Biomaterials xxx (2014) 1e9

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PEGylated PAMAM dendrimeredoxorubicin conjugate-hybridized gold nanorod for combined photothermal-chemotherapy Xiaojie Li, Munenobu Takashima, Eiji Yuba, Atsushi Harada, Kenji Kono* Department of Applied Chemistry, Graduate School of Engineering, Osaka Prefecture University, 1-1 Gakuen-cho, Naka-ku, Sakai, Osaka 599-8531, Japan

a r t i c l e i n f o

a b s t r a c t

Article history: Received 17 March 2014 Accepted 14 April 2014 Available online xxx

We prepared pH-sensitive drugedendrimer conjugate-hybridized gold nanorod as a promising platform for combined cancer photothermal-chemotherapy under in vitro and in vivo conditions. Poly(ethylene glycol)-attached PAMAM G4 dendrimers (PEGePAMAM) were first covalently linked on the surface of mercaptohexadecanoic acid-functionalized gold nanorod (MHA-AuNR), with subsequent conjugation of anti-cancer drug doxorubicin (DOX) to dendrimer layer using an acid-labile-hydrazone linkage to afford PEGeDOXePAMAMeAuNR particles. The particles with a high PEGePAMAM dendrimer coverage density (0.28 per nm2 AuNR) showed uniform sizes and excellent colloidal stability. In vitro drug release studies demonstrated that DOX released from PEGeDOXePAMAMeAuNR was negligible under normal physiological pH, but it was enhanced significantly at a weak acidic pH value. The efficient intracellular acidtriggered DOX release inside of lysosomes was confirmed using confocal laser scanning microscopy analysis. Furthermore, the combined photothermal-chemo treatment of cancer cells using PEGeDOX ePAMAMeAuNR for synergistic hyperthermia ablation and chemotherapy was demonstrated both in vitro and in vivo to exhibit higher therapeutic efficacy than either single treatment alone, underscoring the great potential of PEGeDOXePAMAMeAuNR particles for cancer therapy. Ó 2014 Elsevier Ltd. All rights reserved.

Keywords: Dendrimer Gold nanorods Photothermal therapy Chemotherapy Doxorubicin

1. Introduction The development of near-infrared (NIR) light-responsive gold nanoparticles (AuNPs) for cancer photothermal therapy (PTT) has received much attention in recent years [1]. Because NIR light is minimally absorbed by skin and tissues, it can penetrate tissues noninvasively and more deeply [2e4]. This therapeutic strategy is based on the conversion of absorbed NIR light energy into heat for hyperthermia using various gold nanostructures including gold nanovesicles [5,6], gold nanoshells [7,8], gold nanorods (AuNRs) [9,10], and gold nanocages [11,12], which have well-defined surface plasma resonance (SPR) absorption in the NIR region. Different from traditional hyperthermia using external heating, which probably causes damage to normal tissues, AuNP-based PTT is a minimally invasive, tumor targeted, and localized treatment [13,14]. These AuNPs can accumulate preferentially in tumors through enhanced permeability and retention (EPR) effect. They can release heat inside of the tumors for sufficient tumor ablation by controlling the timing and intensity of NIR light irradiation. In addition, anti-tumor drugs can be bound on or encapsulated in * Corresponding author. E-mail address: [email protected] (K. Kono).

AuNPs platforms [15]. The generated heat is useful to both provide hyperthermia cancer therapy and to trigger drug release for chemotherapeutics. This combination of photothermal therapy with chemotherapy (photothermal-chemotherapy) has been proved to be more effective than respective monotherapies because of additive or synergistic effects [16e24]. However, current systems rely mainly on an NIR light-triggered method to release chemotherapeutic drugs, which are physically mixed or loosely electrostatically bound with AuNPs. Unintended drug release from AuNPs is possible during their circulation in biological systems. Moreover, tumor cells in deep tissues might not be destroyed because of limited laser penetration and drug release. Therefore, the grand challenge of developing a safe and effective strategy for photothermal-chemotherapy remains. AuNRs with tunable longitudinal absorption spectra in NIR region are regarded as offering great potential for cancer photothermal-chemotherapy [25]. However, their low specific surface area limits the loading amount of drugs. Toxicity derived from the cetyltrimethylammonium bromide (CTAB) used during their synthesis severely limits their biomedical application. Dendrimers, a family of synthetic polymers with a regularly branched tree-like structure, abundant surface functional groups, welldefined composition, and non-immunogenicity, are promising

http://dx.doi.org/10.1016/j.biomaterials.2014.04.043 0142-9612/Ó 2014 Elsevier Ltd. All rights reserved.

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candidates to overcome these shortcomings [26e29]. The unique properties of dendrimers make them versatile nanodevices for use in drug delivery applications [30,31]. Dendrimers have been investigated intensively as a versatile platform to form organice inorganic hybrid nanomaterials for potential biomedical applications [32,33]. However, few reports describe the use of dendrimerbased hybrid NPs for drug delivery applications [34]. This study was conducted to develop a dendrimer-modified AuNRs platform with drugecovalent conjugation to dendrimer layer as a smart drug delivery system for combined photothermalchemotherapy. PAMAM dendrimers with abundant surface amino groups were used as main building blocks for the covalent connection of AuNRs for cancer photothermal therapy, doxorubicin (DOX) with acid-labile-hydrazone linkage for cancer chemotherapy, and poly(ethylene glycol) (PEG) chains for improvement of colloidal stability of nanoparticles (Scheme 1). The pH-responsive intracellular release of DOX in acidic organelles is explored for safe in vivo circulation, controlled drug release, and also for effective synergistic effects with cancer photothermal therapy by controlling the laser irradiation timing. Here, the preparation of PEGe PAMAMehydrazone DOX conjugate-hybridized gold nanorod (PEGeDOXePAMAMeAuNR) is described, in addition to its antitumor activity through the synergy of DOX-induced chemo effect and AuNR-induced photothermal effect. 2. Materials and methods 2.1. Materials Gold (III) chloride hydrate (HAuCl4$4H2O), sodium borohydride (NaBH4), silver nitrate (AgNO3), 3-(4,5-dimethyl-2-thiazoryl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC$HCl), and N-hydroxysuccinimide (NHS) were purchased from Wako Pure Chemical (Osaka, Japan). Cetyltrimethylammonium bromide (CTAB, 97%) was obtained from Fluka. Generation 4 PAMAM dendrimer (10 wt.% in methanol), poly(ethylene glycol) monomethyl ether (average Mn w5000), fluorescein isothiocyanate (FITC), 2-iminothiolane hydrochloride, 16-mercaptohexadecanoic acid (MHA), and ascorbic acid were obtained from SigmaeAldrich, Japan (Tokyo, Japan). Hoechst33342 and LysoTracker Green DND-26 were obtained from Invitrogen (Eugegne, Oregon, USA). Opti-MEM without phenol red was obtained from Invitrogen (Carlsbad, USA). Calcein-acetoxymethyl ester (calcein-AM) was from Nacalai Tesque (Kyoto, Japan). Doxorubicin hydrochloride (DOX$HCl) was kindly donated by Kyowa Hakko Kirin Co., Ltd. (Tokyo, Japan). Tetrahydrofuran (THF) and dimethyl sulfoxide (DMSO) were supplied from Kishida Chemical (Osaka, Japan), and were distilled just prior to use. PEG (5000) 4-nitrophenyl carbonate (PEGeNPC) was synthesized using 4-nitrophenyl chloroformate and polyethylene glycol monomethyl ether according to our previous report [35]. The sulfhydryl-reactive (6maleimidocaproyl) hydrazone of doxorubicin (Mal-DOX) were synthesized according to literature procedures [36]. Other reagents without statement were used as received.

2.2. Sample synthesis General approaches employed for the synthesis of pH-sensitive PEGylated PAMAM dendrimer-doxorubicin conjugate-hybridized gold nanorod, PEGeDOXe PAMAMeAuNR, are shown in Scheme 2. 2.2.1. Synthesis of PEGePAMAM dendrimers PEGePAMAM was synthesized by partially modifying the amino groups of generation 4 PAMAM dendrimers with PEGeNPC according to our previous report with a little modification [35]. Typically, G4-PAMAM dendrimer (200.0 mg, 14.07 mmol, 900.5 mmol amino groups) and NPCePEG (1.392 g, 270.1 mmol, 0.3 equivalent to amino groups of PAMAM) were mixed in dry DMSO (8 mL), and this solution was stirred for 5 days at room temperature under N2 atmosphere. The solution was diluted with distilled water and dialyzed using a dialysis bag (molecular weight 12000e14000 cut off) against distilled water for 2 days. The crude compound was lyophilized and then purified by a Sephadex G-75 column [Pharmacia, 4 cm (diameter)  45 cm (length)] using 0.1 M Na2SO4 aqueous solution as the eluent. The eluted dendrimers were detected using an UV detector at a fixed wavelength of 220 nm. The collected fraction was dialyzed using a dialysis bag (molecular weight 12000e14000 cut off) against distilled water for 2 days to remove the salt, and lyophilized to afford the final product PEGePAMAM (1.168 g, yield 73.4%). FITC-labelled PEGePAMAM was synthesized by reacting fluorescein isothiocyanate (FITC) with PEGePAMAM at equal molar ratio in DMSO overnight and purified by dialysis against distilled water for 2 days. After dialysis, the FITCePEGe PAMAM was washed by several times ultrafiltration (Millipore, MWCO, 10,000 DA) until no free FITC can be detected in filtrate using UV-vis spectra, and then lyophilized and used without further characterization. 2.2.2. Synthesis of PEGePAMAMeAuNR conjugate CTABeAuNRs with aspect ratio of w4.1 were synthesized using a one-step seedless growth method [37], and the excess of CTAB was removed by repeated centrifugation (12,000 rpm, 10 min). 10 mg of MHA in 5 mL ethanol was added into 50 mL AuNR aqueous suspension (0.046 mM, pH 10). The mixture was sonicated for 2 h at 50  C using a bath-type sonicator, and then stirred at room temperature for an additional 24 h. The excess reagents were removed by centrifugation at 12,000 rpm for 10 min, and the resulting MHA-AuNR were purified by two washecentrifugation cycles at 12,000 rpm for 10 min and redispersed in distilled water. Subsequently, PEGePAMAMeAuNR was synthesized by conjugation of PEGePAMAM with MHAAuNR using EDC/NHS as coupling reagents. Typically, PEGePAMAM (40.5 mg, 0.387 mmol), EDC$HCl (0.66 mg, 3.47 mmol) and NHS (0.8 mg, 6.94 mmol) were added to an MHA-AuNR solution (0.092 mM, 10 mL). The pH was adjusted to 6.0 using 0.1 N HCl, and the solution was stirred for 30 min at room temperature to activate carboxylic groups of MHA. Then the pH was adjusted to 8.0 using 0.1 M NaOH, and the solution was sonicated for 2 h at 4  C using a bath-type sonicator and stirred overnight at room temperature. Excess reagents were removed by centrifugation at 12,000 rpm for 10 min, and the resulting PEGePAMAMeAuNR was purified by two washecentrifugation cycles at 12,000 rpm for 10 min and redispersed in distilled water. 2.2.3. Synthesis of PEGeDOXePAMAMeAuNR conjugate The conjugation of DOX with PEGePAMAMeAuNR was performed through a two-step synthesis process according to literature methods [38]. To an aqueous solution of PEGePAMAMeAuNR (0.046 mM, 5 mL, pH 6.5), 0.5 equivalent (to amino groups of PEGePAMAM) of 2-iminothiolane hydrochloride (0.18 mg, 1.34 mmol) were added. After stirring at room temperature for 2 h, the mixture was subjected to

Scheme 1. Schematic illustration of pH-sensitive PEGylated PAMAM dendrimer-doxorubicin conjugate-hybridized gold nanorod (PEGeDOXePAMAMeAuNR) for combined photothermal-chemotherapy.

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Scheme 2. Synthetic routes employed for the preparation of PEGeDOXePAMAMeAuNR.

two times centrifugation (12,000 rpm, 10 min) to remove free 2-iminothiolane hydrochloride. Then, Mal-DOX (1.0 mg, 1.34 mmol) was added and the pH was adjusted to 6.7 with 0.1 M NaOH. After stirring for an additional 3 h, unreacted Mal-DOX was removed by two times centrifugation (12,000 rpm, 10 min). The obtained PEGe DOXePAMAMeAuNR conjugate was redispersed in distilled water and stored at 4  C in the dark.

6 W/cm2. A NIR camera (R300W2-R15, NEC Avio Infrared Technologies Co., Ltd., Japan) was placed on top of the suspension, and thermographs were recorded by the NIR camera at an interval of 30 s. The thermographs were analyzed using Infrec analyzer NS9500 professional software to obtain the average temperature of the suspension at each time point. 2.5. In vitro drug release measurement

2.3. General characterizations for polymers and nanoparticles Nuclear magnetic resonance (NMR) spectra were recorded on a JEOL JNM-LA 400 instrument. 1H NMR spectra were recorded in CDCl3, D2O or DMSO-d6 solution. UV-vis absorption spectra were measured with a Jasco V-670 spectrophotometer (Jasco Inc., Japan) at 25  C. Transmission electron microscopy (TEM) observations were performed using an electron microscope (JEOL Ltd., JEM-2000FEX II). A drop of sample aqueous solution was placed on a carbon-coated copper grid (VECO GRID 400mesh) for 10 min, then drawn off with filter paper and allowed to dry. To prepare dendrimer layer-stained samples, a drop of sodium phosphotungstate aqueous solution (2%, pH 7.4) was again deposited on the grid and removed with a filter paper after 10 min. The particle size and zeta potential were measured by dynamic light scattering (DLS) carried out on a Zetasizer Nano ZS90 (Malvern Instruments) with a standard HeeNe 633 nm laser and 173 back scatter. The data were analyzed by Malvern Dispersion Technology Software 7.02. The concentrations of Au elements for samples solutions were determined using inductively coupled plasma mass spectroscopy (ICP-MS, SPS7800, Seiko Instruments Inc., Japan).

The pH-responsive DOX release from PEGeDOXePAMAMeAuNR was estimated in acetate buffer (10 mM, pH 5.0) and phosphate buffer (10 mM, pH 7.4) using a dialysis method. An aliquot (1 mL) of PEGeDOXePAMAMeAuNR (w 0.046 mM) suspension was loaded in a dialysis bag (molecular weight 12000e14000 cut off). The dialysis bag was immediately placed in 50 mL of corresponding buffer at 37  C. Periodically, 2 mL of the external buffer solution was taken out and replaced with equal volume of fresh medium. The amount of DOX was quantified by measuring its absorbance at 485 nm against a standard curve. 2.6. Cell culture HeLa cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, GIBCO), supplemented with 10% fetal bovine serum (FBS, GIBCO), 50 unit/mL penicillin, and 50 mg/mL streptomycin at 37  C under 5% CO2 condition. Cells were cultured for 2 days to achieve approximately same confluence before performing all experiments.

2.4. Photothermal study

2.7. Confocal laser scanning microscopy

1.0 mL of PEGeDOXePAMAMeAuNR aqueous suspensions at various Au element concentrations (0, 1, 5, and 10 mg/mL) was placed in a single well of a 48well plate and irradiated with a NIR laser (AMAKI, l ¼ 808 nm) from the top at

HeLa cells (2  105 cells) were seeded into a 35 mm dish in 2 mL of DMEM supplemented with 10% FBS, and cultured for 24 h. A total of 200 mL of PEGeDOXe PAMAMeAuNR solution (50 mg DOX/mL) in phosphate-buffered saline (PBS, pH 7.4)

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was added. After incubation with sample solutions for 2 h, cells were gently washed with PBS twice, and incubated for another 0, 4, or 22 h in 2 mL of DMEM supplemented with 10% FBS, followed by addition of 30 mL of LysoTracker Green DND-26 (10 pmol/mL in water) and 2 mL of Hoechst33342 (10 mg/mL in water). After 30 min staining, cells were washed with PBS three times. Confocal laser scanning microscopic (CLSM) analysis of these cells was performed on an LSM 5 EXCITER (Carl Zeiss Co. Ltd.). 2.8. In vitro cytotoxicity assay HeLa cells were seeded into a 96-well microplate (1  104 cells/well) in 100 mL of DMEM supplemented with 10% FBS, and cultured for 24 h. A total of 20 mL of PEGe DOXePAMAMeAuNR, PEGePAMAMeAuNR, and free DOX at various concentrations in phosphate-buffered saline (PBS, pH 7.4) were added. After incubated with sample solutions for 3 h, cells were gently washed with PBS twice, and incubated for another 21 h in 100 mL of DMEM supplemented with 10% FBS. Some groups of cells were then irradiated with NIR laser (AMAKI, l ¼ 808 nm) from the top at a power density of 6 W/cm2 for 10 min. Cells without irradiation were used as a control. After irradiation treatment, cells were cultured for another 24 h. Then, the cell viabilities were determined by MTT assay. An aliquot (96 mL) of DMEM and 6 mL MTT solution (10 mg/ mL in PBS) were added as the incubation media. After 3 h, MTT media was removed, and cells were washed two times with PBS. Then, 90 mL of isopropanol with 10 mL of 1 N HCl solution was added, and the absorbance at 490 nm was measured on a VICTOR(3) V 1420 Multilabel Counter (PerkinElmer, USA) to check the cells’ surviving profile. The therapeutic effects of PEGeDOXePAMAMeAuNR on HeLa cells were further evaluated by live cell staining assays using Calcein-AM. HeLa cells (2  105 cells) were seeded into a 35 mm dish in 2 mL of DMEM supplemented with 10% FBS, and cultured for 24 h. A total of 200 mL of PEGeDOXePAMAMeAuNR solution (1.54 mg Au/mL, 110 mg DOX/mL) or PEGePAMAMeAuNR solution (1.54 mg Au/mL) in phosphate-buffered saline (PBS, pH 7.4) was added. After incubated with sample solutions for 3 h, cells were gently washed with PBS twice, and incubated for another 21 h in 2 mL of DMEM supplemented with 10% FBS. Cells were then irradiated with NIR laser (AMAKI, l ¼ 808 nm) from the top at a power density of 6 W/cm2 for various time periods (0, 5, 10 and 15 min). After irradiation treatment, cells were cultured for another 24 h. For live cell staining, 1 mg/mL of Calcein-AM in dimethyl sulfoxide was prepared as stock solution. Calcein-AM (10 mL) stock solutions were added to 15 mL of DMEM supplemented with 10% FBS. Then, cells were washed with PBS and supplemented with 2 mL of Calcein-AM staining solution. After 30 min of incubation, cells were washed with PBS twice. Then, fluorescence images of HeLa cells were observed using an IMT-2 microscope equipped with an IMT-2-RFL fluorescence unit (OLYMPUS). 2.9. Animal model Seven-week-old female BALB/c mice were purchased from Oriental Yeast Co., Ltd. (Tokyo, Japan). Experiments were carried out in accordance with the guidelines for animal experimentation in Osaka Prefecture University. 2.10. In vivo anti-tumor effect Anti-tumor effect of PEGeDOXePAMAMeAuNR was examined according to our previously reported method [39]. Tumor-bearing mice were prepared by inoculating mouse colon carcinoma 26 cells (1  106 cells) into both the left and right flanks of BALB/c mice (female, 7 weeks old) under anesthesia and the tumor was allowed to grow for about 10 days, when the volume was approximately 75 mm3. An aliquot (100 mL) of PEGeDOXePAMAMeAuNR or PEGePAMAMeAuNR (containing 0.5 mg of Au element with/without 36 mg DOX) or PBS was intravenously injected into the mice under anesthesia (n ¼ 3 per group). At 48 h after the injection, the entire region of left tumor was irradiated with NIR laser (AMAKI, l ¼ 808 nm) at 0.24 W/cm2 for 10 min, and the right tumor was used as a control. During irradiation, thermographs were taken using a NIR camera (InfReC Thermography R300, NEC AVIO). The thermographs were analyzed using Infrec analyzer NS9500 professional software to obtain the average temperature of the suspension at each time point. Tumor volumes were monitored by time and determined as Volume ¼

 . L  W2 2;

where L is the longest dimension parallel to the skin surface and W is the dimension perpendicular to L and parallel to the surface.

3. Results and discussion 3.1. Preparation and characterization of PEGeDOXePAMAMeAuNR The PEGylated PAMAM dendrimer-doxorubicin conjugate-hybridized gold nanorod (PEGeDOXePAMAMeAuNR) was prepared through a multiple synthesis process (Scheme 2). To prepare the

PEGylated PAMAM dendrimer-attached gold nanorod (PEGe PAMAMeAuNR), both the bare PAMAM dendrimer and original cetyltrimethylammonium bromide-coated AuNR (CTAB-AuNR) were functionalized in advance. PEGylated PAMAM dendrimer (PEGePAMAM) was synthesized by partially modifying the surface amino groups of G4-PAMAM dendrimers using monomethoxyl poly(ethylene glycol) 4-nitrophenyl carbonate [35]. The average PEG chain number per PEGePAMAM dendrimer was calculated as 18 according to its 1H NMR spectra (Fig. S1). The CTAB-AuNR was functionalized with 16-mercaptohexadecanoic acid (MHA) to obtain MHA-AuNR based on the classic covalent gold-thiol bond chemistry. A particle surface zeta potential changed from 44.4 mV before to 35.4 mV after surface functionalization, indicating a sufficient replacement of the cationic CTAB by anionic MHA. The resulting carboxylic groups on the surface of MHA-AuNR were covalently linked with amino groups of PEGePAMAM dendrimers using EDC and NHS as coupling reagents. Subsequently, residual amino groups of PEGePAMAMeAuNR were partially converted to thiol groups by a reaction with 2-iminothiolane, which was followed by reaction with maleimide hydrazone derivative of DOX (Mal-DOX), affording PEGeDOXePAMAMeAuNR. Based on UV-vis absorption analysis, the average number of PEGePAMAM dendrimers and DOX molecules per AuNR were quantified respectively as 255 and 2877. The PEGePAMAM dendrimer coverage density on AuNR surface was estimated to reach about 0.28 dendrimer and 5.12 PEG chain per nm2 of AuNR (see Supporting Information). UV-Vis-NIR absorption spectra of PEGeDOXePAMAMeAuNR along with its intermediate product were measured to monitor the synthesis process (Fig. 1A). Compared with MHA-AuNR and PEGe PAMAMeAuNR, absorption spectra of PEGeDOXePAMAMeAuNR showed a slight red shift (4 nm) of the longitudinal SPR peak, which resulted from the fact that DOX conjugation increases the local refractive index around AuNR [40]. Neither broadening nor tailing of the SPR band of PEGeDOXePAMAMeAuNR was observed, indicating that no aggregation of AuNRs occurred during the multiple synthesis process. The particle size and morphology of PEGeDOXePAMAMeAuNR in aqueous media were then characterized using dynamic light scattering (DLS) and transmission electron microscopy (TEM) measurements. As shown in Fig. 1B, MHA-AuNR in water had average diameter of 44.47 nm, which is reliable considering that it is close to the average length of AuNR core of 35.0 nm from TEM analysis (Fig. 1C). By covalently attaching PEGePAMAM dendrimers on the surface of MHA-AuNR, the average diameter of PEGe PAMAMeAuNR in PBS increased to 68.15 nm. Considering that PEGePAMAM dendrimer had a diameter of 20.83 nm measured by DLS, it is likely that the PEGePAMAM dendrimer layer formed on the AuNR surface induced a 23.68 nm increase in particle size. After DOX conjugation, PEGeDOXePAMAMeAuNR in PBS had average diameter of 76.17 nm. Reportedly, the PEG chains of PEGePAMAM dendrimers can penetrate into the PAMAM dendrimer moiety in aqueous solutions [41]. Therefore, the 8.02 nm increase in hydrodynamic diameter than PEGePAMAMeAuNR might result from extended PEG chains after DOX loading on the PAMAM dendrimer surface. TEM analysis for PEGePAMAMeAuNR (Fig. 1D) and PEGe DOXePAMAMeAuNR (Fig. 1E, F) with phosphotungstate, which is used for staining of PAMAM dendrimers, indicates that AuNR core was fully covered with a thick polymer layer. To demonstrate the potential application of PEGeDOXe PAMAMeAuNR for cancer photothermal therapy, PEGeDOXe PAMAMeAuNR aqueous suspensions at various concentrations were exposed to NIR laser irradiation at 808 nm with power density of 6 W/cm2 for 5 min. Changes in temperature were recorded using a NIR camera at an interval of 30 s. As shown in Fig. 2A, both the

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Fig. 1. Absorption spectra (A) and DLS measurement (B) of PEGeDOXePAMAMeAuNR along with its intermediate product. TEM images of CTAB-AuNR (C), PEGePAMAMeAuNR (D), and PEGeDOXePAMAMeAuNR (E, F). Scale bar means 50 nm.

temperature increase rate and the final temperature were dependent on the particle concentration; a faster and larger temperature increase was observed for a higher concentration of AuNRs. The temperature of PEGeDOXePAMAMeAuNR aqueous suspension with Au element concentration of 10 mg/mL increased by 30.3  C after 5 min irradiation. The temperature of nanoparticle free distilled water increased by only 5.5  C on the same experiment conditions. It is noteworthy that the continuous laser irradiation caused no loss of the optical properties of AuNR and no change of particle size, indicating that PEGeDOXePAMAMeAuNR was stable under laser irradiation and temperature increase.

DOX was conjugated to the PAMAM dendrimers by an acidlabile-hydrazone bond. Therefore, PEGeDOXePAMAMeAuNR particles should show endo/lysosomal pH-sensitive DOX release. To demonstrate the pH-triggered drug release behavior of PEGeDOXe PAMAMeAuNR, in vitro DOX release was measured at a function of time in pH 7.4 and 5.0 buffers. As shown in Fig. 2B, about 11.5% of DOX was released after 60 h incubation at pH 7.4, indicating that the hydrazone bond was stable at a normal physiological pH value. However, the DOX release rate was much higher at pH 5.0, with 67.6% drug release within 25 h and 88.2% drug release by 60 h, thereby demonstrating the efficacy of pH-sensitive drug release

Fig. 2. Characterization of PEGeDOXePAMAMeAuNR by photothermal study and in vitro drug release measurement. (A) Temperature change of PEGeDOXePAMAMeAuNR aqueous solutions at various Au element concentrations during NIR laser irradiation for 5 min. (B) In vitro DOX release profiles from PEGeDOXePAMAMeAuNR in pH 7.4 and 5.0 buffers.

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from PEGeDOXePAMAMeAuNR at endo/lysosomal pH value. These results agree with our previous studies that the conjugation of DOX with PEG-PAMAM dendrimer by an acid-labile-hydrazone bond as an intracellular acid-sensitive drug delivery system [42]. The fluorescence characteristics of DOX have been used widely to monitor its intracellular distributions. Because the fluorescence of DOX is markedly quenched once conjugated to AuNR as a result of the nanosurface energy transfer (NSET) effect [40], the recovery of fluorescence observed inside cells indicates the intracellular DOX release. Therefore, we next examined intracellular localization of PEGeDOXePAMAMeAuNR and acidtriggered DOX release in HeLa cells using confocal laser scanning microscopy (CLSM). HeLa cells were treated with PEGeDOXe PAMAMeAuNR for 2 h, and were washed with PBS. The cells were then incubated in a culture medium for another 0, 4, or 22 h and observed using CLSM (Fig. 3). In all experiments, the lysosomes and nucleus of cells were stained with LysoTracker green and Hoechst33342 immediately before the CLSM measurements. As expected, a negligible fluorescence of DOX derived from PEGeDOXePAMAMeAuNR was observed after just 2 h incubation. It was attributable to the quenching effect of AuNR core and limited DOX release in such a short time. With incubation time increased to 6 h, weak red fluorescence of DOX was observed in CLSM images. These red fluorescence dots are located in part of green fluorescence places in the overlay image, suggesting that PEGeDOXePAMAMeAuNR was localized in lysosomes and that DOX was released gradually inside of lysosomes. These findings agreed well with the in vitro DOX release results at pH 5.0 using the dialysis method. When the incubation time reached 24 h, clearer fluorescence of DOX was observed. Nearly all green fluorescence-stained places included red fluorescence dots in the overlay image, indicating that PEGeDOXe PAMAMeAuNR was increasingly trafficked into lysosomes, releasing the DOX in lysosomes as time increased. Based on those findings above, pH-responsive PEGeDOXePAMAMeAuNR particles can reduce the undesired drug release effectively during circulation but can also enhance the intracellular drug release in

lysosomes, which might greatly enhance the efficacy of cancer treatment. 3.2. In vitro chemo-, photothermal, and photothermalchemotherapy treatments To investigate the therapeutic efficacy of PEGeDOXePAMAMe AuNR on cancer cells in vitro, HeLa cells were treated with PEGe DOXePAMAMeAuNR with and without laser irradiation. Then cell viability was analyzed using MTT assay. Sufficient drug release from PEGeDOXePAMAMeAuNR inside of lysosomes after 24 h incubation was confirmed using CLSM measurements. We chose this timing for laser irradiation treatment of cells for possible synergistic effect of chemotherapy with photothermal therapy. HeLa cells were incubated with PEGeDOXePAMAMeAuNR at various DOX concentrations for 3 h, and washed with PBS twice. Then they continued to be cultured in medium for a total of 48 h with or without 10 min laser irradiation (808 nm, 6 W/cm2) at the 24 h during incubation. Free DOX and drug-free PEGePAMAMeAuNR were used, respectively, as control with the same DOX dosages or DOX equivalent particle concentrations calculated from PEGe DOXePAMAMeAuNR. As shown in Fig. 4, drug-free PEGePAMAMeAuNR showed negligible cytotoxicity, whereas both free DOX and PEGeDOXe PAMAMeAuNR showed dose-dependent cytotoxicity. The PEGe DOXePAMAMeAuNR was less toxic than free DOX in equivalent DOX concentrations of 5.0e30.0 mg/mL. The IC50 (half maximal inhibitory concentration) of conjugated-DOX was 13.7 mg/mL, although more than 98.5% of cells treated with free DOX were dead at this drug concentration. Free DOX molecules are known to permeate the plasma membrane via simple diffusion. Then they enter the nucleus and intercalate into DNA to express cytotoxic activity. Both time-dependent and pH-dependent drug release characteristics of PEGeDOXePAMAMeAuNR caused a delay effect for conjugated-DOX showing therapeutic efficacy and therefore led to lower cytotoxicity than that of free DOX. However, the cytotoxicity of PEGeDOXePAMAMeAuNR was enhanced significantly with

Fig. 3. CLSM analysis of intracellular DOX release from PEGeDOXePAMAMeAuNR. HeLa cells were treated with PEGeDOXePAMAMeAuNR for 2 h and washed, followed by CLSM observation with time.

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Cell viability (%)

100 80 60 40 20 0

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Equiv. DOX (µg/mL) Fig. 4. In vitro cytotoxicity of PEGePAMAMeAuNR without (black dense column) and with irradiation (black solid column), PEGeDOXePAMAMeAuNR without (red dense column) and with irradiation (red solid column), and free DOX (blue dense column) against HeLa cells by MTT assay at various DOX equivalent concentrations. Data are given as the mean  SD (n ¼ 3).

10 min laser irradiation. The IC50 of conjugated-DOX with laser irradiation was 2.2 mg/mL, which was about 5.2 times lower than that of PEGeDOXePAMAMeAuNR without laser irradiation and even close to that of free DOX with IC50 of 1.0 mg/mL. Similar with that for free DOX, nearly all cells treated with PEGeDOXePAMAMe AuNR at conjugated-DOX dosages higher than 10.0 mg/mL with laser irradiation were dead. Considering that drug-free PEGe PAMAMeAuNR with 10 min laser irradiation showed negligible cytotoxicity, the combination of photothermal therapy from PEGe PAMAMeAuNR carriers and chemotherapy of conjugated-DOX caused a synergistic therapeutic effect that was considerably better than that of either of the two treatments alone. The synergistic photothermal-chemotherapy effects of PEGe DOXePAMAMeAuNR on HeLa cells were also verified using live cell staining assays with Calcein-AM. HeLa cells were treated with PEGeDOXePAMAMeAuNR at a fixed conjugated-DOX dosage of 10 mg/mL for 3 h and washed with PBS twice. Then they continued to be cultured in medium for a total of 48 h without or with 5, 10, and 15 min laser irradiation (808 nm, 6 W/cm2) at the 24 h during incubation. Drug-free PEGePAMAMeAuNR was used as a control. The live cells with green fluorescence were observed using fluorescence microscopy. Fig. 5 shows that no marked loss of cell viability happened in PEGeDOXePAMAMeAuNR-incubated cells without laser irradiation and drug-free PEGePAMAMeAuNRincubated cells with 0, 5, and 10 min laser irradiation. However,

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clearly enhanced cell death was observed for PEGeDOXePAMAMe AuNR-treated cells with laser irradiation time increase to 10 min. The results presented above show good agreement with MTT assay. Based on the fact that neither photothermal treatment nor chemotherapy alone caused any marked cell damage, the synergistic photothermal-chemotherapy was believed to contribute to the enhanced therapeutic effect. As irradiation time increased to 15 min, cells treated with PEGePAMAMeAuNR started to show cell death to some extent, and cells treated with PEGeDOXePAMAMe AuNR showed complete cell death in the laser irradiation area. Reportedly, mesoporous silica-coated gold nanorods can induce strong disruption in the lysosomal membrane [18]. They increased blebbing around the cell membrane above the threshold laser power, which engenders a fast cell death. A reasonable explanation for the complete cell death of PEGeDOXePAMAMeAuNR-treated cells by 15 min laser irradiation is that the longer photothermal treatment time caused an additive hyperthermia effect caused by lysosome membrane disruption. 3.3. In vivo chemo-, photothermal, and photothermalchemotherapy treatments Based on the excellent therapeutic effects of PEGeDOXe PAMAMeAuNR in vitro, we examined their anti-tumor performance further in vivo. We prepared mice bearing tumors with volume of around 75 mm3 by inoculating mouse colon carcinoma 26 cells into both the left and right flanks of BALB/c mice. PEGe DOXePAMAMeAuNR (0.5 mg of Au element and 36 mg DOX per mouse) was injected intravenously into the mouse. To demonstrate the synergistic therapeutic effect of photothermal therapy and chemotherapy in vivo, PEGePAMAMeAuNR with the same dosage and PBS were used as control. At 48 h after the injection, the entire left tumor region was irradiated with an NIR laser (808 nm, 0.24 W/ cm2) for 10 min. The right tumor without laser irradiation was used as a control. During irradiation, thermographs were recorded using an NIR thermal camera. Fig. 6A presents typical thermographs of PEGeDOXePAMAMe AuNR-treated mice with 0, 1, 2, and 5 min laser irradiation to left tumor. Upon laser irradiation, the temperature of the whole left tumor increased rapidly above 45  C, which is a sufficiently high temperature to ablate the tumor cells. The surrounding healthy tissue with laser irradiation exhibited a negligible increase of less than 2  C. No obvious temperature change was observed in the right tumor region without laser irradiation. It is likely that PEGe DOXePAMAMeAuNR might accumulate in the tumor tissue through EPR effects and generate heat under NIR irradiation. Fig. 6B shows the temperature change of the tumor region with laser irradiation. After 10 min of laser irradiation, tumors treated with PEGeDOXePAMAMeAuNR and PEGePAMAMeAuNR had

Fig. 5. Fluorescence images of HeLa cells treated with PEGePAMAMeAuNR (A) or PEGeDOXePAMAMeAuNR (B) and irradiated with NIR laser for various time periods. The live cells were stained with Calcein-AM after a total of 48 h incubation. Scale bar means 1 mm.

Please cite this article in press as: Li X, et al., PEGylated PAMAM dendrimeredoxorubicin conjugate-hybridized gold nanorod for combined photothermal-chemotherapy, Biomaterials (2014), http://dx.doi.org/10.1016/j.biomaterials.2014.04.043

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X. Li et al. / Biomaterials xxx (2014) 1e9

Fig. 6. Synergistic photothermal-chemotherapy effect on colon carcinoma 26 tumor-bearing mice treated with PEGeDOXePAMAMeAuNR and laser irradiation. The mice were intravenously injected with PEGeDOXePAMAMeAuNR, PEGePAMAMeAuNR, or PBS, and were treated with 10 min NIR laser irradiation on the left tumor at 48 h postinjection. (A) Thermographs of PEGeDOXePAMAMeAuNReinjected mouse receiving photothermal treatment for different periods of time. (B) Time courses of temperature variation in the left tumor region under NIR laser irradiation. (C) Tumor growth profiles of tumor-bearing mice after intravenous injection of samples. Tumor volumes were normalized to their initial sizes. Error bars represent the standard deviations (n ¼ 3).

respective average temperatures of 46.9  C and 46.2  C. However, the PBS-treated tumor with the same laser irradiation showed an average temperature of 39.0  C, which is insufficient to damage the tumor effectively. The in vivo therapeutic efficacy of PEGeDOXe PAMAMeAuNR and PEGePAMAMeAuNR with and without laser irradiation was assessed by examination of the tumor growth. The tumor size was normalized to their initial size. Fig. 6C shows that PEGeDOXePAMAMeAuNR (irradiation  ) and PEGePAMAMe AuNR (irradiation þ) groups showed significant tumor growth suppression or delay compared with the control groups (PBS with irradiation) on day 16. In contrast, PEGePAMAMeAuNR without laser irradiation showed no marked changes of the tumor growth rate compared with the control group, indicating the low toxicity of PEGePAMAMeAuNR carriers for in vivo application. It is noteworthy that PEGePAMAMeAuNR (irradiation þ) showed higher therapeutic efficacy than PEGeDOXePAMAMeAuNR (irradiation -) in vivo, which is opposite the in vitro results by MTT assay, where chemotherapy efficacy was stronger than photothermal treatment. A reasonable explanation is that in vivo photothermal therapy with PEGePAMAMeAuNR did not need the intracellular uptake of particles, whereas the in vitro thermotherapy of PEGeDOXePAMAMe AuNR relied only on intracellular acid-triggered drug release. Although PEGeDOXePAMAMeAuNR (irradiation þ) group exhibited similar tumor growth suppression efficacy with PEGePAMAMe AuNR (irradiation þ) group in the first 9 days, remarkable tumor growth delay was observed on day 16. These results demonstrated the synergistic anti-tumor effect of combined photothermalchemotherapy using PEGeDOXePAMAMeAuNR in vivo. 4. Conclusions We have prepared pH-sensitive drugedendrimer conjugatehybridized gold nanorod as a promising platform for combined cancer photothermal-chemotherapy. Compared with unmodified

AuNRs with high cytotoxicity and low specific surface area for drug loading, PEGePAMAM dendrimer-conjugated AuNRs showed negligible toxicity and excellent colloidal stability, and most importantly provided a versatile platform for additional functionalization. By conjugation of DOX to the dendrimer layer of PEGe PAMAMeAuNR through an acid-liable-hydrazone linkage, the obtained PEGeDOXePAMAMeAuNR showed higher therapeutic efficacy to cancer cells attributable to the synergistic photothermal ablation and chemotherapy effect. This work demonstrates the great potential of PEGeDOXePAMAMeAuNR particles for use in cancer therapy applications. The PEGePAMAMeAuNR with a versatile dendrimer layer surface and a multifunctional AuNR core might provide new possibilities for cancer targeting, imaging, and diagnosis applications. Acknowledgments This work was supported in part by a Granteineaid for Scientific Research from the Ministry of Education, Science, Sports, and Culture in Japan (23240075 and 26242049). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.biomaterials.2014.04.043. References [1] Huang XH, Jain P, El-Sayed I, El-Sayed M. Plasmonic photothermal therapy (PPTT) using gold nanoparticles. Lasers Med Sci 2008;23:217e28. [2] Ntziachristos V, Yodh AG, Schnall M, Chance B. Concurrent MRI and diffuse optical tomography of breast after indocyanine green enhancement. Proc Natl Acad Sci U S A 2000;97:2767e72. [3] Weissleder R. A clearer vision for in vivo imaging. Nat Biotechnol 2001;19: 316e7.

Please cite this article in press as: Li X, et al., PEGylated PAMAM dendrimeredoxorubicin conjugate-hybridized gold nanorod for combined photothermal-chemotherapy, Biomaterials (2014), http://dx.doi.org/10.1016/j.biomaterials.2014.04.043

X. Li et al. / Biomaterials xxx (2014) 1e9 [4] Ntziachristos V, Ripoll J, Wang LHV, Weissleder R. Looking and listening to light: the evolution of whole-body photonic imaging. Nat Biotechnol 2005;23: 313e20. [5] Huang P, Lin J, Li WW, Rong PF, Wang Z, Wang SJ, et al. Biodegradable gold nanovesicles with an ultrastrong plasmonic coupling effect for photoacoustic imaging and photothermal therapy. Angew Chem Int Ed 2013;52:13958e64. [6] He J, Huang XL, Li YC, Liu YJ, Babu T, Aronova MA, et al. Self-assembly of amphiphilic plasmonic micelle-like nanoparticles in selective solvents. J Am Chem Soc 2013;135:7974e84. [7] Hirsch LR, Stafford RJ, Bankson JA, Sershen SR, Rivera B, Price RE, et al. Nanoshell-mediated near-infrared thermal therapy of tumors under magnetic resonance guidance. Proc Natl Acad Sci USA 2003;100:13549e54. [8] Bardhan R, Lal S, Joshi A, Halas NJ. Theranostic nanoshells: from probe design to imaging and treatment of cancer. Acc Chem Res 2011;44:936e46. [9] Huang XH, El-Sayed IH, Qian W, El-Sayed MA. Cancer cell imaging and photothermal therapy in the near-infrared region by using gold nanorods. J Am Chem Soc 2006;128:2115e20. [10] Kuo WS, Chang CN, Chang YT, Yang MH, Chien YH, Chen SJ, et al. Gold nanorods in photodynamic therapy, as hyperthermia agents, and in nearinfrared optical imaging. Angew Chem Int Ed 2010;49:2711e5. [11] Chen JY, Wang DL, Xi JF, Au L, Siekkinen A, Warsen A, et al. Immuno gold nanocages with tailored optical properties for targeted photothermal destruction of cancer cells. Nano Lett 2007;7:1318e22. [12] Chen J, Glaus C, Laforest R, Zhang Q, Yang MX, Gidding M, et al. Gold nanocages as photothermal transducers for cancer treatment. Small 2010;6: 811e7. [13] Chatterjee DK, Diagaradjane P, Krishnan S. Nanoparticle-mediated hyperthermia in cancer therapy. Ther Deliv 2011;2:1001e14. [14] Hong C, Kang J, Kim H, Lee C. Photothermal properties of inorganic nanomaterials as therapeutic agents for cancer thermotherapy. J Nanosci Nanotechnol 2012;12:4352e5. [15] Zhang ZJ, Wang J, Chen CY. Near-infrared light-mediated nanoplatforms for cancer thermo-chemotherapy and optical imaging. Adv Mater 2013;25:3869e 80. [16] You J-O, Guo P, Auguste DT. A drug-delivery vehicle combining the targeting and thermal ablation of her2þ breast-cancer cells with triggered drug release. Angew Chem Int Ed 2013;52:4141e6. [17] Hauck TS, Jennings TL, Yatsenko T, Kumaradas JC, Chan WCW. Enhancing the toxicity of cancer chemotherapeutics with gold nanorod hyperthermia. Adv Mater 2008;20:3832e8. [18] Zhang ZJ, Wang LM, Wang J, Jiang XM, Li XH, Hu ZJ, et al. Mesoporous silicacoated gold nanorods as a light-mediated multifunctional theranostic platform for cancer treatment. Adv Mater 2012;24:1418e23. [19] Park H, Yang J, Lee J, Haam S, Choi I-H, Yoo K-H. Multifunctional nanoparticles for combined doxorubicin and photothermal treatments. ACS Nano 2009;3: 2919e26. [20] You J, Zhang GD, Li C. Exceptionally high payload of doxorubicin in hollow gold nanospheres for near-infrared light-triggered drug release. ACS Nano 2010;4:1033e41. [21] Xiao Z, Ji C, Shi J, Pridgen EM, Frieder J, Wu J, et al. DNA self-assembly of targeted near-infrared-responsive gold nanoparticles for cancer thermochemotherapy. Angew Chem Int Ed 2012;51:11853e7. [22] Ren F, Bhana S, Norman DD, Johnson J, Xu LJ, Baker DL, et al. Gold nanorods carrying paclitaxel for photothermal-chemotherapy of cancer. Bioconjugate Chem 2013;24:376e86.

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[23] Song JB, Pu L, Zhou JJ, Duan B, Duan HW. Biodegradable theranostic plasmonic vesicles of amphiphilic gold nanorods. ACS Nano 2013;7:9947e60. [24] Liu HY, Chen D, Li LL, Liu TL, Tan LF, Wu XL, et al. Multifunctional gold nanoshells on silica nanorattles: a platform for the combination of photothermal therapy and chemotherapy with low systemic toxicity. Angew Chem Int Ed 2011;50:891e5. [25] Chen HJ, Shao L, Li Q, Wang JF. Gold nanorods and their plasmonic properties. Chem Soc Rev 2013;42:2679e724. [26] Tomalia DA, Baker H, Dewald J, Hall M, Kallos G, Martin S, et al. A new class of polymers: starburst-dendritic macromolecules. Polym J 1985;17:117e32. [27] Grayson SM, Fréchet JMJ. Convergent dendrons and dendrimers: from synthesis to applications. Chem Rev 2001;101:3819e68. [28] Astruc D, Boisselier E, Ornelas C. Dendrimers designed for functions: from physical, photophysical, and supramolecular properties to applications in sensing, catalysis, molecular electronics, photonics, and nanomedicine. Chem Rev 2010;110:1857e959. [29] Cheng YY, Zhao LB, Li YW, Xu TW. Design of biocompatible dendrimers for cancer diagnosis and therapy: current status and future perspectives. Chem Soc Rev 2011;40:2673e703. [30] Medina SH, El-Sayed MEH. Dendrimers as carriers for delivery of chemotherapeutic agents. Chem Rev 2009;109:3141e57. [31] El Kazzouli S, Mignani S, Bousmina M, Majoral J-P. Dendrimer therapeutics: covalent and ionic attachments. New J Chem 2012;36:227e40. [32] Bronstein LM, Shifrina ZB. Dendrimers as encapsulating, stabilizing, or directing agents for inorganic nanoparticles. Chem Rev 2011;111:5301e44. [33] Shen MW, Shi XY. Dendrimer-based organic/inorganic hybrid nanoparticles in biomedical applications. Nanoscale 2010;2:1596e610. [34] Chang YL, Liu N, Chen L, Meng XL, Liu YJ, Li YP, et al. Synthesis and characterization of DOX-conjugated dendrimer-modified magnetic iron oxide conjugates for magnetic resonance imaging, targeting, and drug delivery. J Mater Chem 2012;22:9594e601. [35] Kojima C, Kono K, Maruyama K, Takagishi T. Synthesis of polyamidoamine dendrimers having poly(ethylene glycol) grafts and their ability to encapsulate anticancer drugs. Bioconjugate Chem 2000;11:910e7. [36] Willner D, Trail PA, Hofstead SJ, King HD, Lasch SJ, Braslawsky GR, et al. (6maleimidocaproyl)hydrazone of doxorubicin. A new derivative for the preparation of immunoconjugates of doxorubicin. Bioconjugate Chem 1993;4: 521e7. [37] Jana NR. Gram-scale synthesis of soluble, near-monodisperse gold nanorods and other anisotropic nanoparticles. Small 2005;1:875e82. [38] Du JZ, Du XJ, Mao CQ, Wang J. Tailor-made dual pH-sensitive polymere doxorubicin nanoparticles for efficient anticancer drug delivery. J Am Chem Soc 2011;133:17560e3. [39] Kono K, Ozawa T, Yoshida T, Ozaki F, Ishizaka Y, Maruyama K, et al. Highly temperature-sensitive liposomes based on a thermosensitive block copolymer for tumor-specific chemotherapy. Biomaterials 2010;31:7096e105. [40] Venkatesan R, Pichaimani A, Hari K, Balasubramanian PK, Kulandaivel J, Premkumar K. Doxorubicin conjugated gold nanorods: a sustained drug delivery carrier for improved anticancer therapy. J Mater Chem B 2013;1:1010e8. [41] Lee H, Larson RG. Effects of pegylation on the size and internal structure of dendrimers: self-penetration of long PEG chains into the dendrimer core. Macromolecules 2011;44:2291e8. [42] Kono K, Kojima C, Hayashi N, Nishisaka E, Kiura K, Watarai S, et al. Preparation and cytotoxic activity of poly(ethylene glycol)-modified poly(amidoamine) dendrimers bearing adriamycin. Biomaterials 2008;29:1664e75.

Please cite this article in press as: Li X, et al., PEGylated PAMAM dendrimeredoxorubicin conjugate-hybridized gold nanorod for combined photothermal-chemotherapy, Biomaterials (2014), http://dx.doi.org/10.1016/j.biomaterials.2014.04.043

PEGylated PAMAM dendrimer-doxorubicin conjugate-hybridized gold nanorod for combined photothermal-chemotherapy.

We prepared pH-sensitive drug-dendrimer conjugate-hybridized gold nanorod as a promising platform for combined cancer photothermal-chemotherapy under ...
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