Copper sulfide nanoparticle-based localized drug delivery system as an effective cancer synergistic treatment and theranostic platform

Accepted Manuscript Copper sulfide nanoparticle-based localized drug delivery system as an effective cancer synergistic treatment and theranostic platform Lin Hou, Xiaoning Shan, Lisha Hao, Qianhua Feng, Zhenzhong Zhang PII: DOI: Reference:

S1742-7061(17)30167-8 http://dx.doi.org/10.1016/j.actbio.2017.03.005 ACTBIO 4773

To appear in:

Acta Biomaterialia

Received Date: Revised Date: Accepted Date:

12 October 2016 4 February 2017 3 March 2017

Please cite this article as: Hou, L., Shan, X., Hao, L., Feng, Q., Zhang, Z., Copper sulfide nanoparticle-based localized drug delivery system as an effective cancer synergistic treatment and theranostic platform, Acta Biomaterialia (2017), doi: http://dx.doi.org/10.1016/j.actbio.2017.03.005

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Copper sulfide nanoparticle-based localized drug delivery system as an effective cancer synergistic treatment and theranostic platform Lin Hou, Xiaoning Shan, Lisha Hao, Qianhua Feng, Zhenzhong Zhang*

School of Pharmaceutical Sciences, Zhengzhou University, 100 Kexue Avenue, Zhengzhou 450001, China Collaborative Innovation Center of New Drug Research and Safety Evaluation, Henan Province, China Key Laboratory of Targeting Therapy and Diagnosis for Critical Diseases, Henan Province

*Corresponding author. Tel: 86-371-67781910; Fax: 86-371-67781908. Email: [email protected].

ϭ 

Abstract Localized cancer treatment with combination therapy has attracted increasing attention for effective inhibition of tumor growth. In this work, we introduced diffusion molecular retention (DMR) tumor targeting effect, a new strategy that employed transferrin (Tf) modified hollow mesoporous CuS nanoparticles (HMCuS NPs) to undergo extensive diffuse through the interstitium and tumor retention after a peritumoral (PT) injection. Herein, HMCuS NPs with strong near-infrared (NIR) absorption and photothermal conversion efficiency could serve as not only a drug carrier but also a powerful contrast agent for photoacoustic imaging to guide chemo-phototherapy. The iron-dependent artesunate (AS), which possessed profound cytotoxicity against tumor cell, was used as model drug. As a result, this AS loaded Tf-HMCuS NPs (AS/Tf-HMCuS NPs) system could specially target to tumor cells and synchronously deliver AS as well as irons into tumor to achieve enhanced antitumor activity. It was found that AS/Tf-HMCuS NPs was taken up by MCF-7 cells via Tf-mediated endocytosis, and could effectively convert NIR light into heat for photothermal therapy as well as generated high levels of reactive oxygen species (ROS) for photodynamic therapy. In addition, in vivo antitumor efficacy studies showed that tumor-bearing mice treated with AS/Tf-HMCuS NPs through peritumoral (PT) injection under NIR laser irradiation displayed the strongest inhibition rate of about 74.8%, even with the reduced frequency of administration. Furthermore, to demonstrate DMR, the optical imaging, photoacoustic tomography and immunofluorescence after PT injection were adopted to track the behavior of AS/Tf-HMCuS NPs in vivo. The results exhibited that Tf-HMCuS NPs prolonged the local accumulation and retention together with slow vascular uptake and extensive interstitial diffusion, which was consistent with the biodistribution studies of AS/Tf-HMCuS NPs. Therefore, the approach of localized delivery through DMR combined with multi-mechanism therapy may be a promising method for cancer treatment.

Keywords:

localized

delivery,

combination

tumor-targeted, hollow mesoporous CuS

Ϯ 

therapy,

diffusion

molecular

retention,

1. Introduction Cancer is a major public health problem all around the world, and currently, the standard clinical treatments include surgery and systemic multiagent chemotherapy [1, 2]. However, systemic administration of most chemotherapeutic drugs can only deliver a limited amount of drug to the tumor site and commonly lead to both short-term and long-term adverse effects [3]. Therefore, localized drug delivery systems which provide both physical targeting to the tumor and the controlled release of drugs may improve patient outcome and potentially overcome the limitations associated with systemic administration [4]. During the past few decades, increasing attention has been paid to injectable thermosensitive hydrogels as a promising approach for local antitumor drug delivery [5-7]. Nevertheless, it is challenging for this kind of system to achieve a high drug loading, overcome biological barriers from the site of injection to the site of action, or entrap drug-loaded particles into a hydrogel without adversely affecting the formation process [7-9]. Especially, the delivery barriers involve accumulation at tumor sites, deep penetration into the tumor interstitium, internalization by cancer cells, and intracellular drug release [10]. To solve these problems as above, a technique termed diffusion molecular retention (DMR) tumor targeting which was proposed by Professor Lee Josephson was introduced in our work [11]. DMR can bypass the delivery barriers encountered with intravenous (IV) administration, through slow vascular uptake and extensive interstitial diffusion by employing the peritumoral (PT) administration. Herein, we employed hollow mesoporous CuS NPs (HMCuS NPs), which were considered as intelligent vehicle preferable to solid nanoparticles due to their uniform pore structure and high surface area for drug encapsulation [12-14], as the carrier for localized drug delivery. Moreover, it has been reported that CuS can not only be selected as photosensitizer for photothermal therapy but also generate cytotoxic reactive oxygen species (ROS) for photodynamic therapy (PDT) under NIR irradiation. Owing to the synergistic effects, this system can achieve enhanced antitumor efficacy with a lower drug dose and mild irradiation conditions [15]. Importantly, CuS also possesses the property suitable for photoacoustic imaging [16], which could help us observe DMR after PT injection. Although conceptually impressive, HMCuS NPs are still lack of active recognition ability with tumor site and the premature drug release should be settled before their application for drug ϯ 

delivery [17]. As is known, transferrin receptor (TfR) is expressed more abundantly in malignant tissues and transferrin (Tf) can be potentially utilized as a cell marker for tumor detection. Tf-TfR interaction has been employed as a potential efficient pathway for cellular uptake of drugs and genes [18]. Consequently, Tf was anchored on the surface of HMCuS NPs as the targeting molecule in our study. What is more, it also could act as a gatekeeper by forming a dense layer around HMCuS NPs, which is responsible for minimizing premature drug release. Most importantly, Tf can transport iron, which is an important regulator of cell growth. Meanwhile, it has been found that artesunate (AS), a partially-synthetic derivative of artemisinin, possesses profound cytotoxicity against tumor cells in vitro and in vivo. And the key anti-tumor mechanism is thought to be iron-mediated endoperoxide bridge cleavage and the formation of toxic free radical [19]. That is to say, AS can be activated by intracellular iron, and the co-delivery of Tf and AS to cancer cells is an attractive strategy to enhance the anticancer activity of AS. Considering the characteristics of Tf and AS, we chose AS as the model drug in this investigation, which could be selectively accumulated in cancer cells through the Tf-TfR interaction. And after endocytosis, iron released from Tf would then readily react with the AS to form free radicals, leading to cell death [20]. In this study, we hypothesized a localized DMR tumor targeting drug delivery system based on nanocarriers entrapment of anticancer drug for the first time (Fig.1), which can achieve high drug loading, improve local drug accumulation and retention, accomplish synergistic combination of chemo-phototherapy, and finally enhance antitumor effect. For the proof of concept, AS-loaded Tf modified HMCuS NPs (AS/Tf-HMCuS NPs) were prepared and characterized using TEM, fluorescence spectrum and ultra-violet-visible (UV-VIS) spectrometer. DMR tumor targeting effect

was

investigated

by

the

optical

imaging,

photoacoustic

tomography

and

immunofluorescence after PT injection of the designed system in vivo. Additionally, the cytotoxicity, receptor-mediated tumor targeting characteristics and synergistic anticancer efficacy were evaluated both in vitro and in vivo. 

2. Experimental section 2.1. Chemicals Artesunate (AS) (Purity > 99.0 %) was purchased from Shanghai Ruiyong Biotech Limited ϰ 

Company. Transferrin (Tf) (purity >98%), fluorescein isothiocyanate (FITC), Rhodamine 6G , Sulforhodamine B (SRB), and dimethyl sulfoxide (DMSO) were obtained from SigmaeAldrich (St Louis, MO, USA). PVPK30 (MW=30000) was bought from Solarbio Science!technology co., Itd. 1,1‘-dioctadecyl-3,3,3’,3‘-tetramethylindotricarbocyanine iodide (DiR) (purity>99%) was bought from Beijing Fanbo Biochemicals co., ltd. Anti-CD31 antibody was bought from Abcam (UK) , Fluorescein-Conjugated AffiniPure Goat Anti-Rabbit IgG was received from ZSGB Biotech Limited Company (Beijing , China) , DAPI Fluoromount-GTM was bought from Shanghai Yisheng Biotech Limited Company. All animal experiments were performed in compliance with the Institutional Animal Care and Use Committee.

2.2. Preparation of AS/Tf-HMCuS NPs HMCuS NPs were prepared as reported in our previous study [14]. In brief, poly-(vinylpyrrolidone) (0.24 g) was dissolved in 25 mL of deionized water firstly, and then CuCl2 solution (100
L, 0.5 mol/L) was added dropwise under stirring at room temperature. Subsequently, NaOH (25 mL, 0.02 mmol/L) as well as moderate hydrazine anhydrous solution were added to form Cu2 O nanospheres, and a stoichiometric amount of Na2S was mixed with above solution. After stirring at 60"for 2 h, the resultants (HMCuS NPs) were collected and purified by washing several times with deionized water. Finally, the obtained products were freeze-dried in vacuum overnight. For AS loading, free AS (5 mg/mL) dissolving in ethanol was mixed with HMCuS NPs (1 mg/mL) in PBS. Then the mixture was ultra-sonicated using an ultrasonic cell disruption system (250 W, 15 times) under ice-bath condition. Subsequently, the solution was magnetically stirred for 24 h. In order to remove residual free AS, the nanosuspension was dialyzed by a dialysis bag (molecular weight cutoff=3500) for 12 h. After preparation of AS/HMCuS NPs, Tf capping was proceeded as follows. Tf (1mg/mL) dissolving in PBS buffer was added dropwise to the AS/HMCuS NPs nanosuspension, and the mixture was stirred at room temperature for 24 h. As an end, the resulting products (AS/Tf-HMCuS NPs) were collected by centrifugation at 12,000 r/min for 10 min and washed with deionized water three times to remove unmodified Tf.Since combining Tf with HMCuS NPs could cause autofluorescence quenching of Tf (ex =280 nm, em = 332 nm), the optimal ϱ 

combining ratio could be determined while the maximum fluorescence quenching of Tf occurred [21]. HMCuS NPs with different concentration of 0, 10.0, 20.0, 30.0 and 40.0
g/ml were prepared and added into Tf solution (10.0
g/ml), respectively. Whereafter, the interaction between HMCuS NPs and Tf was characterized by the fluorescence spectrum (Shimadzu, Tokyo, Japan). With the concentration increased, the fluorescence spectrum was recorded to confirm the best combining ratio. Consequently, the resulting AS/Tf-HMCuS NPs was stored at 4"until use. Tf-HMCuS NPs without AS loading was also prepared in the same method as the control.

2.3. Characterization The average size and zeta potential were determined by dynamic light scattering (DLS) using a ZetaSizer Nano series Nano-ZS (Malvern Instruments Ltd, Malvern, UK). The morphology of HMCuS NPs, TfͲHMCuS NPs and AS/Tf-HMCuS NPs were observed with transmission electron microscopy (TEM) (Tecnai G2 20, FEI).

The extinction spectrum of the NPs was measured

using a UV-Vis spectrophotometer#Lambda 35, Perkin-Elmer, USA$. In addition, the interaction between HMCuS NPs and Tf was characterized by the fluorescence spectrum (Shimadzu, Tokyo, Japan).

2.4. Determination of AS loading on Tf-HMCuS NPs and releasing from AS/Tf-HMCuS NPs The amount of AS in the nanoparticles was determined as follows. Firstly, AS loading in the AS/Tf-HMCuS NPs nanosuspension was extracted by using 3 mL of dichloromethane, dried under a stream of nitrogen and reconstituted in 200
L of ethanol. After that, the solution was hydrolyzed with 2mL of 0.2% NaOH at 50 ± 1"%for 30 min, then cooled down to room temperature and adjusted pH with 1.6 mL of acetic acid (0.08 M) to form a derivative of AS with stable structure (characteristic UV absorption peak of the derivative: 260 nm). Finally, the concentration of AS was measured by high performance liquid chromatography (HPLC, Waters e2695, USA) with the following conditions: a Symmetry® C18 column (150 mm× 4.6 mm, 5.0
m); mobile phase, methanol: 0.01 M sodium acetate-acetic acid buffer solution (pH = 5.8) 62:38 (v/v); column temperature, 30 "; detection wavelength, 260 nm; flow rate, 1.0 mL/min and injection volume 20
L. The release profiles of AS from AS solution, AS/HMCuS NPs and AS/Tf-HMCuS NPs were ϲ 

evaluated by a dialysis method. In brief, samples were placed into dialysis bags (molecular weight cutoff =3500) and tightly sealed. Then, they were immersed in 50 mL of 20% ethanol aqueous buffer solution (ethanol: PBS, v/v = 1:4), and gently shaken at at 37.0 ± 0.5 "%in a water bath with a stirring rate of 100 r/min. The release samples (0.2 mL) were drawn at various time points, and the medium was replaced by the same volume of aqueous buffer solution. The concentration of AS released from AS/Tf-HMCuS NPs was quantified by HPLC under the above conditions.

2.5. Photothermal effects under NIR laser irradiation The photothermal conversion efficiency of the HMCuS NPs and Tf-HMCuS NPs in PBS at different concentrations (HMCuS concentration: 0–200
g/mL) was determined using a continuous–wave NIR laser (808 nm) irradiation with radiant energy at 2 W/cm2. The temperature was detected with an infrared thermometer (HT- 8878, Zhengzhou JinYangGuang Instrument Co. Ltd.) and recorded at 30 s intervals.

2.6. In vitro antitumor effect of AS/Tf-HMCuS NPs 2.6.1. Cellular uptake Cellular uptake was examined using fluorescence microscopy and flow cytometry. Firstly, MCF-7 human breast cancer cells were seeded on culture slides at a density of 3.0× 105 cells per well in 6-well plates and cultured for 24 h to allow the cells to attach to the surface of the wells. Fluorescein isothiocyanate (FITC), a fluorescence probe, was incorporated into HMCuS and Tf- HMCuS to observe their intracellular uptake behavior. FITC was loaded by mixing of the sample with FITC according to the following method: FITC in DMSO (1 mg/mL, 200
l) was added to HMCuS NPs (3.0 mL) and ultrasonicated with ultrasonic cell disruption system. The method of jointing Tf was the same as the preparation of AS/Tf-HMCuS NPs. Excess FITC was removed by Sephadex G-25 column (SigmaeAldrichCo. LLC). Subsequently, MCF-7 cells were treated with FITC, FITC/HMCuS NPs and FITC/ TfHMCuS NPs (HMCuS concentration: 20
g/ml) for 0.5 h, 1 h and 2 h, respectively. At the designated time points, cells were washed three times with PBS and fixed by 4% paraformaldehyde for 20 min. Finally, the samples were mounted in Dako fluorescence mounting medium and observed under fluorescent microscope (LSM 510, Zeiss, Germany). ϳ 

In addition, cell samples were also performed as described above for designated time period (0.5, 1 or 2 h). All the cell samples were washed with PBS, harvested by trypsinization, collected by centrifugation and suspended in PBS buffer for flow cytometry analysis. The detailed method was seen in SI.

2.6.2. In vitro cytotoxicity studies SRB dye reduction assays were carried out to quantify the cytotoxicity of the designed nanoparticles in this study. In a typical procedure, MCF-7 cells were seeded at 3 ×105 cells per well in 96-well plates and cultured for 24 h, followed by incubation with fresh cell medium containing different formulations of HMCuS NPs, AS/ HMCuS NPs, AS, AS-Tf and AS/TfHMCuS NPs (AS concentration: 20
g/mL and HMCuS NPs concentration: 100
g/mL). Then the cells were or were not irradiated with an 808 nm continuous-wave NIR laser with the power density of 2 W/cm2 for 5 min. After that, the cells were incubated at 37  for a further 24, 48 and 72 h. At the end of the incubation time, a standard SRB assay was conducted to measure the cell viability at the given time intervals.

2.6.3. Intracellular reactive oxygen species (ROS) detection Since the anti-tumor mechanisms of AS and HMCuS were both associated with ROS, we examined the degree of ROS generated in the cells. Intracellular ROS was detected by using DCFH-DA Reactive Oxygen Species Assay Kit. MCF-7 cells were seeded in confocal dishes at a density of 5 ×104 cells/dish. Then cells were treated with HMCuS NPs, AS, AS/ HMCuS NPs, AS-Tf (Tf was added an hour earlier than AS) and AS/Tf- HMCuS NPs at the same AS and HMCuS concentrations (AS concentration: 20
g/ml and HMCuS concentration: 100
g/ml) for 4 h. Thereafter, culture medium containing drug was removed and DCFH-DA was incorporated into the cells. Incubated for another 0.5 h later, cells were irradiated by NIR laser (808 nm, 2W/cm2 , 5 min) and fluorescence images of treated cells were acquired using a fluorescence microscope (LSM 510, Zeiss, Germany).

2.7. In vivo evaluation of AS/Tf-HMCuS NPs 2.7.1. In vivo anti-tumor efficacy assay ϴ 

All animal procedures were performed following protocol approved by the Institution Animal Care. For the in vivo anti-tumor experiments, tumor-bearing mice were randomly distributed into twelve groups (six mice per group to minimize the differences of weights and tumor sizes in each group). The mice were administered with (1) saline/PT, (2) Tf-HMCuS NPs/PT, (3) AS/PT, (4) AS/ HMCuS NPs/PT, (5) AS/Tf-HMCuS NPs/PT, (6) saline/laser/PT, (7) Tf-HMCuS NPs/laser/PT, (8)AS/laser/PT, (9) AS/HMCuS NPs/laser/PT, (10) AS/Tf-HMCuS NPs/laser/PT, (11) AS/Tf-HMCuS NPs/IV, (12) AS/Tf-HMCuS NPs/laser/IV. The IV groups received intravenous administration of 100
L formulations every 2 days for 5 times, respectively (AS dose: 35 mg/kg, Tf-HMCuS NPs dose: 100 mg/kg). The PT groups were given to mice peritumorally only twice throughout the experiment. The laser-treated groups were exposed to an 808 nm laser at a power density of 2W/cm2 for 0.5 min at 3 h after injection. Throughout the study, mice were weighed and tumors were measured with calipers every two days. Tumor volume (V) was calculated according to the formula: V & [length×(width)2]/2. At the end of experiment, animals were sacrificed and tumor tissues were taken out and weighed. The inhibition rate was used as another index of antitumor activity and defined by the formula ((Wc -Wt )/Wc)×100% (Wc and Wt stand for the average tumor weight of control group and treatment group, respectively). To further evaluate the anti-tumor effect of above formulations on the animals, the tumors were excised for pathology by hematoxylin and eosin (H&E) staining.

2.7.2. In vivo optical imaging For tracking the trace of HMCuS NPs and Tf-HMCuS NPs in vivo, these nanoparticles were labeled with the near-infrared fluorescence (NIRF) dye, DiR, according to the method for AS/HMCuS NPs and AS/Tf-HMCuS NPs. After formation of DiR/HMCuS and DiR/Tf-HMCuS, the zeta potential increased to -19.8 mV and -9.7 mV, respectively. Noninvasive optical imaging system was utilized. Tumor bearing mice were given PT administration of free DiR, DiR/HMCuS NPs and DiR/Tf-HMCuS NPs at a dose of 100
g DiR/kg. Meanwhile, DiR/Tf-HMCuS NPs were injected by the same dose via tail vein as the control group. All the mice were anesthetized as above described. NIRF imaging experiments were performed at 0.5, 1, 3, 6, 8, 12, 24, 48, 72 and 96 h ϵ 

post-injection using a Kodak in vivo imaging system FX PRO (Kodak, USA) equipped with an excitation bandpass filter at 770 nm and an emission at 830 nm. At last, mice were sacrificed and major organs (liver, spleen, lung, kidney and tumor) were then collected and imaged.

2.7.3. In vivo photoacoustic (PA) imaging For in vivo PA imaging studies, tumor-bearing mice were PT injected with HMCuS NPs and Tf- HMCuS NPs (2.5 mg HMCuS/kg). Meanwhile, Tf-HMCuS NPs were injected by the same dose via tail vein as the control. Subsequently, all the mice were anesthetized by using veterinary anesthesia machine system (VME, Matrx, USA) that delivered 2% isoflurane in oxygen at a flow rate of 2 L/min. In vivo PA images at the tumor site in horizontal plane were acquired at the designated time points post injection (1, 4, 6 and 24 h) using the multispectral optoacoustic tomography (MSOT) imaging system (inVision 128, iThera medical GmbH, Munich, Germany) at a wavelength of 808 nm.

2.7.4. Biodistribution study In order to quantitatively assess the biodistribution of the designed formulations, tumor bearing mice were treated by PT injection with AS solution, AS/ HMCuS NPs or AS/Tf-HMCuS NPs and IV injection with AS/Tf-HMCuS NPs at a matched dose of 35 mg AS/kg. At 0.5, 1, 3, 6, 8, 12, 24, 48, 72, 96 h after injection, five animals per time point were sacrificed and tissues (liver, lung, kidney and tumor) were surgically removed. AS were detected through HPLC method as follows. Ethyl ether (5 mL) was added to the tissue samples and centrifuged after mixing by vortex. The supernatant was entirely taken and dried under nitrogen. The residue was dissolved in 100
L of methanol and hydrolyzed for 30 min at 50 ± 1 " with 0.2% NaOH, cooled down to room temperature and adjusted pH with acetic acid (0.08 M) (methanol: sodium hydroxide: acetic acid = 1: 5: 4). Finally, a 20 ml aliquot of each sample was injected to HPLC to determine the AS concentration under the chromatographic conditions described as above.

2.7.5. Immunofluorescence: evaluation of drug penetration in tumors by DMR For further investigation of the principle of DMR, we employed immunofluorescence assay to track the trace of HMCuS NPs based formulations. Firstly, a fluorescent dye (R6G) was loaded ϭϬ 

into HMCuS NPs in the same method as AS/Tf-HMCuS NPs preparation. Tumor bearing mice were randomly divided into three groups (six mice per group). Then, the mice were administered peritumorally with (1) R6G/Tf-HMCuS NPs, (2) R6G/ HMCuS NPs, (3) R6G (R6G dose: 15 mg/kg). At the designated time points (1, 4, 12 h after injection), mice were sacrificed and tumor as well as peri-tumorous normal tissues were collected and frozen in optimum cutting temperature (OCT) medium (Sakura Finetek, USA) at -80 ". The corresponding slices (6
m) were prepared, air dried for 10 minutes, and fixed with 4% paraformaldehyde (Solarbio, China) for 10 min. For detecting R6G distribution, CD31 (Abcam, UK) was used as a primary antibody to stain the tumor blood vessels. A fluorescein-Conjugated AffiniPure Goat Anti-Rabbit IgG (ZSGB Biotech, China) was

used

as

a

secondary

antibody.

The

nuclei

were

counterstained

with

4’,

6-dia-midino-2-phenylindole (DAPI) (Yisheng Biotech, China). Slices were observed by a fluorescence microscope (LSM 510, Zeiss, Germany).

3. Results and discussion 3.1. Preparation and characterization of AS/Tf-HMCuS NPs The complete procedure for preparing the AS/Tf-HMCuS NPs was shown in Fig. 2. HMCuS NPs based formulations appeared dark green in color and the morphology was characterized with TEM. As shown in Fig. 3A, the transparent core confirmed its hollow structure with particle size of 100 nm while the mesoporous shell thickness was about 20 nm. On account of the hollow porous structure, HMCuS NPs could be used as vehicles in which anticancer drugs AS could be loaded. TEM images of AS/HMCuS NPs demonstrated some morphologically inhomogeneous agglomerates in the hollow structure, which might be attributed to AS filling into the hollow interior. Furthermore, Tf could be served as a capping agent to ensure the water solubility, tumor targetability and drug controlled release of the NPs. Just as the Fig. 3A (c) displayed, HMCuS NPs were wrapped around by shell-liked materials, which were supposed to be Tf, and thus the size of them became larger. DLS was also employed to determine the average particle size of HMCuS NPs, AS/HMCuS NPs and AS/Tf-HMCuS NPs, which were approximately in agreement with that determined by TEM. The mean hydrodynamic diameter of them was 102 nm, 103 nm and 205 nm, respectively, because of the hydrophilic Tf coating present on the HMCuS NPs surface. Additionally, HMCuS ϭϭ 

NPs showed the negative zeta potential of -33.1mV, and on the contrary, zeta potential of Tf was 8.1 mV. After formation of As/HMCuS and As/Tf-HMCuS NPs, the zeta potential increased to -32.3 mV and -25.5 mV, respectively. As a result, Tf modification to the surface of HMCuS NPs might be due to the chelation and electrostatic interactions. Moreover, the HMCuS NPs exhibited an elevated absorption with the extension of wavelength to the NIR region ( = 700–1000 nm) (Fig.4A), which is due to the localized surface plasma resonances (LSPR) originated from the copper vacancy [22, 23]. Such strong absorption of HMCuS NPs in NIR region ensures their potential application as a promising theranostic nanoplatform for NIR laser inducing PA imaging-guided PTT, especially treated with NIR laser at the wavelength near to the absorption peak. For Tf, a strong absorption peak at 282 nm can be clearly found with no absorption in NIR region. After combining Tf with HMCuS NPs, the ultraviolet absorption peak was shifted to 262 nm (Tf- HMCuS NPs), suggesting that a conjugated system was formed. In addition, Tf demonstrated obvious fluorescence absorption, but interaction between Tf and HMCuS NPs could cause autofluorescence quenching of Tf. As a result, the optimal combining ratio could be obtained while the maximum fluorescence quenching of Tf occurred. Fig. 4B showed a hill plot of fluorescence quenching because of the interaction between Tf and HMCuS NPs, and the intensity decreased with the increasing mass concentration of HMCuS NPs. Considering all factors including the cost, we concluded that the most optimal mass ratio of HMCuS NPs to Tf was 2:1. Finally, the stability of AS/Tf-HMCuS NPs was also confirmed and the results were shown in Fig. S2.

3.2. Determination of AS loading on Tf-HMCuS NPs and releasing from AS/Tf-HMCuS NPs To determine the loading capacity and absorption equilibrium level of AS onto Tf-HMCuS NPs, different ratios of AS : Tf-HMCuS NPs were performed, which indicated that AS loading efficiency increased from 9.3% to 28.5% (weight ratio of AS : Tf-HMCuS NPs from 2:1 to 6:1) with the amount of AS increasing (Fig.4C). Therefore, an AS loading of 28% (weight ratio of AS: Tf-HMCuS NPs was 5:1) was chosen for the following experiments. As the control, AS was also loaded into HMCuS NPs without Tf modification. It was found that the drug loading capacity was only 14.2% when the weight ratio of AS : HMCuS NPs was 6:1. The overall results could be attributed to the leakage in the formation process of formulations, in which free AS was removed ϭϮ 

through wash and dialysis. However, after Tf capping, the gatekeeper of Tf was responsible for blocking pores and inhibiting the leakage. Compared to AS/HMCuS NPs, AS/Tf-HMCuS NPs was expected to minimize premature drug release during the drug delivery in vivo, which would extraordinarily enhance tumor accumulation together with reducing the adverse side effects. To further verify our expectation as above, the release profile of AS/Tf-HMCuS NPs, AS solution and AS/HMCuS NPs was investigated. It was observed that over 98% and 88% of AS released from AS solution and AS/HMCuS NPs, respectively, within 24 h, whereas the AS/Tf-HMCuS NPs released only 65% of AS during the same period of time. The sustained release behavior of AS/Tf-HMCuS NPs demonstrated the nanoparticles modified with Tf as a gatekeeper possessed the good capping efficiency, and the interaction of hydrophobic force and Van der Waals force between AS and HMCuS NPs played a critical role. It has been reported that it was necessary to maintain the effective drug level in tumor tissue for a relatively long period of time, which could be achieved by a local depot formulation that continuously released drug locally for prolonged time [24]. As a result, a maintaining of effective drug levels over an extend period of time could be obtained by local delivery of our system, actualizing a better antitumor therapy.

3.3. Photothermal effects under NIR laser irradiation The strong absorption in NIR region of HMCuS NPs encouraged us to determine its photothermal effect in vitro. The light-induced heat generation ability of HMCuS NPs and Tf-HMCuS NPs in solution by measuring the temperature increase upon laser exposure was evaluated in this work. As shown in Fig. 4E, both HMCuS NPs and Tf-HMCuS NPs exhibited concentration and irradiation time-dependent features. The temperature of the aqueous dispersion of nanoparticles at increased Cu concentrations could be elevated up to 54" under NIR laser irradiation at the power density of 2 W/cm2 for 5 min. Similar photothermal conversion profile of HMCuS NPs and Tf-HMCuS NPs displayed that the modification of Tf on HMCuS NPs did not affect their photothermal sensitivity. These results were consistent with several prior researches [25, 26], which ascribed the photothermal effect to the LSPR.

3.4. In vitro antitumor effect of AS/Tf-HMCuS NPs 3.4.1. Cellular uptake ϭϯ 

Cellular uptake behavior of nanoparticles has a great influence on the therapeutic effect, and thus we labeled HMCuS NPs and Tf-HMCuS NPs with FITC to evaluate the internalization by fluorescent microscope and flow cytometry, respectively. In Fig. 5A-B, there were almost no obvious signals in cells throughout the studied period, suggesting that FITC alone could not enter into cancer cells. On the contrary, the fluorescent signals could be observed when MCF-7 cells incubated with HMCuS NPs and Tf-HMCuS NPs for 1 h, and the nanoparticles uptake displayed a time-dependent endocytic process. Furthermore, the fluorescence intensity of FITC-labeled Tf-HMCuS NPs was stronger than that of FITC-labeled HMCuS NPs, implying higher intracellular uptake capabilities of Tf-HMCuS NPs. This result indicated that the modification of Tf on HMCuS NPs had a specific and strong binding ability with TfR (CD71) receptor, which was highly expressed on MCF-7 cells surface [27, 28], and therefore could target the tumor cells via receptor-mediated endocytosis. Flow cytometry was also used to quantitative analysis of cellular uptake for HMCuS NPs and Tf-HMCuS NPs to further analyze the targeting efficiency of Tf, and the results were consistent with those in fluorescence microscope. As shown in Fig. 5C, longer incubation time resulted in high cell uptake, and internalization amount of HMCuS NPs and Tf-HMCuS NPs at 0.5 h was 10.3% and 30.8%, respectively. Moreover, the internalization amount of Tf-HMCuS NPs at 2 h reached up to about 99.9% in comparison with 47% for HMCuS NPs, indicating that Tf-HMCuS NPs could effectively promote cellular uptake and then increase drug intracellular accumulation. Therefore, Tf-HMCuS NPs with high-efficacy of cellular uptake can act as a targeting vehicle for selective delivery of anticancer drugs to tumor cells via specific intracellular signaling pathways.

3.4.2. In vitro cytotoxicity studies Safety of drug carrier must be taken into account before its application in biomedicine. As shown in Fig. 6A, it was clear that the viability of MCF-7 cells treated with HMCuS NPs all exceeded 95% (without laser irradiation) even at the Cu concentration of HMCuS NPs up to 100
g/mL. Therefore, HMCuS NPs was a relatively safe and biocompatible nanocarrier. In addition, according to the literature reports [5, 29], a dose of the copper sulfide NPs around 3 mg/kg is suitable for PA imaging, so the current HMCuS NPs were nearly nontoxic. ϭϰ 

Cytotoxicity study of AS, AS/HMCuS NPs, AS plus Tf solution and AS/Tf-HMCuS NPs was further carried out from 24 h to 72 h for evaluating the antitumor efficacy in vitro. All experiments evidenced an increasing cytotoxicity against MCF-7 cells in a time-dependent manner. Both AS/HMCuS NPs and AS/Tf-HMCuS NPs exhibited an obvious advantage over the AS group in terms of inhibition efficiency at the designated time, which might be due to the metabolism of free drugs in cancer cells. For example, the inhibition ratio of AS, AS/HMCuS NPs and AS/Tf-HMCuS NPs at 72 h was 33.9%, 43.6% and 80.3%, respectively, at the same AS and HMCuS NPs concentration (AS concentration: 20
g/mL and HMCuS NPs concentration: 100
g/mL). As can be seen, the percentage of cell growth inhibition for AS/Tf-HMCuS NPs was much higher than that for AS/HMCuS NPs. It suggested that AS/Tf-HMCuS NPs could efficiently deliver the drug into the cells owing to the high cellular internalization of Tf-modified HMCuS NPs via receptor-binding endocytosis, which was consistent with the results of cellular uptake tests as above. Moreover, AS plus Tf solution also showed higher cytotoxicity than AS and AS/HMCuS NPs did at 24 h, 48 h and 72 h. As reported, the potent anticancer action of AS analogs was attributed to the endoperoxiede bond that could be reduced by iron, leading to cytotoxic carbon-centered radicals [30]. Tf could carry Fe2+ into cancer cells, and so the co-delivery of Tf as well as AS could improve the pharmacological anti-cancer activity of AS (based on Fe2+) [31]. Accordingly, compared with AS/HMCuS NPs, the enhanced efficacy of AS/Tf-HMCuS NPs was also attributed to the reason that iron was transported into cells by iron-carrying holotransferrin (holo-Tf) via a transferrin receptor (TfR)-mediated endocytosis pathway [32] besides the increased intracellular accumulation. Subsequently, a NIR irradiation was conducted to investigate the photothermal sensitivity and photodynamic effect of HMCuS NPs. It was revealed that there was no significant difference for AS and AS plus Tf solution with or without laser irradiation. By contrast, the therapeutic effect of HMCuS NPs, AS/HMCuS NPs and AS/Tf-HMCuS NPs exposed to NIR laser was significantly enhanced with the decreased cell viability of 74.5%, 40.5% and 7.4% at 72 h, respectively, indicative of a dual PTT and PDT cytotoxicity. Meanwhile, the stronger cytotoxicity of AS/HMCuS NPs and AS/Tf-HMCuS NPs under irradiation by NIR laser than HMCuS NPs alone was owing to the synergetic effect from combination of chemo-phototherapy. On the basis of the above analysis, AS/Tf-HMCuS NPs showed potential as a multifunctional ϭϱ 

delivery system, which allowed for enhanced simultaneous treatment by chemotherapy, PTT and PDT with one nanoscale material.

3.4.3. Intracellular reactive oxygen species (ROS) detection Since the anti-tumor mechanism of AS and PDT of HMCuS NPs were both associated with ROS, we examined the ROS level generated in the cells. Herein, dichlorofluorescein diacetate (DCFH-DA) was used to assess intracellular ROS production by fluorescence microscopy. It was found in Fig. 6B that green fluorescence of DCFH was observed in MCF-7 cells incubated with AS, AS/HMCuS NPs, AS plus Tf solution and AS/Tf-HMCuS NPs at the same AS and HMCuS NPs concentrations (AS concentration: 20
g/mL and HMCuS NPs concentration: 100
g/mL) for 4 h even without laser irradiation. This result might be attributed to the presence of AS, which contains an endoperoxide group that breaks up when encounters with iron to generate ROS [33]. Compared with AS, a much higher DCFH fluorescence signal was also found in AS plus Tf solution and AS/Tf-HMCuS NPs treated MCF-7 cells (without laser irradiation). This increase suggested that ROS produced by AS could be enhanced by iron which was transported into cells through Tf, and indicated AS-Tf was a more potent and extremely selective anti-tumor agent compared to AS alone [34], which was in agreement with the cytotoxicity results. The change of iron level in cells before and after treating with AS, HMCuS, AS/HMCuS, AS-Tf, AS/Tf-HMCuS for 48h was shown in Figure S3. What’s more, stronger green fluorescence of DCFH appeared in HMCuS NPs, AS/HMCuS NPs and AS/Tf-HMCuS NPs treated cells under NIR irradiation, in comparison with those corresponding groups treated without laser irradiation. The fluorescence intensity was further analyzed in Fig. 6C. However, for AS and AS plus Tf solution groups, there was no significant difference between with and without laser irradiation, indicating that improved quantity of ROS after 808 nm laser irradiation might be generated by HMCuS. It could be interpreted by the fact that the leakage of copper ions from HMCuS NPs under NIR irradiation could undergo redox reactions with the surrounding environment mainly through a modified Haber-Weiss cycle [15]. As reported, enhanced ROS production could be bound to induce the oxidation of proteins and DNA, as well as strong pro-apoptotic effects, leading to irreversibly cell damage [35, 36]. Therefore, HMCuS NPs made them a promising photodynamic agent that allowed at the same ϭϲ 

time for PTT and PDT. 3.5. In vivo evaluation of AS/Tf-HMCuS NPs 3.5.1. In vivo anti-tumor efficacy assay In order to verify that the localized cancer therapy may provide clinical benefits and low systemic toxicity, we evaluated the in vivo antitumor efficacy of different drug formulations in tumor-bearing mice. The changes of relative tumor volume as a function of time were plotted. As shown in Fig. 7A, the saline and NIR only group exhibited a rapid tumor growth, with relative tumor volume of 5.21±0.27 and 5.17±0.74 at the end of observation. AS/Tf-HMCuS NPs treated through PT administration with laser irradiation presented the best effects on inhibiting the tumor growth, followed by AS/Tf-HMCuS NPs through PT injection without laser irradiation. In detail, after 10 days treatment, the relative tumor volume for the two groups as above(V/V0) was 1.16' 0.36 and 1.92'0.32, respectively. Meanwhile, the inhibition rate was also calculated in Fig. 7B, and the corresponding result for the two groups was 74.8% and 57.9%. The enhanced antitumor activity for AS/Tf-HMCuS NPs through PT injection with laser irradiation compared with no irradiation was due to the synergistic combination of chemo-photothermal and photodynamic therapy of AS/Tf-HMCuS NPs. In addition, the elevated temperatures by PTT effect might facilitate the reactivity of ROS for tumor destruction according to the published protocol [15]. It was noteworthy that AS/Tf-HMCuS NPs through localized administration demonstrated better tumor suppression efficacy than that through intravenous injection, no matter with or without laser irradiation. More importantly, during the whole experiment, PT group was given only twice, less than that of IV group for 5 times. The results indicated that PT injection of AS/Tf-HMCuS NPs could enhance therapeutic effects, reduce frequency of drug administration and improve patient compliance. It might be owing to the high efficiency of tumor targeting and accumulation of drug at tumor site obtained with DMR, which could be evidenced by biodistribution studies. In comparison, systemic administration of most chemotherapeutic drugs could only deliver a limited amount of drug to the tumor site, even using the targeted ligand, and commonly produces severe side effects at high doses [37, 38]. Furthermore, the antitumor activity of free AS, Tf-HMCuS NPs and AS/HMCuS NPs groups through PT injection was also investigated as control. The inhibition rate of them with and without laser irradiation was 18.3%, 19.1%, 32.6% and 19.8%, 2.2%, 20.5%, respectively, which was ϭϳ 

significantly lower than that of AS/Tf-HMCuS NPs. It displayed that Tf-HMCuS NPs was a safe carrier with tumor inhibition efficacy under laser irradiation, while AS and HMCuS NPs could demonstrate synergetic effect with irradiation. The better antitumor effect of AS/Tf-HMCuS NPs could be owing to two main factors. Firstly, Tf could mediate more AS transported into tumor, inducing more drug accumulation at this site. That is to say, DMR was further enhanced by combination with targeting moiety. Besides, more iron could be transported into tumor cells by Tf leading to interacting with AS to generate ROS [39]. This result was consistent with the conclusion in vitro. To further evaluate the antitumor efficacy of free AS, Tf-HMCuS NPs, AS/HMCuS NPs, AS/Tf-HMCuS NPs through PT injection and AS/Tf-HMCuS NPs through IV injection on the animals, the tumor were excised for pathology. As shown in Fig. 7C, the groups injected with saline displayed typical pathological characteristic of tumor, such as closely arranged tumor cells. Tumor tissue in the other groups more or less showed spotty necrosis and intercellular blank. In particular, AS/Tf-HMCuS NPs through PT injection with laser irradiation possessed the most effective tumor inhibiting ability compared to other groups. In addition, the safety evaluation of AS/Tf-HMCuS NPs in vivo has been studied by us and the results were shown in Fig. S4.It was clear to see that all the tissues in the tested group were as normal as that in the control group, which suggested that AS/Tf-HMCuS NPs exhibited good biosafety.

3.5.2. In vivo optical imaging The effective anti-tumor therapeutic efficacy would be achieved when the biodistribution of AS/Tf-HMCuS NPs was clearly understood. To investigate the in vivo behavior and illustrate DMR, we employed a non-invasive near infrared optical imaging technique preliminarily. Fig. 8A showed the real-time images of all samples in the tumor-bearing mice. For DiR/Tf-HMCuS NPs through IV injection group, the fluorescence signals efficiently accumulated at the tumor site at just 1 h post injection, and the DiR signal maintained up to 24 h, which might be owing to the long circulation and Tf-mediated targeting ability of this system. By comparison, DiR/Tf-HMCuS NPs and DiR/HMCuS NPs through PT injection presented a superior tumor targeting and retention capacity. In the DiR/Tf-HMCuS NPs/PT group, there still was clear NIR ϭϴ 

signal in the tumor region at 96 h and the decay of the signal was much slower than that of DiR/HMCuS NPs/PT group, which might be due to the Tf modification. Additionally, although free DiR could also target the tumor site quickly after PT injection, the signal decreased rapidly and was nearly not detected at 12 h as expected. Based on these results, the advantages of DiR/Tf-HMCuS NPs through PT injection in reducing times of drug administration with still strong antitumor efficacy could be interpreted. Ex vivo fluorescence evaluation of dissected tumor at 96 h post-injection also revealed that the tumor accumulation of DiR/Tf-HMCuS NPs through PT injection was much more than that of the other three groups (Fig.8B). Moreover, semi-quantitative biodistribution of each excised organ was performed by using quantitative region-of-interest analysis. It showed that signals of DiR/Tf-HMCuS NPs/PT group were increased in tumor tissues and kidney, but decreased significantly in liver, spleen and lung compared with DiR/Tf-HMCuS NPs/IV group. We supposed that the enhanced accumulation in kidney might indicate the excretion of DiR/Tf-HMCuS NPs/PT through kidney. These results demonstrated that DMR was an efficient targeting method for tumor targeting, compared to the relatively inefficient targeting obtained with IV administration. Furthermore, DiR/Tf-HMCuS NPs following the PT injection used by DMR enhanced and prolonged the local accumulation and retention of drugs, which lead to synergetic treatment outcomes in vivo safely.

3.5.3. In vivo photoacoustic (PA) imaging PA tomography is a promising modality possessing strong biochemical contrast and high spatial resolution, compared with the optical imaging methods. As CuS could convert the absorbed photothermal energy into acoustic signal under NIR irradiation, it was a good contrast agent for PAT. A set of PA images of the tumor region acquired before and at different time points post-injection are presented in Fig. 8C. PA signals could be hardly detected before injection, and upon intravenous injection, the intensity of PA signals exhibited 4.35 times enhancement at 1 h, which was consistent with the results of optical imaging. It was owing to the prolonged circulation in blood and effective accumulation within tumor. Afterwards, the intensity decreased with time passed on, and was nearly same to that of pre-injection at 24 h. For the PT injection groups of Tf-HMCuS NPs and HMCuS NPs, stronger PA signals were ϭϵ 

observed compared to that of IV injection group. The signals were quantified as 13.57 times and 11.38 times higher in the post-injection of Tf-HMCuS NPs and HMCuS NPs for 1 h, respectively, and could maintain high level even post 24 h injection (about 9.42 times and 4.45 times stronger than that of pre-injection). This result confirmed that Tf-HMCuS NPs and HMCuS NPs for PT injection possessed a significantly long retention effect and effective contrast enhancement for PA imaging by DMR, which made it sufficient to guide cancer treatment by providing the identification of the location and morphology as well as size of tumor. Besides, Tf-HMCuS NPs showed better tumor targeting ability than HMCuS NPs because of the modification of Tf. In such settings, Tf-HMCuS NPs for DMR could be used as a targeting technique for either diagnostic or therapeutic agents.

3.5.4. Biodistribution study After evaluating in vivo behavior of the vehicle (Tf-HMCuS NPs), the drug (AS) distribution in main tissues (liver, lung and kidney) and tumor was further measured to determine if the AS/Tf-HMCuS NPs formulation could adjust the biodistribution of AS. The results were presented in Fig. 9. Notably, the AS concentration of formulations through PT administration was significantly higher than that of AS/Tf-HMCuS NPs through IV administration, proving that the PT injection led to preferential accumulation in the tumor. However, the AS in tumor from free AS group by PT injection decayed fast, reaching the examination limit in less than 36 hours. By contrast, AS/Tf-HMCuS NPs for PT injection demonstrated the best tumor targeting ability and retention effect compared to the other two PT injection groups, which was owing to the Tf modification as well as the sustained release profile of AS from Tf-HMCuS NPs. Most importantly, the amount of AS in tumor could be still detected until 96 h, leading to a promising antitumor activity even with reduced frequency of administration, which was corresponding to the in vivo anti-tumor efficacy assay. The higher efficiency of tumor targeting obtained with DMR, relatively to the standard IV method, was evident by these results. AS/Tf-HMCuS NPs through IV administration exhibited relatively higher accumulation in liver and lung for AS, which were consistent with the results of in vivo optical imaging. It was because most macromolecules larger than the renal cut-off size (10 nm) were eliminated predominately via reticuloendothelial system uptake such as Kupffer cells in the liver, and the ϮϬ 

lung capillary bed possessed filtration effect [40]. In comparison, AS/Tf-HMCuS NPs through PT administration could decrease the maximum AS concentration in normal tissues, indicating of the low systemic toxicity. However, higher concentration of AS in kidney was observed in PT injection groups, which might be due to the elimination of AS mainly through kidney.

3.5.5. Immunofluorescence: evaluation of drug penetration in tumors by DMR To further illustrate DMR, we employed immunofluorescence analysis as described earlier. Fig.10 showed the time course of diffusion and elimination of RG6 labeled Tf-HMCuS NPs, RG6 labeled HMCuS NPs and RG6 after PT administration. It was found that RG6 labeled Tf-HMCuS NPs exhibited stronger fluorescent intensity and larger penetration distribution than that of the other two groups. This was probably caused by Tf-mediated tumor targeting efficiency together with slow vascular uptake and extensive interstitial diffusion, which could be called as diffusion molecular retention (DMR).

4. Conclusions In this study, we have developed a multi-functional tumor-targeted AS/Tf-HMCuS NPs localized delivery system which possessed photoacoustic imaging, PTT and PDT properties for multimodality theranostic applications in cancer treatment. It has been shown that AS/Tf-HMCuS NPs have good photothermal conversion efficiency and unique photodynamic capability under NIR laser light illumination. Both in vitro and in vivo evaluation demonstrated that the system maintained a high intracellular drug concentration within tumor cells, displayed an efficient intracellular delivery and rendered a high drug accumulation in the tumor tissue, thereby yielding the elevated therapeutic treatment. We can ascribe the enhanced effect to the combination of Tf and DMR, rather than targeting modification alone. It is worthy to mention that, with the help of DMR, the frequency of administration for AS/Tf-HMCuS NPs by this route is significantly reduced, but the antitumor efficiency is improved. This strategy provides a great potential for clinically cancer treatment, diagnosis, drug delivery and so on.

Acknowledgement This work was supported by grants from the National Natural Science Foundation of China Ϯϭ 

(81273451) and Outstanding Young Talent Research Fund of Zhengzhou University (51099255).  

References  ΀ϭ΁>ƵĞƚŬĞ͕DĞLJĞƌƐW͕>ĞǁŝƐ/͕:ƵĞƌŐĞŶƐ,͘KƐƚĞŽƐĂƌĐŽŵĂƚƌĞĂƚŵĞŶƚͲtŚĞƌĞĚŽǁĞƐƚĂŶĚ͍ƐƚĂƚĞ ŽĨƚŚĞĂƌƚƌĞǀŝĞǁ͘ĂŶĐĞƌdƌĞĂƚZĞǀϮϬϭϰ͖ϰϬ͗ϱϮϯͲϯϮ͘ ΀Ϯ΁DĂ,͕,Ğ>͕ŚĞŶŐz>͕zĂŶŐD͕ĂŶŐ:d͕>ŝƵ:'͕ŚĞŶy^͘>ŽĐĂůŝnjĞĚŽͲĚĞůŝǀĞƌLJŽĨŽdžŽƌƵďŝĐŝŶ͕ ŝƐƉůĂƚŝŶ͕ ĂŶĚ DĞƚŚŽƚƌĞdžĂƚĞ ďLJ dŚĞƌŵŽƐĞŶƐŝƚŝǀĞ ,LJĚƌŽŐĞůƐ ĨŽƌ ŶŚĂŶĐĞĚ KƐƚĞŽƐĂƌĐŽŵĂ dƌĞĂƚŵĞŶƚ͘ ĐƐƉƉůDĂƚĞƌ/ŶƚĞƌϮϬϭϱ͖ϳ͗ϮϳϬϰϬͲϴ͘ ΀ϯ΁ >ŝŶ Y͕ 'ĂŽ t͕ ,Ƶ ,y͕ DĂ <͕ ,Ğ ͕ Ăŝ t͕ tĂŶŐ yY͕ tĂŶŐ :͕ ŚĂŶŐ y͕ ŚĂŶŐ Y͘ EŽǀĞů ƚŚĞƌŵŽͲƐĞŶƐŝƚŝǀĞ ŚLJĚƌŽŐĞů ƐLJƐƚĞŵ ǁŝƚŚ ƉĂĐůŝƚĂdžĞů ŶĂŶŽĐƌLJƐƚĂůƐ͗ ,ŝŐŚ ĚƌƵŐͲůŽĂĚŝŶŐ͕ ƐƵƐƚĂŝŶĞĚ ĚƌƵŐ ƌĞůĞĂƐĞ ĂŶĚ ĞdžƚĞŶĚĞĚ ůŽĐĂů ƌĞƚĞŶƚŝŽŶ ŐƵĂƌĂŶƚĞĞŝŶŐ ďĞƚƚĞƌ ĞĨĨŝĐĂĐLJ ĂŶĚ ůŽǁĞƌ ƚŽdžŝĐŝƚLJ͘ : ŽŶƚƌŽů ZĞůĞĂƐĞϮϬϭϰ͖ϭϳϰ͗ϭϲϭͲϳϬ͘ ΀ϰ΁ <ĂŶŐ zD͕ <ŝŵ ',͕ /ů <ŝŵ :͕ <ŝŵ z͕ >ĞĞ E͕ zŽŽŶ ^D͕ <ŝŵ :,͕ <ŝŵ D^͘ /Ŷ ǀŝǀŽ ĞĨĨŝĐĂĐLJ ŽĨ ĂŶ ŝŶƚƌĂƚƵŵŽƌĂůůLJ ŝŶũĞĐƚĞĚ ŝŶ ƐŝƚƵͲĨŽƌŵŝŶŐ ĚŽdžŽƌƵďŝĐŝŶͬƉŽůLJ;ĞƚŚLJůĞŶĞ ŐůLJĐŽůͿͲďͲƉŽůLJĐĂƉƌŽůĂĐƚŽŶĞ ĚŝďůŽĐŬ ĐŽƉŽůLJŵĞƌ͘ŝŽŵĂƚĞƌŝĂůƐϮϬϭϭ͖ϯϮ͗ϰϱϱϲͲϲϰ͘ ΀ϱ΁ůĞdžĂŶĚĞƌ͕ũĂnjƵĚĚŝŶ͕ <ŚĂŶ:͕ ^ĂƌĂĨ ^͕^ĂƌĂĨ^͘ WŽůLJ;ĞƚŚLJůĞŶĞŐůLJĐŽůͿʹƉŽůLJ;ůĂĐƚŝĐͲĐŽͲŐůLJĐŽůŝĐ ĂĐŝĚͿ ďĂƐĞĚ ƚŚĞƌŵŽƐĞŶƐŝƚŝǀĞ ŝŶũĞĐƚĂďůĞ ŚLJĚƌŽŐĞůƐ ĨŽƌ ďŝŽŵĞĚŝĐĂů ĂƉƉůŝĐĂƚŝŽŶƐ͘ : ŽŶƚƌŽů ZĞůĞĂƐĞ ϮϬϭϯ͖ϭϳϮ͗ϳϭϱͲϮϵ͘ ΀ϲ΁tĂŶŐtt͕^ŽŶŐ,:͕ŚĂŶŐ:͕>ŝW͕>ŝ͕tĂŶŐ͕<ŽŶŐ>͕ŚĂŶŐY͘ŶŝŶũĞĐƚĂďůĞ͕ƚŚĞƌŵŽƐĞŶƐŝƚŝǀĞ ĂŶĚ ŵƵůƚŝĐŽŵƉĂƌƚŵĞŶƚ ŚLJĚƌŽŐĞů ĨŽƌ ƐŝŵƵůƚĂŶĞŽƵƐ ĞŶĐĂƉƐƵůĂƚŝŽŶ ĂŶĚ ŝŶĚĞƉĞŶĚĞŶƚ ƌĞůĞĂƐĞ ŽĨ Ă ĚƌƵŐ ĐŽĐŬƚĂŝůĂƐĂŶĞĨĨĞĐƚŝǀĞĐŽŵďŝŶĂƚŝŽŶƚŚĞƌĂƉLJƉůĂƚĨŽƌŵ͘:ŽŶƚƌŽůZĞůĞĂƐĞϮϬϭϱ͖ϮϬϯ͗ϱϳͲϲϲ͘ ΀ϳ΁ tĂŶŐ tt͕ĞŶŐ >͕ yƵ ^y͕ ŚĂŽ yD͕ >ǀ E͕ ŚĂŶŐ 'y͕'Ƶ E͕ ,Ƶ Z:͕ ŚĂŶŐ :,͕ >ŝƵ ::͕ ŽŶŐ :͘  ƌĞĐŽŶƐƚŝƚƵƚĞĚΗƚǁŽŝŶƚŽŽŶĞΗƚŚĞƌŵŽƐĞŶƐŝƚŝǀĞŚLJĚƌŽŐĞůƐLJƐƚĞŵĂƐƐĞŵďůĞĚďLJĚƌƵŐͲůŽĂĚĞĚĂŵƉŚŝƉŚŝůŝĐ ĐŽƉŽůLJŵĞƌŶĂŶŽƉĂƌƚŝĐůĞƐĨŽƌƚŚĞůŽĐĂůĚĞůŝǀĞƌLJŽĨƉĂĐůŝƚĂdžĞů͘:DĂƚĞƌŚĞŵϮϬϭϯ͖ϭ͗ϱϱϮͲϲϯ͘ ΀ϴ΁ EŝĞ ^&͕ ,ƐŝĂŽ t>t͕ WĂŶ t^͕ zĂŶŐ :͘ dŚĞƌŵŽƌĞǀĞƌƐŝďůĞ WůƵƌŽŶŝĐ ;ZͿ &ϭϮϳͲďĂƐĞĚ ŚLJĚƌŽŐĞů ĐŽŶƚĂŝŶŝŶŐ ůŝƉŽƐŽŵĞƐ ĨŽƌ ƚŚĞ ĐŽŶƚƌŽůůĞĚ ĚĞůŝǀĞƌLJ ŽĨ ƉĂĐůŝƚĂdžĞů͗ ŝŶ ǀŝƚƌŽ ĚƌƵŐ ƌĞůĞĂƐĞ͕ ĐĞůů ĐLJƚŽƚŽdžŝĐŝƚLJ͕ ĂŶĚƵƉƚĂŬĞƐƚƵĚŝĞƐ͘/Ŷƚ:EĂŶŽŵĞĚϮϬϭϭ͖ϲ͗ϭϱϭͲϲϲ͘ ΀ϵ΁ >ŝ ,:͕ Ƶ :͕ Ƶ y:͕ yƵ &͕ ^ƵŶ z͕ tĂŶŐ ,y͕ ĂŽ d͕ zĂŶŐ y͕ ŚƵ z,͕ EŝĞ ^D͕ tĂŶŐ :͘ ^ƚŝŵƵůŝͲƌĞƐƉŽŶƐŝǀĞĐůƵƐƚĞƌĞĚŶĂŶŽƉĂƌƚŝĐůĞƐĨŽƌ ŝŵƉƌŽǀĞĚƚƵŵŽƌƉĞŶĞƚƌĂƚŝŽŶĂŶĚ ƚŚĞƌĂƉĞƵƚŝĐ ĞĨĨŝĐĂĐLJ͘ WEĂƚůĐĂĚ^Đŝh^ϮϬϭϲ͖ϭϭϯ͗ϰϭϲϰͲϵ͘ ΀ϭϬ΁ ^ĂŶŚĂŝ tZ͕ ^ĂŬĂŵŽƚŽ :,͕ ĂŶĂĚLJ Z͕ &ĞƌƌĂƌŝ D͘ ^ĞǀĞŶ ĐŚĂůůĞŶŐĞƐ ĨŽƌ ŶĂŶŽŵĞĚŝĐŝŶĞ͘ EĂƚ EĂŶŽƚĞĐŚŶŽůϮϬϬϴ͖ϯ͗ϮϰϮͲϰ͘ ΀ϭϭ΁'ƵŽzz͕zƵĂŶ,^͕ŚŽ,^͕<ƵƌƵƉƉƵ͕:ŽŬŝǀĂƌƐŝ<͕ŐĂƌǁĂů͕^ŚĂŚ<͕:ŽƐĞƉŚƐŽŶ>͘,ŝŐŚĨĨŝĐŝĞŶĐLJ ŝĨĨƵƐŝŽŶDŽůĞĐƵůĂƌZĞƚĞŶƚŝŽŶdƵŵŽƌdĂƌŐĞƚŝŶŐ͘WůŽƐKŶĞϮϬϭϯ͖ϴ͘ ΀ϭϮ΁ ŽŶŐ <͕ >ŝƵ ͕ >ŝ ,͕ ZĞŶ :^͕ YƵ y'͘ ,LJĚƌŽƉŚŽďŝĐ ŶƚŝĐĂŶĐĞƌ ƌƵŐ ĞůŝǀĞƌLJ ďLJ Ă ϵϴϬ Ŷŵ >ĂƐĞƌͲƌŝǀĞŶWŚŽƚŽƚŚĞƌŵĂůsĞŚŝĐůĞĨŽƌĨĨŝĐŝĞŶƚ^LJŶĞƌŐŝƐƚŝĐdŚĞƌĂƉLJŽĨĂŶĐĞƌĞůůƐ/ŶsŝǀŽ͘ĚǀDĂƚĞƌ ϮϬϭϯ͖Ϯϱ͗ϰϰϱϮͲϴ͘ ΀ϭϯ΁ <Ƶ '͕ ŚŽƵ D͕ ^ŽŶŐ ^>͕ ,ƵĂŶŐ Y͕ ,ĂnjůĞ :͕ >ŝ ͘ ŽƉƉĞƌ ^ƵůĨŝĚĞ EĂŶŽƉĂƌƚŝĐůĞƐ Ɛ Ă EĞǁ ůĂƐƐ ŽĨ WŚŽƚŽĂĐŽƵƐƚŝĐŽŶƚƌĂƐƚŐĞŶƚĨŽƌĞĞƉdŝƐƐƵĞ/ŵĂŐŝŶŐĂƚϭϬϲϰŶŵ͘ĐƐEĂŶŽϮϬϭϮ͖ϲ͗ϳϰϴϵͲϵϲ͘ ΀ϭϰ΁ &ĞŶŐ Y͕ ŚĂŶŐ z͕ ŚĂŶŐ t͕ ^ŚĂŶ y͕ zƵĂŶ z͕ ŚĂŶŐ ,͕ ,ŽƵ >͕ ŚĂŶŐ ͘ dƵŵŽƌͲƚĂƌŐĞƚĞĚ ĂŶĚ ŵƵůƚŝͲƐƚŝŵƵůŝ ƌĞƐƉŽŶƐŝǀĞ ĚƌƵŐ ĚĞůŝǀĞƌLJ ƐLJƐƚĞŵ ĨŽƌ ŶĞĂƌͲŝŶĨƌĂƌĞĚ ůŝŐŚƚ ŝŶĚƵĐĞĚ ĐŚĞŵŽͲƉŚŽƚŽƚŚĞƌĂƉLJ ĂŶĚƉŚŽƚŽĂĐŽƵƐƚŝĐƚŽŵŽŐƌĂƉŚLJ͘ĐƚĂŝŽŵĂƚĞƌŝĂůŝĂϮϬϭϲ͖ϯϴ͗ϭϮϵͲϰϮ͘ ϮϮ 

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΀ϯϮ΁ĂY͕ŚŽƵE͕ƵĂŶ:͕ŚĞŶd͕,ĂŽD͕zĂŶŐy͕>ŝ:͕zŝŶ:͕ŚƵZ͕tĂŶŐ,͘ŝŚLJĚƌŽĂƌƚĞŵŝƐŝŶŝŶĞdžĞƌƚƐ ŝƚƐ ĂŶƚŝĐĂŶĐĞƌ ĂĐƚŝǀŝƚLJ ƚŚƌŽƵŐŚ ĚĞƉůĞƚŝŶŐ ĐĞůůƵůĂƌ ŝƌŽŶ ǀŝĂ ƚƌĂŶƐĨĞƌƌŝŶ ƌĞĐĞƉƚŽƌͲϭ͘ WůŽƐ KŶĞ ϮϬϭϮ͖ϳ͗ĞϰϮϳϬϯ͘ ΀ϯϯ΁ 'ĂůĂů D͕ ZŽƐƐ ^͕ ů^ŽŚůLJ D͕ ů^ŽŚůLJ ,E͕ ůͲ&ĞƌĂůLJ &^͕ ŚŵĞĚ D^͕ DĐWŚĂŝů d͘ ĞŽdžLJĂƌƚĞŵŝƐŝŶŝŶ ĚĞƌŝǀĂƚŝǀĞƐ ĨƌŽŵ ƉŚŽƚŽŽdžLJŐĞŶĂƚŝŽŶ ŽĨ ĂŶŚLJĚƌŽĚĞŽdžLJĚŝŚLJĚƌŽĂƌƚĞŵŝƐŝŶŝŶ ĂŶĚ ƚŚĞŝƌ ĐLJƚŽƚŽdžŝĐĞǀĂůƵĂƚŝŽŶ͘:ŽƵƌŶĂůŽĨŶĂƚƵƌĂůƉƌŽĚƵĐƚƐϮϬϬϮ͖ϲϱ͗ϭϴϰͲϴ͘ ΀ϯϰ΁>Ăŝ,͕^ĂƐĂŬŝd͕^ŝŶŐŚEW͕DĞƐƐĂLJ͘ĨĨĞĐƚƐŽĨĂƌƚĞŵŝƐŝŶŝŶͲƚĂŐŐĞĚŚŽůŽƚƌĂŶƐĨĞƌƌŝŶŽŶĐĂŶĐĞƌĐĞůůƐ͘ >ŝĨĞƐĐŝĞŶĐĞƐϮϬϬϱ͖ϳϲ͗ϭϮϲϳͲϳϵ͘ ΀ϯϱ΁,ĂŵĂĐŚĞƌͲƌĂĚLJ͕^ƚĞŝŶ,͕dƵƌƐĐŚŶĞƌ^͕dŽĞŐĞů/͕DŽƌĂZ͕:ĞŶŶĞǁĞŝŶE͕ĨĨĞƌƚŚd͕ŝůƐZ͕ƌĂĚLJ EZ͘ ƌƚĞƐƵŶĂƚĞ ĂĐƚŝǀĂƚĞƐ ŵŝƚŽĐŚŽŶĚƌŝĂů ĂƉŽƉƚŽƐŝƐ ŝŶ ďƌĞĂƐƚ ĐĂŶĐĞƌ ĐĞůůƐ ǀŝĂŝƌŽŶͲĐĂƚĂůLJnjĞĚ ůLJƐŽƐŽŵĂů ƌĞĂĐƚŝǀĞŽdžLJŐĞŶƐƉĞĐŝĞƐƉƌŽĚƵĐƚŝŽŶ͘dŚĞ:ŽƵƌŶĂůŽĨďŝŽůŽŐŝĐĂůĐŚĞŵŝƐƚƌLJϮϬϭϭ͖Ϯϴϲ͗ϲϱϴϳͲϲϬϭ͘ ΀ϯϲ΁DĞƌĐĞƌ͕ŽƉƉůĞ/D͕DĂŐŐƐ:>͕KΖEĞŝůůWD͕WĂƌŬ<͘dŚĞƌŽůĞŽĨŚĞŵĞĂŶĚƚŚĞŵŝƚŽĐŚŽŶĚƌŝŽŶŝŶ ƚŚĞ ĐŚĞŵŝĐĂů ĂŶĚ ŵŽůĞĐƵůĂƌ ŵĞĐŚĂŶŝƐŵƐ ŽĨ ŵĂŵŵĂůŝĂŶ ĐĞůů ĚĞĂƚŚ ŝŶĚƵĐĞĚ ďLJ ƚŚĞ ĂƌƚĞŵŝƐŝŶŝŶ ĂŶƚŝŵĂůĂƌŝĂůƐ͘dŚĞ:ŽƵƌŶĂůŽĨďŝŽůŽŐŝĐĂůĐŚĞŵŝƐƚƌLJϮϬϭϭ͖Ϯϴϲ͗ϵϴϳͲϵϲ͘ ΀ϯϳ΁^ĂĨĂǀLJ͘ZĞĐĞŶƚĚĞǀĞůŽƉŵĞŶƚƐŝŶƚĂdžĂŶĞĚƌƵŐĚĞůŝǀĞƌLJ͘ƵƌƌĞŶƚĚƌƵŐĚĞůŝǀĞƌLJϮϬϬϴ͖ϱ͗ϰϮͲϱϰ͘ ΀ϯϴ΁ĂĞz,͕WĂƌŬ<͘dĂƌŐĞƚĞĚĚƌƵŐĚĞůŝǀĞƌLJƚŽƚƵŵŽƌƐ͗ŵLJƚŚƐ͕ƌĞĂůŝƚLJĂŶĚƉŽƐƐŝďŝůŝƚLJ͘:ŽŶƚƌŽůZĞůĞĂƐĞ ϮϬϭϭ͖ϭϱϯ͗ϭϵϴͲϮϬϱ͘ ΀ϯϵ΁ĨĨĞƌƚŚd͕ĞŶĂŬŝƐ͕ZŽŵĞƌŽDZ͕dŽŵŝĐŝĐD͕ZĂƵŚZ͕^ƚĞŝŶďĂĐŚ͕,ĂĨĞƌZ͕^ƚĂŵŵŝŶŐĞƌd͕KĞƐĐŚ &͕ <ĂŝŶĂ ͕ DĂƌƐĐŚĂůů D͘ ŶŚĂŶĐĞŵĞŶƚ ŽĨ ĐLJƚŽƚŽdžŝĐŝƚLJ ŽĨ ĂƌƚĞŵŝƐŝŶŝŶƐ ƚŽǁĂƌĚ ĐĂŶĐĞƌ ĐĞůůƐ ďLJ ĨĞƌƌŽƵƐ ŝƌŽŶ͘&ƌĞĞƌĂĚŝĐĂůďŝŽůŽŐLJΘŵĞĚŝĐŝŶĞϮϬϬϰ͖ϯϳ͗ϵϵϴͲϭϬϬϵ͘ ΀ϰϬ΁ ,ŽƵ >͕ zĂŽ :͕ ŚŽƵ :͕ ŚĂŶŐ Y͘ WŚĂƌŵĂĐŽŬŝŶĞƚŝĐƐ ŽĨ Ă ƉĂĐůŝƚĂdžĞůͲůŽĂĚĞĚ ůŽǁ ŵŽůĞĐƵůĂƌ ǁĞŝŐŚƚ ŚĞƉĂƌŝŶͲĂůůͲƚƌĂŶƐͲƌĞƚŝŶŽŝĚ ĂĐŝĚ ĐŽŶũƵŐĂƚĞ ƚĞƌŶĂƌLJ ŶĂŶŽƉĂƌƚŝĐƵůĂƚĞ ĚƌƵŐ ĚĞůŝǀĞƌLJ ƐLJƐƚĞŵ͘ ŝŽŵĂƚĞƌŝĂůƐ ϮϬϭϮ͖ϯϯ͗ϱϰϯϭͲϰϬ͘

Ϯϰ 

Figure captions Fig.1. Schematic illustration of the localized delivery for synergistic tumor treatment. Fig.2. Scheme for preparation of AS/Tf-HMCuS NPs. Fig.3. Characterization of HMCuS based formulations. A: TEM images: (a) HMCuS, (b) AS/ HMCuS, (c) AS/Tf-HMCuS; B: Size distribution of HMCuS (a) and AS/Tf-HMCuS (b); C: Zeta potential analysis: (a) HMCuS, (b) Tf, (c) AS/Tf-HMCuS. Fig.4. Characterization of Tf-HMCuS and HMCuS based formulations with or without AS. A: UV spectrum of (a) HMCuS, (b) Tf and (c) Tf-HMCuS; B: The fluorescence spectroscopy with increased mass concentration ratio of HMCuS :Tf from 0:1 to 4:1; C: AS loading at different amounts of Tf-HMCuS and HMCuS; D: Release profile of AS from AS solution, AS/ HMCuS and AS/ Tf-HMCuS; E: The photothermal heating curves of HMCuS and Tf-HMCuS with different concentrations by an 808 nm laser irradiation. Fig.5. Cellular uptake studies of HMCuS and Tf-HMCuS. A: Fluorescence microscopic images of (a) FITC, (b) FITC labeled HMCuS, (c) FITC labeled Tf-HMCuS; B: Average fluorescence intensity of FITC, FITC labeled HMCuS and FITC labeled Tf-HMCuS; C: Flow cytometric assay for (a) FITC, (b) FITC labeled HMCuS, (c) FITC labeled Tf-HMCuS at the designate time. Fig.6. In vitro antitumor activity studies. A: Cell viability of different treatments on MCF-7 cells with or without laser irradiation; B: Intracellular ROS detection in MCF-7 cells of (a) control group, (b) AS, (c) HMCuS, (d) AS/ HMCuS, (e) AS-Tf and (f) AS/Tf-HMCuS; C: Analysis of average fluorescence intensity according to section B. Fig.7. In vivo antitumor activity studies. A: Relative tumor volumes of tumor-bearing mice in different treatment groups as a function of time; B: Tumor inhibition rates in tumor-bearing mice treated with Tf-HMCuS, AS, AS/HMCuS, AS/Tf-HMCuS through PT injection and AS/Tf-HMCuS through IV injection; C: HE stained tumor tissues harvest from the mice with different treatments: (a) Control group, (B) Tf-HMCuS/PT, (c) AS/PT, (d) AS/HMCuS/PT, (e) AS/Tf-HMCuS/IV and (f) AS/Tf-HMCuS/PT. Fig.8. A: In vivo optical imaging of tumor-bearing mice treated with (a) DiR/Tf-HMCuS/IV, (b) DiR/Tf-HMCuS/PT, (c) DiR/HMCuS/PT and (d) DiR/PT; B: Ex vivo optical imaging of the main organs (heart, liver, spleen, lung, kidney and tumor); C: In vivo photoacoustic images acquired pre- and at different time points after IV injection of (a) Tf-HMCuS and PT injection of (b) Tf-HMCuS, (c) HMCuS. Fig.9. Biodistribution studies of AS/Tf-HMCuS/IV, AS/Tf-HMCuS/PT, AS/HMCuS/PT and AS/PT for main organs (A: liver, B: lung, C: kidney, D: tumor). Fig.10. Evaluation of (a) R6G labeled Tf-HMCuS, (b) R6G labeled HMCuS and (c) R6G penetration in tumors by Ϯϱ 

DMR using immunofluorescence assay at designated times (A: 1 h, B: 4 h, C: 12 h, D: 48 h, E: 96 h). F: Average fluorescence intensity of different groups as above.

Ϯϲ 

  &ŝŐ͘ϭ͘^ĐŚĞŵĂƚŝĐŝůůƵƐƚƌĂƚŝŽŶŽĨƚŚĞůŽĐĂůŝnjĞĚĚĞůŝǀĞƌLJĨŽƌƐLJŶĞƌŐŝƐƚŝĐƚƵŵŽƌƚƌĞĂƚŵĞŶƚ͘

Ϯϳ 

  

  &ŝŐ͘Ϯ͘^ĐŚĞŵĞĨŽƌƉƌĞƉĂƌĂƚŝŽŶŽĨ^ͬdĨͲ,DƵ^EWƐ͘

Ϯϴ 

 



&ŝŐ͘ϯ͘ŚĂƌĂĐƚĞƌŝnjĂƚŝŽŶŽĨ,DƵ^ďĂƐĞĚĨŽƌŵƵůĂƚŝŽŶƐ͗͘dDŝŵĂŐĞƐ͗;ĂͿ,DƵ^͕;ďͿ^ͬ,DƵ^͕ ;ĐͿ^ͬdĨͲ,DƵ^͖͗^ŝnjĞĚŝƐƚƌŝďƵƚŝŽŶŽĨ,DƵ^;ĂͿ͕^ͬ,DƵ^;ďͿĂŶĚ^ͬdĨͲ,DƵ^;ĐͿ͖͗ĞƚĂ ƉŽƚĞŶƚŝĂůĂŶĂůLJƐŝƐ͗;ĂͿ,DƵ^͕;ďͿdĨ͕;ĐͿ^ͬ,DƵ^͕;ĚͿ^ͬdĨͲ,DƵ^͘

Ϯϵ 



  &ŝŐ͘ϰ͘ ŚĂƌĂĐƚĞƌŝnjĂƚŝŽŶ ŽĨ dĨͲ,DƵ^ ĂŶĚ ,DƵ^ ďĂƐĞĚ ĨŽƌŵƵůĂƚŝŽŶƐ ǁŝƚŚ Žƌ ǁŝƚŚŽƵƚ^͘ ͗ hs ƐƉĞĐƚƌƵŵŽĨ;ĂͿ,DƵ^͕;ďͿdĨĂŶĚ;ĐͿdĨͲ,DƵ^͖͗dŚĞĨůƵŽƌĞƐĐĞŶĐĞƐƉĞĐƚƌŽƐĐŽƉLJǁŝƚŚŝŶĐƌĞĂƐĞĚ ŵĂƐƐ ĐŽŶĐĞŶƚƌĂƚŝŽŶ ƌĂƚŝŽ ŽĨ ,DƵ^ ͗dĨ ĨƌŽŵ Ϭ͗ϭ ƚŽ ϰ͗ϭ͖ ͗ ^ ůŽĂĚŝŶŐ Ăƚ ĚŝĨĨĞƌĞŶƚ ĂŵŽƵŶƚƐ ŽĨ dĨͲ,DƵ^ĂŶĚ,DƵ^͖͗ZĞůĞĂƐĞƉƌŽĨŝůĞŽĨ^ĨƌŽŵ^ƐŽůƵƚŝŽŶ͕^ͬ,DƵ^ĂŶĚ^ͬdĨͲ,DƵ^͖ ͗dŚĞƉŚŽƚŽƚŚĞƌŵĂůŚĞĂƚŝŶŐĐƵƌǀĞƐŽĨ,DƵ^ĂŶĚdĨͲ,DƵ^ǁŝƚŚĚŝĨĨĞƌĞŶƚĐŽŶĐĞŶƚƌĂƚŝŽŶƐďLJĂŶ ϴϬϴŶŵůĂƐĞƌŝƌƌĂĚŝĂƚŝŽŶ͘

ϯϬ 



  &ŝŐ͘ϱ͘ĞůůƵůĂƌƵƉƚĂŬĞƐƚƵĚŝĞƐŽĨ,DƵ^ĂŶĚdĨͲ,DƵ^͗͘&ůƵŽƌĞƐĐĞŶĐĞŵŝĐƌŽƐĐŽƉŝĐŝŵĂŐĞƐŽĨ;ĂͿ &/d͕;ďͿ&/důĂďĞůĞĚ,DƵ^͕;ĐͿ&/důĂďĞůĞĚdĨͲ,DƵ^͖͗ǀĞƌĂŐĞĨůƵŽƌĞƐĐĞŶĐĞŝŶƚĞŶƐŝƚLJŽĨ&/d͕ &/d ůĂďĞůĞĚ ,DƵ^ ĂŶĚ &/d ůĂďĞůĞĚ dĨͲ,DƵ^͖ ͗ &ůŽǁ ĐLJƚŽŵĞƚƌŝĐ ĂƐƐĂLJ ĨŽƌ ;ĂͿ &/d͕ ;ďͿ &/d ůĂďĞůĞĚ,DƵ^͕;ĐͿ&/důĂďĞůĞĚdĨͲ,DƵ^ĂƚƚŚĞĚĞƐŝŐŶĂƚĞƚŝŵĞ͘

ϯϭ 



  &ŝŐ͘ϲ͘ /Ŷ ǀŝƚƌŽ ĂŶƚŝƚƵŵŽƌ ĂĐƚŝǀŝƚLJ ƐƚƵĚŝĞƐ͘ ͗ Ğůů ǀŝĂďŝůŝƚLJ ŽĨ ĚŝĨĨĞƌĞŶƚ ƚƌĞĂƚŵĞŶƚƐ ŽŶ D&Ͳϳ ĐĞůůƐ ǁŝƚŚŽƌǁŝƚŚŽƵƚůĂƐĞƌŝƌƌĂĚŝĂƚŝŽŶ͖͗/ŶƚƌĂĐĞůůƵůĂƌZK^ĚĞƚĞĐƚŝŽŶŝŶD&ͲϳĐĞůůƐŽĨ;ĂͿĐŽŶƚƌŽůŐƌŽƵƉ͕ ;ďͿ ^͕ ;ĐͿ ,DƵ^͕ ;ĚͿ ^ͬ ,DƵ^͕ ;ĞͿ ^ͲdĨ ĂŶĚ ;ĨͿ ^ͬdĨͲ,DƵ^͖ ͗ ŶĂůLJƐŝƐ ŽĨ ĂǀĞƌĂŐĞ ĨůƵŽƌĞƐĐĞŶĐĞŝŶƚĞŶƐŝƚLJĂĐĐŽƌĚŝŶŐƚŽƐĞĐƚŝŽŶ͘

ϯϮ 



  &ŝŐ͘ϳ͘ /Ŷ ǀŝǀŽ ĂŶƚŝƚƵŵŽƌ ĂĐƚŝǀŝƚLJ ƐƚƵĚŝĞƐ͘ ͗ ZĞůĂƚŝǀĞ ƚƵŵŽƌ ǀŽůƵŵĞƐ ŽĨ ƚƵŵŽƌͲďĞĂƌŝŶŐ ŵŝĐĞ ŝŶ ĚŝĨĨĞƌĞŶƚƚƌĞĂƚŵĞŶƚŐƌŽƵƉƐĂƐĂĨƵŶĐƚŝŽŶŽĨƚŝŵĞ͖͗dƵŵŽƌŝŶŚŝďŝƚŝŽŶƌĂƚĞƐŝŶƚƵŵŽƌͲďĞĂƌŝŶŐŵŝĐĞ ƚƌĞĂƚĞĚ ǁŝƚŚ dĨͲ,DƵ^͕ ^͕ ^ͬ,DƵ^͕ ^ͬdĨͲ,DƵ^ ƚŚƌŽƵŐŚ Wd ŝŶũĞĐƚŝŽŶ ĂŶĚ ^ͬdĨͲ,DƵ^ ƚŚƌŽƵŐŚ/sŝŶũĞĐƚŝŽŶ͖͗,ƐƚĂŝŶĞĚƚƵŵŽƌƚŝƐƐƵĞƐŚĂƌǀĞƐƚĨƌŽŵƚŚĞŵŝĐĞǁŝƚŚĚŝĨĨĞƌĞŶƚƚƌĞĂƚŵĞŶƚƐ͗ ;ĂͿ ŽŶƚƌŽů ŐƌŽƵƉ͕ ;Ϳ dĨͲ,DƵ^ͬWd͕ ;ĐͿ ^ͬWd͕ ;ĚͿ ^ͬ,DƵ^ͬWd͕ ;ĞͿ ^ͬdĨͲ,DƵ^ͬ/s ĂŶĚ ;ĨͿ ^ͬdĨͲ,DƵ^ͬWd͘

ϯϯ 



  &ŝŐ͘ϴ͘ ͗ /Ŷ ǀŝǀŽ ŽƉƚŝĐĂů ŝŵĂŐŝŶŐ ŽĨ ƚƵŵŽƌͲďĞĂƌŝŶŐ ŵŝĐĞ ƚƌĞĂƚĞĚ ǁŝƚŚ ;ĂͿ ŝZͬdĨͲ,DƵ^ͬ/s͕ ;ďͿ ŝZͬdĨͲ,DƵ^ͬWd͕ ;ĐͿ ŝZͬ,DƵ^ͬWd ĂŶĚ ;ĚͿ ŝZͬWd͖ ͗ dž ǀŝǀŽ ŽƉƚŝĐĂů ŝŵĂŐŝŶŐ ŽĨ ƚŚĞ ŵĂŝŶ ŽƌŐĂŶƐ ;ŚĞĂƌƚ͕ ůŝǀĞƌ͕ ƐƉůĞĞŶ͕ ůƵŶŐ͕ ŬŝĚŶĞLJ ĂŶĚ ƚƵŵŽƌͿ͖ ͗ /Ŷ ǀŝǀŽ ƉŚŽƚŽĂĐŽƵƐƚŝĐ ŝŵĂŐĞƐ ĂĐƋƵŝƌĞĚ ƉƌĞͲ ĂŶĚ Ăƚ ĚŝĨĨĞƌĞŶƚ ƚŝŵĞ ƉŽŝŶƚƐ ĂĨƚĞƌ /s ŝŶũĞĐƚŝŽŶ ŽĨ ;ĂͿ dĨͲ,DƵ^ ĂŶĚ Wd ŝŶũĞĐƚŝŽŶ ŽĨ ;ďͿ dĨͲ,DƵ^͕;ĐͿ,DƵ^͘

ϯϰ 

   %           %    

%

%

      &ŝŐ͘ϵ͘ŝŽĚŝƐƚƌŝďƵƚŝŽŶƐƚƵĚŝĞƐŽĨ^ͬdĨͲ,DƵ^ͬ/s͕^ͬdĨͲ,DƵ^ͬWd͕^ͬ,DƵ^ͬWdĂŶĚ^ͬWdĨŽƌ ŵĂŝŶŽƌŐĂŶƐ;͗ůŝǀĞƌ͕͗ůƵŶŐ͕͗ŬŝĚŶĞLJ͕͗ƚƵŵŽƌͿ͘

ϯϱ 



  &ŝŐ͘ϭϬ͘ǀĂůƵĂƚŝŽŶŽĨ;ĂͿZϲ'ůĂďĞůĞĚdĨͲ,DƵ^͕;ďͿZϲ'ůĂďĞůĞĚ,DƵ^ĂŶĚ;ĐͿZϲ'ƉĞŶĞƚƌĂƚŝŽŶ ŝŶƚƵŵŽƌƐďLJDZƵƐŝŶŐŝŵŵƵŶŽĨůƵŽƌĞƐĐĞŶĐĞĂƐƐĂLJĂƚĚĞƐŝŐŶĂƚĞĚƚŝŵĞƐ;͗ϭŚ͕͗ϰŚ͕͗ϭϮŚ͕͗ ϰϴŚ͕͗ϵϲŚͿ͘&͗ǀĞƌĂŐĞĨůƵŽƌĞƐĐĞŶĐĞŝŶƚĞŶƐŝƚLJŽĨĚŝĨĨĞƌĞŶƚŐƌŽƵƉƐĂƐĂďŽǀĞ͘           

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Graphical Abstract

Copper sulfide nanoparticle-based localized drug delivery system as an effective cancer synergistic treatment and theranostic platform Lin Hou, Xiaoning Shan, Lisha Hao, Qianhua Feng, Zhenzhong Zhang*

  A multi-functional tumor-targeted AS/Tf-HMCuS NPs localized delivery system, whichemployed the diffusion molecular retention (DMR) tumor targeting effect, possessed photoacoustic imaging, PTT and PDT properties for multimodality theranostic applications in cancer treatment

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Statement of Significance

In recent years, localized cancer treatment using different biomaterials has attracted increasing attention for effective inhibition of tumor growth. However, it is still challenging for this kind of system to achieve a high drug loading, overcome biological barriers from the site of injection to the site of action, and combine synergetic therapy with diagnosis without adversely affecting the formation process. This study provides a localized diffusion molecular retention (DMR) tumor targeting drug delivery system based on hollow mesoporous copper sulfide nanoparticles (HMCuS NPs) entrapment of anticancer drug for the first time, which can achieve high drug loading, improve local drug accumulation and retention, accomplish synergistic combination of chemo-phototherapy, and finally enhance antitumor effect. In addition, HMCuS NPs also possesses the property suitable for photoacoustic imaging, which could offer us a theranostic platform.

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