Polypeptide-engineered Hydrogel Coated Gold Nanorods for

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Polypeptide-engineered Hydrogel Coated Gold Nanorods for Targeted Drug Delivery and Chemo-photothermal Therapy Jie Yang, Ming-Hao Yao, Rui-Mei Jin, Dong-Hui Zhao, Yuan-Di Zhao, and Bo Liu ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.7b00359 • Publication Date (Web): 28 Aug 2017 Downloaded from http://pubs.acs.org on August 31, 2017

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ACS Biomaterials Science & Engineering

Polypeptide-engineered Nanorods

for

Hydrogel

Targeted

Drug

Coated

Gold

Delivery

and

Chemo-photothermal Therapy Jie Yang1,2, Ming-Hao Yao1,2, Rui-Mei Jin1, Dong-Hui Zhao1, Yuan-Di Zhao1,2, and Bo Liu1,2* 1

Britton Chance Center for Biomedical Photonics at Wuhan National Laboratory for

Optoelectronics - Hubei Bioinformatics & Molecular Imaging Key Laboratory, Collaborative Innovation Center for Biomedical Engineering, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan 430074, Hubei, P. R. China 2

Key Laboratory of Biomedical Photonics (HUST), Ministry of Education, Huazhong University

of Science and Technology, Wuhan 430074, Hubei, P. R. China

ACS Paragon Plus Environment

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ABSTRACT: A new hybrid nanogel system using polypetide-engineered coated gold nanorods has been developed for targeted drug delivery and tumor chemo-photothermal therapy. A triblock engineered polypeptide PC10A(RGD) was immobilized on the surface of gold nanorods by the electrostatic adsorption. The immobilized PC10A(RGD) formed hydrogel by self-assembly to load doxorubicin for chemotherapy. Coating polypeptide-engineering hydrogel on gold nanorods enhanced the stability in high-salt media and significantly reduced the cytotoxicity. An arginine-glycine-aspartic acid motif was introduced into the polypeptide on the surface of hybrid nanogels to promote cellular uptake through receptor-mediated endocytosis in αvβ3 overexpressing HeLa cells. In addition, compared with single chemotherapy and near-infrared photothermal therapy, the combination therapy has a synergistic effect on the cancer cells. Thus, the chemo-photothermal therapy based on polypeptide-engineered hydrogel coated gold nanorods and doxorubicin is expected to have great potential impact on cancer therapy.

KEYWORDS: Hybrid nanogels, polypeptide-engineered, targeting, chemo-photothermal therapy, synergistic effect


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INTRODUCTION Photothermal therapy, especially induced by near-infrared (NIR) light, as a noninvasive therapeutics has attracted considerable attention, which has deep-tissue penetration with minimal normal tissue damage.1 To date, organic dyes and nanoparticles are two major photothermal agents. Compared with traditional organic dyes, nanoparticles possess larger absorption cross-sections, higher stability, and lower photobleaching.2,

3

Recently, many NIR absorbing

nanoparticles, such as hollow gold nanoshells, gold nanorods (GNRs), quantum dots, carbon nanotubes, and graphene nanosheets, have been developed for localized hyperthermia tumor therapy.4-9 Among these, GNRs as photothermal agents have been extensively studied due to their advantages of excellent photothermal property, high electron density, and tunable longitudinal surface plasmon resonance (LSPR) from visible to NIR wavelength.10-12 However, single photothermal therapy is difficult to eradicate completely tumor cells because of uneven heat distribution in the treatment volume.13,

14

Therefore, in order to eradicate

completely tumor cells, photothemal therapy need to be assisted in other therapy methods. Although chemotherapy is one of the most important measures in the treatment of malignant tumors, chemotherapy is generally faced with a series of problems, such as poor water solubility, poor targeting, and strong side effects.15 Nanoscale drug delivery systems have been applied in cancer therapeutics.16 In particular, target drug delivery is one of the most active areas in the study of nano drugs because of enhancing the efficiency of chemotherapeutics and minimizing the toxicity of drugs for normal cells. Recently, some studies have shown that the combination of photothermal therapy and targeted chemotherapy within functionalized GNRs has been demonstrated to result in synergistic anticancer efficacy in both in vitro and in vivo models.17-19 Various stimuli-responsive strategies, such as pH, light, heat, glutathione, and enzymes, have


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been applied to triggering release of the payloads from the functionalized GNRs.20-24 Among all, NIR light-triggered payloads releasing from the loaded complexes in a location area has attracted much attention because it can lead to the noninvasive release.25 Although many polymer molecules such as poly(sodium 4-styrenesulfonate) (PSS), polyethylene glycol (PEG), poly(allyamine hydrochloride) (PAH), PAMAM dendrimer, and silicon have been applied in modification of GNRs for passivation of the toxic effect of the surfactant cetyltrimethylammonium bromide (CTAB) and loading drugs,26-30 proteins as an efficient drug carrier have also drawn much attention

because of their biocompatible nature.

For example, GNRs coated with coronas of serum proteins or BSA has been reported.31-33 However, the sizes of these coronas are large, and targeting ligands need to be introduced by the further chemical conjugation or modification. The recombinant DNA technology can be used to prepare polypeptides with designed structures at the molecular level. Sequences with certain functions, such as targeted peptides or therapeutic peptides can be introduced into engineered polypeptides, which could skip further coupling of the biomolecules.34 Therefore, the application of GNRs coated with engineered polypeptides can be a new approach to cancer therapy. Herein, we developed a cancer therapeutic system by the combination of targeted chemotherapy and photothermal therapy using multifunctional GNRs-polypeptide hybrid nanogels (Scheme 1). A triblock engineered polypeptide PC10ARGD was modified on the surface of GNRs by the electrostatic adsorption. The immobilized PC10ARGD formed hydrogel on the surface of GNRs through self-assembly. The doxorubicin (DOX) as model drug was loaded into the hydrogel of PC10ARGD for chemotherapy. After hybrid nanogels accumulating in the targeted cancer cells or tumors, DOX was released for chemotherapy through a photothermal mechanism of GNRs and pH. In addition, synergistic effects of chemo-photothermal therapy on


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cancer cells treated with hybrid nanogels were observed. Thus, the design reported here may provide a unique opportunity for cancer therapy.

Scheme

1.

Schematic

illustration

of

GNRs-polypeptide@DOX

hybrid

nanogels

for

combined

chemo-photothermal therapy (a) and the sequences of PC10A and PC10ARGD (b).

EXPERIMENTAL SECTION Materials. Restriction endonuclease BamHI, SpeI, NheI, and T4 DNA ligase were obtained from New England Biolabs Inc. (Beijing, China). Chloroauric acid (HAuCl4·4H2O), isopropyl-β-D-thiogalactoside (IPTG), calcein AM, Ethidium homodimer-1 (EthD-1), ampicillin, kanamycin, CTAB, ascorbic acid (AA), and silver nitrate (AgNO3) were obtained from Sigma-Aldrich (Shanghai, China). Nickelnitrilotriacetic acid (Ni-NTA) separation column was


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purchased from Qiagen China (Shanghai) Co., Ltd. DOX and sodium borohydride (NaBH4) were purchased from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). Synthesis and purification of the polypeptides. The pQE9PC10A plasmid was a gift from Prof. D. Tirrell (California Institute of Technology, Pasadena, CA). PC10A and PC10ARGD were prepared according to our previously reported protocol.34 The MALDI-TOF spectra of purified polypeptides PC10A and PC10ARGD were recorded on a Bruker Reflex III instrument (Figure S1). PC10A (MS: 20932.6 Da, molecular weight (MW): 20858.5 Da), PC10ARGD (MS: 22371.4 Da, MW: 22295.9 Da). Synthesis and characterization of GNRs. GNRs were synthesized according to previously reported literature.34, 35 Briefly, CTAB (0.2 M, 1 mL) was added into 1 mL HAuCl4 (0.5 mM), and the mixture was stirred on the room temperature. Ice-cold NaBH4 (0.01 M, 0.12 mL) was added, and the mixture was stirred for another 5 min. The solution was stored at 28 ºC for 2 h to prepare seed solution. HAuCl4 (1 mM, 30 mL), AgNO3 (0.04 M, 130 µL), and H2SO4 (0.5 M, 120 µL) were added in CTAB (0.2 M, 30 mL), and the mixture was reduced by AA (0.0788 M, 420 µL). Seed solution (72 µL) was added, and the mixture solution was stirred for 10 s. The growth solution was placed at 28 ºC for 10-15 h. The absorbance spectra of the GNRs were recorded on a UV-2550 UV-vis spectrophotometer (Shimadzu, Japan) at room temperature. The sizes and morphologies of GNRs were determined by a transmission electron microscope (TEM, Tecnai G2 20 U-Twin). Preparation

of

GNRs@polypeptide

hybrid

nanogels.

For

the

preparation

of

GNRs@polypeptides (PC10A or PC10ARGD)@DOX hybrid nanogels, PC10A or PC10ARGD (0.1 µmol) was dissolved in 2 mL ultrapure water. The pH of the polypeptide solution was adjusted to


6

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7.5. GNRs (50 µg mL-1, 2 mL) were centrifuged twice at 12000 g for 15 min. GNRs precipitate were resuspended with the polypeptide solution, and the mixture was shaken at 200 rpm to react for 12 h. The mixture was centrifuged at 9000 g for 15 min, and the precipitate was resuspended in PBS. Preparation

of

GNRs@polypeptide@DOX

hybrid

nanogels.

The

GNRs@PC10ARGD@DOX hybrid nanogels were prepared through the similar method in the above section. The difference is mixture 10-40 µg DOX/50 µg GNRs in PC10A or PC10ARGD before using polypeptides for resuspension. Thermal treatment of each sample was performed to quantify the DOX initially loaded into the GNRs@PC10A(RGD) nanogels. In order to do that, 500 µL GNRs@PC10A(RGD)@DOX were heated in a boiling water bath for 30 min and centrifuged at 14000 g for 10 min. The concentration of DOX in the supernatant was quantified by using a LS-55 spectrophotometer (PerkinElmer, USA). Stability of GNRs@polypeptide@DOX hybrid nanogels. GNRs (50 µg mL-1) or GNRs equivalent concentrations of GNRs@PC10ARGD@DOX hybrid nanogels was centrifuged at 9000 g for 15 min. The precipitate was resuspended in different aqueous media (ultrapure water, PBS, Serum-free DMEM, or DMEM with serum). The solutions were placed in a 37 ºC incubator. The UV-visible absorbance spectra of the solutions were measured at room temperature to monitor their stability. Photothermal effects of GNRs@PC10ARGD@DOX. To measure the temperature changes in response to NIR light absorption by GNRs@PC10ARGD@DOX, 100 µg mL-1 GNRs@ PC10ARGD@DOX was diluted to 80, 40, 20, or 10 µg mL-1 with ultrapure water. Each sample (200 µL) was added into a 0.5 mL PE tube. The NIR laser was placed 5 cm over the test tube.


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Tubes with different concentrations of GNRs@PC10ARGD@DOX were directly exposed for 8 min to a continuous NIR light laser (Changchun New Industries Optoelectronics Technology, Changchun, China). Temperature changes were monitored using a Guide EasIR-9 IR thermal camera (Wuhan Guide Infrared Co., Ltd, China). Different laser powers were used. Drug release from GNRs@PC10ARGD@DOX. The GNRs@PC10ARGD@DOX (50 µg mL-1, 10 mL) was added in a dialysis bag (MW cutoff: 5 kDa) and immersed in 50 mL acetate buffer (pH 5) and PBS (pH 7.4). The samples were stirred at room temperature. The amount of DOX released from the GNRs@PC10ARGD@DOX hybrid nanogels was quantified by fluorescence spectroscopy. In addition, at each time point, the samples were irradiated with an 810 nm NIR laser (2 W cm-2) for 4 min. Cellular uptake. Fluorescein isothiocyanate (FITC) was first labelled onto the PC10A(RGD) with green fluorescence to observe the cellular uptake of GNRs@PC10A(RGD)@DOX. Briefly, 4 mL of PC10A(RGD) sodium bicarbonate buffer (450 µM) was mixed with 2 mL sodium bicarbonate buffer containing FITC (5400 µM). The mixture was wrapped with aluminum foil and stirred for 12 h. The mixture was dialyzed against ultrapure water for three days. The solution in the dialysis bag was lyophilized. FITC-labelled GNRs@PC10A@DOX and FITC-labelled GNRs@PC10ARGD@DOX were prepared through the similar method in the above section. The difference is using FITC-labelled PC10A(RGD) instead of PC10A(RGD). HeLa cells were seeded in glass bottom culture dishes (35 mm; MatTek) and cultured in a cell incubator (5% CO2, 37 ºC) for 24 h. Cells were washed twice with PBS. Hybrid nanogels (FITC-labelled

GNRs@PC10A@DOX

or

FITC-labelled

GNRs@PC10ARGD@DOX)

in


8

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serum-free DMEM at 5 µg mL-1 were added and incubated for another 3 h. DMEM was discarded, and cells were washed twice with PBS. The cells were fixed with formaldehyde solution (4% w/v) for 20 min. The cells were stained with 4’, 6-diamidino-2-phenylindole (DAPI, 0.1 µg mL-1) for 10 min. Subsequently, the cells were washed twice with PBS and imaged by using a 60× objective (1.35 numerical apertures) on an Olympus FLUOVIEW FV1000 confocal laser scanning microscope. In vitro cytotoxicity. HeLa cells were seeded in 96-well plates (5000 cells per well) and cultured in a cell incubator (5% CO2, 37 °C) for 24 h. The medium was discarded, and the cells were incubated with CTAB-GNRs (35 µg mL-1), GNRs@PC10ARGD (35 µg mL-1), DOX (1 µg mL-1 and 4 µg mL-1), and various DOX equivalent concentrations of GNRs@PC10ARGD@DOX in serum-free DMEM for another 30 h. The cells were washed twice with PBS. MTT (20 µl, 5 mg mL-1) solution was added into each well, and the cells were cultured in the cell incubator for 4 h. The medium was discarded, and DMSO (150 µL) was added in each well to dissolve the insoluble purple formazan crystals. The absorbance at 490 nm was measured with a microplate reader (BioTek ELX808IU, USA). Cell viability was calculated by assuming the control with a viability of 100%. In vitro chemo-photothermal therapy. HeLa cells were seeded in 6-well plates and cultured in a cell incubator (5% CO2, 37 ºC). After incubation for 20 h, the cells were washed twice with DMEM. A series of concentrations of GNRs@PC10ARGD@DOX (0-50 µg mL-1) were added and incubated for another 30 h. The samples were divided into two groups. The first group of cells were stained with calcein AM/EthD-1 for 15 min. The cells were washed with PBS and imaged immediately on the inverted microscope (Olympus IX71, Japan) with a 20 × objective. The second group of cells were washed with PBS three times. PBS (200 µL) was added in each


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well. Subsequently, cells were irradiated with the 810 nm laser at a power density of 3.0 W cm-2 for 5 min, and cells were stained with calcein AM/EthD-1 for 15 min. The cells were washed with PBS and imaged immediately on the inverted microscope. In addition, a series of concentrations of GNRs@PC10ARGD (0-50 µg mL-1) were added, and the cells were incubated for another 30 h. The cells were washed with PBS three times. PBS (200 µL) was added in each well. Subsequently, cells were irradiated with the 810 nm laser at a power density of 3.0 W cm-2 for 5 min, and cells were stained with calcein AM/EthD-1 for 15 min. The cells were washed with PBS and imaged immediately on the inverted microscope.

RESULTS AND DISCUSSION The triblock genetically engineered polypeptides PC10A and PC10ARGD were expressed in E. coli and purified according to previously reported literatures.36 The sequences of PC10A and PC10ARGD were shown in Scheme 1b. An integrin-targeted tripeptide arginine-glycine-aspartic acid (RGD), which promotes selective cellular uptake and imaging specificity, was successfully incorporated into the polypeptide PC10A to construct PC10ARGD through genetic engineering methods. This result indicates that other interesting peptide sequences can also be introduced in the engineered polypeptides. PC10A can form stable physical hydrogel through the self-assembly of A domain and coiled-coil P domain, which the P domain assembles into a pentameric physical association and the A domain assembles into a tetrameric physical association.37, 38 The erosion rate of PC10A hydrogel (7% w/v) is 9.6 × 10-5 mg cm-2 min-1 at room temperature. Namely, 1-mm-thick PC10A hydrogel (7% w/v) dissolves completely in open aqueous solution within 50 days. The stability of PC10A hydrogel is due to the fact that coiled-coil domains P and A cannot assemble with each other.37


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GNRs with dimensions of 50.9 ± 6.6 nm by 14.2 ± 2.1 nm (aspect ratio = 3.6) (Figure 1a) and a LSPR at 780 nm (Figure 1c) were prepared according to previously reported synthesis method.34,

35

The hybrid nanogels of GNRs@PC10ARGD and GNRs@PC10ARGD@DOX were

prepared by simple mixing of PC10ARGD or the mixture of PC10ARGD and DOX with GNRs for 12 h at room temperature. Hybrid nanogels were prepared by the electrostatic adsorption of positively charged GNRs and negatively charged polypeptides. A PC10ARGD layer with a thickness of about 3-4 nm (Figure 1a) was observed on the surface of the GNRs, which did not exist in GNRs prior to PC10ARGD coating (Figure 1a inset). The result of atomic force microscope also confirmed that the GNRs were coated with a layer polypeptide of about 3-4 nm (Figure S2). The absorptive PC10ARGD formed a layer of physical hydrogel on the surface of GNRs by the self-assemble of PC10ARGD. Zeta potentials of GNRs and GNRs-polypeptide hybrid nanogels were measured and shown in Figure 1b. The zeta potential of GNRs decreased about 20 mV after centrifuge twice. As expected, the strong negative zeta potentials were observed after coating with PC10A(RGD). The surface charge of GNRs changed from positive to negative possibly due to some glutamic acid and aspartic acid residues in the engineered polypeptides, further demonstrating that negative polypeptide has successfully coated onto the surface of positive GNRs through electrostatic adsorption. The zeta potential of GNRs@PC10A (-30.5 mV) is lower than that of GNRs@PC10ARGD (-26.3 mV) due to the positive RGD sequences. The zeta potential of GNRs@PC10ARGD@DOX is slightly larger than that of the GNRs@PC10ARGD, indicating that the positively charged DOX are successfully loaded into the PC10ARGD hydrogel and is not adsorbed on the surface of GNRs@PC10ARGD nanogels. With the increase in the concentration of DOX, the amount of loaded DOX in hybrid nanogels increases first, then plateau and finally


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decreases (Figure S3). The maximum DOX payload was 0.16 µg µg-1 GNRs (quantified from thermal treatment at 100 ºC for 30 min). In addition, it is worth pointing out that the sequential assembly (Figure S4), which involved coating PC10ARGD onto the GNRs and adding DOX, failed to form hybrid nanogels due to formation of precipitation. It is most likely due to DOX adsorption on the surface of the GNRs@PC10ARGD and result in an increase of zeta potential.

Figure 1. Characterization of the GNRs-polypeptide hybrid nanogels. (a) TEM image of the GNRs@PC10ARGD@DOX nanogels and GNRs (inset). (b) Zeta-potential of GNRs, GNRs@polypeptide hybrid nanogels, and GNRs@PC10ARGD@DOX nanogels. Error bars indicate standard deviations (n =3). (c) The absorption spectra of GNRs, GNRs@PC10ARGD, and GNRs@PC10ARGD@DOX. (d) Agarose gel electrophoresis of GNRs (lane 1), GNRs@PC10ARGD@DOX (0.4 µg DOX/ µg GNRs) (lane 2), GNRs@PC10ARGD@DOX (0.6 µg DOX/ µg GNRs) (lane 3), and GNRs@PC10ARGD@DOX (0.8 µg DOX/ µg GNRs) (lane 4). (e, f) The photothermal images and temperature changes of PC10ARGD and hybrid nanogels were recorded at different concentrations under irradiation by a NIR laser (810 nm, 2 W cm-2) with an IR thermal camera.


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PC10ARGD hydrogel on the surface of GNRs resulted in a red-shift (15 nm) of the LSPR band of the GNRs, whereas the transverse SPR remained unchanged. The red-shift of the LSPR after polypeptide modification may be due to the differences of the local refractive indexes between polypeptide and water relative to GNRs (Figure 1c).39 No obvious change of the LSPR of the GNRs after loading DOX in the hydrogel was observed. In addition, the LSPR of the functionalized GNRs was not broadened after PC10ARGD coating and DOX loading, indicating no aggregation. Coating of PC10ARGD and loading of DOX in GNRs@PC10ARGD hybrid nanogels were further confirmed by the agarose gel electrophoresis (Figure 1d). Almost no migration of GNRs was observed because GNRs precipitated in the TAE running buffer. Compared with GNRs, GNRs@PC10ARGD hybrid nanogels migrated toward the positive electrode, whereas the loaded DOX in GNRs@PC10ARGD@DOX hybrid nanogels migrated to the opposite direction. To evaluate the photothermal effect of functionalized GNRs, GNRs@PC10ARGD@DOX solution with various concentrations was exposed to NIR laser irradiation (810 nm, 2 W cm-2), and the photothermal images were recorded by a IR thermal camera

(Figure

1e,

f).

Compared

with

the

original

GNRs

(Figure

S5),

GNRs@PC10ARGD@DOX remains high photothermal efficiency. When the irradiation power density was set at 2 W cm-2, the temperature of GNRs@PC10ARGD@DOX (20 µg mL-1) could reach about 55 ºC within 5 min. The temperature rising rate depended on the concentration of GNRs (Figure 1f) and laser power (Figure S5).

The stabilities of GNRs and GNRs@PC10ARGD@DOX in different media were examined by following the LSPR of the absorption spectra as a function of time (Figure 2). GNRs treated with two rounds of centrifugations were stable only in DMEM with serum and water (Figure 2e, f), while they aggregated in PBS and serum-free DMEM (Figure 2a, b). The sharp decrease of


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longitudinal peak in PBS and serum-free DMEM may be due to the higher salt concentration of these media.40 However, the position and intensity of the LSPR of GNRs@PC10ARGD@DOX in PBS and serum-free DMEM did not change after incubation for 7 days, indicating that GNRs@PC10ARGD@DOX stably dispersed in the media (Figure 2c, d). It is possible that stable hydrogel forming from polypeptide PC10ARGD on the surface of GNRs resulted in enhanced stability of GNRs in the media. The absorbance value of GNRs@PC10ARGD@DOX in PBS decreased at day 7, indicating that complex slightly aggregated. GNRs@PC10ARGD@DOX also presented good stability in water and DMEM with serum (Figure 2g, h). Therefore, PC10ARGD coated GNRs can be dispersed and sustained in both PBS and serum-free DMEM for a long time, which facilitates their use in biological applications.


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Figure 2. Optical stability of GNRs (a, b, e, and f) and GNRs@PC10ARGD@DOX (c, d, g, and h) were monitored by the LSPR in PBS, serum-free DMEM, water, and DMEM with serum, respectively. The concentrations of GNRs and GNRs equivalent concentration of GNRs@PC10ARGD@DOX are 50 µg mL-1.

PC10A hydrogel has been demonstrated that the conformation is responsive to temperature and pH.41 GNRs@PC10ARGD@DOX hybrid nanogels are expected to show the same properties, which facilitate the controlled release of the drug from the hybrid nanogels. To demonstrate the drug release property of GNRs@PC10ARGD@DOX triggered by pH- and temperature, the release of DOX from the GNRs@PC10ARGD@DOX hybrid nanogels was examined at 37 ºC in pH (7.4) and pH 5 buffer using a dialysis bag (MW cutoff: 5 kDa). As shown in Figure 3, the


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DOX release was very quick within 10 h, and then the rate drop. The release amounts of the DOX from the GNRs@PC10ARGD@DOX hybrid nanogels at pH 5 were significantly faster than those at pH 7.4, indicating that these hybrid nanogels are pH sensitive. High drug release in acidic pH environments is beneficial to hybrid nanogels for cancer chemotherapy. 42 In addition, the release rate is significantly enhanced by laser irradiation, the DOX release amount with a continuous wave NIR laser irradiation (810 nm, 2 W cm-2, 4 min) is twice the amount released without laser irradiation at pH 7.4 (Figure 3), indicating that these hybrid nanogels are temperature-responsive. The high release rate with laser irradiation is probably due to dissociation of the P and A domain by temperature elevation.43

Figure 3. In vitro DOX release from GNRs@PC10ARGD@DOX nanogels (50 µg mL-1) with or without NIR laser irradiation in different pH buffer (acetate buffer, pH 5, and PBS buffer, pH 7.4).

High nanoparticle uptake is a prerequisite for chemotherapy and photothermal therapy. Our previous report showed that an integrin-targeted RGD in the C-terminus of the polypeptide PC10ARGD rendered efficient αvβ3-mediated endocytosis.41 The GNRs@PC10ARGD@DOX is expected to exhibit targeted uptake property. To directly observe the cellular uptake of


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GNRs@PC10A(RGD)@DOX,

the

PC10A(RGD)

was

labelled

with

FITC.

The

αvβ3

overexpressing human cervical carcinoma HeLa cells were separately incubated with FITC-labelled GNRs@PC10A@DOX and FITC-labelled GNRs@PC10ARGD@DOX for 3 h and investigated by a confocal laser scanning microscopy. As expected, HeLa cells treated with FITC-labelled GNRs@PC10ARGD@DOX exhibit strong green fluorescence (FITC) in the cytoplasm and red fluorescence (DOX) in the nucleus (Figure 4), implying that a substantial amount of the FITC-labelled GNRs@PC10ARGD@DOX hybrid nanogels have entered the cells, and DOX was released from hybrid nanogels. In contrast, almost no green fluorescence was observed in HeLa cells treated with FITC-labelled GNRs@PC10A@DOX, indicating that FITC-labelled GNRs@PC10A@DOX can not be internalized into HeLa cells. HeLa cells incubation with FITC-labelled GNRs@PC10A@DOX showed very weak red fluorescence, which is probably caused by the released DOX from extracellular FITC-labelled GNRs@PC10A@DOX (Figure 4). This result suggests that the GNRs and DOX can be delivered into targeted cells via receptor-mediated endocytosis. These results are consistent with our previous study.41

Figure

4.

Confocal

fluorescence

images

of

HeLa

cells

incubated

with

the

FITC-labelled

GNRs@PC10A@DOX and FITC-labelled GNRs@PC10ARGD@DOX for 3 h. The nucleus of HeLa cells was stained with DAPI. A 60× objective was used. The scale bars are 20 µm.


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To evaluate the chemo therapeutic efficacy of GNRs@PC10ARGD@DOX hybrid nanogels in vitro, HeLa cancer cells were treated with GNRs@PC10ARGD@DOX at various concentrations for 30 h, and the cell viabilities were assessed with MTT assay. The cytotoxicity of CTAB-GNRs, free DOX, and GNRs@PC10ARGD was measured and shown in Figure 5. Previous studies have shown that CTAB on the surface of GNRs are toxic to many types of cells.44, 45 The viability of the HeLa cells incubated with 20 µM PC10ARGD was about 100%, indicating that the PC10ARGD is no toxic to HeLa cells. The viability of the HeLa cells incubated with 35 µg mL-1 CTAB-GNRs decreased about 85%, which may be due to the cytotoxicity caused by CTAB.44,

45

Compared to CTAB-GNRs, PC10ARGD-functionalized

GNRs showed little cytotoxicity at a wide range of concentrations (0 - 50 µg mL-1), which may be attributed to toxic CTAB have been coated into the nontoxic polypeptide. The cytotoxicity of the DOX-loaded hybrid nanogels sharply increased due to released DOX. Compared with free DOX, GNRs@PC10ARGD@DOX with an equivalent dose of DOX had the more highly cytotoxic effect. The viabilities of free DOX and GNRs@PC10ARGD@DOX were dependent on the dose of DOX.


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Figure 5. Cell viability of HeLa cells treated with free PC10ARGD polypeptide, CTAB-GNRs, GNRs@PC10ARGD, free DOX, and various DOX equivalent concentrations of GNRs@PC10ARGD@DOX. The concentration of the free PC10ARGD polypeptide, CTAB-GNRs, and GNRs@PC10ARGD is 20 µM, 35 µg mL-1, and 50 µg mL-1, respectively.

To

evaluate

the

efficacy

of

chemotherapy

and

photothermal

therapy

of

GNRs@PC10ARGD@DOX, HeLa cells were incubated with GNRs@PC10ARGD or GNRs@PC10ARGD@DOX hybrid nanogels at various concentrations. The cell viability was measured with and without NIR laser irradiation using live/dead cell staining assays with calcein Am/EthD-1 homodimer. From the above-mentioned results we knew that the cytotoxicity is related with the dose of DOX (Figure 6a-c). In addition, the efficacy of the photothermal therapy of GNRs@PC10ARGD showed dose-dependent cytotoxicity (Figure 6d-f). Although DOX is an effective chemotherapeutic agent, we found most of the cells are alive at a low DOX dose (Figure

6c).

Compared

with

treated

with

GNRs@PC10ARGD@DOX

alone,

GNRs@PC10ARGD@DOX-treated HeLa cells exhibited destruction within the laser spots after exposure to NIR laser, whereas the cells not exposed to NIR laser were alive (Figure 7b). Few


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damaged cells were observed for control cells after laser irradiation (Figure 7a), indicating that cell damage (Figure 7b) is due to the combined effect of the cytotoxicity of DOX and the photothermal effect of GNRs. To verify whether the result of cytotoxicity in Figure 7b is most due to the photothermal effect of GNRs, the cells in the control that incubated with GNRs@PC10ARGD alone at the GNRs@PC10ARGD equivalent concentration in Figure 7b after exposure to the NIR laser (810 nm) are alive (Figure 6f). These results clearly demonstrate that the combined treatment of chemo-photothermal therapy showed a synergistic effect for cancer therapy.

Figure 6. Fluorescence microscope images of HeLa cells treated with different concentration (a-c: 50 µg mL-1, 25 µg mL-1, and 8 µg mL-1) of GNRs@PC10ARGD@DOX for 30 h and incubated GNRs@PC10ARGD (d-f: 50 µg mL-1, 25 µg mL-1, and 8 µg mL-1) for 30 h and irradiated with 810 nm laser at a power density of 3.0 W cm-2 for 5 min (d-f). The dashed curves indicate the region exposed by NIR laser.


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Figure 7. Fluorescence microscope images of control HeLa cells (a) and HeLa cells treated with GNRs@PC10ARGD@DOX (8 µg mL-1) that were irradiated with a NIR laser (810 nm, 3.0 W cm-2) for 5 min (b). The dashed curves indicate the region exposed by NIR laser.

CONCLUSIONS

A new hybrid nanogel system based on the positively charged GNRs and the negatively charged polypeptide PC10ARGD was fabricated by electrostatic adsorption. The immobilized PC10ARGD on the surface of GNRs formed physical hydrogel by self-assembly. DOX can be loaded into PC10ARGD formed hydrogel. The GNRs@PC10ARGD@DOX hybrid nanogels exhibited high stability in water, PBS, serum-free DMEM, and DMEM with serum. GNRs@ PC10ARGD@DOX still remained excellent photothermal effect. DOX release is able to be stimulated by the temperature and pH. Compared with CTAB coated gold nanorods, polypeptide formed hydrogel on the surface of GNRs enhanced the biocompatility. Chemotherapy and photothermal therapy experiments showed that the combination therapy treated with GNRs@PC10ARGD@DOX was superior to individual chemotherapy or photothermal therapy. These results indicate that GNRs@ PC10ARGD@DOX provides a desirable strategy for tumor-targeted drug delivery and synergistic combined therapy.

ASSOCIATED CONTENT Supporting Information The MS spectra of polypeptides. DOX loading into GNRs@PC10ARGD nanogels for different initial DOX concentration. Schematic illustration of two different preparation routes of hybrid


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nanogels. Photothermal effect of GNRs. (PDF) The Supporting Information is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author *B. Liu. E-mail: [email protected]. Fax: +86 27 87792202; Tel: +86 27 87793863 Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (Grant No. 81471697), Yellow Crane Talent (Science & Technology) Program of Wuhan City and Applied Basic Research Program of Wuhan City (2016060101010044), the Fundamental Research Funds for the Central Universities (Hust, 2016YXMS253). The authors thank the Analytical and Testing Center (HUST) for the help of measurement.

REFERENCES (1) Shanmugam, V.; Selvakumar, S.; Yeh, C. Near-infrared light-responsive nanomaterials in cancer therapeutics. Chem. Soc. Rev. 2014, 43, 6254-6287. (2) Song, X.; Chen Q.; Liu Z. Recent advances in the development of organic photothermal nano-agents. Nano Res. 2015, 8, 340-354. (3) Zou, L.; Wang, H.; He, B.; Zeng, L.; Tan, T.; Cao, H.; He, X.; Zhang, Z.; Guo, S.; Li, Y. Current approaches of photothermal therapy in treating cancer metastasis with nanotherapeutics. Theranostics 2016, 6, 762-772.


22

Page 23 of 28

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


(4) Wang, S.; Zhang, Q.; Yang, P.; Yu X.; Huang, L.; Shen, S.; Cai, S. Manganese oxide-coated carbon nanotubes as dual-modality lymph mapping agents for photothermal therapy of tumor metastasis. ACS Appl. Mater. Interfaces 2016, 8, 3736-3743. (5) Ali, M. R.; Ali, H. R.; Rankin, C. R.; EI-Sayed, M. A. Targeting heat shock protein 70 using gold nanorods enhances cancer cell apoptosis in low dose plasmonic photothermal therapy. Biomaterials 2016, 102, 1-8. (6) Zhang, L.; Chen, Y.; Li, Z.; Li, L.; Saint-Cricq, P.; Li, C.; Lin, J.; Wang, C.; Su, Z.; Zink, J. I. Tailored synthesis of octopus-type janus nanoparticles for synergistic actively-targeted and chemo-photothermal therapy. Angew. Chem. 2016, 128, 2158-2161. (7) Chen, Y.; Su, Y.; Hu, S.; Chen, S. Functionalized graphene nanocomposites for enhancing photothermal therapy in tumor treatment. Adv. Drug Deliver. Rev. 2016, 105, 190-204. (8) Gao, J.; Wu, C.; Deng, D.; Wu, P.; Cai, C. Direct synthesis of water-soluble aptamer-Ag2S quantum dots at ambient temperature for specific imaging and photothermal therapy of cancer. Adv. Healthc. Mater. 2016, 5, 2437-2449. (9) Guo, M.; Xiang, H.; Wang, Y.; Zhang, Q.; An L.; Yang, S.; Ma, Y.; Wang, Y.; Liu, J. Ruthenium nitrosyl functionalized graphene quantum dots as an efficient nanoplatform for NIR-light-controlled and mitochondria-targeted delivery of nitric oxide combined with photothermal therapy. Chem. Commun. 2017, 53, 3253-3256. (10) Huang, X.; EI-Sayed, H.; Qian, W.; EI-Sayed, M. A. Cancer cell imaging and photothermal therapy in the near-infrared region by using gold nanorods. J. Am. Chem. Soc. 2006, 128, 2115-2120.


23


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

Page 24 of 28

(11) Wang, B.; Wang, J.; Liu, Q.; Huang, H.; Chen, M.; Li, K.; Li, C.; Yu, X.; Chu, P. K. Rose-bengal-conjugated gold nanorods for in vivo photodynamic and photothermal oral cancer therapies. Biomaterials 2014, 35, 1954-1966. (12) Chen, J.; Liang, H., Lin, L.; Guo, Z.; Sun, P.; Chen, M.; Tian, H.; Deng, M.; Chen, X. Gold-nanorods-based gene carriers with the capability of photoacoustic imaging and photothermal therapy. ACS Appl. Mater. Interfaces 2016, 8, 31558-31566. (13) Zhang, Z.; Wang, L.; Wang, J.; Jiang, X.; Li, X.; Hu, Z.; Ji, Y.; Wu, X.; Chen, C. Mesoporous silica-coated gold nanorods as a light-mediated multifunctional theranostic platform for cancer treatment. Adv. Mater. 2012, 24, 1418-1423. (14) Shen, S.; Tang, H.; Zhang, X.; Ren, J.; Pang, Z.; Wang, D.; Gao, H.; Qian, Y.; Jiang, X.; Yang, W. Targeting mesoporous silica-encapsulated gold nanorods for chemo-photothermal therapy with near-infrared radiation. Biomaterials 2013, 34, 3150-3158. (15) Pakunlu, R. I.; Wang, Y.; Tsao, W.; Pozharov, V.; Cook, T. J.; Minko, T. Enhancement of the efficacy of chemotherapy for lung cancer by simultaneous suppression of multidrug resistance and antiapoptotic cellular defense. Cancer Res. 2004, 64, 6214-6224. (16) Yang, G.; Liu, J.; Wu, Y.; Feng, L.; Liu, Z. Near-infrared-light responsive nanoscale drug delivery systems for cancer treatment. Coordin. Chem. Rev. 2016, 320-321, 100-117. (17) Liao, J.; Li, W.; Peng, J.; Yang, Q.; Li, H.; Wei, Y.; Zhang, X.; Qian, Z. Combined cancer photothermal-chemotherapy

based

on

doxorubicin/gold

nanorod-loaded

polymersomes.

Theranostics 2015, 5, 345-356. (18) Chen, H.; Di, Y.; Chen, D.; Madrid, K.; Zhang, M.; Tian, C.; Tang, L.; Gu, Y. Combined chemo- and photo-thermal therapy delivered by multifunctional theranostic gold nanorod-loaded microcapsules. Nanoscale 2015, 7, 8884-8897.


24

Page 25 of 28

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


(19) Yuan, A.; Huan, W.; Liu, X.; Zhang, Z.; Zhang, Y.; Wu, J.; Hu, Y. NIR light-activated drug release for synergetic chemo-photothermal therapy. Mol. Pharmaceutics 2017, 14, 242-251. (20) Kah, J. C. Y.; Chen, J.; Zubieta, A.; Hamad-Schifferli, K. Exploiting the protein corona around gold nanorods for loading and triggered release. ACS Nano 2012, 6, 6730-6740. (21) Kramer, J. R.; Deming, T. J. Glycopolypeptides with a redox-triggered helix-to-coil transition. J. Am. Chem. Soc. 2012, 134, 4112-4115. (22) Zhang, Z.; Wang, J.; Nie, X.; Wen, T.; Ji, Y.; Wu, X.; Zhao, Y.; Chen, C. Near infrared laser-induced targeted cancer therapy using thermoresponsive polymer encapsulated gold nanorods. J. Am. Chem. Soc. 2014, 136, 7317-7326. (23) Zhang, W.; Wang, F.; Wang, Y.; Wang, J.; Yu, Y.; Guo, S.; Chen, R.; Zhou, D. pH and near-infrared light dual-stimuli responsive drug delivery using DNA-conjugated gold nanorods for effective treatment of multidrug resistant cancer cells. J. Control. Release 2016, 232, 9-19. (24) Nguyen, S. C.; Zhang, Q.; Manthiram, K.; Ye, X.; Lomont, J. P.; Harris, C. B.; Weller, H.; Alivisatos, A. P. Study of heat transfer dynamics from gold nanorods to the environmentvia time-resolved infrared spectroscopy. ACS Nano 2016, 10, 2144-2151. (25) Kuo, T.; Hovhannisyan, V. A.; Chao, Y.; Chao, S.; Chiang, S.; Lin, S.; Dong, C.; Chen, C.; Multiple release kinetics of targeted drug from gold nanorod embedded polyelectrolyte conjugates induced by near-infrared laser irradiation. J. Am. Chem. Soc. 2010, 132, 14163-14171. (26) Li, X.; Takashima, M.; Yuba, E.; Harada, A.; Kono, K. PEGylated pAMAM dendrimer-doxorubicin

conjugate-hybridized

gold

nanorod

for

combined

photothermal-chemotherapy. Biomaterials. 2014, 35, 6576-6584.


25


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

Page 26 of 28

(27) Chen, J.; Jackson, A. A.; Rotello, V. M.; Nugen, S. R. Colorimetric detection of escherichia coli based on the enzyme-induced metallization of gold nanorods. Small 2016, 12, 2469-2475. (28) Burrows, N. D.; Lin, W.; Hinman, J. G.; Dennison, J. M.; Vartanian, A. M.; Abadeer, N. S.; Grzincic, E. M.; Jacob, L. M.; Li, J.; Murphy, C. Anisotropic nanoparticles and anisotropic surface chemistry. Langmuir 2016, 32, 9905-9921. (29) Ruff, J.; Steitz, J.; Buchkremer, A.; Noyong, M.; Hartmann, H.; Besmehn, A.; Simon, U. Multivalency of PEG-thiol ligands affects the stability of NIR-absorbing hollow gold nanospheres and gold nanorods. J. Mater. Chem. B 2016, 4, 2828-2841. (30) Kong, F.; Zhang, H.; Qu, X.; Zhang, X.; Chen, D.; Ding, R.; Makila, E.; Salonen, J.; Santos, H. A.; Hai, M. Gold nanorods, DNA origami, and porous silicon nanoparticle-functionalized biocompatible double emulsion for versatile targeted therapeutics and antibody combination therapy. Adv. Mater. 2016, 28, 10195-10203. (31) Huang, H.; Walker, C. R.; Nanda, A.; Rege, K. Laser welding of ruptured intestinal tissue using plasmonic polypeptide nanocomposite solders. ACS Nano 2013, 7, 2988-2998. (32) Yasun, E.; Li, C.; Barut, I.; Janvier, D.; Qiu, L.; Cui, C.; Tan, W. BSA modification to reduce CTAB induced nonspecificity and cytotoxicity of aptamer conjugated gold nanorods. Nanoscale 2015, 7, 10240-10248. (33) Li, Z.; Huang, H.; Tang S.; Li, Y.; Yu, X.; Wang, H.; Li, P.; Sun, Z.; Zhang, H.; Liu, C.; Chu, P. Small gold nanorods laden macrophages for enhanced tumor coverage in photothermal therapy. Biomaterials 2016, 74, 144-154. (34) Yang, J.; Yao, M.; Du, M.; Jin, R.; Zhao, D.; Ma, J.; Ma, Z.; Zhao, Y.; Liu, B. A near-infrared light-controlled system for reversible presentation of bioactive ligands using polypeptide-engineered functionalized gold nanorods. Chem. Commun. 2015, 51, 2569-2572.


26

Page 27 of 28

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


(35) Nikoobakht, B.; EI-Sayed, M. A. Preparation and growth mechanism of gold nanorods (NRs) using seed-mediated growth method. Chem. Mater. 2003, 15, 1957-1962. (36) Petka, W. A.; Harden, J. L.; McGrath, K. P.; Wirtz, D.; Tirrell, D. A. Reversible hydrogels from self-assembling artificial proteins. Science 1998, 281, 389-392. (37) Shen, W.; Zhang, K. C.; Kornfield, J. A.; Tirrell, D. A. Tuning the erosion rate of artificial protein hydrogels through control of network topology. Nat. Mater. 2006, 5, 153-158. (38) Yao, M.; Yang, J.; Song, J.; Zhao, D.; Du, M.; Zhao, Y.; Liu, B. Directed self-assembly of polypeptide-engineered physical microgels for building porous cell-laden hydrogels. Chem. Commun. 2014, 50, 9405-9408. (39) Mei, Z.; Tang, L. Surface-plasmon-coupled fluorescence enhancement based on ordered gold nanorod array biochip for ultrasensitive DNA analysis. Anal. Chem. 2017, 89, 633-639. (40) Huang, H.; Barua, H. S.; Kay, S. B.; Rege, K. Simultaneous enhancement of photothermal stability and gene delivery efficacy of gold nanorods using polyelectrolytes. ACS Nano 2009, 3, 2941-2952. (41) Yang, J.; Yao, M.; Wen, L.; Song, J.; Zhang, M.; Zhao, Y.; Liu, B. Multifunctional quantum dot-polypeptide hybrid nanogel for targeted imaging and drug delivery. Nanoscale 2014, 6, 11282-11292. (42) Du, J.; Du, X.; Mao, C.; Wang, J. Tailor-made dual pH-sensitive polymer-doxorubicin nanoparticles for efficient anticancer drug delivery. J. Am. Chem. Soc. 2011, 133, 17560-17563. (43) Gunasekar, S. K.; Asnani, M.; Limbad, C.; Haghpanah, J. S.; Hom, W.; Barra, H.; Nanda, S.; Lu, M.; Montclare, J. K. N-Terminal aliphatic residues dictate the structure, stability, assembly, and small molecule binding of the coiled coil region of cartilage oligomeric matrix protein. Biochemistry 2009, 48, 8559-8567.


27


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Page 28 of 28

(44) Su, L.; Hu, S.; Zhang, L.; Wang, Z.; Gao, W.; Yuan, J.; Liu, M. A fast and efficient replacement of CTAB with MUA on the surface of gold nanorods assisted by a water-immiscible ionic liquid. Small 2016, 13, DOI: 10.1002/smll.201602809. (45) Zhu, X.; Fang, C.; Jia, H.; Huang, Y.; Cheng, C. H. K.; Ko, C.; Chen, Z.; Wang, J.; Wang, Y. Cellular uptake behaviour, photothermal therapy performance, and cytotoxicity of gold nanorods with various coatings. Nanoscale 2014, 6, 11462-11472.

Table of Contents Graphic We developed a new hybrid nanogel system based on gold nanorods (GNRs) functionalized with polypeptide-engineered and doxorubicin for combined cancer chemo-photothermal therapy.


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Polypeptide-engineered Hydrogel Coated Gold Nanorods for

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