Intraorgan Targeting of Gold Conjugates for Precise Liver Cancer

Aug 25, 2017 - §School of life sciences and ∥State Key Laboratory of Analytical Chemistry for Life Science and Collaborative Innovation Center of C...
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Intraorgan Targeting of Gold Conjugates for Precise Liver Cancer Treatment Yuan-Yue Gao,†,# Huan Chen,‡,# Ying-Ying Zhou,† Lin-Tao Wang,§ Yanglong Hou,*,⊥ Xing-Hua Xia,∥ and Ya Ding*,† †

State Key Laboratory of Natural Medicines, Department of Pharmaceutical Analysis, and ‡Department of Biochemistry, School of Life Science and Technology, China Pharmaceutical University, Nanjing 210009, China § School of life sciences and ∥State Key Laboratory of Analytical Chemistry for Life Science and Collaborative Innovation Center of Chemistry for Life Sciences, School of Chemistry and Chemical Engineering, Nanjing University, 22 Hankou Road, Nanjing 210093, China ⊥ Department of Materials Science and Engineering, College of Engineering and Beijing Key Laboratory for Magnetoelectric Materials and Devices, Peking University, Beijing 100871, China S Supporting Information *

ABSTRACT: Intraorgan targeting of chemical drugs at tumor tissues is essential in the treatment of solid tumors that express the same target receptor as normal tissues. Here, asialoglycoprotein receptor (ASGP-R)-targeting paclitaxel-conjugated gold nanoparticles (Gal/ PTX-GNPs) are fabricated as a demonstration to realize the precise treatment of liver cancer. The enhanced biological specificity and therapeutic performance of drugs loaded on nanoparticles not only rely on the ligands on carriers for receptor recognition but are also determined by the performance of gold conjugates with designed structure. The tumor cell selectivity of the designed conjugates in liver tumor (HepG2) cells is close to six times of that incubated with control conjugates without galactose modification in liver normal (L02) cells. The drug level in tumor versus liver of Gal/PTX-GNPs is 121.0% at 8 h post injection, a 15.7fold increase in the tumor specificity compared to that of GNPs conjugated with PTX only. This intraorgan-targeting strategy results in a considerable improvement of performance in treating both Heps heterotopic and orthotopic xenograft tumor models, which is expected to be used for the enhanced antitumor efficacy and reduced hepatotoxicity in liver cancer treatment. KEYWORDS: intraorgan targeting, paclitaxel-conjugated gold nanoparticles, structure design, tumor penetration, liver cancer treatment (PTX)-conjugated GNPs23 and 13.1% for liposome-encapsulating PTX-conjugated GNPs at 8 h post injection.25 This phenomenon of liver selection stemmed from the role of reticuloendothelial system (RES), which possessed endothelial and reticular attributes as well as the ability to take up colloidal particles.26−28 Thus, there is a misconception in the treatment of liver cancer that “most NPs end up in the liver, so who needs targeting”, and liver targeting is considered to be a promising strategy to increase the drug accumulation in liver tumor. However, the treatment of liver cancer is different from that of liver diseases; liver targeting simultaneously increases drug levels in normal liver tissue. Therefore, an intraorgan targeting of gold conjugates at tumor tissues in liver is of great significance to improve the antitumor efficacy and reduce hepatotoxicity in hepatic carcinoma treatment. Galactose-functionalized nanoparticles, especially GNPs, provide a promising strategy called cluster effect29−31 for

1. INTRODUCTION In the receptor-mediated delivery of chemical drugs for cancer therapy, the targeting capacity is mainly determined by the differences in receptor expression between normal and tumor cells and the affinity of the receptors with its substrate.1−3 However, in the treatment of solid tumors that express the same target receptor as normal tissues, such as the asialoglycoprotein receptor (ASGP-R) in hepatic carcinoma,4−6 the efficacy and safety of this strategy remain to be further explored.7,8 Gold nanoparticles (GNPs) have been successfully used as an excellent model system for understanding the biological effects of nanomaterials9−12 and as promising and efficient nanocarriers for numerous chemical drugs in the fundamental science13−19 and clinical trials.20,21 It has been reported that surface-functionalized GNPs display enhanced permeability and retention (EPR) effect, which makes drugs covalently linked to their surface and apparently accumulated in tumor compared with free drugs.22−25 Nevertheless, most of the loaded drugs were collected in the liver22−25 and the concentration ratio of drugs in tumor to the liver was as low as 7.7% for paclitaxel © 2017 American Chemical Society

Received: June 22, 2017 Accepted: August 25, 2017 Published: August 25, 2017 31458

DOI: 10.1021/acsami.7b08969 ACS Appl. Mater. Interfaces 2017, 9, 31458−31468

Research Article

ACS Applied Materials & Interfaces

Figure 1. Schematic illustration of ASGP-R-targeting PTX-conjugated GNPs for intraorgan-targeting treatment of liver cancer. (a) Structure of Gal/ PTX-GNPs and PTX-GNPs; (b) orthotopic hepatic xenograft model and drug accumulation in tumor; (c) therapeutic mechanism of PTX delivered and released by Gal/PTX-GNPs in liver tumor cells.

circulation aroused from the PEG molecules, (2) clearance-free RES and enhanced drug accumulation in tumor via the EPR effect due to the suitable size (Figure 1b), (3) selective targeting of liver tumor cells on the basis of the recognition of ASGP-R by galactose moiety (Figure 1c),31−34 and (4) high stability in circulation and specific drug release of PTX responded to high glutathione (GSH) and esterase concentrations inside tumor cells (Figure 1c).23 This strategy promises new perspectives on the accuracy improvement of chemical drugs in hepatic carcinoma treatment and their intact effect on normal liver.

treating liver because the high density and multiantennary modification of galactose increased the dissociation constant (Kd value) of particles with ASGP-R. Herein, we propose that the biological specificity of drugs to treat hepatic carcinoma will not only rely on the ligands on the carriers for receptor recognition but also be determined by the performance of gold conjugates with designed structure. To prove our hypothesis, GNPs, PTX, and galactose (Gal) are employed as the model carrier, drug, and targeting ligand to fabricate ASGP-R-targeting PTX-conjugated GNPs for a precise treatment of liver cancer. In the designed system, ASGP-R-targeting ligand (galactose− poly(ethylene glycol)−lipoic acid, Gal−PEG−LA) and therapeutic compound (paclitaxel−poly(ethylene glycol)−thioglycolic acid, PTX−PEG−TA) are simultaneously incorporated on the surface of GNPs (Figure 1a). This structure possesses unique features, including: (1) high dispersion and long-time

2. MATERIALS AND METHODS 2.1. Materials. Hydrogen tetrachloroaurate hydrate (HAuCl4· 4H2O) was purchased from Shanghai Chemical Reagent Company (China). Poly(ethylene glycol) (molecular weights of 400 and 1000 Da), N,N′-dicyclohexylcarbodiimide (DCC), 4-dimethylamiopryidine 31459

DOI: 10.1021/acsami.7b08969 ACS Appl. Mater. Interfaces 2017, 9, 31458−31468

Research Article

ACS Applied Materials & Interfaces (DMAP), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC·HCl), and N-hydroxysulfosuccinimide (NHS) were provided by Sigma-Aldrich. Lipoic acid (LA, 99%) was obtained from Nanjing Zelang Pharmaceutical Technology (China). PTX was obtained from Yew Pharmaceutical Co. (Jiangsu, China). PEG−LA and Gal−PEG−LA were synthesized according to the procedures described in the Supporting Information. All starting materials were obtained from commercial suppliers and used without further purification unless otherwise stated. Deionized water (>18 MΩ, Purelab Classic Corp.) was used in the preparation of all aqueous solutions. 2.2. Apparatus. The chemical structures of the organic ligands in KBr disks were determined using a Tensor-27 Fourier transform IR spectrometer (Bruker). 1H and 13C NMR spectra were recorded on a Bruker Avance AV-400 spectrometer. Chemical shifts (δ) were listed in parts per million (ppm) using tetramethylsilane as an internal reference. UV−vis spectra were detected with a UV-3600 spectrophotometer (Shimadzu, Japan). A Zetasizer 3000HS instrument (Malvern Instruments, Malvern, UK) with 633 nm He−Ne lasers at 25 °C was used to measure the hydrodynamic diameters and ζ potentials. With an acceleration voltage of 200 kV, gold conjugates were imaged using a JEM-200CX transmission electron microscope (JEOL). Gold concentration was measured by Optima 5300DV inductively coupled plasma mass spectrometry (ICP-MS) (PE). Surface element analysis of gold conjugates was carried out using a PHI 5000 Versaprobe X-ray photoelectron spectroscope (UlVAC-PHI, Japan). A Shimadzu LC20A series with a Hypersil BDS C18 reversed-phase chromatography column (150 × 4.6 mm2) and a UV detector set at 227 nm were employed to perform analytical reversed-phase high-performance liquid chromatography (RP-HPLC). The mobile phase of methanol/ water (70:30, v/v) was used at the flow rate of 0.8 mL/min (35 °C). 2.3. Preparation of the Conjugates. Citrate-protected GNPs were synthesized in a single-phase system according to the previous reports.35 The molar ratio of Gal−PEG400−LA to PTX−PEG1000−TA in Gal/ PTX-GNPs was set at 1:1. For example, Gal−PEG400−LA (0.14 mmol) was dissolved in methanol (1 mL), 1.5 equiv sodium borohydride (NaBH4, 0.21 mmol, 1 mg/mL) in ice water was added, the mixed solution was stirred for 1 h at 4 °C, and the pH value was adjusted to 6 by adding HCl (1 mol/L). A methanol solution containing PTX−PEG1000−TA (0.14 mmol) was added into the above solution and stirred for 30 min for sufficient mixing. Then, citrateprotected GNPs (3 mL) were added to the mixture solution and the mixture was stirred for 1 h to obtain the gold conjugate solution. The gold conjugate solutions were then aged overnight and dialyzed in a fresh deionized water solution for 72 h (molecular weight cutoff, MWCO = 7000 Da). After freeze drying, the products were readily dispersed in water and phosphate-buffered saline (PBS). PTX-GNPs were prepared according to the method described above using the same molar number of PEG400−LA instead of Gal−PEG400−LA. 2.4. Stability Studies. PTX-GNPs and Gal/PTX-GNPs were incubated at 37 °C in different media: (1) 0.03 M PBS at pH 7.4, (2) 0.03 M PBS at pH 5.5, (3) higher ionic strength of 0.2 M PBS at pH 7.4, and (4) 0.03 M PBS at pH 7.4 with 2% serum. Absorbance of the conjugates at 520 nm was determined at the indicated time points. The stability (%) was calculated by the equation: stability (%) = 1 − | Ai − A0|/A0, where Ai and A0 are the absorbances at the detected time point and the beginning time, respectively. 2.5. In Vitro Drug Release. The release of PTX from gold conjugates was investigated by a dialysis method.22,24 Gal/PTX-GNPs containing 1 mg of PTX was dissolved in 1 mL of PBS at pH 7.4 and placed in a dialysis bag (MWCO = 7000 Da). To investigate the drug release behavior, the PBS solution containing Tween 80 (0.1%, w/v) at pH 7.4 was employed as the release medium. GSH concentrations in the release medium were set at 2 μM (extracellular level) and 10 mM (intracellular level), and esterase from porcine liver (20 units/ mL) was added. The release medium was stirred at 100 rpm at 37 °C. At predetermined time intervals, 200 μL aliquots of the release medium were withdrawn and the same volume of a fresh medium was added. The in vitro release behavior of the Gal/PTX-GNPs was

measured by RP-HPLC analysis. All assays were performed in triplicate. 2.6. Cell Culture. HepG2 and L02 cell lines were obtained from the China Center for Type Culture Collection (Shanghai, China). The cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum (FBS) and incubated at 37 °C in 5% CO2 humidified atmosphere. 2.7. Detection of Intracellular Gold and Drug Contents. HepG2 and L02 were separately seeded in a 100 mm dish. After 80% overspread, the cells were incubated with PTX, PTX-GNP, and Gal/ PTX-GNP solutions containing the same amount of PTX (4 μg/mL) at 37 °C for an additional 4 or 8 h. The cells were subsequently washed with PBS twice and lysed with a cell lysis buffer. The resulting supernatant was then treated with methanol for protein precipitation. After removal of precipitates by centrifugation, a sample of 20 μL of the supernatant was analyzed by the RP-HPLC method to determine the amount of PTX in cells. The concentration of gold in the supernatant of PTX-GNP and Gal/PTX-GNP groups was detected by the ICP-MS method. 2.8. Intracellular Distribution of Gal/PTX-GNPs. HepG2 cells were seeded in six-well plates at a density of 8 × 105 cells per well and incubated for 24 h to reach 80% confluence. The cells were then incubated for additional 4 or 8 h with Gal/PTX-GNPs at a PTX dose of 4 μg/mL. Subsequently, transmission electron microscopy (TEM) samples were prepared and imaged according to our previous report.25 2.9. Cytotoxicity. HepG2 cells were seeded in a 96-well plate (1 × 104/well). After incubation for 24 h, the cells were separately treated with different concentrations of PTX, PTX-GNP, and Gal/PTX-GNP solutions for 48 h. The cell viability was determined by 3-(4, 5dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assay as described in the previous report.25 Data are presented as mean ± standard deviation (SD) (n = 5). 2.10. Animals and Tumor Model Establishment. Healthy Sprague-Dawley (SD) rats (male, 200 ± 20 g) and Institute of Cancer Research (ICR) mice (male, 20 ± 2 g) were purchased from the Experimental Animal Center of Zhejiang Province (Certificate number: SCXK (Zhe) 2014-0001) and Comparative Medicine Centre of Yangzhou University (Certificate number: SCXK (Su) 2012-0004), respectively, and received care in compliance with the guidelines outlined in the Guide for the Care and Use of Laboratory Animals. Mouse hepatoma (Heps, Hepa 1−6, ATCCCRL-1830) cells were obtained from Stem Cell Bank, China Academy of Sciences, and maintained by intraperitoneal passage in ICR mice. Heps cells were collected from the peritoneal cavity, washed, and diluted in RPMI1640 medium. To establish the heterotopic tumor model, Heps cells were injected subcutaneously in the left armpits of mice at the concentration of 1 × 106 cells per 0.1 mL of PBS. The orthotopic model of liver cancer was generated by direct intrahepatic injection of Heps cells. Healthy male ICR mice were anesthetized, and the abdomen was opened through a midline incision to expose the liver. Then, 10 μL of Heps cell suspension (2.5 × 107 cells/mL) was implanted into a lobe of the liver using a microinjector. Gentle compression was applied for 20 s using gelatin sponge to avoid bleeding and reflux of the cells. The abdomen was closed and the mice were transferred to a warm cage for full recovery. Penicillin was administered in drinking water for 3 days. 2.11. Pharmacokinetic Studies. Healthy SD rats (male, 200 ± 20 g) were randomly divided into three groups (n = 4). Taxol, PTXGNPs, and Gal/PTX-GNPs were intravenously administered through the tail vein with an equivalent dose of 7 mg/kg of PTX. Blood samples (0.5 mL) were collected from the venous plexus at various times (0.08, 0.17, 0.25, 0.5, 1, 2, 4, 8, 12, 24, and 48 h) into heparinized tubes. The drug concentrations of plasma were determined according to our previous report.25 2.12. In Vivo Antitumor Efficacy. To evaluate the in vivo antitumor efficacy of different PTX formulations on Heps tumor xenograft models, mice with a tumor volume of 200 mm3 after Heps tumor inoculation were divided into four groups (n = 5): saline, Taxol, PTX-GNP, and Gal/PTX-GNP solutions containing PTX concentration of 1 mg/mL. Each treatment group was composed of five 31460

DOI: 10.1021/acsami.7b08969 ACS Appl. Mater. Interfaces 2017, 9, 31458−31468

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ACS Applied Materials & Interfaces

Figure 2. Characterization and in vitro drug release of gold conjugates. (a) Hydrodynamic diameters, (b) ζ potentials, (c) XPS images and atomic concentrations, and (d) TEM images of PTX-GNPs and Gal/PTX-GNPs. (e) Drug release profiles of PTX from Gal/PTX-GNPs in (I) 0.03 M PBS at pH 7.4 with intracellular and extracellular concentration of GSH (10 mM and 2 μM, respectively), (II) 0.03 M PBS at pH 7.4 with and without esterase from porcine liver (20 units/mL), and (III) 0.03 M PBS at pH 7.4 vs the same medium containing GSH (10 mM) and esterase from porcine liver (20 units/mL). Data are presented as mean ± SD (n = 3). tumor-bearing mice that were injected through the tail vein with 20 mg/kg of the indicated formulation in 2 day intervals. Tumor volume was calculated from the following equation: tumor volume (mm3) = (length × width2)/2. Finally, tumor tissues were excised, washed with saline, and weighed. The therapeutic effect of the formulations was also determined in the orthotopic liver cancer model. Seven days after intrahepatic tumor cell implantation (day 0), the mice were divided into four groups and injected intravenously with saline, Taxol, PTX-GNPs, or Gal/PTXGNPs at 20 mg of PTX/kg body weight once every day. After 10 days, the liver tissues bearing tumors were excised, washed with saline, and weighed. 2.13. Liver and Tumor Distribution. ICR male mice (20 ± 2 g) with a tumor volume of 200 mm3 after Heps tumor inoculation were randomly divided into three groups (n = 3): Taxol, PTX-GNPs, and Gal/PTX-GNPs. Each group was injected through the tail vein with the indicated formulation at a dose of 20 mg/kg. After administration, the mice were sacrificed to excise liver and tumor tissues at predetermined time points (0.5, 1, 4, 8, 12, and 24 h) and the content of PTX in the tissues was determined according to the previous literature.25 2.14. Histological Analysis. The excised tumor tissues or liver tissues were weighed and stained with hematoxylin and eosin (H and

E) for histological observation (Olympus TH4-200, Olympus Optical Co. Ltd., Japan). The tissue sections from orthotopic liver-tumorbearing mice were additionally stained with TdT-mediated dUTP nick end labeling (TUNEL) using the TUNEL Apoptosis Detection Kit (Roche). 2.15. Immunofluorescence Analysis. For immunofluorescence (IF) analysis, paraffin-embedded tissue sections were deparaffinized and hydrated and then slides were incubated in 1× citrate buffer for 10 min for antigen retrieval. Afterward, the sections were blocked with 5% bovine serum albumin in PBS for 30 min and stained with an anti-αTublin antibody (ab80779, Abcam, Shanghai, China) at 4 °C overnight. Then, a 30 min incubation with Alexa Fluor 546-labeled donkey antimouse secondary antibody (Life Technologies) was followed by nuclear staining with 4,6-diamidino-2-phenylindole (DAPI). All images were captured on a Nikon fluorescent microscope equipped with a digital camera (TE2000-U, Nikon) and analyzed using NIS-Elements Basic Research (Nikon) software with a magnification of 400×. 2.16. Statistical Analysis. All of the results are presented as mean ± SD. The significance of the differences between groups was evaluated using the Student t test when only two groups were compared or using one-way ANOVA when there are more than two groups. P ≤ 0.05 was considered to be statistically significant. 31461

DOI: 10.1021/acsami.7b08969 ACS Appl. Mater. Interfaces 2017, 9, 31458−31468

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ACS Applied Materials & Interfaces

Figure 3. Cellular selectivity, intracellular distribution, drug release, and cytotoxicity. (a) Intracellular gold contents of PTX-GNPs and Gal/PTXGNPs in L02 and HepG2 cells after 4 and 8 h incubation (mean ± SD, n = 3) detected by the ICP-MS assay. *P < 0.05, **P < 0.01 (one-sample ttest). (b) TEM images of HepG2 cells incubated with drug-free medium for 4 h (I) and Gal/PTX-GNPs for 4 h (II) and 8 h (III); (I′), (II′), and (III′) are the magnified images of (I), (II), and (III), respectively. (c) Intracellular PTX amount of PTX solution, PTX-GNPs, and Gal/PTX-GNPs in L02 and HepG2 cells after 4 and 8 h incubation (mean ± SD, n = 3) measured by the RP-HPLC method. *P < 0.05, **P < 0.01 (one-sample ttest). (d) Cytotoxicity of PTX solution, PTX-GNPs, and Gal/PTX-GNPs with varying PTX concentrations after 48 h incubation with HepG2 cells (mean ± SD, n = 5).

3. RESULTS AND DISCUSSION 3.1. Preparation and Characterization. For the fabrication of gold conjugates, citrate-protected GNPs of ca. 3 nm were prepared first.35,36 Two types of molecules for GNP surface modification were designed: Gal−PEG−LA and PTX− PEG−TA (Figure 1a). The synthesis and characterization of Gal−PEG−LA and PTX−PEG−TA are presented in the Supporting Information (Figures S1−S4) and our previous report,23 respectively. After the performance evaluation of the conjugates only bearing targeting ligand or therapeutic compound, Gal−PEG400−LA showed the highest targeting ability (Figures S5−S7, Supporting Information) and PTX− PEG1000−TA exhibited the best antitumor efficacy.23 To balance the ability of tumor cell targeting and killing for gold conjugates, Gal−PEG400−LA and PTX−PEG1000−TA are

simultaneously conjugated on GNPs at a molar ratio of 1:1, denoted as Gal/PTX-GNPs (Figure 1a and Figure S8, Supporting Information). At the same time, the control conjugates PTX-GNPs, shown in Figure 1a, are prepared using PEG400−LA and PTX−PEG1000−TA at the same molar ratio described above. The surface composition of gold conjugates was characterized to evaluate their receptor-binding capacity because the exposure of sugar moiety is essential for the recognition by ASGP-R.37,38 The addition of galactose does not increase the hydrodynamic diameter of gold conjugates. On the contrary, dynamic light scattering measurements (Figure 2a) show that Gal/PTX-GNPs are 16.4 nm smaller (234.7 ± 3.8 nm) than PTX-GNPs (251.1 ± 2.3 nm), suggesting the organic shell shrink due to the hydrogen-bonding interactions between 31462

DOI: 10.1021/acsami.7b08969 ACS Appl. Mater. Interfaces 2017, 9, 31458−31468

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ACS Applied Materials & Interfaces saccharides on the surface of GNPs.39 The attachment of galactose results in a slight increase in surface charge, with the ζ potential changing from −26.86 ± 3.98 mV (PTX-GNPs) to −18.93 ± 5.35 mV (Gal/PTX-GNPs) (Figure 2b). To directly confirm the galactose position in the conjugates, surface elemental analysis of both conjugates is performed by X-ray photoelectron spectroscopy (XPS, Figure 2c). Gal/PTX-GNPs have higher nitrogen percentage (1.33 atom %) than PTXGNPs (0.42 atom %) (Figure 2c) due to the attachment of ethylenediamine spacer connecting galactose with PEG (Figure 1a), indicating the exposure of galactose on the surface of the conjugates. It can be explained by the coassembly of the rigid short PEG (Gal−PEG400−LA)40 and the flexible PTX− PEG1000−TA that possesses a coiled PEG geometry,41,42 as shown in Figure 1a. 3.2. Dispersity, Stability, and in Vitro Drug Release. Different from the GNPs modified by PTX−PEG1000−TA only,23 both Gal/PTX-GNPs and PTX-GNPs in this work are well dispersed in aqueous solution. TEM images show that the gold cores in the conjugates have an average diameter of ca. 3 nm and good dispersity (Figure 2d). It can be due to the coassembly of Gal−PEG400−LA or PEG400−LA with PTX− PEG1000−TA, which breaks the hydrophobic interaction between PTX molecules and improves the dispersion of the conjugates. This break also results in a high in vitro stability of Gal/PTX-GNPs (Figure S9, Supporting Information). Within 10 h measurements, the change of the ultraviolet−visible (UV− vis) absorption intensity for the conjugates at 520 nm is less than 30% in various simulated environments, such as physiological condition, high salt, low pH, and the presence of serum. The release profiles of Gal/PTX-GNPs exhibit a similar drug release behavior to the GNPs connected with only PTX−PEG1000−TA (Figures 1c and 2e).23 Free PTX tends to be released under the conditions of both high concentrations of GSH (such as intracellular GSH concentration of 10 mM)43 and esterase (e.g., esterase from porcine liver)44,45 after the internalization of conjugates by tumor cells. 3.3. Cellular Uptake and Selectivity. Thus, cellular internalization and selectivity experiments of Gal/PTX-GNPs were conducted against the human hepatic cells (L02) and human hepatocellular carcinoma cell line (HepG2). After incubation of the test samples for 4 h, the gold element in cells was detected by inductively coupled plasma mass spectrometry (ICP-MS) (Figure 3a). It is worth noting that Gal/PTX-GNPs show the highest uptaking property as revealed by the gold content in HepG2 cells (18.03 ± 6.01 μg/L), which is 5.85 times of that for PTX-GNPs in L02 cells (3.08 ± 0.44 μg/L). This enhancement of cell selectivity comes from two aspects. The first is differences in ASGP-R expression between normal and tumor liver cells. Because HepG2 cells express approximately 2 times higher ASGP-R than L02 cells,33 Gal/PTXGNPs show a consistent increase, approximately 2 times, in intracellular gold in HepG2 cells (18.03 ± 6.01 μg/L) compared to L02 cells (9.90 ± 2.25 μg/L). The second is sensitivity differences of cell lines toward gold conjugates. The gold uptake of PTX-GNPs in HepG2 cells (10.34 ± 6.89 μg/L) is more than 3 times of that in L02 cells (3.08 ± 0.44 μg/L), even though the particle surface is absent of galactose moiety. These data demonstrate the sensitivity of HepG2 cells toward gold conjugates, which can be ascribed to the different membrane composition and structure of tumor cells from normal liver L02 cells as well as the nature of gold conjugates, although it is not clear yet. Taking the above two factors

together, the selectivity of tumor liver cells for Gal/PTX-GNPs is magnified close to 6 times of PTX-GNPs in normal liver cells. Moreover, after 8 h incubation, the gold amount in both cells decreases, which might imply the possible efflux or digestion of GNPs from 4 to 8 h. 3.4. Intracellular Distribution, Drug Release, and Cytotoxicity. To reveal the possible treatment mechanism, the intracellular distribution of Gal/PTX-GNPs was investigated by TEM (Figure 3b). Compared to HepG2 cells incubated with cell culture medium (Figure 3b,I,I′), Gal/PTXGNPs are found in some small vesicles present in the cytoplasm after 4 h incubation (Figure 3b,II,II′). After 8 h incubation (Figure 3b,III,III′), gold cores of the conjugates remain in the cytoplasm. However, it is important to note that a juxtanuclear movement is found at this time (Figure 3b,III,III′), demonstrating an endocytosis uptake and lysosomal location of Gal/PTX-GNPs in HepG2 cells,46,47 although the escape mechanism is not clear now. Thus, the intracellular distribution of the designed conjugates reveals the drug release site, cytoplasm, where Tubulin exists, responsible for the biological activity of PTX.48−50 After confirming the drug release site, intracellular drug concentrations of samples were detected by the reversed-phase high-performance liquid chromatography (RP-HPLC) method using PTX solution as the control. Because free PTX in solution moves rapidly across the cell membrane followed by a concentration gradient, high concentration of PTX inside both L02 and HepG2 cells after 4 and 8 h incubation can be observed (Figure 3c). In L02 cells, PTX levels for PTXconjugated GNPs with and without galactose modification are very low after both 4 and 8 h incubation (Figure 3c, columns for L02 cells). These values are equivalent to 1/5−1/3 of the PTX solution group, indicating that the galactose modification has little influence on the uptake of conjugates against cell lines with low expression of ASGP-R. In the case of HepG2 cells, intracellular PTX amount of PTX-GNPs keeps almost unchanged at a low level (Figure 3c, columns of PTX-GNPs for HepG2 cells). However, a rapid rising of PTX is observed in Gal/PTX-GNP-incubated group at two time points (Figure 3c, columns of Gal/PTX-GNPs for HepG2 cells), which are equal to 4.7−5.6 folds of PTX-GNPs and exceed the level of PTX solution group after 8 h incubation. These comparable drug levels of Gal/PTX-GNPs to PTX solution indicate the effective delivery and release of drug by the Gal/PTX-GNPs in their selected tumor cells. Interestingly, contrary to the intracellular gold content within the incubation for 8 h (Figure 3a, columns of Gal/PTX-GNPs for HepG2 cells), the drug concentration remains stable and slightly increases (Figure 3c, columns of Gal/PTX-GNPs for HepG2 cells). This inconsistency in concentration changes of gold and PTX inside tumor cells implies that the reflux or digestion of gold core occurs after the drug release from the conjugates. On the basis of the high cell selectivity and effective drug release of the designed conjugates, the cytotoxicity of PTXconjugated GNPs against HepG2 cells can be a strong cytological evidence to confirm their tumor-cell-killing ability. As shown in Figure 3d, in a relatively high concentration range of PTX (20−50 μg/mL), the cell viability is significantly reduced by Gal/PTX-GNPs as compared to PTX solution and PTX-GNPs. This concentration-dependent decrease in cell viability exhibits a dosage effect on the cell-killing ability of Gal/ PTX-GNPs. Therefore, the accumulation of conjugates around 31463

DOI: 10.1021/acsami.7b08969 ACS Appl. Mater. Interfaces 2017, 9, 31458−31468

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ACS Applied Materials & Interfaces

Figure 4. In vivo antitumor efficacy in Heps heterotopic xenograft models. (a) Tumor volume changes, (b) typical photos of tumor tissues, (c) tumor weight, (d) H&E-stained tumor tissue sections with magnification of 400× (the scale bar is 50 μm), and (e) body weight changes of saline, Taxol, PTX-GNPs, and Gal/PTX-GNPs in Heps tumor xenograft ICR mouse models during the 10 day treatment or at 10 days post injection. Each point in (a, c, and e) represents mean ± SD (n = 5). Biodistribution profiles of PTX in (f) tumor and (g) liver tissues of Heps tumor-bearing mice following intravenous injection of Taxol, PTX-GNPs, and Gal/PTX-GNPs at PTX dose of 20 mg/kg at different times. Data represent mean value ± SD (n = 3). The PTX concentration ratio of tumor to liver for PTX-GNPs and Gal/PTX-GNPs is also presented in (h). *P < 0.05, **P < 0.01 (onesample t-test).

Gal/PTX-GNPs show the highest area under the plasma concentration−time curve (AUC0→48, 26.04 ± 0.94 μg/mL/ kg), t1/2 (43.83 ± 7.26 h), and MRT (59.26 ± 8.42 h) values among test samples, indicating their good stability in long circulation time. At the same time, Gal/PTX-GNPs also show the lowest Vd (9.67 ± 1.15 L) and CL (0.15 ± 0.01 L/h) data, confirming the low elimination rate of PTX in the conjugates. This long-time blood circulation of the conjugates will raise the possibility of the PTX retention in the tumor when the conjugate passes through the tumor vasculature via EPR effect. 3.6. In Vivo Antitumor Efficacy in Heps Heterotopic Xenograft Models. The liver-tumor-targeting ability of Gal/ PTX-GNPs was then tested using Heps heterotopic xenograft mice as the evaluation model, and the results are presented in Figure 4. From the second day, after treated with Gal/PTXGNPs, the mice with transplant Heps tumors begin to show the lowest tumor growth rate and the smallest tumor size among all test groups (Figure 4a,b). After the 10 day treatment, the tumor weight of the mice treated by Gal/PTX-GNPs is the lowest, which is 0.35, 0.77, and 0.75 folds of that of the mice treated

tumor cells in the tumor tissue is of great importance for their therapeutic performance. 3.5. Pharmacokinetic Studies. To accumulate the drug in the tumor by the conjugates, the long-time circulation in the blood compartments and the entrance of tumor vasculature via EPR effect are equally important.48 The plasma concentration− time profiles of PTX after intravenous administration of Gal/ PTX-GNPs were detected to investigate their in vivo circulation characteristics and were compared to results from the commercial formulation of PTX (Taxol) and PTX-GNPs (Figure S10a, Supporting Information). Taxol shows the lowest plasma concentration from 0.5 to 48 h, indicating a high plasma protein-binding rate and rapid elimination of PTX in the blood. Both Gal/PTX-GNPs and PTX-GNPs show higher PTX plasma concentrations than Taxol, but there is no significant difference between two conjugates. It indicates that these two conjugates are stable in the flow of blood arising from the modified PEG molecules, and the surface modification of galactose will not change this stability in vivo. In addition, the main pharmacokinetic parameters are shown in Figure S10b. 31464

DOI: 10.1021/acsami.7b08969 ACS Appl. Mater. Interfaces 2017, 9, 31458−31468

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Figure 5. In vivo antitumor efficacy in Heps orthotopic xenograft models. (a) Photos of liver tissues bearing tumors excised from mice treated with (I) saline, (II) Taxol, (III) PTX-GNPs, and (IV) Gal/PTX-GNPs. (b) Weight ratio of tumor: liver (g/g, %) on day 10. (c) Body weight changes and (d) survival rate during the 10 day treatment (n = 10). Each point in (b) and (c) represents mean ± SD (n = 5). Histological observation of the liver and tumor tissue sections stained with (e) hematoxylin and eosin (the scale bar is 100 μm) and (f) TdT-mediated dUTP nick end labeling using TUNEL kit, Roche (the scale bar is 50 μm). (g) Immunofluorescence assay of (I) normal liver tissue, (II) Heps orthotopic liver tumor tissue, and (III) tumor tissue treated with Gal/PTX-GNPs was performed to determine the expression of α-tublin (the scale bar is 50 μm). Red and blue fluorescences represent α-tublin and DAPI nuclear staining, respectively; (IV) quantitative immunofluorescence results showing the protein levels of α-Tublin in images I−III. The data are presented as mean ± SD (n = 6). *p < 0.05, **p < 0.01 (one-sample t-test).

with saline, Taxol, and PTX-GNPs, respectively (Figure 4c). The significant reduction in the volume and weight of tumor treated with Gal/PTX-GNPs can be ascribed to the accumulation and retention capacities of the conjugates in tumor tissues and the subsequent cell-killing effect by the release of drug. Therefore, histological examination of H&Estained tumor tissue sections at 10 days post injection was performed to observe the tumor damage (Figure 4d). Comparing to the dotted absence of nucleus in the tissue

section of other groups, the maximum area of necrosis is observed in the group of Gal/PTX-GNPs, suggesting an effective killing of tumor cells in heterogeneous tumor community. During the 10 day treatment, the body weight of the mice treated with Gal/PTX-GNPs gradually increases and becomes the heaviest group on the 10th day (33.0 ± 2.4 g, Figure 4e). Its increment (10.0 g) exceeds that of other groups, indicating an improved quality of life for the mice due to the low 31465

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Furthermore, to reveal the effectiveness of Gal/PTX-GNPs that interference with the normal function of microtubules in tumor cells, immunofluorescence analysis of α-Tubulin in normal liver and untreated and Gal/PTX-GNP-treated Heps orthotopic liver tumor has been carried out (Figure 5g). Because of the rapid cell division of tumor cells, the expression of α-Tubulin in tumor tissues is higher (Figure 5g, image II and histogram II) than that in normal liver tissues (Figure 5g, image I and histogram I). However, after the treatment of Gal/PTXGNPs, the fluorescence intensity in tumor tissues decreases obviously (Figure 5g, image III and histogram III). The drops of the fluorescence intensity indicate that the PTX released from Gal/PTX-GNPs exhibits a higher affinity than the primary antibody (α-Tubulin mAb) and competitively binds with αTubulin in the cytoplasm of tumor cells. This result confirms that the antitumor mechanism of Gal/PTX-GNPs occurs through the stabilization of the microtubule polymer and protecting it from disassembly by free PTX.52−54

hepatotoxicity of Gal/PTX-GNPs compared to other formulations. As shown in Figure 4f,g, Gal/PTX-GNPs show a sustained PTX concentration and the highest value among the test formulations in tumor from 4 to 12 h (Figure 4f), providing the reasonable explanation for the best tumor suppression in Figure 4a−c. The PTX concentration ratio of Gal/PTX-GNPs in tumor to liver is in the range of 59.3−121.0%, which is much higher than that of PTX-GNPs (41.0−88.0%, Figure 4h). These data are also much higher than the ones reported previously on PTX-conjugated GNPs (7.7%)23 and liposome-encapsulating PTX-conjugated GNPs (13.1%).25 This is a remarkable fact that the conjugates designed in this work can effectively accumulate PTX in tumors. In contrast, Taxol displays rapid location and elimination in both tumor and liver (Figure 4f,g). Its high concentrations at 0.5 and 1 h in liver (37.1 ± 6.5 and 26.8 ± 13.5 μg/g, respectively) explain the high toxicity of this commercial formulation (Figure 4e), especially in the clinical application. 3.7. In Vivo Antitumor Efficacy in Heps Orthotopic Xenograft Models. Because the above solid tumors are ectopically implanted in other sites rather than the liver that is highly vascular, ectopic transplantation can hardly reflect the actual microenvironment of the cancer located in the liver. Thus, a Heps orthotopic xenograft mouse model was established to further evaluate the in vivo antitumor activity of Gal/PTX-GNPs and their possible damage to normal liver. After 10 day treatment, all tumors were harvested with their surrounding liver tissue (Figure 5a). Both the volume and weight of tumors are further suppressed in the group treated with Gal/PTX-GNPs. The tumor volumes are too small (less than 1 mm3) to be measured (Figure 5a, the last row). The percentage of final weight of the tumor vs. liver (%, g/g) after 10 day treatment with Gal/PTX-GNPs is also quite low (9.1 ± 2.5%, Figure 5b). It is 0.18-, 0.43-, and 0.37-folds of that of saline, Taxol, and PTX-GNPs groups, respectively, demonstrating the superior tumor suppression of Gal/PTX-GNPs. Similar to the result in Heps heterotopic xenograft mice model (Figure 4e), the heaviest body weight (48.2 ± 5.9 g) and the fastest weight increment (15.8 g) of the mice are found in the Gal/ PTX-GNPs group (Figure 5c). It reflects the good quality of life in Heps orthotopic xenograft mice, which ensures a high survival rate. No mice died after 10 day treatment with Gal/ PTX-GNPs, whereas the survival rate of saline- and PTX-GNPtreated groups is 40% and no mouse survived in the Taxoltreated group on the 10th day (Figure 5d). Therefore, the changes of body weight and survival rate in Heps orthotopic xenograft mice clearly demonstrate a lower hepatotoxicity of Gal/PTX-GNPs than other test formulations. To confirm this hypothesis, histological sections are stained by hematoxylin and eosin (Figure 5e) and TdT-mediated dUTP nick end labeling (Figure 5f) to detect the cell-killing and apoptosis degree of liver and tumor tissues in Heps orthotopic liver tumor mice. Gal/PTX-GNPs result in the largest region of necrosis and the most significant apoptosis in the tumor tissue, but no apparent damage and apoptosis are found in the normal liver tissue (Figure 5e,f). It suggests that even though the tumor is located on liver Gal/PTX-GNPs are able to precisely target and effectively kill the tumor cells. Thus, the precise intraorgan target confirmed here, combining with the high renal clearance of gold nanoparticles with ultrasmall size reported previously,51 that Gal/PTX-GNPs would be safe in the liver cancer treatment.

4. CONCLUSIONS In the present study, we developed a PTX-conjugated GNP system to achieve precise treatment of liver cancer via an ASGP-R-mediated delivery. The designed system possesses a unique molecule structure, which offers an intraliver targeting ability and considerably improved efficacy in treating both Heps heterotopic and orthotopic xenografts tumors. Taking advantages of the nature of the conjugates and the expression differences of ASGP-R between liver normal and tumor cells, (1) the selectivity of tumor cells and (2) the ratio of drug concentrations in tumor to liver are significantly increased by the designed gold conjugates, and no obvious toxicity is found in normal liver. This work demonstrates the significance of the rational design and regulation of the conjugate structure, which allows the fine control of performance and toxicity in antitumor therapy systems.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.7b08969. Synthetic procedures and characterization of PEG−LA and Gal−PEG−LA; preparation and characterization of Gal−PEG-GNPs, GNPs conjugated with PTX−PEG− TA only, PTX-GNPs, and Gal/PTX-GNPs; and pharmacokinetic studies of Taxol, PTX-GNPs, and Gal/PTX-GNPs (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. Phone/Fax: +86-10-62753115 (Y.H.). *E-mail: [email protected]. Phone/Fax: +86-25-83271326 (Y.D.). ORCID

Yanglong Hou: 0000-0003-0579-4594 Xing-Hua Xia: 0000-0001-9831-4048 Ya Ding: 0000-0001-6214-5641 Author Contributions #

Y.-Y.G. and H.C. contributed equally to this work.

Notes

The authors declare no competing financial interest. 31466

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ACKNOWLEDGMENTS This work was supported by grants from the National Natural Science Foundation of China (30900337, 31470916, and 51672010), the Fundamental Research Funds for the Central Universities (2015PT036), and the Priority Academic Program Development of Jiangsu Higher Education Institutions, and the Open Project Program of MOE Key Laboratory of Drug Quality Control and Pharmacovigilance (DQCP2015MS01).

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