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Hyaluronic acid-functionalized gold nanorods with pH/NIR dual-responsive drug release for synergetic targeted photothermal-chemotherapy of breast cancer Weijun Xu, Junmin Qian, Guanghui Hou, Aili Suo, Yaping Wang, Jinlei Wang, Tiantian Sun, Ming Yang, Xueli Wan, and Yu Yao ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b08700 • Publication Date (Web): 04 Oct 2017 Downloaded from http://pubs.acs.org on October 5, 2017

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ACS Applied Materials & Interfaces

Hyaluronic acid-functionalized gold nanorods with pH/NIR dual-responsive drug release for synergetic targeted photothermal-chemotherapy of breast cancer

Weijun Xu†, Junmin Qian†,*, Guanghui Hou†, Aili Suo‡,*, Yaping Wang†, Jinlei Wang†, Tiantian Sun†, Ming Yang†, Xueli Wan†, Yu Yao‡

†

State Key Laboratory for Mechanical Behavior of Materials, Xi’an Jiaotong University, Xi’an 710049,

China ‡

Department of Oncology, The First Affiliated Hospital of Xi’an Jiaotong University, Xi’an 710061, China

*Corresponding Authors: E-mail address: [email protected] (J.M. Qian); [email protected] (A.L. Suo);

Keywords: Gold nanorods; Hyaluronic acid; Targeted delivery; Dual-responsive drug release; Synergistic therapy; Breast cancer

ACS Paragon Plus Environment


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Abstract: Tumor targeted delivery of photothermal agent and controlled release of concomitant chemotherapeutic drug are two key factors for combined photothermal-chemotherapy. Herein, we developed a pH/near-infrared (NIR) dual-triggered drug release nanoplatform based on hyaluronic acid (HA)-functionalized gold nanorods (GNRs) for actively targeted synergetic photothermal-chemotherapy of breast cancer. Targeting folate (FA), dopamine and adipic acid dihydrazide tri-conjugated HA was firstly synthesized and used to decorate GNRs via Au-catechol bonds, and then an anticarcinogen doxorubicin (DOX) was conjugated onto HA moieties via an acid-labile hydrazone linkage, resulting in multifunctional nanoparticles GNRs-HA-FA-DOX. The nanoparticles exhibited excellent stability and had a pH and NIR dual-responsive drug release behavior. In vitro studies showed that the nanoparticles could be efficiently internalized into breast cancer MCF-7 cells and kill them under NIR irradiation in a synergistic fashion via inducing cell apoptosis. Pharmacokinetics and biodistribution studies in tumor-bearing mice indicated that the nanoparticles had a long blood circulation with a half-life of 2.4 h and exhibited a high accumulation of 11.3% in tumor site. The tumors of mice treated with combined chemotherapy and photothermal therapy were completely suppressed without obvious systemic toxicity after 20 days of treatment. These results demonstrated a great potential of GNRs-HA-FA-DOX nanoparticles for targeted synergistic therapy of breast cancer.


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1. Introduction Breast cancer ranks second most prevalent cancer worldwide among women after lung cancer, and about one in every eight women is expected to develop breast cancer during her lifetime1. As one of the most common therapeutic approaches to breast cancer, current chemotherapy suffers greatly from limited therapeutic efficacy and toxic side effects in patients2. To overcome these problems, researchers have presented various strategies, including improving tumor-targeting ability of carriers3, enhancing the cellular uptake of chemotherapeutics4,5, and developing novel therapeutic technologies for combined modality treatments6,7. Among various combination therapies, the combination of chemotherapy and near-infrared (NIR) light-mediated hyperthermia has drawn attention due to the good tissue penetration of NIR light (up to 10 cm)2,8-10. NIR-induced phtotothermal heating can not only directly destroy cancer cells9-10 but also remarkably improve chemotherapeutic efficacy by triggering drug release or enhancing the toxicity of chemotherapeutics to cancer cells10. Therefore, combined photothermal-chemotherapy may be a promising alternative approach to overcoming the limitations of current treatments for breast cancer. To obtain highly effective photothermal therapy (PTT), enable deep penetration into biological tissues and avoid nonspecific heating, desirable photothermal agents should exhibit high absorption in the NIR region and can be selectively uptaken by tumor cells11. Some studies suggest that gold nanostructures, particularly gold nanorods (GNRs) with strong surface plasmon resonance absorption in the NIR region, have great potential in photothermal-chemotherapy applications 12-14. The GNRs can absorb NIR light and convert it into heat with nearly 100% efficiency 15 when their characteristic absorption peak matches the wavelength of NIR light. NIR-induced heat can directly kill cancer cells by hyperthermia and stimulate the release of drugs from nanocarriers12. On the other hand, rod-like nanoparticles show prolonged circulation time16,17 and can more effectively accumulate in tumors than spherical ones18,19 This makes GNRs particularly suitable for photothermally modulated drug delivery and release. However, their practical applications as drug carriers are severely restricted by limited drug loading capacity9,20, instability under physiological conditions20 and toxicity of cetyltrimethylammonium bromide (CTAB) which is a surfactant commonly used in the synthesis of GNRs21. To overcome the above-mentioned drawbacks, considerable efforts have been devoted to the surface modification of GNRs with various materials such as chitosan22, peptide23, DNA and/or PEG12,24,25, polyamidoamine9 and mesoporous silica20,26. These functionalized GNRs can be used for simultaneous hyperthermia and drug delivery to efficiently inhibit tumor growth. Although the promising results are obtained, they have two imperfectnesses, namely nonspecific delivery



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and unintended drug release. Current GNRs-based delivery systems rely mainly on simple passive targeting through the enhanced permeability and retention (EPR) effect. This cannot reach an enough accumulation of GNRs and chemotherapeutic drug in tumors due to the lack of specific interaction between GNRs and cancer cells27, leading to a limited therapeutic efficacy. In addition, in GNRs-based drug delivery systems, chemotherapeutic drugs are usually loaded on GNRs surface via physical adsorption or electrostatic interaction. Undesired drug release from GNRs is highly possible in the blood circulation9, which might result in adverse effects to normal tissues and even evoke innate drug resistance in cancer cells28. Hence, elaborate surface decoration of GNRs is still highly desirable for the development of tumor-targeted and stimuli-responsive drug delivery systems. Hyaluronic acid (HA), a natural negatively charged linear hydrophilic polysaccharide, has shown great potential in pharmaceutical applications due to its non-immunogenicity, non-toxicity, and biodegradability29 as well as plentiful functional groups available for chemical modification and drug loading30. Furthermore, HA can act as a powerful targeting moiety because it specifically interacts directly with CD44 receptor, which is significantly over-expressed in breast cancer31,32. Importantly, HA functionalized gold nanostructures (i.e. nanosphere33, nanostar34 and nanocage35) can effectively reach the tumor site by a combination of EPR-mediated passive targeting and HA-mediated active targeting. Thus, the integration of the superior properties of HA and the unique advantages of GNRs is highly expected to provide the potential for tumor targeted delivery of GNRs and site-specific release of chemotherapeutics. As far as we know, there are still very few studies on the construction of HA-modified GNRs for the combination therapy of cancer. Herein, we developed a pH and NIR dual-responsive nanoplatform GNRs-HA-FA-DOX for actively targeted synergetic photothermal-chemotherapy of breast cancer, as shown in Figure 1. To prepare the nanoplatform, dopamine (DA), adipic acid dihydrazide(ADH) and folate (FA) tri-functionalized HA was firstly synthesized by successive carbodiimide reaction and used to decorate GNRs via Au-catechol bonds, and then doxorubicin (DOX) was chemically conjugated onto HA moieties via an acid-labile hydrazone linkage, resulting in multifunctional nanoparticles GNRs-HA-FA-DOX. The tri-functionalized HA coating was designed to endow the nanoparticles with enhanced stability, prolonged blood circulation and tumor targetability. By combination of DOX-mediated chemotherapy and PTT based on the hyperthermia of GNRs, the GNRs-HA-FA-DOX under NIR irradiation was expected to improve the tumor therapeutic efficiency and reduce undesirable side effects. The physicochemical properties of the nanoparticles,


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including morphology, size, zeta potential, optical stability, photothermal conversion ability and drug release kinetics were examined. The cytotoxicity, cellular uptake, targeting ability of the nanoparticles, and in vitro therapeutic effect of combined photothermal-chemotherapy were evaluated in MCF-7 cells under NIR irradiation. The in vivo pharmacokinetics, biodistribution, anticancer effect and systemic toxicity were investigated

in

tumor-bearing

mice.

The

results

demonstrated

that

the

combined

photothermal-chemotherapy significantly improved the therapeutic efficacy in a synergistic fashion.

2. Materials and methods 2.1. Materials Hyaluronic acid (HA, molecular weight: 8000 Da), hydrogen tetrachloroaurate (III) trihydrate (HAuCl4·3H2O), adipic acid dihydrazide (ADH), silver nitrate (AgNO3), N-hydroxysuccinimide (NHS), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC·HCl), L-ascorbic acid (AA) and dopamine hydrochloride (DA·HCl) were purchased from Aladdin Reagent Inc. (Shanghai, China). Doxorubicin (DOX) was purchased from Meilunbio Co., Ltd (Dalian, China). Folate (FA), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl

tetrazolium

bromide

(MTT),

calcein

AM,

fluorescein

isothiocyanate labeled phalloidin (Phalloidin-FITC) and cetyltrimethylammonium bromide (CTAB, Cat. No.:H6269) were purchased from Sigma-Aldrich (Shanghai, China). Cyanine 7.5 (Cy7.5) was purchased from Amersham Biosciences (Piscataway, NJ, USA). Dulbecco's modified Eagle's medium (DMEM), penicillin-streptomycin, 0.25% (w/v) trypsin and fetal bovine serum (FBS) were purchased from Procell (Wuhan, China). Athymic nude mice (BALB/c-nu) and human breast cancer MCF-7 cell line were supplied by Medical Center of Xi'an Jiaotong University (Xi'an, China). Triton X-100, 4',6-diamidino-2-phenylindole (DAPI), crystal violet and bovine serum albumin (BSA) were purchased from Dingguochangsheng Biotechnology Co. Ltd. (Beijing, China). An Annexin V-FITC apoptosis detection kit was purchased from 7-Sea Biotech Co., Ltd. (Shanghai, China) and used according to the manufacturer’s protocol. All other reagents were of analytical grade and obtained from Sinopharm Chemical Reagent Co. Ltd. (Xi'an, China). Ultrapure water (18.25 MΩ cm) was used to prepare various aqueous solutions. 2.2. Synthesis of DA/ADH/FA tri-functionalized HA DA and ADH di-functionalized HA was synthesized following the procedures described in the literature with some modifications28,36,37. DA and ADH were grafted on HA through carbodiimide reaction



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between both the amine groups of DA and the hydrazide groups of ADH and the carboxyl groups of HA. Briefly, HA (1.0 g，2.5 mmol carboxyl group) was first dissolved in 30 mL of degassed phosphate buffered saline solution (PBS, 0.01 M, pH 5.0), and then DA·HCl (0.095 g, 0.5 mmol), EDC·HCl (0.096 g, 0.5mmol) and NHS (0.059 g, 0.5 mmol) were added. After the mixture solution was stirred for 12 h at room temperature under the atmosphere of nitrogen, ADH (1.742 g, 10 mmol), EDC·HCl (0.192 g, 1.0 mmol) and NHS (0.119 g, 1.0 mmol) were added. The solution was stirred for a further 12 h and then dialyzed (MWCO 1000 Da) against acidified water (pH < 2) for 48 hours and lyophilized. A white floccular intermediate product was obtained. The intermediate product was further modified with FA via carbodiimide reaction as follows. A mixture of FA (0.11 g, 0.25 mmol), NHS (0.03 g, 0.25 mmol) and EDC·HCl (0.048 g, 0.25 mmol) were added into 30 mL of degassed PBS solution (0.01 M, pH 5.0) and the mixture was stirred for 0.5 h. After 1 g of intermediate product was added, the solution was stirred at room temperature for 12 h under the protection of nitrogen, dialyzed and then lyophilized, obtaining the final product DA/ADH/FA tri-functionalized HA. 2.3 Decoration of GNRs with tri-functionalized HA (GNRs-HA-FA) CTAB coated GNRs (GNRs-CTAB) with predetermined maximal longitudinal surface plasmon resonance (LSPR) were prepared according to the seed-mediated growth method38. The conversion of GNRs-CTAB to GNRs-HA-FA was accomplished by replacing CTAB molecules on GNRs with tri-functionalized HA via Au-catechol bonds. Briefly, 1 mL of solution of tri-functionalized HA in water (8 mg/mL) was added to 30 mL of GNRs-CTAB suspension (20 µg Au/mL) and the mixture was mildly stirred for 24 h. The suspension was centrifuged at 14, 000 g for 10 min, and the collected precipitate was ultrasonically dispersed (Figure S1) in 30 mL of standard PBS (pH 7.4) and stored at 4 ºC for use. Non-FA-decorated GNRs, GNRs-HA, was prepared following the same procedure to serve as a control. 2.4 Characterization 1

H NMR analysis was performed using a Bruker spectrometer (400 MHz) in D2O. The conjugation of

tri-functionalized HA onto GNRs was verified by Fourier transform infrared spectroscopy (FT-IR) (Prestige-21, Shimadzu, Japan). The UV-Vis-NIR absorption spectra were recorded using a TU-1810 spectrophotometer (Purkinje General Instrument Co. Ltd. Beijing, China). High resolution transmission electron microscopy (HRTEM) images of the nanoparticles were taken with a JEM-2010 transmission electron microscope. The corresponding zeta potential (ζ) and hydrodynamic size were measured using dynamic light scattering method (DLS, Nano ZS90, Malvern Instruments, UK) at 25 ºC. The concentration


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of GNRs-CTAB was measured by an inductively coupled plasma mass spectrometry (ICP-MS, Spectro Arcos, Germany). The temperature of solutions was monitored by a Fluke Ti32 infrared thermal camera (Infrared Cameras, Fluke, USA). The stability of the nanoparticles was monitored by UV-Vis-NIR spectroscopic method. 2.5 DOX loading and release The covalent conjugation of DOX onto GNRs-HA-FA was accomplished by the formation of hydrazone linkage between DOX molecules and tri-functionalized HA. In a typical procedure, 10 mL of dimethyl sulfoxide (DMSO) containing DOX (0.5 mg) was mixed with 20 mL of aqueous suspension of GNRs-HA-FA (30 µg Au/mL) overnight at room temperature. DOX loaded GNRs (GNRs-HA-FA-DOX) were obtained by centrifugation (14,000 g, 10 min) and ultrasonically re-dispersed in PBS (pH = 7.4). The DOX loading content (DLC), defined as the weight percentage of DOX in the GNRs-HA-FA-DOX, was determined by a fluorescence spectrometer (LS55, Perkin Elmer, USA). Briefly, DOX was released completely from the carrier in a 0.1 mol/L HCl solution, and then the DLC was obtained by measuring the fluorescence intensities at excitation/emission wavelengths of 480/590 nm and comparing with a standard calibration curve. To reveal DOX release profiles, GNRs-HA-FA-DOX in PBS in a 1 kDa MWCO dialysis tube was placed in PBS at pH 5.0, 6.0, or 7.4. At each predetermined time point, the samples required to be irradiated were irradiated by an 808 nm NIR laser (Changchun Femtosecond Technology Co. Ltd., China. 2.5 W/cm2, 5 min). 0.5 mL of dialysis solution was extracted before and after the irradiation, and the equal volume of fresh PBS was replenished to it. The amount of DOX released was determined using a fluorescence spectrometer. 2.6 Photothermal conversion measurements GNRs-CTAB, GNRs-HA-FA and GNRs-HA-FA-DOX in PBS solutions (3 mL, 20 µg Au/mL) were placed in quartz cuvettes and irradiated with 2.0 W/cm2 of NIR energy for 10 min. During the irradiation, temperature was monitored with a Fluke Ti32 infrared thermal camera every 2 min. To clarify the influence of Au concentration on the photothermal heating effect, GNRs-HA-FA-DOX with different concentrations (20, 30 and 40 µg Au/mL) were conducted as above. 2.7 Stability of GNRs-HA-FA-DOX GNRs-HA-FA-DOX were incubated in ultrapure water, PBS (0.01 M, pH 7.4) or culture medium supplemented with 10% FBS at 37 °C. At preset time points, the sample suspensions were monitored using a UV-Vis-NIR spectrometer for the examination of stability of GNRs-HA-FA-DOX. GNRs-HA-FA-DOX



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suspension was exposed to NIR irradiation (2.0 W/cm2) to investigate its photo-stability. At preset temperatures, the suspensions were examined by the UV-Vis-NIR spectrophotometer. 2.8 Cell culture MCF-7 cells were incubated in DMEM medium supplemented with 10% FBS and 1% penicillin-streptomycin in a humidified incubator at 37 ºC in air with 5% CO2. 2.9 In vitro photothermal-chemotherapy Cytotoxicity assay, photothermal ablation and apoptosis assay were employed to reveal the synergistic effect between chemotherapy and PTT in MCF-7 cells. The cytotoxicity of GNRs-HA-FA, GNRs-HA-DOX, GNRs-HA-FA-DOX, and free DOX in MCF-7 cells was evaluated by the MTT assay. MCF-7 cells were seeded into 96-well plate at a density of 104 per well in 200 µL of culture medium. After 24 h of incubation, the formulations with different Au concentrations were added and the cells were cultured for a further 24 h. Each group was divided into two subgroups with or without NIR irradiation. The medium was replaced with 200 µL of fresh medium, and cells in irradiation groups were irradiated for 5 min with an NIR laser (2.0 W/cm2) and incubated for a further 24 h. The cell viability was tested using the MTT assay. To perform photothermal ablation study, MCF-7 cells were seeded in 24-well tissue culture plates at a density of 105/well and cultured for 24 h. The cells were then incubated with GNRs-HA-FA, GNRs-HA-DOX, GNRs-HA-FA-DOX and free DOX for 24 h. The Au concentration was 10 µg/mL and corresponding DOX content was 2.5 µg/mL. The medium was then replaced with fresh medium and the cells were treated with NIR irradiation (2.0 W/cm2) for 5 min. After incubation for 24, live cells were stained with calcein-AM and studied by a confocal laser scanning microscope (CLSM, Leica, TCS SP5 II, Germany). To investigate cellular apoptosis and necrosis, MCF-7 cells were incubated for 24 h in 6-well plates at a density of 5×105 cells per well and treated following the procedure described in above-mentioned photothermal ablation study. Finally, the cells were trypsinized, washed with PBS and stained with Annexin V-FITC/PI. After that, the flow cytometry analysis was immediately conducted on the Becton Dickson FACS Canto II flow cytometer (BD Biosciences, USA). 2.10 In vitro cell uptake studies MCF-7 cells were first incubated for 24 h in 24-well plates at a density of 5×104/well and then incubated with GNRs-HA-DOX and GNRs-HA-FA-DOX (10 µg Au/mL) for 1, 4 and 24 h, respectively. Subsequently, the cells were stained using FITC-phalloidine and DAPI to visually observe cellular F-actin


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and cell nuclei by CLSM. For TEM examination, after 24 h of culture, MCF-7 cells (5×105/well) were treated with GNRs-HA-DOX and GNRs-HA-FA-DOX (10 µg Au/mL) for 1 or 24 h following our previously reported procedure6 and then examined using TEM (Hitachi H-7650, Japan). To quantitatively evaluate the cellular uptake of GNRs-HA-DOX and GNRs-HA-FA-DOX (defined as mean number of GNRs per cell), MCF-7 cells were incubated for 1, 4 and 24 h with the formulations at a fixed Au concentration of 10 µg/mL. Afterward, the cells were thoroughly washed with PBS, trypsinized, counted, centrifuged at 1000 rpm for 10 min at 4 ºC. Live cells were sorted and quantified by flow cytometry, and then digested with 1 mL of aqua regia. The amount of gold was analyzed by ICP-MS and converted into the concentration of GNRs by the following method: the atom number of each Au rod was calculated based on the estimated volume with TEM images and the assumed gold unit cell edge (4.0786 Å)21. Each test was done in triplicate. 2.11 Tumor xenograft Animal experiments were carried out according to the protocol approved by the office of Scientific Research Management of Xi’an Jiaotong University. To prepare in vivo xenografted tumors, 200 µL of MCF-7 cell suspension (1×107 cells) was subcutaneously injected into 6-8 week-old female BALB/c nude mice. Tumor sizes were measured every two days. When the tumor volume increased to about 250 mm3 (approximately 2 weeks after inoculation), the animals were subjected to in vivo experiments. 2.12 Blood circulation and biodistribution The blood circulation time of GNRs-HA-FA-DOX was investigated by ICP-MS. 200 µL of GNRs-HA-FA-DOX suspension (1 mg Au/mL) was injected intravenously into the tumor-bearing mice from the tail. At 5 min, 30 min, 1 h, 4 h, 12h and 24 h post administration, blood samples were collected from mouse tail vein and stored at -20 °C before ICP-MS analysis. To investigate the biodistribution of GNRs-HA-FA, Cy7.5-NHS (a NIR imaging agent) instead of DOX, was loaded onto GNRs-HA-FA (called GNR-HA-FA-Cy7.5) according to a reported method

39

.

NIR fluorescence images were acquired on an IVIS Lumina at different times after intravenous injection (1, 4, and 24 h). At the end point, mice were sacrificed and the major organs as well as tumors were imaged ex vivo. The amount of Au in the major organs and tumors was quantitatively measured by ICP-MS as follows. 200 µL of GNRs-HA-DOX and GNRs-HA-FA-DOX (1 mg Au/mL) was injected intravenously into tumor-bearing mice. The mice were sacrificed 1, 4 and 24 h after the injection,. The major organs and



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tumors were collected, wet-weighed, lyophilized and grinded into powder. For ICP-MS experiments, the powder samples were predigested overnight with 5.0 mL aqua regia, completely digested in open vessels at 140 °C for 2 h, and then cooled down to room temperature. Each solution was diluted with water to 10 mL before ICP-MS analysis. 2.13 In vivo synergetic photothermal-chemotherapy Thirty tumor-bearing mice were randomly divided into six groups (n=5 per group): PBS, free DOX, GNRs-HA-DOX, GNRs-HA-FA + NIR irradiation, GNRs-HA-DOX + NIR irradiation and GNRs-HA-FA-DOX + NIR irradiation. Mice in each group were intravenously injected with 200 µL of PBS, DOX, GNRs-HA-DOX or GNRs-HA-FA-DOX solutions (1 mg Au/mL, 0.25 mg DOX/mL). The tumors in irradiation groups were irradiated with the laser (1.5 W cm–2) for 5 min 24 h after the injection, and temperature was monitored with the infrared thermal camera. The tumor volume and body weight were measured every two days with a caliper and an electronic balance, respectively. In the meantime the photographs of the mice were taken with a digital camera. The tumor volume was determined using the formula V= L × W2 /2 (L, longest dimension; W, shortest dimension). All mice were sacrificed 20 days after the first injection, and tumors and major organs were collected for weighting, photo imaging and histological examination. 5-μm sections were visualized on a Leica SCN400 Slide Scanner (Leica, Germany), and the images were taken with a Leica Digital Image Hub (Leica, Germany). Histological analysis was performed using SlidePath Gateway Client software. 2.14 Statistical analysis Data were presented as mean ± standard deviation. Statistical analysis was performed using Student’s t-test. p < 0.05 was considered statistically significant and p < 0.01 was considered statistically very significant.

3. Results and discussion 3.1 Preparation and characterization of GNRs-HA-FA-DOX Figure 1A schematically shows the preparation process of GNRs-HA-FA-DOX. Firstly, DA, ADH and FA tri-functionalized HA was synthesized by successive grafting of DA, ADH and FA onto HA via carbodiimide reaction. The chemical structure of the tri-functionalized HA was verified by 1H NMR spectra (Figure 2A)40,41. Given that substitution of carboxylic acid groups in HA would compromise its targeting ability to breast cancer42, FA was used to decorate HA to compensate for the reduced targeting


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capacity via its specific interaction with folate receptors overexpressed in breast cancer cells6. On the other hand, the total substitution degree of carboxylic acid groups in HA was controlled to be less than 25% to avoid the adverse effect of the group substitution on its targeting ability. The grafting degrees of DA and ADH onto HA were determined to respectively be about 6% and 13% by calculating the ratios of the methylene protons of both DA and ADH to the methyl protons of acetyl groups of HA in the 1H NMR spectrum. Moreover, only a single peak at 280 nm was observed in the UV-Vis-NIR spectrum of tri-functionalized HA (Figure S2), indicating that the grafted DA was not oxidized29. Secondly, the tri-functionalized HA was used to replace toxic CTAB molecules on GNRs-CTAB surface through the formation of mussel-inspired Au-catechol bonds43 between GNRs and the DA moieties of tri-functionalized HA. The catechol groups can strongly bind onto the surface of Au through coordination, and the Au-catechol bonds exhibited good stability under various conditions. For example, in a highly reductive intracellular microenvironment, Au-catechol bonds were far more stable than Au-thiol bonds50. Therefore, GNRs-HA-FA was hypothesized to have excellent stability under physiological conditions. By comparing the FT-IR spectra (Figure 2B), it was found that the characteristic peaks of methine groups of CTAB at 2923 and 2845 cm-1 disappeared in the spectrum of GNRs-HA-FA and the peaks at 1150 and 1086 cm-1 due to the stretching vibrations of -C-O-C- bonds in HA appeared. These results clearly demonstrated the successful replacement of CTAB molecules on the GNRs surface by the tri-functionalized HA. Finally, DOX was bonded onto HA molecules via an acid liable hydrazone linkage with a loading content of about 7.1 wt.%, and the schematic chemical structure of GNRs-HA-FA-DOX was shown in Figure 1B. Figure 1C shows the in vivo four-step targeting process of GNRs-HA-FA-DOX and the synergetic photothermal-chemotherapy of breast cancer. Firstly, GNRs-HA-FA-DOX can efficiently reach and accumulate in tumor tissue via the EPR effect due to their good stability and prolonged blood circulation after systemic administration44. Secondly, they may easily enter cancer cells via receptor-mediated endocytosis. Thirdly, pH-sensitive hydrazone bonds would be cleaved in intracellular acidic environment, and DOX is released and enters nuclei for chemotherapy. Finally, NIR irradiation can promote the release of DOX and simultaneously induce hyperthermia for PTT.



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Figure 1. (A) Illustration of preparation process of GNRs-HA-FA-DOX. (B) Chemical structure of GNR-HA-FA-DOX. (C) Schematic illustration of pH/NIR dual-responsive GNRs-HA-FA-DOX for targeting delivery and synergetic photothermal-chemotherapy of breast cancer. I: Entrance of GNRs-HA-FA-DOX into tumor tissue via the EPR effect due to their enhanced stability and prolonged blood circulation; II: Internalization of GNRs-HA-FA-DOX by tumor cells via receptor-mediated endocytosis; III: Endosomal escape of GNRs-HA-FA-DOX into cytoplasm; IV: Release of DOX triggered by acidic intracellular microenvironment; V: Hyperthermia and enhanced release of DOX induced by NIR irradiation.


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Figure 2. (A) 1H NMR spectra of tri-functionalized HA (a) and HA (b). (B) FT-IR spectra of GNRs-HA-FA, HA and GNRs-CTAB.

The physicochemcial properties of the as-synthesized GNRs-HA-FA-DOX were investigated and the results are displayed in Figure 3. As indicated in Figure 3A, the longitudinal absorption peak of GNRs-HA-FA-DOX showed a little blue-shift (~23 nm) as compared to that of GNRs-CTAB, and the peak intensity decreased due to repeated centrifuge purification. Similar phenomena were observed in HA-conjugated gold nanocages35. This might be attributed to the excellent dispersion of the nanoparticles. The size distribution and morphology of different forms of GNRs were characterized by DLS and HRTEM. As indicated in Figure 3B, the nanorods showed multi-peak size distributions. Considering the rotational diffusion of the nonspherical nanorods, the small size peaks less than 10 nm were thought to be moot and the big size peaks were used for further studies

45

. The average size (70.9 ± 1.4 nm) of

GNRs-HA-FA-DOX was much larger than that (24.4 ± 0.5 nm) of GNRs-CTAB (Figure 3B). Compared with HRTEM image of GNRs-CTAB (Figure 3C), a polymer layer (about 1.9 nm thickness) was seen around the GNRs in the image of GNRs-HA-FA (Figure 3D), which confirmed the existence of HA on GNRs surface. In addition, the zeta-potential of GNRs with different coatings was monitored. As shown in Figure 3E, the parental GNRs-CTAB displayed a positive surface charge of 43.2 ± 2.3 mV because of the existence of the CTAB bilayer, while the GNRs-HA-FA had a negative zeta potential of -11.4 ± 0.9 mV. This provided a proof of the replacement of CTAB on the surface of GNRs by tri-functionalized HA. It was noted that the chemical conjugation of positively charged DOX molecules onto GNRs-HA-FA had a



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negligible effect on the zeta potential. The stability of GNRs-HA-FA-DOX in various aqueous solutions was characterized by UV-Vis-NIR spectroscopy (Figure 3F and Figure S3). The LSPR peak is a good indicator of shape change of GNRs, because the peak position is decided by the aspect ratio of GNRs10,46. Figure 3F shows the absorption spectra of GNRs-HA-FA-DOX dispersed in complete culture medium (90% DMEM + 10% FBS) for different time periods. After standing for 7 days, the LSPR bands of GNRs-HA-FA-DOX and their intensities remained unchanged, demonstrating their excellent stability. Additionally, GNRs-HA-FA-DOX had also excellent stability in water and PBS solution (pH 7.4). In contrast, GNRs-CTAB were unstable in PBS solution (Figure S3).

Figure 3. UV-Vis-NIR spectra (A) and size distributions (B) of GNRs-CTAB, GNRs-HA-FA and GNRs-HA-FA-DOX. HRTEM images of GNRs-CTAB (C) and GNRs-HA-FA (D). (E) Zeta potentials of


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GNRs-CTAB, GNRs-HA-FA and GNRs-HA-FA-DOX. (F) Absorption spectra of GNRs-HA-FA-DOX dispersed in complete culture media for different standing time periods at room temperature.

3.2 Photothermal effect and dual-responsive drug release GNRs are considered as a desirable photothermal agent because of their exactly adjustable LSPR peaks in the NIR region9. The photothermal conversion performance of different forms of GNRs is a critically important parameter for PTT of tumors. To evaluate the photothermal conversion abilities of GNRs-CTAB, GNRs-HA-FA and GNRs-HA-FA-DOX, aqueous suspensions of different forms of GNRs with a fixed Au concentration of 20 µg/mL as well as PBS solution were continuously irradiated by an 808 nm laser at an irradiation energy density of 2.0 W/cm2 for 10 min. The temperature changes during the irradiation period were monitored with an infrared thermal camera. As shown in Figure 4A, the temperature of all suspensions containing GNRs increased by about 30 ºC after irradiation for 10 min, and surface modifications had a negligible effect on the photothermal conversion capability of the GNRs. In contrast, no obvious temperature change was observed for PBS solution. Infrared thermal images (Figure 4B) showed the visually displayed temperature change of GNRs-HA-FA-DOX suspension over the 10 min irradiation period. It was found that the photothermal heating effect of GNRs-HA-FA-DOX was concentration-dependent (Figure 4C), which is in accordance with previously reported results10,13,47. The temperature of GNRs-HA-FA-DOX suspension could be elevated to 70 °C within 10 min when the concentration of Au was preset at 40 µg/mL. We further investigated the photo-stability of GNRs-HA-FA-DOX under NIR irradiation (Figure S4). After one heating-cooling circle, though a bit red shift (~8 nm) of the longitudinal absorption peak was observed due to the rapid release of DOX10, the photo-stability of GNRs-HA-FA-DOX was still good enough for PTT. High DLC controlled drug release are the two most important factors for drug delivery systems. In the present study, DOX was effectively loaded onto GNRs-HA-FA via an acid liable hydrazone bond, and the DLC was about 7.1 wt.%. As displayed in Figure 4D, the release of DOX from the GNRs-HA-FA-DOX was accelerated at lower pH or under NIR irradiation. Almost 50% of encapsulated DOX was released at pH 5.0 within 24 h, while only about 15% was released at pH 7.4. Such a pH-sensitive DOX release property is of great significance in cancer therapy because the extracellular microenvironment and intracellular lysosomes of tumor tissue have acidic pH values much lower than that of normal tissue and biological fluids48. This can reduce and even avoid undesired drug release during transportation in blood circulation. It was ever reported that the



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NIR irradiation-induced heating could accelerate the release of DOX from nanocarriers

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49

. However, the

existing NIR-triggered drug release is mostly observed in those delivery systems where DOX molecules are physically loaded, i.e. via electrostatic interaction

12,13,50

or direct encapsulation into hollow

nanostructures20,35,48. To determine whether our DOX-conjugated delivery system had similar behavior, the release kinetics of DOX was investigated under NIR irradiation. It was found that NIR irradiation significantly enhanced the release of DOX regardless of pH13. In the case of total irradiation time of 25 min within 24 h, the rate of DOX release was about 1.3-times faster than that without NIR irradiation. This was ascribed to the heating of the fluid surrounding GNRs induced by NIR irradiation. This heating effect loosened the structure of HA layer and decreased the viscosity of local HA solution35, leading to exposure of more DOX molecules to external environment. At the same time, the heating effect promoted the diffusion of DOX released. These lead to the accelerated DOX release. Therefore, the release of DOX from the GNRs-HA-FA-DOX could be adjusted by exposing to NIR irradiation and changing pH.

Figure 4. (A) Photothermal heating curves of PBS, GNRs-CTAB, GNRs-HA-FA and GNRs-HA-FA-DOX irradiated at 2.0 W/cm2 by an 808 nm laser. The equivalent Au concentration was 20 µg/mL. (B) Corresponding infrared thermal images of GNRs-HA-FA-DOX suspension at 0-, 2-, 4-, 6-, 8- and 10-min intervals. (C) Photothermal heating curves of GNRs-HA-FA-DOX suspensions at equivalent Au concentrations of 20, 30 and 40 µg/mL after exposure to 808 nm NIR irradiation (2.0 W/cm2) for different


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times. (D) Release behaviors of DOX from GNRs-HA-FA-DOX at pH 5.0, 6.0 and 7.4 in the presence and absence of NIR irradiation. “on” indicates NIR irradiation (5 min, 2.0 W/cm2) at each time point.

3.3 In vitro cellular uptake study Cellular uptake of GNRs-HA-DOX and GNRs-HA-FA-DOX was observed by TEM, and the results are shown in Figure 5A-D. We observed that the cellular uptake process of GNRs-HA-FA-DOX included the following several steps (Figure S5). Firstly, the GNRs were captured by MCF-7 cells. Secondly, the captured GNRs were internalized by MCF-7 cells and transited by vesicles. Thirdly, the GNRs entered endosomes. Finally, the GNRs escaped from the endosomes into the cytoplasm. The whole process was similar to that observed by Qiu et al21. It can be seen from Figure 5A-D that the two kinds of GNRs could be efficiently taken up by MCF-7 cells regardless of the presence of FA and the number of GNRs in MCF-7 cells significantly increased with increasing culture time. Moreover, GNRs-HA-FA-DOX were more abundant in MCF-7 cells than GNRs-HA-DOX after the same period of incubation, suggesting that the targeting moiety FA allowed the GNRs to achieve cell-specific internalization via receptor-mediated endocytosis. The high-magnification images revealed that the GNRs in cells were still monodispersed and maintained their original morphologies, which were different from GNRs-CTAB that would aggregate seriously in cells and thus cause a distinct mitochondrial damage21. To detect the concentration of gold atoms in MCF-7 cells, a quantitative analysis was conducted by ICP-MS. The number of GNRs per cell was calculated according to the dimensions of a gold nanorod in Figure 3D (60 nm in length, 15 nm in diameter) and the number of cells in the solution. As shown in Figure 5E, the number of GNRs uptaken by cells in FA-decorated GNRs group was nearly the same as that in FA-undecorated GNRs group after 1 h of incubation. In contrast, after incubation for 24 h, the number of FA-decorated GNRs uptaken by cells was 3.4 × 105/cell that was 2.27-fold higher than that of FA-undecorated GNRs (1.5 × 105/cell) (p < 0.05). These results were in agreement with TEM observations. The entry of GNRs into cells is usually considered to be a receptor-mediated endocytosis process51,52 and strongly dependent on their aspect ratio and surface coating21. In the present study, FA-decorated and undecorated GNRs had the same aspect ratio and polymeric coating layer. These results further confirmed that FA decoration significantly promoted GNRs uptake by MCF-7 cells. In addition, it is well-known that MCF-7 cells overexpress CD44 receptor that can specifically bind to HA, so the uptake of HA-decorated nanoparticles by MCF-7 cells is far greater than that in normal tissues53,54. Previous studies have indicated



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that HA-coated gold nanoparticles exhibited greater cellular uptake than non-coated ones13,35. Therefore, HA could not only protect GNRs from aggregation in aqueous solutions but also act as a targeting agent to improve the cellular uptake of GNRs. As a dual-targeted nanoplatform, our GNRs-HA-FA-DOX showed a cell uptake efficiency of up to 70%, which is much higher than that (30.84%) of GNRs-HA-DOX with single HA targeting.

Figure 5. TEM images of MCF-7 cells incubated with GNRs-HA-DOX (A and B) and GNRs-HA-FA-DOX (C and D) for 2 (A and C) and 24 h (B and D). Black arrows indicate the GNRs. (E) Number of GNRs in MCF-7 cells treated with GNRs-HA-DOX and GNRs-HA-FA-DOX for 1, 4 and 24 h. n=3, *p < 0.05.

The GNRs-mediated intracellular DOX delivery was studied in MCF-7 cells by CLSM and flow cytometry, and the results are shown in Figure 6. It was seen that the MCF-7 cells treated with either GNRs-HA-DOX or GNRs-HA-FA-DOX for 1 h displayed weak red fluorescence from DOX. This implied that the GNRs-based carriers were taken up by MCF-7 cells irrespective of the presence of folate. The fluorescence intensity significantly increased with prolonged incubation time and obvious nuclei accumulation of DOX was found, indicating the continuous cellular internalization of nanoparticles. Importantly, the intensity of DOX fluorescence in the MCF-7 cells treated with GNRs-HA-FA-DOX was considerably higher than that treated with GNRs-HA-DOX at the same time points. After incubation for 24


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h, the fluorescence intensity of DOX of FA-decorated GNRs was 1.71-fold higher than that of FA-undecorated GNRs. This is attributed to the specific interaction between FA-decorated GNRs and folate receptors in MCF-7 cells, facilitating cellular uptake of GNRs-HA-FA-DOX. These findings agreed well with the qualitative and quantitative results from TEM and ICP-MS studies.

Figure 6. Cellular uptake and intracellular distribution of GNRs-HA-DOX (A) and GNRs-HA-FA-DOX (B) after culture for 1, 4 and 24 h, as displayed by CLSM. The concentration of DOX was 2 µg/mL. For each row, images from left to right show nuclei stained with DAPI (blue), DOX fluorescence (red), phalloidin-FITC fluorescence (green) and overlays of three images. Scale bars represent 25 µm. (C) Flow cytometry histogram profile and (D) intracellular mean fluorescence intensity of MCF-7 cells incubated with GNRs-HA-DOX and GNRs-HA-FA-DOX for 1, 4 and 24 h. 3.4 In vitro photothermal-chemotherapy studies To explore the synergistic therapeutic effect of GNRs-mediated PTT and DOX-mediated chemotherapy, the viability of MCF-7 cells treated with different formulations was determined by both MTT and live-cell staining assays. As displayed in Figure 7A, GNRs-HA-FA displayed a negligible toxicity on MCF-7 cells in the whole concentration range studied, indicating their good cytocompatibility. In the case of NIR



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irradiation, cell viability remarkably decreased to 32% when the Au concentration was 40 µg/mL, demonstrating the high PTT efficacy of GNRs-HA-FA. It was found that, at an Au concentration of 10 µg/mL, GNRs-HA-DOX, GNRs-HA-FA-DOX or GNRs-HA-FA + NIR irradiation groups could inhibit MCF-7 cells growth and the GNRs-HA-FA-DOX group exhibited the lowest cell viability of 65%. In stark contrast to them, the cell viability of GNRs-HA-DOX + NIR irradiation and GNRs-HA-FA-DOX + NIR irradiation groups was 42% and 31%, respectively. In comparison to free DOX, a slight reduction in the cytotoxicity of GNRs-HA-DOX was not surprising since DOX was gradually released inside the cells. Similar phenomenon was reported in the literature50. Regardless of NIR irradiation, GNRs-HA-FA-DOX always exhibited greater toxicity than GNRs-HA-DOX, which suggested that FA-decoration enhanced cellular

internalization

of

the

GNRs.

Figure

7B

shows

the

therapeutic

efficacies

of

photothermal-chemotherapy combination (GNRs-HA-FA-DOX + NIR irradiation group, calculated by subtracting the cell viability from 100%) and the additive therapeutic efficacies of chemotherapy (GNRs-HA-FA-DOX group) and PTT (GNRs-HA-FA + NIR irradiation group), which were estimated using the formula of Tadditive = 100 − (fchemo × fphotothermal) × 100, where f is the fraction of surviving cells after each treatment55. At the same Au concentration, the former was higher than the latter, demonstrating the excellent synergistic anticancer effect of PTT and chemotherapy. To intuitively observe the cytotoxicity of different formulations, MCF-7 cells treated with free DOX, GNRs-HA-DOX, GNRs-HA-DOX with NIR irradiation and GNRs-HA-FA-DOX with NIR irradiation were stained with calcein AM. As indicated in Figure 7C, the change trend of florescence pattern was similar to that of MTT results. GNRs-HA-DOX group showed a higher cytotoxicity than PBS control group but lower than free DOX group. As expected, NIR irradiation caused obvious death of cells treated with GNRs-HA-DOX and GNRs-HA-FA-DOX, and a clear demarcation line was observed between dead and live cell (green) regions. Moreover, GNRs-HA-FA-DOX + NIR irradiation group had higher lethality. The death mechanism of MCF-7 cells treated with different formulations was investigated by means of flow cytometric analysis of annexin V-FITC/PI double stained cells, and the results are shown in Figure 7D. It can be seen from Figure 7D that GNRs-HA-FA-DOX + NIR irradiation group showed a higher percentage of total apoptotic cells (66.00%) than GNRs-HA-DOX + NIR irradiation (37.17%) and GNRs-HA-DOX

(20.37%)

groups,

which

further

confirmed

the

synergistic

effect

of

photothermal-chemotherapy and targeting effect of FA decoration. In contrast, PBS-treated cells did not uptake annexin V or PI, suggesting that they were healthy. Free DOX exhibited more cytotoxicity on


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MCF-7 cells than the DOX-loaded GNRs because of its faster entry into the nucleus. Results of in vitro studies demonstrated that MCF-7 cells could be induced by GNRs-HA-FA-DOX with NIR irradiation to undergo apoptosis. Apoptotic cells are generally marked with "eat me" signals like phosphatidyl serine for phagocytosis by phagocytes56. Apoptosis, being different from necrosis, is controlled cell death and does not release harmful cellular waste, which does not cause obvious damage to adjacent cells57. It followed that our pH/NIR dual-responsive and active targeting nanoplatform was effective in inhibiting MCF-7 cells growth by combined photothermal chemotherapy.

Figure 7. In vitro photothermal-chemotherapy assay. (A) Viability of MCF-7 cells incubated with different concentrations of free DOX, GNRs-HA-FA, GNRs-HA-DOX or GNRs-HA-FA-DOX for 24 h in the presence and absence of NIR irradiation (2.0 W/cm2, 5 min). (B) Comparison of synergistic efficacy of photothermal-chemotherapy (GNRs-HA-FA-DOX + NIR irradiation) and additive efficacy of independent PTT (GNRs-HA-FA + NIR irradiation) and chemotherapy (GNRs-HA-FA-DOX) using t-tests with all p-values < 0.05. Single asterisk indicates p < 0.05, and double asterisk indicates p < 0.01. (C) CLMS photographs of MCF-7 cells treated with PBS (a), free DOX (b), GNRs-HA-DOX (c), GNRs-HA-DOX



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with NIR irradiation (d) and GNRs-HA-FA-DOX with NIR irradiation (e). Green dots represent calcein AM-stained viable cells. Scale bars represent 100 µm. (D) Apoptotic effects of different treatments in (C).

3.5 In vivo biodistribution studies Long blood circulation time is indispensable for successful nanoparticle-based targeted drug delivery and anti-cancer treatment, since it promises sustained passive and active accumulation of nanocarriers into tumor50,58. On the basis of change of Au contents determined by ICP-MS, GNRs-HA-FA-DOX showed a relatively long blood circulatory half-life of about 2.4 h (Figure 8A). Moreover, at 24 h after injection, GNRs-HA-FA-DOX exhibited higher blood retention (about 8.1% ID) than the reported PEG-59, SiO2-50 and CS-22 decorated GNRs (≤3.0% ID). The superior blood lifetime and retention of GNRs-HA-FA-DOX were mainly due to the hydrophilicity and immunosuppressive effect of HA 60. The in vivo biodistribution and tumor targeting ability of GNRs-HA-FA-DOX were assessed qualitatively using an NIR fluorescence imaging system after Cy7.5-labeled GNRs were intravenously injected into tumor-bearing mice. As shown in Figure 8B, fluorescence was observed in the whole body at the 1-h time point resulting from the blood circulation of GNRs, and then gradually became weak with prolonged circulation time. In contrast, the fluorescence intensity at the tumor site rapidly increased over the time period, implying effective tumor accumulation of GNRs-HA-FA-Cy7.5. It could be seen from the fluorescence photographs of ex vivo tumor and major organs that the tumor showed the strongest fluorescence, followed by the kidney, liver, spleen, lung and heart. The quantitative results from ICP-MS analysis indicated that uptake efficacy of GNRs-HA-FA-DOX by tumor was 11.3%, nearly 2-fold higher than that of GNRs-HA-DOX, confirming the in vivo tumor targeting ability of FA. There was no significant difference in uptake of GNRs-HA-DOX and GNRs-HA-FA-DOX in the five kinds of organs, which was consistent with the result of ex vivo NIR fluorescence analysis. It was noted that GNRs mainly existed in the liver and spleen due to their strong phagocytosis as reticuloendothelial system (RES) organs10,61. In addition, kidney exhibited stronger fluorescence than spleen, but the Au content of kidney was much lower than that of spleen. The possible reason is that Cy7.5 in GNRs-HA-FA-Cy7.5 might gradually dissociate from GNRs after injection and then excreted from the kidney via the “renal excretion” pathway7,62. These results indicated that GNRs-HA-FA-DOX could effectively accumulate in tumor, primarily owing to their enhanced stability, long circulation time in blood and specific binding to receptors on tumor cells. The above-mentioned results showed that the biodistribution of GNRs-HA-FA-DOX was far from being


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optimum because they nonspecifically accumulated in the liver and spleen. Similar phenomenon was found in the PEG-59, polyamidoamine-63 or CS-22 coated GNRs. However, this would not be a serious issue in PTT, since only tumor sites are exposed to NIR light. Considering that the ‘stealth coating’ materials of GNRs are significantly different, the nonspecific accumulation of various functionalized GNRs in the RES system may be attributed to the intrinsic rigidity and rod morphology of GNRs. To improve the biodistribution of GNRs, modulating GNR dimensions, exploring new ‘stealth coating’ material and ligand decoration will be major research directions.

Figure 8. (A) Blood circulation time of GNRs-HA-FA-DOX determined by quantification of Au in blood by ICP-MS, expressed as percentage of injected dose (ID) (mean ± SD, n=3). (B) NIR fluorescence images of tumor-bearing mice taken at 1, 4 and 24 h after injection of Cy7.5 labeled GNRs (a) and NIR fluorescence images of excised major organs and tumor at 24 h post-injection of Cy7.5 labeled GNRs (b). (C) Biodistribution of GNRs-HA-DOX and GNRs-HA-FA-DOX in tumor-bearing mice at 24 h after injection (n = 3, **p < 0.01, expressed as percentage of ID per organ.

3.6 In vivo synergetic photothermal-chemotherapy The minimum threshold temperature for hyperthermia therapy of tumors is usually regarded as 45 °C 64, so the effects of in vivo photothermal heating of our GNRs-based system were firstly investigated. Mice bearing tumors were injected intravenously with GNRs-HA-DOX or GNRs-HA-FA-DOX suspensions,



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using the same volume of PBS and DOX solution as controls. Tumor regions at 24 h post-injection were irradiated with 808 nm laser at 1.5 W/cm2 for 5 min. The temperature of tumors was measured by infrared thermal imaging and the results are shown in Figure 9A. The relationship between tumor temperature and irradiation time is displayed in Figure 9C. Tumor temperature in mice treated with PBS or DOX showed little change within 5 min of irradiation and its maximum value was lower than 40 ºC which was insufficient

to

irreversibly

damage

tumor

cells65.

In

contrast,

both

GNRs-HA-DOX

and

GNRs-HA-FA-DOX groups exhibited an obvious photothermal heating effect, and tumor temperature just after 1 min of irradiation could reach 45.9 and 48.6ºC, respectively, exceeding the minimum threshold temperature. After 5 min of irradiation, the tumor temperature of mice treated with GNRs-HA-DOX and GNRs-HA-FA-DOX increased to 62.3 and 67.5 ºC, respectively. Temperatures above 60 ºC can activate cellular degradation pathways, such as denaturation and folding of proteins and cross-linking of DNA, causing instantaneous coagulative necrosis and irreversible tumor cell death66. The higher temperature elevation in GNRs-HA-FA-DOX group compared to GNRs-HA-DOX group was due to the improved cellular uptake of GNRs-HA-FA-DOX in tumor tissue, which agreed well with the results from the above-mentioned cellular uptake and biodistribution studies. Owing to their excellent in vitro synergistic effect, long circulation time in plasma, in vivo tumor-targeting capacity and high-performance photothermal heating, GNRs-HA-FA-DOX might contribute to superior antitumor effects. The antitumor efficacy of GNRs-HA-FA-DOX was assessed in tumor-bearing mice. The efficacy was gauged by monitoring the tumor volume and body weight during therapeutic period. Tumors of mice treated with GNRs-HA-DOX + NIR irradiation or GNRs-HA-FA-DOX + NIR irradiation showed severe necrosis, and the original tumors turned into black scars (Figure 9B), similar to the phenomena observed in previously reported PTTs35. The scars diminished gradually and fell off after 16 days. However, the tumor recurred at day 18 in GNRs-HA-DOX + NIR irradiation treatment group and grew to about 150 mm3, indicative of incomplete tumor ablation. In contrast, the tumor in GNRs-HA-FA-DOX + NIR irradiation group was completely eliminated, further reaffirming the enhanced tumor uptake of GNRs-HA-FA-DOX. No obvious therapeutic effect was observed in PBS, DOX or GNRs-HA-DOX treatment groups. Administrations of DOX, GNRs-HA-DOX or GNRs-HA-FA + NIR alone showed moderate inhibitory effect on the tumors only in the early days (Figure 9B and D). Although DOX is a wide-used chemotherapeutic drug against breast cancer, a high dosage is usually required to achieve a satisfying therapeutic result. In this study, we found that treating mice with only a single dose of


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DOX was certainly not sufficient to suppress tumor growth in vivo. It was noteworthy that GNRs-HA-DOX significantly inhibited tumor growth as compared to DOX, most likely due to their enhanced tumor-targeting ability and gradual drug release. In addition, for the mice treated with GNRs-HA-FA + NIR, the tumor volume decreased within the first 4 days, and then increased again and reached to 500 mm3 after 20 days, indicating that the incomplete tumor ablation would lead to tumor recurrence. Collectively, the following conclusions were obtained: (1) Each drug treatment group had higher efficacy than PBS group (p < 0.05), (2) there was a significant difference between DOX-loaded GNRs groups and free DOX group (p < 0.05), (3) combined photothermal-chemotherapy showed synergistic effect and (4) GNRs-HA-FA-DOX + NIR irradiation group showed greater antitumor efficacy than other groups (p < 0.01). The excised major organs and tumors were processed for histological analysis. The tumors were weighed and the results were presented in Figure 9E. The tumors in mice treated with GNRs-HA-DOX + NIR irradiation had the lowest weight among all five groups. As displayed in the H&E images of sections of tumors treated with different formulations (Figure 9F), the tumor cells in PBS-treated mice retained normal cell morphology and fine structure. In contrast, in the drug-treated groups, the loss of membrane integrity was obvious in most of tumor cells, the intercellular gap junctions were ambiguous, and tissue necrosis of different extent as well as extensive nuclear shrinkage, fragmentation and even absence appeared. In addition, some tumor cells existed around blood vessels in GNRs-HA-DOX + NIR irradiation-treated tumor, indicating that the tumor was not completely damaged.



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Figure 9. In vivo antitumor effect against MCF-7 breast tumors in nude mice. (A) Photothermal images of tumor-bearing mice treated with PBS (a), DOX (b), GNRs-HA-DOX (c) or GNRs-HA-FA-DOX (d) under 808 nm laser irradiation at 1.5 W cm−2 for 5 min. (B) Representative photographs of mice bearing MCF-7 tumors before and after PBS (a), DOX (b), GNRs-HA-DOX (c), GNRs-HA-DOX + NIR irradiation (d) or GNRs-HA-FA-DOX + NIR irradiation (e) treatments for 4, 8, 12, 16 and 20 days. Red circle indicates recurrent tumor. (C) Temperature change curves of tumor corresponding to infrared thermal images of mice in (A). Red dot line indicates 45 ºC, the lowest temperature required for thermal ablation of tumors in minutes. (D) Change in tumor volume over time post-treatment. (n = 5, **indicates p < 0.01, compared to all other groups; * indicates p < 0.05). (E) Tumor weight of each group. (n = 5, ** indicates p < 0.01, compared to all other groups). (F) Images of H&E stained tissue sections of tumors from each group.

Systemic toxicity is a major concern with the in vivo use of nano-therapeutics, and high toxicity usually


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leads to weight loss. The body weights of the mice were measured every two days during the 20-day treatment period (Figure S6). All mice were alive throughout the study period, and the differences in the body weight and physical activity level between the groups were not significant, implying that the toxicity of these formulations was well tolerated. An obvious weight loss in the mice treated with GNRs-HA-FA-DOX + NIR irradiation or GNRs-HA-DOX + NIR irradiation within the first two days was due to photothermal tumor ablation. To examine the potential toxic effects of different formulations on major organs including heart, liver, spleen, lung and kidney, these organ samples were analyzed histologically with H&E staining and the results are shown in Figure 10. A lot of swelling and vacuolated cells and myocardial fiber rupture were observed in the heart tissue in free DOX group, which was ascribed to the cardiac toxicity of DOX20. In the GNRs-HA-DOX and GNRs-HA-FA-DOX groups, neither adverse side effect on heart nor noticeable lesions in other organs were observed. These results demonstrated that the photothermal-chemotherapy of breast cancer bearing mice based on GNRs-HA-FA-DOX did not cause any serious side effects.

Figure 10. Images of H&E stained tissue sections of heart, liver, spleen, lung and kidney of mice treated with different formulations.

4. Conclusions A novel nanoplatform GNRs-HA-FA was successfully developed for active targeting delivery and



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synergetic photothermal-chemotherapy of breast cancer. The nanoplatform could chemically load DOX via a pH-sensitive hydrazone linkage to form versatile nanoparticles GNRs-HA-FA-DOX. The negatively charged nanoparticles showed enhanced stability in serum medium and high photothermal heating capacity, and displayed pH and NIR irradiation dual-responsive drug release. The nanoplatform exhibited favorable biocompatibility. Compared with non-FA-decorated nanoparticles, FA-decorated nanoparticles exhibited a significantly higher capacity of delivering GNRs and DOX into MCF-7 cells via folate receptor-mediated endocytosis and could more efficiently kill them under NIR irradiation by inducing cell apoptosis. Pharmacokinetics and biodistribution results indicated the nanoparticles possessed a long blood circulation time and prominent good tumor-specific accumulation. The in vivo combined photothermal-chemotherapy studies showed that tumors were completely eliminated without recurrence and severe side effects to normal tissues. What's more, the combination of PTT and chemotherapy exhibited a synergistic therapeutic efficacy both in vitro and in vivo, which was better than chemotherapy or PTT alone. This work demonstrated the great potential of GNRs-HA-FA-DOX in breast cancer therapy and may provide insights for the design of multifunctional nanocarriers to maximize the therapeutic efficacy of combined anti-cancer therapy.

Supporting Information Photographs of GNRs-HA-FA before and after centrifugation; UV-Vis-NIR spectrum of DA/ADH/FA tri-functionalized HA; UV-Vis-NIR spectra of GNRs-HA-FA-DOX and GNRs-CTAB in different media after different standing time periods; Photographs of GNRs-CTAB and GNRs-HA-FA-DOX after standing in different solutions for 7 days; Absorption spectra of GNRs-HA-FA-DOX solution exposed to 808 nm laser; TEM images showing the cellular uptake process; Changes in body weight of the mice treated with various formulations.

Author Information Corresponding Authors *E-mail address: [email protected] (J. M. Qian); [email protected] (A. L. Suo). Notes The authors declare no competing ﬁnancial interest.

Acknowledgements


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The authors gratefully acknowledge financial support from the Key Research and Development Program of Shaanxi Province, China (2017SF-179 and 2017SF-190) and the National Natural Science Foundation of China (50603020 and 50773062).

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