Synthesis of Cisplatin(IV) Prodrug -Tethered CuFeS Nanoparticles in

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Synthesis of Cisplatin(IV) Prodrug -Tethered CuFeS2 Nanoparticles in Tumor-Targeted Chemotherapy and Photothermal therapy Wubshet Mekonnen Girma, Shin-Hwa Tzing, Po-Jen Tseng, Chih-Ching Huang, Yong-Chien Ling, and Jia-Yaw Chang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b19640 • Publication Date (Web): 16 Jan 2018 Downloaded from http://pubs.acs.org on January 16, 2018

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ACS Applied Materials & Interfaces

Synthesis of Cisplatin(IV) Prodrug -Tethered CuFeS2 Nanoparticles in Tumor-Targeted Chemotherapy and Photothermal therapy Wubshet Mekonnen Girma,a Shin-Hwa Tzing,b Po-Jen Tseng,c Chih-Ching Huang,d Yong-Chien Ling,b Jia-Yaw Changa* a. Department of Chemical Engineering, National Taiwan University of Science and Technology, Taipei, 10607, Taiwan, Republic of China b. Department of Chemistry, National Tsing Hua University, Hsinchu, 30013, Taiwan, Republic of China c. Department of Safety Health and Environmental Engineering, National Yunlin University of Science and Technology, Yunlin, 64002, Taiwan, Republic of China d. Institute of Bioscience and Biotechnology, National Taiwan Ocean University, Keelung, 20224, Taiwan, Republic of China

*Corresponding author: Jia-Yaw Chang Department of Chemical Engineering, National Taiwan University of Science and Technology, 43, Section 4, Keelung Road, Taipei, 10607, Taiwan, Republic of China E-mail: [email protected] Tel.: +886-2-27303636. Fax: +886-2-27376644.

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ABSTRACT: In this study, for the first time, CuFeS2 nanocrystals were successfully prepared through a facile noninjection-based synthetic strategy, by reacting Cu and Fe precursors with dodecanethiol in a 1-octadecene solvent. This one-pot noninjection strategy features easy handling, large-scale production, and high synthetic reproducibility. Following hyaluronic acid (HA) encapsulation, CuFeS2 nanocrystals coated with HA (CuFeS2@HA) not only readily dispersed in water and showed improved biocompatibility, but also possessed a tumor-specific targeting ability of cancer cells bearing the cluster determinant 44 (CD44) receptors. The encapsulated CuFeS2@HA showed broad optical absorbance from the visible to the near-infrared (NIR) region, and high photothermal conversion efficiencies of about 74.2%. It can, therefore, be utilized for the photothermal ablation of cancer cells with NIR light irradiation. In addition, toxicity studies in vitro (B16F1 and HeLa) and in vivo (zebrafish embryos), as well as in vitro blood compatibility studies, indicated that CuFeS2@HA show low cytotoxicity at the doses required for photothermal therapy. More importantly, CuFeS2@HA can be used as delivery vehicles for the chemotherapy prodrug, cisplatin (IV), forming CuFeS2@HA-Pt(IV). Their release profile revealed pH- and glutathione-mediated drug release from CuFeS2@HA-Pt(IV), which may minimize the side effects of the drug to normal tissues during therapy. Subsequent in vitro experiments confirmed that the use of CuFeS2@HA-Pt(IV) provides an enhanced and synergistic therapeutic effect compared with the use of either chemotherapy or photothermal therapy alone.

KEYWORDS: CuFeS2, noninjection approach, photothermal therapy, cisplatin prodrug, chemotherapy


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INTRODUCTION Worldwide, cancer has been the most common disease-related cause of death for decades.1 Among the various approaches to cancer treatment, photothermal therapy (PTT) has recently attracted interest and is widely applied in clinical settings.2 PTT uses photothermal agents that induce hyperthermia by absorbing near-infrared region (NIR) laser energy and dissipating it in the form of heat.3 This efficiently ablates tumor cells and tissues. When the photothermal agent is near the tumor site, the increase in temperature tips to a level that can cause cancer cell death. Compared with conventional therapeutic methods, PPT displays unique benefits in tumor cell therapy, including precise spatial-temporal selectivity, high specificity, and only slight invasiveness.4-7 By assimilating therapeutic and imaging tools to create distinct nanoconstructions, nanomedicines allow the diagnosis of particular disease and tumor treatment.8 The therapeutic efficacy of PTT normally relies on the conversion of light to adequate heat via nanoscale photothermal agents. To date, a variety of nanotherapeutics such as gold nanoparticles,9 metal sulfides,10-13 nanocarbons,14 and organic nanoagents15 has been widely studied. Unremarkably, the use of nanoparticles (NPs) as PTT agents has attracted particular attention, due to the easy availability of their precursors, their simple preparation strategies and easy functionalization, their large absorption range in NIR, their solubility in biocompatible solvents, and their high photothermal conversion efficiencies.16-18 The challenge of eradicating cancer cells using only PTT is well recognized, as the energy of the NIR region steadily decreases as the depth of the tissue increases, due to light scattering and absorption.18,19 The heat produced from PTT can increase the metabolism in cells and the penetrability of cell membranes, which in turn enhance the drug intake of cancer cells and advance the effects of treatment in the tumor cells.20 Therefore, assimilating nanomaterial-based PTT with chemotherapy is vital for cancer therapy, and opens up new possibilities for the treatment of cancer. The dual-modality of PTT and chemotherapy can inspire synergetic effects, which improve


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treatment efficacy compared with the use of each treatment independently. Chemotherapy is the most commonly used modality in cancer cell treatment, as it is efficient in the treatment of both primary stage tumors and metastatic cells in distinct organs.21-23 Chalcopyrite I–III–VI2 semiconductors, such as CuInS2, CuInSe2, AgInS2, and AgInSe2, play important roles in the fabrication of photovoltaic devices and in biomedical applications.24-28 However, less attention has been paid to chalcopyrite CuFeS2, which crystallizes in a tetragonal structure, with the space group I42d. It can be obtained by doubling the unit cell of a zinc blende structure, with the S atom residing in the tetrahedral void formed by the Cu and Fe atoms.29 CuFeS2 is an n-type semiconductor that exhibits low band gaps (~0.6 eV for the bulk), large thermoelectric power, and antiferromagnetic behavior, with a relatively high Neel temperature of 550 °C.30,31 Recently, CuFeS2 has been studied as a potentially photothermal material, due to its broad spectral range (UV-visible-NIR regions), and high molar attenuation coefficient (ε = 5.2 × 106 M−1cm−1).32 Thus far, reports on the preparation of CuFeS2 NPs have been rather limited. The hot-injection method32-36 is the most widely used in the synthesis of CuFeS2 NPs; however, this approach lacks synthetic reproducibility and is not suitable for the large-scale production of NPs with narrow size distribution requirements. Therefore, finding an effective and reliable method of producing monodispersed CuFeS2 NPs has become increasingly urgent. In this study, we report for the first time the synthesis of CuFeS2 NPs using a one-pot noninjection method. The CuFeS2 NPs exhibited good dispersibility in aqueous mediums through hyaluronic acid (HA) modification assisted by ultrasonic irradiation. HA has several advantages including its hydrophilic properties and biocompatibility, the abundance of functional groups for further modification, and its ability to target ligands that bind to determinant 44 (CD44) receptors with high affinity and specificity. The resulting CuFeS2 NPs with the HA modification (CuFeS2@HA NPs) present negligible in vitro and in vivo cytotoxicity and good in vitro blood


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compatibility. They have also shown good photothermal destruction of cells under irradiation with an 808 nm NIR laser. In addition, cisplatin(IV) prodrugs conjugated with CuFeS2@HA (CuFeS2@HA-Pt(IV)) showed an effective chemotherapeutic effect. To the best of our knowledge, this is the first example of attaching drug molecules to CuFeS2 NPs, and represents a new drug delivery vehicle. Importantly, the combination of PTT and chemotherapy demonstrates more efficient destruction of tumor cells compared with any individual treatment alone, due to the synergistic effects of this combination.


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EXPERIMENTAL SECTION Chemicals. Copper (I) acetate (CuAc, 97%, Sigma-Aldrich), anhydrous iron (III) chloride (FeCl3, 98%, Alfa-Aesar), cis-dichlorodiamine platinum (II) (cis-PtCl2(NH2)2, 99.99%, Acros Organics), succinic anhydride (99%, Alfa-Aesar), hydrogen peroxide (30% H2O2, Sigma-Aldrich), WST-1 (4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate) assay was purchased from Roche Applied Science (Penzberg, Germany). Sodium hyaluronate (HA, Research grade, 100–150 kDa, Lifecore biomedical), N-hydroxy sulfosuccinimide sodium salt (Sulfo-NHS, 97%, Alfa-Aesar), ethyl (dimethyl aminopropyl) carbodiimide (EDC, 99%, AlfaAesar), reduced L-glutathione (GSH > 98%) 1-dodecathiol (98%, Acros Organics), 1-octadecene (ODE, 90%, Acros Organics), oleic acid (OA, 65-88%, Sigma-Aldrich). All other chemicals and reagents used were analytical grade. Synthesis of CuFeS2 NPs. CuAc (0.2 mmol), FeCl3 (0.2 mmol), 3 mL 1-dodecanethiol, and 5 mL ODE were loaded onto a 50 mL, four-necked, round-bottomed flask equipped with a magnetic stirrer, a heating mantel, a condenser, and temperature control and placed under an argon gas flow. The mixture was degassed in a vacuum system and purged four times under the argon gas flow to remove the oxygen inside the reaction system. The reaction was then heated to 40 °C and left until it became stable at this temperature. Subsequently, the temperature was increased to 240 °C and the mixture was heated for 10 min. After the reaction was complete, the heating mantel was removed, and the mixture was cooled to room temperature and centrifuged at 6000 rpm for 40 min. The supernatant was discarded. Finally, the precipitate was purified by adding hexane and methanol (1/2, v/v) and by centrifuging at 6000 rpm for 40 min. The product was dried and labeled CuFeS2 NPs. Phase Transfer of CuFeS2 NPs to an Aqueous Phase. Briefly, 0.03 mL of OA was added to 5 mg of CuFeS2 NPs, and the mixture sonicated for 30 min. Chloroform (1 mL) was then added, and


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the resulting mixture was sonicated for 1 h. The solution was mixed with 5 mL of 2-(Nmorpholino)ethanesulfonic (MES) (pH 7.4) buffer containing 5 mg HA and was ultra-sonicated for 2 min (20 KHz, 130 W). Finally, the aqueous part of the solution was centrifuged at 6000 rpm for 20 min and filtered using Millipore syringe filters with a pore size of 0.22 µm. Cell Culture and Cell Viability Test. Human cervical cancer cells (HeLa), and mouse melanoma cancer cells (B16F1) were cultivated in Dulbecco’s Modified Eagle’s Medium (DMEM, Hyclone), supplemented with 10% fetal bovine serum, 1% non-essential amino acids, 1% antibiotic antimycotic formulation, 1% sodium pyruvate, and 1% L-glutamine at 37 °C in a humidified 5% CO2 atmosphere. The cytotoxicity of NPs was assessed using WST-1 assays. Cells (~1.0 × 104/well) were seeded in a 96-well plate. After 24 h, the medium was replaced with a fresh culture medium containing different concentrations of Cu in the CuFeS2@HA solution (0–100 µg/mL), and the cells were incubated at 37 °C for 24 h. Next, the medium in each well was replaced with fresh medium (0.01 mL of WST-1 and 0.1 mL culture medium). The plate was incubated for another 2 h at 37 °C. Cell viability was monitored by measuring light absorbance at 450 nm using a microplate reader (BioTek, Synergy H1, USA). Loading Anticancer Drugs into CuFeS2@HA NPs. The drugs were loaded into CuFeS2@HA NPs to obtain CuFeS2@HA-Pt(IV) using the following procedures. To activate the carboxylic groups of the cisplatin(IV) prodrug, EDC (5 mg) and sulfo-NHS (5 mg) in 0.5 mL of MES buffer were added to 1 mL of 10 mg cisplatin(IV) prodrug solution and then stirred at room temperature for 30 min. Subsequently, 0.068 mmol of 2,2′-(Ethylenedioxy)bis(ethylamine) was added to the activated cisplatin(IV) prodrug solution, and the mixture was stirred gently at room temperature for 24 h. In another vial, the carboxylic groups in 2 mL of a CuFeS2@HA NP solution (10 mg/mL) were activated by adding EDC (5 mg) and sulfo-NHS (5 mg) in 0.5 mL of MES buffer solution


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and stirring for 30 min. The amine-functionalized cisplatin(IV) prodrug solution was added to the CuFeS2@HA NPs and stirred for 6 h at room temperature. Finally, the mixture was purified by centrifugation (3000 MWCO, Macrosep Advance Centrifugal Device) at 6000 rpm for 20 min to obtain CuFeS2@HA-Pt(IV) NPs. The loading efficiency of the cisplatin(IV) prodrug is expressed as a percentage. Loading Efficency % =

Weight of cisplatinIV prodrug in CuFeS @HA × 100% Weight of cisplatinIV prodrug in feed

Drug Release from CuFeS2@HA-Pt(IV) NPs. The Pt release was carried out as follows. Dialysis bags of CuFeS2@HA-Pt(IV) in a phosphoric acid buffer solution (PBS, 2 mg/mL) at pH 5 and 7.4 were immersed in 250 mL of PBS (at pH 5 and 7.4) with and without 10 mM GSH. The dialysis was performed with continuous stirring at 37 °C. 1 mL of aliquot was withdrawn at selected time intervals within a period of 72 h and replaced by an equal volume of fresh PBS to keep the total volume of the outside phase constant. The Pt concentration was determined using an inductively coupled plasma atomic emission spectrometer (ICP-AES). The amount of Pt released from the NPs was stated as the cumulative amount (%) of Pt in the outer phase of the dialysis bag, in relation to the total amount of Pt in the NPs. Measurements of the CuFeS2@HA NPs’ Photothermal Effects. To measure the photothermal effects of CuFeS2@HA NPs, 1 mL of an aqueous solution of NPs was injected in to a quartz cuvette with different concentrations of Cu (0, 25, 50, 75 and 100 µg/mL) and were irradiated by NIR laser (808 nm, 2 W/cm2) for 10 min. The Cu concentrations were determined using ICP-AES measurements. A digital thermometer with an accuracy of 0.1 °C was used to read the temperature of the solution. The temperature was recorded every 30 s. When the temperature rose, the laser turned off and the solution cooled naturally. The photothermal conversion efficiency was obtained by a series of calculations, as presented in the supporting information.


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In Vitro Experiment on the Photothermal Ablation of Cancer Cells. WST-1 was used to assess the in vitro photothermal ablation potential of CuFeS2@HA NPs against cancer cells. B16F1 cells (~1.0 × 104/well) were seeded in a 96-well plate in a culture medium for 24 h. The cultured cells were washed with PBS (pH 7.4) and 0.1 mL of a fresh culture medium containing NPs with different Cu concentrations (0, 25, 50, 75, 100 µg/mL) was added. After incubation for another 24 h, cells were again washed with PBS (pH 7.4), to remove non-internalized NPs, and a fresh 0.1 mL of culture medium was added. The cells were irradiated by NIR laser (808 nm, 2 W/cm2) for 10 min. The irradiated cells were again washed with PBS (pH 7.4), re-cultured by adding a fresh cell culture medium, and incubated for another 24 h. For control purposes, the same experiment was performed without irradiation with the laser. Finally, the cell viability was determined by measuring light absorbance at 450 nm, using a microplate reader (BioTek, Synergy H1, USA). For the anticancer drug-loaded CuFeS2@HA-Pt(IV) NPs, the same experiment was performed except that the different concentrations of the culture media were based on Pt concentrations (0, 15, 30, 45, 60 µg/mL). Zebra Fish Culture and Embryonic Toxicity of CuFeS2@HA NPs. Wild-type AB strains of zebrafish (Danio rerio) were raised and maintained at 28 ± 2 °C, under a 10h/13h light/dark cycle. Adult zebrafish, at a ratio of two males to one female, were kept in a tank under a 12 h light/dark cycle. Brine shrimp were used as feed twice a day. Fertilized eggs were collected and washed several times with oxygenated water. After washing, 10 healthy zebrafish embryos were incubated in CuFeS2@HA solution at five different concentrations of Cu (0, 25, 50, 75 and 100 µg/mL) in a 24-well culture plate. Development of zebrafish embryos and larvae were continuously monitored, from 3 h post-fertilization (hpf) to 96 hpf, using an inverted microscope (Olympus IX73) equipped with a digital camera. The percentage of completely hatched zebrafish embryos out of the total


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number of zebrafish embryos by 96 hpf was used to estimate hatching rate. The survival rate was calculated as the percentage of viable embryos out of the total number of embryos by 96 hpf. Each set included 10 embryos per condition, and three replicate trials were carried out for all experiments. The use of animals in the experiments was reviewed and approved by the Animal Experimentation Committee of National Tsing Hua University. Hemolysis. Ethylenediamine tetraacetic acid (EDTA)-stabilized human blood samples were freshly collected according to a protocol approved by the Review Board of the National Taiwan Ocean University. First, 4 mL samples of whole blood were added to 8 mL PBS (pH 7.4), and the red blood cells (RBCs) were isolated from the serum by centrifugation at 3000 rpm for 10 min. The RBCs were washed five times with PBS solution. Subsequently, the RBCs were diluted to 8% using PBS. Then, 0.5 mL of the diluted RBC suspension was added to 0.5 mL of a CuFeS2@HA NPs PBS suspension, at concentrations of Cu between 25 and 100 µg/mL. All samples were prepared in triplicate, and incubated to 37 °C for 1 h. RBCs incubated in deionized water and PBS (pH 7.4), at the same volume as used for the NPs suspensions, were used as positive and negative controls, respectively. The suspension was centrifuged for 3000 rpm for 10 min. After that, 0.2 mL of the supernatant from the sample tube was transferred to a 96-well plate. The absorbance of hemoglobin was measured at 576 nm. The percentage of hemolysis was calculated as follows: Hemolysis % = [(sample absorbance - negative control)/ (positive control - negative control)] × 100%. Trypan Blue Staining Experiments. B16F1 cells, with a density of approximately 1.0 × 104 per well, were cultured in a 96-well plate for 24 h at 37 °C and 5% CO2. Next, the cells were washed with PBS (pH 7.4) and cultured in a 37 °C incubator with 0.1 mL of new culture media containing different concentrations of Cu in CuFeS2@HA (100 µg/mL). After incubating for 24 h, the cells were washed with PBS (pH 7.4) to remove the non-internalized NPs, and fresh culture


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medium was added. Subsequently, the cells were irradiated with 808 nm laser (2 W/cm2) for 10 min. The cells were then stained with 0.1 mL trypan blue (0.4 wt%) for 5 min, after which the culture medium was removed and the cells were washed with PBS (pH 7.4) three times. Finally, the resulting number of viable and dead cells were observed under the 40× magnification of an inverted microscope (IX73; Olympus, Japan). The same procedure was applied for CuFeS2@HAPt(IV) NPs except that the concentration was based on Pt. Characterization. The absorbance spectra were recoded using UV-visible spectrometer (JASCO V-630). Transmission electron microscopy (TEM) images were captured using FEI Tecnai G2 F20 microscope (Philips, Holland). X-ray diffraction (XRD) patterns were obtained by using Bruker D8 X-ray diffractometer. X-ray photoelectron spectroscopy (XPS) analysis were performed by using ESCALAB 250 photoelectron spectrometer. Metallic concentrations were determined by using inductively coupled plasma atomic emission spectrometer (ICP-AES, JY 2000-2, Jobin Yvon Horiba). A Nicolet 5700 FT-IR spectrometer was used to obtain Fourier transform infrared (FT-IR) spectra.


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Scheme 1. Schematic representations of (a) the synthesis of CuFeS2 nanoparticles using a heating (non-injection) approach and (b) the phase transfer mechanism from oil-soluble CuFeS2 NPs to water-soluble CuFeS2@HA NPs using HA.


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(C)

(d)

(e)

Figure 1. (a) TEM and (b) HR-TEM images of the obtained CuFeS2 NPs. The inset in Figure 1b shows the HR-TEM image of one individual NP. (c) EDS spectrum of CuFeS2 NPs (d) Representative XRD patterns of CuFeS2 NPs. The XRD pattern of bulk chalcopyrite CuFeS2 (JCPDS no. 37-0471) is shown at the bottom for comparison. (e) The optical UV-vis-NIR absorbance spectrum of the as-prepared CuFeS2 NPs.


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RESULTS AND DISCUSSION Synthesis and characterization of the CuFeS2 NPs. The one-pot noninjection-based synthetic strategy for the preparation of CuFeS2 NPs is illustrated in Scheme 1a. Mixtures of CuAc and FeCl3 in noncoordinating solvents (ODE) containing 1-dodecanethiol were used as sources of copper and iron, respectively. 1-Dodecanethiol was included both as a reactant for the sulfur source and as a source of capping ligands, due to its strong coordination with metal cations. During heat treatment, precursors thermally decomposed and monomers successively accumulated in the solution. When the monomer concentration rose above a critical level of nucleation, burst nucleation occurred, followed by the growth of CuFeS2 NPs. The as-synthesized NPs were soluble in nonpolar solvents such as hexane, toluene, and chloroform. The morphology of the as-prepared NPs was investigated by TEM. As shown in Figure 1a-b, the TEM images showed quasi-spherical, monodispersed nanocrystals. Figure S1 displayed the corresponding size distribution histograms, and the average particle sizes of the as-prepared NPs are 6.7 nm. The HR-TEM image (Figure 1b, inset) clearly showed that the distance between the adjacent lattice fringes was 0.31 nm, which corresponds to the (112) d-spacing of CuFeS2. The EDS analysis (Figure 1c) revealed the presence of Cu, Fe, and S elements in the as-prepared CuFeS2 NPs. The presence of Au peaks is due to the Au TEM grid used for measurement. The powder XRD pattern (Figure 1d) of the obtained CuFeS2 NPs clearly showed four diffraction peaks that match well with the (112), (200), (204), and (312) lattice planes of chalcopyrite CuFeS2 (JCPDS no. 37-0471). The optical properties of the as-prepared CuFeS2 NPs were investigated using UV-vis absorption spectroscopy. As shown in Figure 1e, when dissolved in hexane, they showed a broad absorbance spectrum extending from 300 to 1000 nm with a long tail on the longer-wavelength side.


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The successful formation and valance states of CuFeS2 NPs were further studied with XPS analysis. The XPS results revealed that the NPs were mainly composed of Cu, Fe, and S, as shown in Figure 2. Figure 2a displays the XPS survey analysis of CuFeS2 NPs. The Cu 2p, Fe 2p, and S 2p core levels were examined. The Cu 2p level split into 2 p 3/2 (932.9 eV) and 2 p 1/2 (952.7 eV) peaks (Figure 2b), suggesting that the Cu valance state of CuFeS2 NPs is +1, as reported in the literature.37 Likewise, Fe 2p (Figure 2c) split into two peaks at 713.9 and 727.3 eV, consistent with the +3 valance of Fe. The spectrum of S 2p (Figure 2d) showed doublet peaks at 164.8 and 169.6 eV, which assigned a -2 valance state to S. (b)

(a)

Intensity (a.u)

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0

200

(c)

400 600 800 Binding Energy (eV)

1000

1200

(d)

Figure 2. (a) XPS survey spectrum of the as-prepared CuFeS2 NPs. High-resolution (b) Cu 2p, (c) Fe 2p, and (d) S 2p XPS spectra of the as-prepared CuFeS2 NPs, respectively. Synthesis and characterization of HA-capped CuFeS2 NPs. To make CuFeS2 NPs suitable


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for biological applications, these hydrophobic NPs needed to be transferred to an aqueous phase. Scheme 1b illustrates the synthetic procedure used to synthesize CuFeS2@HA NPs. HA and OA were used to make the hydrophobic CuFeS2 NPs water soluble via a self-organized encapsulation by means of ultrasonication.38 First, the aliphatic chains of OA interdigitated with the pristine capping ligands (1-dodecanethiol) of CuFeS2 NPs through hydrophobic interactions. HA subsequently wrapped around the outmost surface of the CuFeS2 NPs, as a result of the formation of hydrogen bonds between the carbonyl terminal groups of OA and the amide and hydroxyl groups of HA. HA served not only to supply the wrapping molecules for the transfer of the hydrophobic NPs to the aqueous phase, but also as a targeting ligand due to its high affinity for CD44 receptors. Moreover, its carboxylic functional groups enabled conjugation with a wide variety of molecules. The conjugation of HA with CuFeS2 NPs was confirmed using FT-IR analysis. In Figure S2, free HA shows a characteristic IR absorption peaks of 3404, 2886, 1675, 1549, 1409, 1160, 1036, 622 cm-1. The peaks appearing above 3000 cm-1 are assigned to the N-H and -OH stretching vibration bands. The HA characteristic C-H stretching mode appears at 2886 cm-1. The peak appearing at 1675 cm-1 is the stretching vibrations of carbonyl C=O bonds. The peaks appearing at 1607, 1549 and 1409 cm-1 are assigned to the bending vibrations of N-H, the stretching vibrations of C-C and CH2, respectively. Further, the peaks at 1160 and 1036 cm-1 are from C-O stretching. The peak at 622 cm-1 is assigned for C-H bending vibrations. The FT-IR spectrums of CuFeS2 without HA modification exhibits indicative peaks at 2918 cm-1 for stretching vibrations of C-H arise from the pristine capping ligand 1-dodecathiol, and peak appearing at 2346 cm-1 is assigned for stretching of C-S bonded to nanocrystal. Peaks at 1554 and 1446 cm-1 are stretching vibrations of C-C and –CH2 respectively. Peaks at 670 cm-1 is assigned to the C-H bending vibrations. After conjugations with CuFeS2, the broad band peak at 3404 cm-1, which is assigned to stretching


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vibrations of N-H and –OH in free HA shifted to 3433 cm-1 in CuFeS2@HA sample. Peaks of CuFeS2@HA at 2924 and 2849 cm-1 attributed to C-H stretching of HA shifted from 2886 cm-1and CuFeS2 from 2918 cm-1, respectively. The C=O stretching peak of free HA also shifted from 1675 cm-1 to 1639 cm-1 in CuFeS2@HA, whereas no peaks were observed in CuFeS2. The stretching vibrations of C-C and –CH2 displays a peak shift to 1552 and 1459 cm-1, respectively. Likewise, the C-O stretching peaks of HA observed at 1186 and 1041 cm-1 in CuFeS2@HA. Therefore, FTIR results reveal the successful conjugations of HA with CuFeS2 NPs via self-organized encapsulation by means of ultrasonication. Photographs of the initial hydrophobic CuFeS2 NPs dispersed in chloroform and of the corresponding CuFeS2@HA NPs dispersed in water are displayed in Figure 3a. The results illustrate that CuFeS2 NPs were successfully transferred from an organic medium to an aqueous phase after HA surface modification. The optical properties of the as-prepared CuFeS2@HA NPs were studied using UV-vis absorption spectroscopy (Figure 3b). As shown in Figure 3c, the hydrodynamic diameter of CuFeS2@HA NPs obtained from DLS measurements was about 78.1 nm, which is larger than the size observed by TEM. This may be attributed to the formation of HA and OA coatings and a hydration layer surrounding the surface of the CuFeS2 NPs. From the TEM and HR-TEM images (Figures 3d-f), it was found that HA surface modification did not alter the size and crystallinity of pristine CuFeS2 NPs, and no observable agglomeration or alteration of NPs occurred during the phase transfer from organic solvent to water. Furthermore, we examined the colloidal stability of CuFeS2@HA NPs in different media, including deionized water, PBS solution, and DMEM culture medium. As shown in Figure S3, the NPs were stable and well dispersible in different media for over one week.


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40

(a)

(c)

(b) 30

20

H2O

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CHCl3 0 1

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100 Size (d.nm)

1000

(f)

Figure 3. (a) Photographs of CuFeS2 NPs and CuFeS2@HA NPs dispersed in chloroform and water, respectively. (b) The UV-vis-NIR absorption spectrum and (c) the hydrodynamic diameters of CuFeS2@HA NPs dispersed in aqueous medium. (c) TEM and (d) HR-TEM images of CuFeS2@HA. (f) The lattice-spacing profile of one individual NP measured from panel (e). Photothermal properties of CuFeS2@HA NPs. Inspired by the pronounced absorption by CuFeS2@HA NPs in the near-IR region, we further investigated the photothermal properties of aqueous dispersions of as-prepared NPs under NIR laser irradiation (808 nm) with a power density of 2 W cm−2. As presented in Figure 4a, the temperature of droplets of CuFeS2@HA NPs solution was increased under continuous irradiation by an NIR laser. After 10 min of irradiation (808 nm, 2 W/cm2) the NPs containing 100 µg/mL of Cu showed a dramatic temperature change of 35.8 °C, while no significant temperature change was observed in deionized water. In addition, as the concentration of Cu decreased to 75, 50 and 25 µg/mL, the temperature change in 10 min was 31.9, 26.4 and 24.3 °C respectively. The photothermal conversion efficiency was further assessed based on literature reports.39 Temperature change against irradiation time was recorded until the change reached steady state and the laser was shut off, allowing the solution cooled naturally


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(Figure 4b). Figure 4c displays linear time data against –lnθ obtained from the cooling periods displayed in Figure 4b. The photothermal conversion efficiency of CuFeS2@HA NPs was obtained using the following equation.

η=

hS(Tmax − Tsur ) − Qdis I(1−10 -A )

Where η is the photothermal conversion efficiency, h is the heat transfer coefficient, S is the surface area of the container during irradiation, Tmax is the maximum equilibrium temperature reached by the solution, Tsur is the room temperature of the surroundings, Qdis is the heat dissipation during light absorption associated with the solvent and the cuvette, I represents the incident laser power, and A is the absorbance of the sample at 808 nm. According to the equation, the photothermal conversion efficiency of CuFeS2@HA NPs under 808 nm laser irradiation was determined to be 74.2%, which is significantly higher than that of Au nanorods (22%),40 Cu2-xSe (22 %),41 black phosphorus quantum dots (28.4%),42 and Cu9S5 nanocrystals (25.7%),13 and comparable to that of Cu7.2S4 nanocrystals (56.7%).43 We further examined the photostability of CuFeS2@HA NPs (100 µg/mL) by irradiating a sample under the same conditions until it reached steady state and turned the laser off, cooling the solution naturally. A temperature generation-dissipation curve was plotted for four heating/cooling cycles, as displayed in Figure 4d. Almost identical temperature variation was achieved during each laser on/off cycle. These results confirmed the photothermal stability of the as-prepared CuFeS2@HA NPs under laser irradiation, making it a potential PTT agent for the photothermal ablation of cancer cells. In vitro and in vivo biocompatibility tests of CuFeS2@HA NPs. The in vitro cytotoxicity of CuFeS2@HA NPs against CD44 receptor-deficient HeLa cell lines and CD44 receptor-bound B16F1 cell lines was examined using WST-1 assays. As shown in the Figure 5a, after incubation


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for 24 h in CuFeS2@HA NP solutions at Cu concentrations ranging from 0 to 100 µg/mL, the viability of cells was not affected in either of the two cancer cell lines. Thus, low cytotoxicity was observed even at higher concentrations of Cu, warranting potential use in future biomedical applications. 70

(a)

(b)

60 50 40 30 0

100

200

300

400

500

600

Time (s)

(d

(c)

Figure 4: The photothermal properties of CuFeS2@HA NPs dispersed in aqueous medium: (a) temperature evaluation curves of different concentrations of Cu under laser irradiation as a function of irradiation time, (b) the photothermal response under irradiation for 10 min and with the laser turned off and natural cooling, (c) the linear time data versus –lnθ obtained from the cooling period of Figure 4b, and (d) the temperature evaluation of Cu concentrations of 100 µg/mL over four complete laser on/off irradiation cycles. Hemocompatibility is another major concern for the material’s safety because blood compatibility is an essential requirement for in vivo application. The hemocompatibility of the assynthesized CuFeS2@HA NPs was evaluated using a hemolytic assay. In contrast to the positive


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control (water), CuFeS2@HA PBS (pH 7.4) solution containing Cu concentrations of 0−100 µg/mL did not show significant hemolytic activities on human red bold cells, as shown in Figure 5b. For example, quantitative analyses displayed that the highest hemolytic efficiency was 10.7% when the Cu concentration reached 100 µg/mL. The results indicated that CuFeS2@HA NPs possesses good hemocompatibility, which makes this nanomaterial suitable for future in vivo applications. To evaluate in vivo biocompatibility, Zebrafish (D. rerio) are a good animal model.44,45 Zebrafish have numerous advantages, such as being easy to care for, having a high fertility rate and fast embryonic growth, being genetically similar to humans and being transparent, allowing tissue visualization via optical microscopy.46,47 The in vivo biocompatibility of CuFeS2@HA NPs was assessed using the embryonic development of zebrafish, including their phenotypic development, their embryonic survival rate, and hatching interferences. The embryonic hatching and survival rates of the zebrafish are displayed in Figure 5c. The control, without CuFeS2@HA showed a 100% survival rate at 96 hpf. The survival and hatching rates were calculated as the number of embryos capable of becoming zebrafish larvae and the number of hatching embryos at 96 hpf, respectively, out of the total number of embryos. After exposure to different concentrations of CuFeS2@HA, zebrafish embryos showed no mortality within 96 hpf, even at higher concentrations (Figure 5c). In addition, as displayed in Figure 5c, no hatching delay was recorded at any concentration. Figure S4 shows the embryonic developmental stages of zebrafishes incubated with different amounts of CuFeS2@HA (Cu concentration: 0, 25, 50, 75, 100 µg/mL). Control group zebrafish embryos showed normal developmental stages, from cleavage (3 hpf), segmentation (24 hpf), and hatching (48 hpf), to fully developed larvae (96 hpf). Zebrafish embryos treated with CuFeS2@HA also showed normal phenotypic development.


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(a)

(b)

(c)

Figure 5. (a) Viability of HeLa and B16F1 cancer cell lines incubated with CuFeS2@HA NPs at different Cu concentrations (0, 25, 50, 75, and 100 µg/mL) for 24 h at 37 °C, measured by WST-1 assay. (b) Hemolytic assay of CuFeS2@HA NPs with human red blood cells. (Inset photographs: Water (+) and PBS (-) represent positive and negative controls, respectively, and CuFeS2@HA PBS (pH 7.4) solution containing different Cu concentrations (25, 50, 75, and 100 µg/mL)). (c) Hatching and survival rates of zebrafish embryos incubated with CuFeS2@HA NPs containing


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different concentrations of Cu. All experiments were repeated in triplicate in three independent experiments. In vitro drug loading and release and cytotoxicity studies of CuFeS2@HA-Pt(IV). It is known that deep-seated tumors are impossible to eradicate just with PTT. As the tissue depth increases, the power of the NIR laser steadily decreases and near large blood vessels the heat generated can be easily dissipated by the blood.48 Hence combining chemotherapy with PTT can motivate synergetic effects and raise the tumor ablation efficacy. To confer additional chemotherapeutic capabilities on CuFeS2@HA, the as-prepared NPs were covalently tethered to cisplatin(IV) prodrugs with amide bond connections in the presence of EDC and sulfo-NHS (Figure S5). Cisplatin(IV) was selected as a model drug because it has been reported to be reduced intracellularly and extracellularly by biological reductants (e.g., glutathione), transforming it into the more cytotoxic Pt(II) species.49,50 The Pt(II) species subsequently forms 1,2-d(GpG) intrastrand cross-links with nuclear DNA, resulting in cell death. The proposed synergistic therapeutic mechanism of the as-prepared CuFeS2@HA-Pt(IV) is shown in Scheme 2. First, CuFeS2@HAPt(IV) specifically attached to the membrane of cancerous cells and was then internalized into the cytoplasm through an HA receptor-mediated endocytosis process. Subsequently, under laser irradiation, CuFeS2@HA-Pt(IV) converted light energy into heat, resulting in an increase of the internal temperature of the cell and leading to cancer cell death. Simultaneously, the cisplatin(IV) prodrugs were cleaved from the CuFeS2@HA-Pt(IV) under reductive conditions, such as the GSH51-53 and acidic microenvironments54,55 in endosomes/lysosomal compartments. The released cisplatin(IV) prodrugs were reduced to active cisplatin(II) by biological reductants (GSH, ascorbic acid, and cysteine) and could then intercalate nuclear DNA, inhibiting DNA replication and cell proliferation. To confirm the conjugation of cisplatin(IV) prodrugs with CuFeS2@HA NPs, we carried out


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FT-IR characterization, as well as morphological studies. To justify conjugation of cisplatin(IV) prodrugs to CuFeS2@HA NPs, we carried out FT-IR experiments. As shown in Figure S6, the peak found above 3429 cm-1 can be assigned to OH functional groups, whereas peaks around 2936 cm-1 arise from HA CH2 groups. The peak ranging from 1642 and 1407 cm-1 was assigned to stretching vibrations of COO- and -NH2 in the spectra of CuFeS2@HA-Pt(IV). The peak shifts were observed after conjugation. Generally, the peak intensities in CuFeS2@HA-Pt(IV) NPs increased, confirming the conjugation of cisplatin(IV) prodrugs to CuFeS2@HA. Figure S7 shows a TEM image and an EDS spectrum of the CuFeS2@HA-Pt(IV). An ICP-AES was used to measure the amount of Pt in CuFeS2@HA-Pt(IV), and the loading efficiency was 29.7%.

Scheme 2. Schematic illustration of the possible mechanisms of photothermal ablation and drug delivery using CuFeS2@HA-Pt(IV) in B16F1 cells. It was reported that GSH exists in millimolar concentrations (1-10 mM) in the intracellular microenvironment (in the cytoplasm, mitochondria, and nucleus), whereas it is rarely present in


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blood plasma (2–20 µM).56,57 The cytosolic GSH level in some cancer cells is reported to be at least four-times higher than that in normal cells.58 Furthermore, extracellular cancer tissues and the intracellular conditions (e.g., lysosomes and endosomes) in tumor cells are acidic microenvironments.59 Therefore, to investigate the pH and glutathione-mediated release of cisplatin(IV) prodrugs from the CuFeS2@HA-Pt(IV), the releasing process was carried out at different pH values with and without GSH. The amount of Pt released from the CuFeS2@HAPt(IV) was determined by ICP-AES. Figure 6a shows the cumulative release (as a function of time) of cisplatin(IV) prodrugs from CuFeS2@HA-Pt(IV) in different pH media, with and without adding GSH. At pH 5.0 in the absence of GSH (Figure 6a), less than 20% of the drug was released within the experimental time of 72 h. An even slower release rate is observed under physiological conditions (pH 7.4), suggesting that cisplatin(IV) prodrugs could be steadily conjugated in CuFeS2@HA-Pt(IV) during circulation in the bloodstream (pH 7.4) in vivo. Upon the addition of GSH at pH 7.4, CuFeS2@HA-Pt(IV) exhibited an initial drug release of about 30% during the initial 12 h and 36.7% after 72 h. When the pH was changed from 7.4 to 5.0 with GSH addition, mimicking the intracellular tumor environment, 50% of the drug was released from CuFeS2@HAPt(IV) during the initial 12 h and 58% drug release was achieved at 72 h. As shown in Figure S5, cisplatin(IV) prodrug contains hydrolysable ester bonds at the axial positions in CuFeS2@HAPt(IV), which could be hydrolyzed at acidic and neutral conditions. In comparison with neutral conditions (pH 7.4), the faster-releasing rate of a drug from CuFeS2@HA-Pt(IV) at acidic condition (pH 5.0) possibly could be because of faster hydrolysis rate of the ester bonds. Similar treatments under acidic conditions have already proven to enhance cisplatin release from the nanocarriers due to easier and faster hydrolysis of the ester bonds.55,60,61 All above results demonstrate that CuFeS2@HA-Pt(IV) possesses pH- and glutathione-mediated drug release, which is highly desirable for cancer therapy because it can minimize the side effects of cisplatin(IV)


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prodrugs to healthy tissue. The cytotoxic effects of free cisplatin(IV) and CuFeS2@HA-Pt(IV) on the B16F1 cell line was investigated by conducting WST-1 assays. Figure 6b shows the viability of cells after being cultured for 24 h with free cisplatin(IV) and CuFeS2@HA-Pt(IV) at different concentrations. Cell viability against CuFeS2@HA-Pt(IV) decreased as the concentration of Pt increased, similar to that against the free cisplatin(IV). It was found that free cisplatin(IV) caused a slightly higher number of B16F1 cell deaths than CuFeS2@HA-Pt(IV) with an equivalent concentration of Pt. This is probably due to different cellular uptake mechanisms. For example, free cisplatin(IV) enters cells by passively diffusing through the cell membrane, resulting in rapid accumulation in the nucleus, whereas the cellular uptake of CuFeS2@HA-Pt(IV) is via receptor-mediated endocytosis.62 Another possibility is that the difference results from the slow and continuous release of the drug from CuFeS2@HA-Pt(IV). 63


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100

(a)

80 60 40 20 0 0

10

20

30

40

50

60

70

80

Time (h)

(b)

Figure 6. (a) The cumulative release profile of Pt for the CuFeS2@HA-Pt(IV) measured in phosphate-buffer saline (pH 7.4 and pH 5.0) with and without GSH at room temperature. (b) The cytotoxicity of B16F1 cells after treatment with cisplatin(IV) prodrugs and CuFeS2@HA-Pt(IV). In vitro feasibility of photothermal- and chemo-therapy. The biocompatibility, high photothermal conversion efficiency, and remarkable photostability of the NPs inspired us to further evaluate the in vitro cytotoxicity of CuFeS2@HA-Pt(IV). In vitro experiments were carried out to investigate the photothermal and chemotherapeutic efficacy of CuFeS2@HA-Pt(IV) NPs against cancer cells using WST-1 assays and trypan blue tests. First, the PTT efficacy of CuFeS2@HA on B16F1 cells was quantified using a WST-1 assay method under NIR laser irradiation. After incubation with CuFeS2@HA NPs at different concentrations for 24 and 48 h, the NP-treated cells were irradiated with NIR laser (808 nm, 2 W/cm2) for 10 min and incubated for an


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additional 24 h. As expected, the cells incubated with CuFeS2@HA NPs and NIR irradiation showed decreasing cell viability as the concentration of the NPs increased (Figure 7a and S8a). To directly visualize the PTT therapeutic efficacy, the viability of treated cells incubated under different conditions was reconfirmed by trypan blue staining experiments. As observed in optical microscopic images (the upper left and upper right sections in Figure 7c and S8c), there were no observable blue-stained cells when B16F1 cells were cultured with and without NIR laser irradiation. Similar results (shown in the lower left section of Figure 7c and S8c) indicated that CuFeS2@HA NP-treated cells were viable in the absence of laser irradiation, revealing that the asprepared CuFeS2@HA NPs alone are biocompatible and did not induce cell death. In contrast, the lower right section of Figure 7c and S8c show that most of the B16F1 cells were stained blue after treating them with a combination of CuFeS2@HA NPs and NIR laser irradiation. These results suggest that the CuFeS2@HA NPs are capable of the selective photothermal killing of cancer cells, controlled through the application of NIR laser treatment. To further compare the effects of chemotherapy and the combination of PTT/chemotherapy in vitro, B16F1 cells were treated with different concentrations of CuFeS2@HA-Pt(IV) in the presence and absence of NIR laser irradiation. As shown in Figure 7b, at all Pt concentrations, incubation with CuFeS2@HA-Pt(IV) along with NIR irradiation reduced cell viability more than CuFeS2@HA-Pt(IV) treatment alone. For example, quantitative evaluation showed that more than 80% of the cells treated with the combination of CuFeS2@HA-Pt(IV) (Pt concentration of 30 µg/mL) and NIR irradiation were killed, but the viability of the cells treated with CuFeS2@HAPt(IV) alone remained 50% at the same Pt concentration. The cell death induced by CuFeS2@HAPt(IV)-mediated chemotherapy and the combination of PTT/chemotherapy was further investigated by trypan blue staining. As displayed in the upper left and upper right sections of Figure 7d, the cells survive almost completely when incubated with and without NIR laser


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irradiation in the absence of CuFeS2@HA-Pt(IV). The lower left section of Figure 7d displays some cancer cells stained blue, indicating that several cells were killed by CuFeS2@HA-Pt(IV) alone, because of chemotherapy. Almost all the cancer cells were killed, under the combined CuFeS2@HA-Pt(IV) and NIR laser irradiation treatment (lower right section, Figure 7d). These results indicate that CuFeS2@HA-Pt(IV) combined with NIR laser irradiation could achieve synergistic therapeutic efficacy. Moreover, the trypan blue staining experiment showed that cancer cells incubated with different concentrations of CuFeS2@HA-Pt(IV) were killed more efficiently in a concentration-dependent manner (Figure S9), consistent with the results obtained by the WST1 assay. To further evaluate the uptake of CuFeS2@HA-Pt(IV) through CD44 receptor-mediated cellular uptake, competitive inhibition studies were performed by addition of excess free HA to the culture medium prior to incubating cells with the as-prepared NPs. As shown in Figure 7b, the CuFeS2@HA-Pt(IV) alone and the combination of CuFeS2@HA-Pt(IV) and NIR laser irradiation both had limited cytotoxic effects on the B16F1 cells in an excess free HA medium. This is because CD44 receptors on the surface of B16F1 cells were pre-blocked with free HA, resulting in decreased internalization of CuFeS2@HA-Pt(IV), and supporting a specific CD44 receptormediated endocytosis mechanism. Similar experiments were also carried out by using HeLa cells, a CD44 receptor-deficient cell line (negative control). Figure S10a shows that the HeLa cells incubated with CuFeS2@HA NPs and NIR irradiation led to less cytotoxicity in comparison to B16F1 cells (Figure 7a). Moreover, lower in vitro therapeutic effects were observed (Figure S10b) for the HeLa cells treated with CuFeS2@HA-Pt(IV) alone or CuFeS2@HA-Pt(IV) combined with NIR laser irradiation, compared to the cytotoxic effects of CuFeS2@HA-Pt(IV) alone or with NIR laser irradiation on B16F1 cells (Figure 7b). For example, almost 70% of B16F1 cancer cells died when treated with CuFeS2@HA-Pt(IV) (Pt concentration of 60 µg/mL), whereas CuFeS2@HAPt(IV) did not cause significant cytotoxicity in HeLa cells under the same conditions. The B16F1


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cell viability decreased to 50% when cultured with a combination of CuFeS2@HA-Pt(IV) (Pt concentration of 15 µg/mL) and NIR irradiation; however, the HeLa cell viability was still ~90% under identical conditions. Based on these results, the targeting properties of CuFeS2@HA-Pt(IV) was verified. The half-maximal inhibitory concentration (IC50) value of CuFeS2@HA-Pt(IV) combined with NIR laser irradiation was found to be ~14.88 µg/mL (in terms of the concentration of Pt), which was lower than that of CuFeS2@HA-Pt(IV) without NIR laser irradiation (~37.08 µg/mL) (Figure 7b), which suggests a reduced dose of anticancer drug. The above result could be attributed to the synergistic effects of PTT/chemotherapy. To investigate the cytotoxic effect of longer time cell incubation with NPs, B16F1 cells incubated with CuFeS2@HA-Pt(IV) for 48 h, and then treated in the absence and presence of NIR laser for 10 min. Figure S8b displayed that the cells treated with 48 h incubation of CuFeS2@HA-Pt(IV) shows much stronger cytotoxicity than that of 24 h incubation (Figure 7b). The viability of treated cells incubated under different conditions was reconfirmed by trypan blue staining experiments as shown in Figure S8d.


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Figure 7. (a) Cell viability of B16F1 cells incubated with CuFeS2@HA for 24 h, and then treated in the absence and presence of NIR laser (808 nm, 2 W/cm2) for 10 min. The relative viabilities of the cells are expressed as a function of the Cu concentration. (b) Cell viability of B16F1 cells incubated with CuFeS2@HA-Pt(IV) for 24 h, and then in the absence and presence of NIR laser treatments (808 nm, 2 W/cm2) for 10 min. The relative viabilities of the cells are expressed as a function of the Pt concentration. Data are presented as the mean ± standard deviation (SD; n = 3). Optical micrographs of trypan blue-stained B16F1 cells with scale bar 100 nm: (c) B16F1 cells only (upper left section), B16F1 cells irradiated with NIR laser (upper right section), B16F1 cells incubated with CuFeS2@HA nanoparticles (Cu concentration: 100 µg/mL, lower left section), and B16F1 cells treated with CuFeS2@HA nanoparticles (Cu concentration: 100 µg/mL) and NIR laser (lower right section). (d) B16F1 cells only (upper left section), B16F1 cells irradiated with NIR laser (upper right section), B16F1 cells incubated with CuFeS2@HA-Pt(IV) (Pt concentration: 60 µg/mL, lower left section), and B16F1 cells treated with CuFeS2@HA-Pt(IV) (Pt concentration: 60 µg/mL) and NIR laser (lower right section).


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CONCLUSION In summary, we have successfully developed a multifunctional nanomaterial based on CuFeS2 NPs. To the best of our knowledge, it is the first time that this has been attempted using a facile noninjection-based synthetic strategy of preparation. By using HA encapsulation, the as-prepared CuFeS2@HA NPs not only displayed good in vitro blood compatibility and low in vitro and in vivo cytotoxicity, but also exhibited highly efficient photothermal effects when exposed to an NIR laser. Moreover, the CuFeS2@HA NPs can serve as drug carriers for cisplatin(IV) prodrugs, releasing them only when triggered by a more acidic environment and GSH conditions. Compared with single treatments (chemotherapy or PTT alone), proof-of-principle in vitro cell viability tests revealed that CuFeS2@HA-Pt(IV) can significantly enhance therapeutic efficacy due to the synergistic effects of PTT and chemotherapy.


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ASSOCIATED CONTENT Supporting information The particle size distribution of the as-prepared CuFeS2 NPs, FT-IR spectra of CuFeS2, HA and CuFeS2@HA, photographs of CuFeS2@HA NPs dispersed in different media, developmental stages of zebrafish embryos cultured with CuFeS2@HA. FT-IR spectra, TEM image, and EDS spectrum of CuFeS2@HA-Pt(IV), the cell viability of B16F1 cells treated with CuFeS2@HAPt(IV) in the absence and presence of NIR laser, trypan blue staining and cell viability of HeLa cells incubated with CuFeS2@HA and CuFeS2@HA-Pt(IV) under different conditions. This information is available free of charge via the Internet at http://pubs.acs.org/.

AUTHOR INFORMATION Corresponding author *(J.-Y.C.) E-mail [email protected]; telephone +886-2-27303636; fax +886-2-27376644. ORCID Wubshet Mekonnen Girma: 0000-0003-3370-6731 Jia-Yaw Chang: 0000-0002-4172-6612 Conflict of interest The authors declare no competing ﬁnancial interest. ACKNOWLEDGMENTS This work was supported by the Ministry of Science and Technology of the Republic of China under Contract No. MOST 106-2113-M-011-002.


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Graphic Abstract


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Synthesis of Cisplatin(IV) Prodrug -Tethered CuFeS Nanoparticles in

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