy conversion efficiency is similar for Au1-4@CuS YSNPs but the crystalline plane-mediated ROS generation ability is different. Furthermore,


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Figure S12 and Figure S13 also indicate that the photothermal properties of Au2@CuS YSNPs were superior to the physical mixture, further corroborating that the importance of yolk-shell structure for the photothermal performance enhancement of Au@CuS YSNPs. NO generation from NIR laser irradiated AAu@CuS YSNPs. ROS potentially can convert L-Arg into NO during the RET process that releases high energy. The L-Arg was efficiently loaded into Au1-4@CuS YSNPs to form various AAu1-4@CuS YSNPs with the loading capacities of ~7% based on the measurement of guanidyl groups on L-Arg. NO generation of AAu1-4@CuS YSNPs was assessed by using Griess reagents. Figure 2E shows that AAu2@CuS YSNPs among various AAu1-4@CuS YSNPs could produce the highest NO level, followed by AAu3@CuS, AAu4@CuS,

and

AAu1@CuS

YSNPs, which coincides with the ROS production profile of

Au@CuS YSNPs (Figure 2C), demonstrating the stronger ROS production of Au@CuS YSNPs can facilitate the higher NO generation. In comparison, a physical mixture of Au2@CuS YSNPs and L-Arg was found to generate much lower NO level than AAu2@CuS YSNPs (Figure S14), further revealing the importance of yorlk-shell structure for NO conversion from L-Arg. Sequential release of NO and Dox from ADLAu2@CuS YSNPs. The efficient NO generation performance of AAu2@CuS YSNPs suggests their potentials as NO-responsive drug delivery nanosystem. Both L-Arg and Dox were loaded in Au2@CuS YSNPs to form

ADAu2@CuS

YSNPs, with the Dox loading capacity of 9.11% based on UV-Vis absorption measurement. Then, a NO-responsive liposome was used to encapsulate ADLAu2@CuS

ADAu2@CuS

YSNPs to form

YSNPs. For preparation of the NO-responsive liposome, o-phenylenediamine–

containing lipid was first synthesized through treating 1,2-diamino benzene with myristic acid in the

presence

of

O-(6-chlorobenzotriazol1yl)-N,N,N’,N’-tetramethyluronium

hexafluorophosphate (HCTU) and triisopropylamine.32 The chemical structure of o-


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phenylenediamine–containing lipid was identified by 1H nuclear magnetic resonance (1H NMR) spectroscopic analysis (Figure S15A). o-phenylenediamine–containing lipid can react with NO to form a water-soluble nitrogen-containing heterocycle (benzotriazole) and a fatty acid (myristic acid). Figure S15B shows the 1H NMR spectroscopy of o-phenylenediamine–containing lipid after reaction with NO. The chemical shift of hydrogen (a, b, c) of o-phenylenediamine– containing lipid shifted from T 6.72~7.25 ppm to T 7.5~8.5 ppm, which is attributed to hydrogen (a, b, c) of formed benzotriazole; simultaneously, the chemical shift of hydrogen (e) of ophenylenediamine–containing lipid was found to shift from T 2.4 ppm to T 3.4 ppm, which is attributed to hydrogen (e) of formed myristic acid. All these results prove the NO-responsive ability of o-phenylenediamine–containing lipid. o-phenylenediamine–containing lipid was mixed with soya lecithin, cholesterol, and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-Nmethoxy(polyethyleneglycol) (DSPE-PEG2000) to form dry film of NO-responsive liposome, where hydrophobic o-phenylenediamine–containing lipid was embedded in the phospholipid layer of liposome. Figure S16 shows the 1H NMR spectroscopy of NO-responsive liposome before and after reaction with NO, indicating the occurring of hydrolysis of ophenylenediamine–containing lipid. After sonicating the dry film with

ADAu2@CuS

YSNPs, the

achieved ADLAu2@CuS YSNPs had a hydrodynamic size of 274.2 ± 9.7 nm (Figure S17A) and a zeta potential of -23.5 ± 1.1 mV in water (Figure S17B). The TEM image showed that the NOresponsive liposomes well wrapped the surface of the particles (Figure 3A). Thermogravimetric analysis (TGA) indicated the mass content of NO-responsive liposomes on Au@CuS YSNPs was 8.8% (Figure S18). After 10 min of 808 nm laser irradiation (0.5 W cm-2), both NO and Dox could be released from ADLAu2@CuS YSNPs in sustained styles, however, there existed an obvious time gap between NO and Dox release (Figure 3B), where the initial NO release


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significantly preceded the Dox release. This releasing time gap should be ascribed to NOresponsive liposome-mediated differential escaping ability between NO and Dox. Upon NIR laser irradiation, NO is produced in ADLAu2@CuS YSNPs and has opportunity to hydrolyze the o-phenylenediamine groups of liposome layer, regionally destabilizing the phospholipid bilayer structure, as a result of NO release. o-phenylenediamine–containing lipid has previously been used to form a NO-responsive lipid-based liquid crystalline (LLC) nanosystem.32 The hydrolysis of o-phenylenediamine–containing lipid in response to NO can change the hydrophobicity/hydrophilicity property of LLC and induce a turn in the curvature of the lipid-bilayer, causing the destabilization and a phase inversion of LLC. In the present study, o-phenylenediamine–containing lipid should locate at the hydrophobic region of liposome. After reaction with NO, the hydrolyzed hydrophilic benzotriazole tends to escape the hydrophobic region, leading to the curvature change and regional destabilization of the lipid-bilayer, which potentially can destroy the assembly structure of liposome and result in breakage of liposome membrane. However, although NO can escape from liposome encapsulation, Dox is still blocked inside at this initial stage because its large molecular scaffold cannot go through the narrow pore created by NO. As the reaction progresses, the stability of liposome is aggravated by the large amount of NO and the pore is gradually enlarged, which allows Dox to get out of ADLAu2@CuS YSNPs. Thus, a significant releasing time gap is created and will be beneficial for NO and Dox sequential release in MDR cells, where the early release of NO can reduce the P-gp expression and facilitate the accumulation of subsequently released Dox for killing MCF-7/ADR cells. In comparison, ADAu2@CuS YSNPs were also encapsulated with NO-nonresponsive liposome to achieve ADWAu2@CuS YSNPs, which showed similar hydration particle size and zeta potential


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with those of

ADLAu2@CuS

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YSNPs but low NO and Dox release, meaning both NO and DOX

are sealed inside the particle without significant escape (Figure 3C). Moreover, the surface of ADAu2@CuS

YSNPs were fabricated with polyethylene glycol (PEG), and the formed

ADPAu2@CuS

YSNPs showed the rapid NO and Dox release profile within 15 min and a

sustained release profile in the following ~3 h after NIR laser irradiation. This sustained release style of ADPAu2@CuS YSNPs potentially is ascribed to their porous structure of CuS shell, which can provide a large surface area for NO absorption and desorption. However, there was no time gap between NO and Dox release (Figure 3D), which further highlights the critical role of NOresponsive liposome layer of ADLAu2@CuS YSNPs. Herein, it has to be mentioned, if the power density was elevated to 1 W cm-2, the NO and Dox release of

ADLAu2@CuS

YSNPs did not have the time gap anymore (Figure 3E), which is

because the dramatically produced NO at the high NIR laser power density can rapidly break up the liposome layer and leads to the rapid release of both NO and Dox without the pore size limit. So, the low power density is very necessary for

ADLAu2@CuS

YSNPs to exhibit this NO and

Dox sequential release behavior. Therapeutic effect and underlying mechanism of cells. The sequential NO and Dox release of

ADLAu2@CuS

ADLAu2@CuS

YSNPs to MCF-7/ADR

YSNPs potentially can be used for

treatment of Dox-resistant MCF-7/ADR cells. The resistance feature of MCF-7/ADR cells has been confirmed according to their viability examination to Dox (Figure S19). In vitro biocompatibility of

ADLAu2@CuS, ADPAu2@CuS,

and

ADWAu2@CuS

YSNPs were evaluated in

MCF/ADR cells at a wide dose range (according to CuS content) by 3-(4,5-dimethylthiazol-2yl)-2,5 diphenyltetrazolium bromide (MTS) assay. All these YSNPs did not exhibit toxicity in


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MCF-7/ADR cells (Figure S20), exhibiting excellent biocompatibility. Under 808 nm laser irradiation (0.5 W cm-2, 10 min), ADLAu2@CuS YSNPs could remarkably reduce the viability of MCF-7/ADR cell, showing the most potent injury to MCF-7/ADR cells (Figure 4A), which is mainly ascribed to the NO and Dox releasing time gap. Although

ADPAu2@CuS

YSNPs could

also reduce the viability of MCF-7/ADR cells (Figure 4A), the reduction level was much lower than that of ADLAu2@CuS YSNPs because NO and Dox release of

ADPAu2@CuS

YSNPs occurs

at the same time range and NO cannot precede Dox to optimize cell microenvironment. Moreover,

ADWAu2@CuS

YSNPs triggered very low reduction in cell viability because of their

low NO and Dox release. Live/Dead cell viability analysis also achieved the consistent results (Figure 4B and S21). The therapeutic effects of these YSNPs were further reflected by analyzing the apoptotic cell population through flow cytometry and Annexin V-FITC/Propidium Iodide (PI) cell apoptosis assay kit. Without subjected to NIR laser, these YSNPs induced little apoptotic cells (Figure S22). With 808 nm laser irradiation, ADWAu2@CuS


YSNPs could induce 22.6, 11.0, and 13. 7 % of the early apoptotic cells and 29.2,

12.6, and 0.067% of the late apoptotic cells, respectively (Figure 4C), revealing

ADLAu2@CuS

YSNPs among these YSNPs have the most efficient ability to induce apoptosis. Taken all together, ADLAu2@CuS YSNPs have an overwhelming advantage for MCF-7/ADR cell killing. To clarify the underlying mechanism of effective

ADLAu2@CuS

YSNPs, the cellular uptake,

NO level, P-gp protein expression, and Dox accumulation profiles were systematically studied. Cy7-labeled


and

ADWAu2@CuS

YSNPs were used for cellular

uptake studies. The fluorescence microscopy images (Figure 4D) indicated that the cells treated with these Cy7-labeld YSNPs could exhibit more promising NIR fluorescence than untreated cells, suggesting these YSNPs can be effectively internalized into MCF-7/ADR cells, which was


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further corroborated by flow cytometry analysis (Figure S23). To further clarify the cellular uptake approach and cellular distribution, Cy7-labeled

ADLAu2@CuS

YSNPs as representative

YSNPs were used to investigate the relationship with the endosomes of cells that were stained by LysoTracker Green. Figure S24 shows

ADLAu2@CuS

YSNPs could colocalize with endosomes

at the early stage (2 h incubation), meaning these YSNPs are internalized into cells through the endocytosis approach, but escaped endosomes into cytosol at the late stage (6 h of incubation). Cellular NO production was evaluated by fluorescence microscopy using NO fluorescence indicator. Figure 4E shows that, when irradiated by 808 nm laser at 0.5 W cmU' for 10 min, both ADLAu2@CuS

and

ADPAu2@CuS

YSNPs could significantly elevate the cellular NO levels in

MCF-7/ADR cells, while ADWAu2@CuS YSNPs had relatively little effect on the NO level. P-gp expression of MCF-7/ADR cells was analyzed by a western blot method. Figure 4F reveals that, when irradiated by 808 nm laser at 0.5 W cmU' for 10 min, both ADLAu2@CuS and ADPAu2@CuS YSNPs could significantly reduce P-gp expression, where advantageous than

ADPAu2@CuS

YSNPs, but

ADLAu2@CuS

ADWAu2@CuS

YSNPs were more

YSNPs did not affect the P-gp

abundance. Moreover, Dox accumulation was evaluated by determining cellular Dox fluorescence intensity through V = cytometry. As observed in Figure 4G, after 10 min of irradiation with an 808 laser at 0.5 W cmU',

ADLAu@CuS

YSNPs among these YSNPs could

induce the most prominent Dox fluorescence enhancement in cells. Taken all together, NOresponsive liposome layer endows

ADLAu2@CuS

YSNPs with evident NO and Dox releasing

time gap, resulting in elevated NO level, reduced P-gp protein expression, and enhanced Dox accumulation in the cells, which facilitates ADLAu2@CuS YSNPs to exhibit the potent therapeutic effect in MCF-7/ADR cells.


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image analysis of cellular NO level based on DAF-FA staining; (F) Western blot of P-gp protein; the protein density was calculated by Image J software; (G) Flow cytometry analysis of cellular Dox levels. For (A) to (G), cells were incubated with various concentrations (A) or 100 µg mL-1 (B) or 25 µg mL-1 YSNPs (equivalent to CuS) (C-G) for 6 h, and then irradiated with an 808 nm laser at 0.5 W cm-2 for 10 min, followed with additional incubation. *p < 0.05; **p < 0.01.

Tumor accumulation, biodistribution, biocompatibility, and therapeutic effect of ADLAu2@CuS

YSNPs in MCF-7/ADR tumor-bearing mice. Encouraged by in vitro

therapeutic assessments, the in vivo therapeutic effect of

ADLAu2@CuS

YSNPs was further

examined in mice bearing MCF-7/ADR tumor. Fluorescence imaging and inductively coupled plasma-optical emission spectroscopy (ICP-OES) analysis were first employed to explore the biodistribution of ADLAu2@CuS

ADLAu2@CuS

YSNPs in mice. After intravenous injection with Cy7-labeled

YSNPs, the fluorescence images of mice were taken at 1-24 h post-injection.

Figure 5A shows that the tumor area displayed the obvious fluorescence after injection, revealing the significant tumor accumulation ability of Cy7-ADLAu2@CuS YSNPs. Au@CuS YSNPs have exhibited an adjustable, intense and narrow absorption peak in NIR region,33, 34 and have been used as molecular probes for tumor photoacoustic imaging (PA) imaging. Further PA image of tumor region at 24 h post-injection displayed the even distribution of

ADLAu2@CuS

YSNPs in the tumor tissue (Figure 5B). Moreover, at 24 h post-injection, the Cu element of the major organs and tumor tissues dissected from mice was analyzed by ICP-OES to further corroborate the high accumulation of Cu element in tumor tissues (Figure 5C).


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Nano Letters 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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under 808 nm laser irradiation at 0.5 W cm-2 for10 min; (F) Representative photos of mice and (G) H&E stained tumor sections of mice at the end of treatment as described in (E). All the doses of YSNPs were equivalent to 20 mg CuS/kg mouse. *p < 0.05; **p < 0.01.

The effective accumulation of

ADLAu2@CuS

YSNPs in the tumor region provides a powerful

basis for in vivo treatment. After intravenous injection of

ADLAu2@CuS

YSNPs into mice, the

tumor area was irradiated by an 808 nm laser at 0.5 W cm-2for 10 min at 24 h post-injection. The mice were imaged by an infrared camera to observe the changes in tumor temperature. Figure 5D presents the tumor temperature could increase from ~ 28 ºC to ~ 45 ºC under 808 nm laser irradiation, which potentially can induce cellular hyperthermia. The therapeutic effect of ADLAu2@CuS

YSNPs was evaluated by assessment of tumor growth inhibition. After 24 h of

intravenous injection of

ADLAu2@CuS, ADPAu2@CuS, ADWAu2@CuS

YSNPs into mice with or

without the following irradiation by 808 nm laser at 0.5 W cm-2for 10 min at 24 h post-injection, the tumor growth rate was monitored every day through measurement of tumor sizes. Figure 5E reveals the highest inhibition efficiency of ADLAu2@CuS YSNPs in tumor growth under 808 nm laser irradiation, followed by

ADPAu2@CuS

and

ADWAu2@CuS

YSNPs. After treatment, the

representative photos of mice were displayed in Figure 5F. In contrast, if not subjected to NIR laser irradiation (Figure S25), these YSNPs showed little inhibition effect on tumor growth, demonstrating their nontoxicity to tumor cells. To further evaluate the therapeutic effect of

ADLAu2@CuS

YSNPs, haematoxylin and eosin

(H&E) stained tumor sections were employed to investigate the pathological structure evolution of the tumors at the end of treatments. If not subjected to NIR laser irradiation, the mice treated with


or

ADWAu2@CuS

YSNPs showed little pathological change

(Figure S26). However, upon 808 nm laser irradiation,

ADLAu2@CuS

YSNPs were able to


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induce the most severe damage in the tumor tissue, followed by ADPAu2@CuS and ADWAu2@CuS YSNPs (Figure 5G). This histological evaluation corroborates the promising therapeutic effect of ADLAu2@CuS YSNPs for MCF-7/ADR tumor. The biocompatibility of these YSNPs was investigated by recording the body weight and evaluating the pathological structure of main organs of mice. The mice treated with PBS or YSNPs did not significantly differ in their body weights no matter whether subjected to NIR laser or not (Figure S27). Histological analysis of the main organs excised from the mice showed that these YSNPs could not induce significant abnormalities or damage in major organs with or without NIR laser irradiation (Figure S28). These results demonstrate the excellent biocompatibility of ADLAu2@CuS YSNPs. CONCLUSIONS In summary, NIR laser-activated RET process and ROS generation in the confined space of Au@CuS YSNPs can convert L-Arg into NO, where facet Au2 NRs can more significantly promote ROS generation and NO formation due to their active crystalline plane. L-Arg/Doxloaded and NO-responsive liposome encapsulated Au2@CuS YSNPs (ADLAu2@CuS YSNPs) can sequentially release NO and Dox in response to NO generation, leading to a promising therapeutic effect in MCF-7/ADR cells, where the early-stage NO release can reduce P-gp expression and create a preferential microenvironment for the late-stage Dox accumulation. In MCF-7/ADR tumor-bearing mice,

ADLAu2@CuS

YSNPs exhibit excellent tumor accumulation

ability, therapeutic effect, and biocompatibility, presenting great potential for Dox-resistant cacer therapy.


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MATERIALS AND METHODS Chemicals. All chemicals were purchased from Sigma-Aldrich and the reagents could be used directly. The water used was purified reagent grade water (Millipore, Bedford, USA). Preparation of conventional and faceted Au NRs. Preparation of conventional Au1 NRs. Au1 NRs were prepared in an aqueous solution by a seed-mediated method.35 A seed solution was prepared as follows: 5 mL of hexadecyl trimethyl ammonium bromide (CTAB) aqueous solution (0.2 mol L-1) was mixed with 5 mL of gold chloride hydrate (HAuCl4) aqueous solution (0.5 mmol L-1) under vigorous stirring for 1 min, followed by addition of 0.6 mL of borohydride (NaBH4) aqueous solution (0.01 mol L-1). The resulting seed solution was stored at 37 ºC for 1 h before use. To prepare Au1 NRs, CTAB (1.4 g) and sodium oleate (NaOL) (0.25 g) were mixed in 50 mL of water, and then, 4.8 mL of 4 mmol L-1 silver nitrate (AgNO3) was transferred to the mixture under stirring at 35 °C, followed by addition of 50 mL of 1 mmol L-1 HAuCl4 aqueous solution after 15 minutes of reaction. With the solution getting colorless, 0.36 mL of concentrated HCl (37 wt %) and 0.26 mL of 64 mmol L-1 ascorbic acid (AA) solution were added to the mixture. Finally, 0.08 mL of the seed solution was added. After standing at 35 °C for 12 h, the achieved suspension was centrifuged (8000 rpm, 10 min), and the resulting Au1 NRs as pellets were re-dispersed in 10 mL of 20 mmol L-1 CTAB. Preparation of faceted Au2 NRs. Au2 NRs were prepared using Au1 NRs as a seed. The above Au1 NR suspension was washed once with water, and re-dispersed in 100 P! of 0.10 mol L-1 CTAB. To prepare a growth solution, 7.40 mL of H2O, 0.2 mL of HAuCl4 (10 mmol L-1), and 1.0 mL of AA (0.10 mol L-1) into 1.30 mL of CTAB (0.10 mol L-1) were mixed first. 100 µL of the above Au1 NR suspension was added to the growth solution, and the achieved solution was allowed to stand at 35 °C for 1 h, and the products then collected by centrifugation (8000 rpm, 10 min). The pellet as formed Au2 NRs was re-dispersed in 10 mL of 20 mmol L-1 CTAB. Preparation of faceted Au3 and Au4 NRs. Over growth of Au1 NRs was used to form Au3 and Au4 NRs. The above Au1 NR suspension was washed once with water, and re-dispersed in 100 P! of 0.10 mol L-1 CTAB. To prepare a growth solution, 7.395 mL of H2O, 0.2 mL of HAuCl4 (10 mmol L-1), 5 P! of Cu(NO3)2 (10 mmol L1),

and 1.0 mL of AA (0.10 mol L-1) into 1.30 mL of CTAB (0.10 mol L-1) solution were mixed first. 100 µL of

the above Au1 NR suspension was transferred to the growth solution, and the achieved solution was allowed to


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stand at 35 °C for 1 h, and the products then collected by centrifugation (8000 rpm, 10 min). The pellet as formed Au3 NRs was re-dispersed in 1 mL of 20 mmol L-1 CTAB. To achieve Au4 NRs, the similar procedure was carried out using the similar growth solution containing 5 µL of Cu (NO3)2 (70 mmol L-1) instead of Cu (NO3)2 (10 mmol L-1). Preparation of Au1-4@CuS YSNPs. 1 mL of copper nitrate trihydrate (Cu(NO3)2•3H2O) solution (0.1 mol L-1) incubated in a 40 °C coil bath was added to 9 mL of polyvinylpyrrolidone (PVP, K-30) (0.2 g) aqueous solution, followed by addition of 5 mL of above prepared Au1-4 nanomaterial solution and 5 P! of hydrazine monohydrate (N2H4•H2O) solution (35 wt %). After 10 min of reaction, 0.3 mL of sodium sulfide (Na2S) solution (0.2 mol L-1) was added and the resulting mixture was stirred for 2 h. Au1-4@CuS YSNPs were collected by centrifugation (8000 rpm, 10 min). Preparation of Arg-loaded Au1-4@CuS YSNPs (AAu1-4@CuS YSNPs). 5 mL of 2 mg mL-1 L-Arg in PBS (20 mM, pH=7.4) was mixed with 5 mL of 5 mg mL-1 Au1-4@CuS YSNP suspension in PBS, and the achieved suspension was stirred for 24 h at 42 ºC for sufficiently loading L-Arg inside Au1-4@CuS YSNPs. Ultimately, the final AAu1-4@CuS YSNPs were collected via centrifugation, and the unloaded L-Arg was assessed by determining the guanidyl groups of Arg.36 Determination of guanidyl groups on arginine. Solution 1: 0.24 g of phenol was dissolved in 3 mL of npropanol; solution 2: 15 µl of diacetyl was mixed with 3 ml of n-propanol; solution 3:1 mol L-1 NaOH. The three solutions were mixed in equal proportions. 130 µl of above solution was further mixed with 20 µl of the test sample, and the achieved solution was kept at room temperature for 20 minutes and subjected to measurement of absorbance at 540 nm to determine the L-Arg loading amount. The L-Arg loading capacity was calculated as following: LC = (Ctotal Arg-Csupernatant Arg) / (Ctotal particle) × 100%; Ctotal Arg is the total mass concentration of Arg; Csupernatant Arg is the Arg mass concentration in supernatant; Ctotal particle is the total mass concentration of AAu1-4@CuS YSNPs. Preparation of L-Arg- and Dox-loaded Au2@CuS YSNPs (ADAu2@CuS YSNPs). 5 mL of L-Arg (2 mg mL-1) in PBS , 5 mL of Dox (1 mg mL-1 ) in PBS, and 5 mL of Au@CuS YSNP suspension (5 mg mL-1 ) in PBS were completely mixed, and the resulting suspension was kept at 42 °for 24 h under vigorous stirring. Then, the suspension was centrifuged (8000 rpm, 10 min) to remove unloaded L-Arg and Dox, and the pellet


23

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as

ADAu2@CuS

Page 24 of 34

YSNPs was washed with PBS three times. The Dox concentration in the supernatant was

determined by measurement of the absorbance at 480 nm. Dox loading capacity (LC) was calculated as following: LC = (Ctotal Dox-Csupernatant Dox) / (Ctotal particle) × 100%. Ctotal Dox is the total mass concentration of Dox; Csupernatant

Dox

ADAu2@CuS

is the Dox mass concentration in supernatant; Ctotal

particle

is the total mass concentration of

YSNPs.

Synthesis of NO-responsive and -nonresponsive liposome. Synthesis of a NO-responsive lipid small molecule. 10 mmol of myristic acid dissolved in 150 mL of tetrahydrofuran (THF) was mixed with 12 mmol of HCTU under stirring. 20 ml of acetonitrile containing 2.163 g of 1,2-diamino benzene was added to the mixture. After 15 min of reaction, 4.3 ml of triisopropanolamine was finally added. After overnight stirring, the product was extracted by column chromatography using chloroform/methanol (3/1, v / v) as eluent. 1H NMR was performed to determine the purity of the final product. 1H

NMR (500 MHz, CDCl3): 7.16–7.22 (d, 1H, -C6H4-);7.02–7.12 (t, 1H, -C6H4-); 6.78–6.85 (t, 2H, -

C6H4-);2.38–2.45 (t, 2H, -CH2-); 1.73–1.79 (m, 2H, -CH2-); 1.25–1.30 (m, 20H, -CH2-); 0.87–0.94 (t, 3H, CH3). Synthesis of NO-responsive liposome. The soya lecithin/cholesterol/DSPE-PEG2000/NO response lipid small molecule at a molar ratio of 33:59:2:6 were mixed in chloroform/methanol (3:1, v/v) solution in a round bottom flask, which was further slowly rotated at 40 ° C to form a film through evaporation . Synthesis of NO-nonresponsive liposome. NO-nonresponsive liposome was synthesized using the similar procedure except removing NO response lipid small molecule. Preparation of

ADLAu2@CuS

and

ADWAu2@CuS

YSNPs. 5 mL of 1 mg mL-1

ADAu2@CuS

YSNPs

aqueous suspension was added to the above spin-dried NO-responsive or –nonresponsive liposome film flask, where the weight ratio of Au2@CuS YSNPs to liposome was fixed at 1:1.5. After two hours of sonication at room temperature,

ADLAu2@CuS

or

ADWAu2@CuS

YSNPs were harvested by centrifuging the suspension

(8000 rpm, 7 min), followed by washing with water for three times. Preparation of 1SH-PEG

ADPAu2@CuS

YSNPs. 1 mg mL-1

ADAu2@CuS

YSNPs (5 mL) was mixed with 2 mg mL-

(3.4 kDa) (5 mL), and the achieved suspension remained stirred for 24 h. Through centrifuging the

suspension (8000 rpm, 7 min) and washing with water, ADPAu2@CuS YSNPs as the pellet was obtained.


24

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Preparation of Cy7-labeled Cy7-labeled

ADLAu2@CuS

ADLAu2@CuS

YSNPs,

ADWAu2@CuS

YSNPs were prepared through sonicating

YSNPs, and

ADAu2@CuS

ADPAu2@CuS

YSNPs.

YSNPs with Cy7-labeled

NO-responsive liposome film that containing the soya lecithin, cholesterol, DSPE-PEG2000, NO response lipid small molecule, DSPE-PEG-Cy7 (Shanghai Jinpan Biotech Co Ltd) at a molar ratio of 31:59:2:6:2. Similar procedure was used to prepare Cy7-labeled lipid small molecule. Cy7-labeled

ADPAu2@CuS

ADWAu2@CuS

YSNPs except removing the NO response

YSNPs were prepared through incubating ADAu2@CuS with

SH-PEG containing 2% of SH-PEG-Cy7 (Shanghai Jinpan Biotech Co Ltd). Characterization. TEM images were collected using a JEOL microscope (1200 EX II). The crystalline structure was evaluated by XRD analysis on a Rigaku-Dmax 2500 diffractometer. UV-Vis-NIR spectroscopy were collected on a Cary V-550 UV-Vis-NIR spectrometer. Measurement of photothermal performance. To analyze the photothermal performance of Au1-4@CuS YSNPs, a quartz cuvette containing 1.5 mL of 100 P of mL-1 YSNP aqueous suspension was exposed to an 808 nm laser (0.5 W cm-2, 10 min). During the experimental process, the temperature was recorded every 30 seconds using a high-precision thermocouple sensor. To calculate the photothermal conversion efficiency, the suspension cooled down naturally after removing the laser. The photothermal conversion efficiency was determined by the method reported by Roper and co-workers.29 ROS detection. ROS were specifically determined by 2',7'-dichlorofluorescein (DCF) assay. 20 P! of YSNP aqueous solution (equivalent to 100 P mL-1 CuS) was mixed with 80 P! of 10 P

DCF, followed by

10 min of exposure to an 808 nm laser at the power density of 0.5 W cm-2 and another 2 h of incubation. DCF fluorescence emission spectra with excitation at 455 nm were measured by a Spectra Max M5 microplate reader. NIR laser-triggered NO formation of AAu1-4@CuS YSNPs. 5 mL of AAu1-4@CuS YSNPs aqueous suspension (1 mg mL-1) was irradiated by 10 min of 808 nm laser (0.5 W cm-2). The formed NO amount was determined by a Griess reagent kit (Beyotime Biotechnology) through the detection of nitrite ions based on measurement of absorbance at 540 nm. NIR laser-triggered NO and Dox sequential release of and

ADPAu2@CuS

YSNPs. 5 mL of

ADLAu2@CuS

YSNPs,

ADLAu2@CuS

YSNPs,

ADWAu2@CuS

ADWAu2@CuS

YSNPs, or

YSNPs,

ADPAu2@CuS

YSNP


25

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Page 26 of 34

aqueous suspension (1 mg mL-1) was exposed to 10 min of 808 nm laser irradiation (0.5 W cm-2). The release of NO and DOX was measured at different time periods within 15 hours. At various time points, 200 P of the composite dispersion was taken from the total solution. After centrifugation (8000 rpm, 7 min), Dox in supernatant as the released Dox was measured based on its absorbance at 480 nm. Then, a NO detection reagent Griess reagent kit (Beyotime Biotechnology) was added to the supernatant to measure the amount of NO released. Cell culture. Human breast cancer adriamycin resistant cell line MCF-7/ADR cells were cultured in 1640 Medium (containing 12% fetal bovine serum, 100 units mL-1 penicillin and 100 mg mL-1 streptomycin). The cells were incubated in a humidified 5% CO2 incubator at 37 °C. Subculture was carried out when the cells grew to 80%-90% of the surface area. Cellular uptake of YSNPs. Cellular uptake of various YSNPs in MCF-7/ADR cells was investigated by fluorescence microscopy and flow cytometry. For fluorescence microscopy study, a coverslip was placed in one well of a six-well plate, and 1.6 x 105 MCF-7/ADR cells were placed in each well and grown for 16 h. Thereafter, the cells were incubated with Cy7-labeled YSNPs (25 µg mL-1) in culture medium for 6 h. After washing by PBS and fixation with 4% paraformaldehyde, the nuclei were stained with 1 µmol L-1 Hoechst 33342 for 30 min. Finally, the coverslips were mounted on glass slides and imaged on a Zeiss confocal microscope (LSM 700, Carl Zeiss, Germany). For flow cytometry study, a similar process was performed as described above. After treatment with Cy7-labeled YSNPs, cells were washed and collected by trypsin, and their fluorescence intensity was detected by a FACS-Calibur flow cytometer (BD Biosciences). Cytotoxicity measurements of YSNPs with or without NIR laser irradiation. 1 × 104 cells were seeded in each well of a 96-multiwell microplate for 16 h of growth. The original medium was then replaced with 100 µL of medium containing 6.25, 12.5, 25, 50, 100 and 200 µg mL-1 YSNPs (according to CuS content). For the irradiation group, after 6 h of incubation with above particles, cells were irradiated by 10 min of 808 nm laser (0.5 W cm-2) and incubated for additional 18 h. For the non-irradiation group, cells were incubated with above particles for 24 h without any laser treatment. Finally, cell viability was determined by MTS assay kit (Promega, Shanghai, China) according to manufacturer’s instruction.


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Live/dead cell staining analysis. 2 × 104 cells were seeded in each well of a 48-multiwell microplate for 16 h of growth. The original medium was then replaced with 200 µL of medium containing 100 µg mL-1 YSNPs (according to CuS content). For the irradiation group, after 6 h of incubation with above particles, cells were irradiated by 10 min of 808 nm laser (0.5 W cm-2) and incubated for additional 18 h. For the non-irradiation group, cells were incubated with above particles for 24 h without any laser treatment. Then, the cell staining was carried out using calcein-AM (1 Pmol L-1) and PI (1 µmol L-1). After washing with PBS and fixation with 4% paraformaldehyde, cells were imaged by Olympus BX-51 microscope (Tokyo, Japan). Apoptosis analysis by flow cytometry. 1.6 x 105 MCF-7/ADR cells were placed in each well of a 6multiwell plate for 16 h of growth. The original medium was then replaced with 1.6 mL of medium containing 25 µg mL-1 YSNPs (according to CuS content). For the irradiation group, after 6 h of incubation with above particles, cells were irradiated by 10 min of 808 nm laser (0.5 W cm-2) and further incubated for additional 18 h. For the non-irradiation group, cells were incubated with above particles for 24 h without any laser treatment. Then, after washing with PBS, the cells were collected by trypsinization, and stained by Annexin V-FITC and PI, and analyzed on a BD Accuri C6 flow cytometer. Intracellular nitric oxide staining. Cellular NO production was studied by staining the cells with a NO indicator, 3 amino-4-aminomethyl-2',7'-di-fluorescein diacetate (DAF-FM) (Biyuntian, Nanjing, China). The procedure was as follows: 1.6 x 105 MCF-7/ADR cells were placed in each well of a 6-multiwell plate for 16 h of growth; then, the cells were stained with 5 P

L-1 DAF-FM solution (1 mL) in a 37 °C incubator for 30

min, and treated with 25 µg mL-1 YSNPs for 6 h; the cells were subjected to 808 nm laser irradiation (0.5 W cm-2, 10 min); the fluorescence cell image was taken on a fluorescence microscope (Nikon Ti-S microscope, Tokyo, Japan). Determination of intracellular Dox accumulation. Intracellular Dox level in MCF-7/ADR cells was determined by flow cytometry. 1.6 x 105 MCF-7/ADR cells were placed in each well of a six-multiwell plate for 16 h of growth. The original medium was then replaced with 1.6 ml of medium containing 25 µg mL-1 YSNPs (according to CuS content). For the irradiation group, after 6 h of incubation with above particles, cells


27

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Page 28 of 34

were irradiated by 10 min of 808 nm laser (0.5 W cm-2) and incubated for additional 6 h. Then, after washing with PBS, the cells were collected by trypsinization and analyzed on a BD Accuri C6 flow cytometer. Western blot analysis of P-gp expression. 1.6 x 105 MCF-7/ADR cells were placed in each well of a sixmultiwell plate for 16 h of growth. The original medium was then replaced with 1.6 mL of medium containing 25 µg mL-1 YSNPs (according to CuS content). For the irradiation group, after 6 h of incubation with above particles, cells were irradiated by 10 min of 808 nm laser (0.5 W cm-2) and incubated for additional 6 h. For the non-irradiation group, cells were incubated with above particles for 12 h without any laser treatment. West blot analysis was performed as previously described37 except that anti-P-gp monoclonal antibody (abcam Anti-PGP antibody (ab103477)) (1:1000) was used. Animals. Female BALB/c athymic nude mice weighing approximately 20 g were purchased from Beijing Vital River Experiment Animal Technology Co. Ltd. All experiments were conducted in Center for Experimental Animals, Jilin University, in accordance with the requirements of the Animal Ethics Committee of Jilin University. In vivo fluorescence imaging and biodistribution analysis. 100 µL of PBS (containing 1 × 107 MCF7/ADR cells) was subcutaneously implanted into the back of female nude mice for tumor growth. When the tumor grew to a size of approximately 150 mm3, the mice were intravenously injected with 200 P! of Cy7labeled

ADLAu2@CuS

YSNP (equivalent to 20 mg CuS/kg mice). After certain period of time, the mice were

anesthetized for in vivo fluorescence imaging (Maestro In Vivo Imaging System (CRi Inc)). At the end of the treatment, the mice were sacrificed to collect major organ tissues for Cu element-based ICP-OES (Thermo Scientific ICAP 6300) analysis. In vivo photoacoustic (PA) imaging. MCF-7/ADR tumor–bearing nude mice were intravenously injected with 200 µL of

ADLAu2@CuS

YSNPs (equivalent to 20 mg CuS/kg mice). PA imaging of mice were

accomplished at 24 h post-injection by a MOST system (MOST in Vision 128). In vivo therapeutic evaluation. 100 P! of PBS (containing 1 × 107 MCF-7/ADR cells) was subcutaneously injected into the back of female nude mice for tumor growth. When the tumor grew to a size of approximately 75 mm3, the mice were divided into eight groups: (a) PBS; (b) ADLAu2@CuS;

(e) PBS (NIR); (f)

ADWAu2@CuS

(NIR); (g)

ADWAu2@CuS;

ADPAu2@CuS

(c)

(NIR); (h)

ADPAu2@CuS;

ADLAu2@CuS

(d)

(NIR).


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Mice were intravenously injected with 200 µL of various YSNP suspensions (equivalent to 20 mg CuS/kg mice). For NIR irradiation group (e)-(h), the tumor area was exposed to 808 nm laser irradiation (0.5 W cm-2, 10 min) at 24 h post-injection. The tumor temperature was monitored by an IR thermal camera (FLIR, USA). Tumor size and body weighs of mice were recorded in the following days. The tumor volume (V) was calculated by a formula of V= ab2/2, where a and b are the tumor length and width, respectively. Histology analysis. After therapeutic evaluation, the main organs of the mouse (heart, liver, spleen, lung, kidney and tumor) were removed, immobilized in 10% neutral buffered formalin, and then encapsulated in paraffin. The sample was cut into sections having a thickness of 5 µm, and stained with H&E. Finally, the pathology was examined by a microscopy.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.nanolett.XX. AUTHOR INFORMATION Corresponding Author * E-mail: [email protected] * E-mail: [email protected] Author Contributions L. Wang, H. Zhang and X. Li contributed to the design and implementation of the research. L. Wang, Y. Chang, Y. Cheng and Y. Feng conducted the preparation of nanomaterials. L. Wang, H. Jian, X. Ma, R. Zheng, X Wu and K. Xu carried out the cell and animal examination. L. Wang, X. Li and H. Zhang wrote the manuscript. X. Li and H. Zhang oversaw the whole project. Notes


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Page 30 of 34

The authors declare no competing financial interest. ACKNOWLEDGMENT This study was funded by National Natural Science Foundation of China (21573216, 21703232, 21777152)

and

Jinlin

Provincial

Science

and

Technology

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Program

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Nitric Oxide Stimulated Programmable Drug Release of Nanosystem

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