Intraorgan Targeting of Gold Conjugates for Precise Liver Cancer

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Intra-organ 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 ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b08969 • Publication Date (Web): 25 Aug 2017 Downloaded from http://pubs.acs.org on August 27, 2017

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ACS Applied Materials & Interfaces

Intra-organ targeting of gold conjugates for precise liver cancer treatment Yuan-Yue Gao,†,⊥ Huan Chen,‡,⊥ Ying-Ying Zhou,† Lin-Tao Wang,⸹ Yang-Long Hou,*,ξ Xing-Hua Xia,∞ Ya Ding*,†

†

State Key Laboratory of Natural Medicines, Department of Pharmaceutical Analysis, China

Pharmaceutical University, Nanjing 210009, China ‡

Department of Biochemistry, School of Life Science and Technology, China Pharmaceutical

University, Nanjing 210009, China ⸹

School of life sciences, Nanjing University, 22 Hankou Road, Nanjing 210093, China

ξ

Department of Materials Science and Engineering, College of Engineering, Peking

University, Beijing Key Laboratory for Magnetoeletric Materials and Devices, Beijing 100871, China ∞

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, Nanjing 210093, China ⊥

The first two authors contributed equally to this work.

* Corresponding author: E-mail: [email protected] (Y.-L. Hou), [email protected] (Y. Ding)

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ABSTRACT: Intra-organ 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 also are 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 6 times of that incubated with control conjugates without galactose modification in liver normal (L02) cells. The drug level in tumor vs. liver of Gal/PTX-GNPs is 121.0% at 8 h post-injection, a 15.7fold increase in the tumor specificity compared with that of GNPs conjugated PTX only. This intra-organ targeting strategy results in a considerable improvement of performance in treating both Heps heterotopic and orthotopic xenografts tumor models, which is expected to be used for the enhanced antitumor efficacy and reduced hepatotoxicity in liver cancer treatment.

KEYWORDS: Intra-organ targeting, Paclitaxel-conjugated gold nanoparticles, Structure design, Tumor penetration, Liver cancer treatment


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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 nanomaterials,9-12 and as the promising and efficient nanocarriers for numerous chemical drugs in the fundamental science13-19 and clinical trials.20, 21

It has reported that surface functionalized GNPs display enhanced permeability and

retention (EPR) effect, which makes drugs covalently link on their surface and apparently accumulate in tumor compared with free drugs.22-25 Nevertheless, most of the loaded drugs were collected in liver22-25 and the concentration ratio of drugs in tumor vs. liver was as low as 7.7% for paclitaxel (PTX)-conjugated GNPs23 and 13.1% for liposome-encapsulating PTXconjugated GNPs at 8 h post-injection.25 This phenomenon of liver selection is stemmed from the role of reticuloendothelial system (RES) possessed the 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, “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 those of liver diseases; Liver targeting simultaneously increases drug levels in normal liver tissue. Therefore, an intra-organ targeting of gold conjugates at tumor tissues in liver is greatly significance to improve the antitumor efficacy and reduce hepatotoxicity in hepatic carcinoma treatment. 3 ACS Paragon Plus Environment


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Galactose-functionalized nanoparticles, especially GNPs, provide a promising strategy for treating liver, since the high density and multi-antennary modification of galactose increased the dissociation constant (Kd value) of particles with ASGP-R, called cluster effect.29-31 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-polyethylene glycol-lipoic acid, Gal-PEG-LA) and therapeutic compound (paclitaxel-polyethylene glycolthioglycolic acid, PTX-PEG-TA) are simultaneously incorporated on the surface of GNPs (Figure 1a). This structure possesses unique feature, including: (1) high dispersion and longtime circulation characters aroused from the PEG molecules, (2) clearance-free by 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 based on the recognition of ASGP-R by galactose moiety (Fig. 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.


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

2. Materials and methods 2.1 Materials. Hydrogen tetrachloroaurate hydrate (HAuCl4⋅4H2O) was purchased from Shanghai Chemical Reagent Company (China). Polyethylene glycol (molecular weights of 400 and 5 ACS Paragon Plus Environment


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1,000 Da), N, N’-dicyclohexylcarbodiimide (DCC), 4-dimethylamiopryidine (DMAP), 1-(3dimethylaminopropyl)-3-ethylcarbodiimide

hydrochloride

(EDC·HCl),

and

N-

hydroxysulfosuccinimide (NHS) were provided by Sigma-Aldrich (USA). 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., USA) was used in all aqueous solutions preparation.

2.2 Apparatus. The chemical structures of the organic ligands in KBr discs were determined using a Tensor-27 Fourier IR spectrometer (Bruker, USA). 1H and 13C NMR spectra were recorded on a Bruker (AVACE) 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 UV3600 spectrophotometer (Shimadzu, Japan). A Zetasizer 3000HS instrument (Malvern Instruments, Malvern, UK) with 633 nm He-Ne lasers at 25 oC was used to measure the hydrodynamic diameters and zeta potentials. With an acceleration voltage of 200 kV, gold conjugates were imaged using a JEM-200CX TEM (JEOL). Gold concentration was measured by Optima 5300DV ICP-MS (PE, USA). Surface element analysis of gold conjugates was carried out using a PHI 5000 Versaprobe XPS (UlVAC-PHI, Japan). A Shimadzu LC-20A series with a Hypersil BDS C18 reversed-phase chromatography column (150 × 4.6 mm) and a UV detector set at 227 nm were employed to perform analytical reverse-phase high performance liquid chromatography (RP-HPLC). The mobile phase of methanol/water (70/30, v/v) was used with the flow rate at 0.8 mL/min (35 oC). 6 ACS Paragon Plus Environment

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2.3 Preparation of the conjugates. Citrate-protected GNPs were synthesized in a single-phase system referred to the previous reports.35 The molar ratio of Gal-PEG400-LA: PTX-PEG1000-TA in Gal/PTX-GNPs was set to be 1: 1. For example, Gal-PEG400-LA (0.14 mmol) was dissolved in methanol (1 mL). 1.5 equiv. of sodium borohydride (NaBH4, 0.21 mmol, 1 mg/mL) in ice water was added. The mixed solution was stirred for 1 h at 4 oC and the pH value was adjusted to pH 6 with HCl (1 mol·L1

). A methanol solution containing PTX-PEG1000-TA (0.14 mmol) was added into the above

solution and stirred for 30 min for sufficient mixing. And then, citrate-protected 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 cut off, MWCO = 7,000 Da). After freeze drying, the products were readily dispersed in water and in phosphate-buffered solutions (PBS). PTXGNPs were prepared referred to the method described above by 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 oC 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 of Stability (%)=1-|Ai-A0|/A0, where Ai and A0 were the absorbance at the detected time points and at the beginning time, respectively. 7 ACS Paragon Plus Environment


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2.5 In vitro drug release. The release of PTX from gold conjugates was investigated by a dialysis method.22, 24 The Gal/PTX-GNPs containing 1 mg PTX was dissolved in 1 mL PBS at pH 7.4 and placed in a dialysis bag (MWCO = 7,000 Da). To investigate 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 oC. At predetermined time intervals, 200 µL aliquots of release medium were withdrawn, and the same volume of 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 (DMEM) supplemented with 10% fetal bovine serum (FBS) and incubated at 37oC in humidified atmosphere with 5% CO2.

2.7 Detection of intracellular gold and drug contents. HepG2 and L02 were seeded in 100 mm dish, respectively. After 80% overspread, the cells were incubated with PTX, PTX-GNPs, and Gal/PTX-GNPs solutions containing the same amount of PTX (4 µg/mL) at 37 oC for an additional 4 or 8 h. The cells were subsequently washed with PBS twice and lysed with cell lysis buffer. The resulting 8 ACS Paragon Plus Environment

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supernatant was then treated with methanol for protein precipitation. After removal of precipitates by centrifugation, a sample of 20 µL of the supernatant was applied to RP-HPLC to determine the amount of PTX in cells. The gold concentration in the supernatant of PTXGNPs and Gal/PTX-GNPs groups was detected by ICP-MS method.

2.8 Intracellular distribution of Gal/PTX-GNPs. HepG2 cells were seeded in 6-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 an additional 4 or 8 h with Gal/PTX-GNPs at a PTX dose of 4 µg/mL. Subsequently, the TEM sample 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 treated with different concentrations of PTX, PTX-GNPs, and Gal/PTX-GNPs solutions for 48 h, respectively. The cell viability was determined by MTT assay as described in the previous report. 25 Data are presented as the means ± SD (n=5).

2.10 Animals and tumor model establishment. Healthy Sprague-Dawley rats (SD rats, male, 200 ± 20 g) and Institute of Cancer Research mice (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. 9 ACS Paragon Plus Environment


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Mouse hepatoma cells (Heps, Hepa 1-6, ATCC®CRL-1830™) 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 RPMI-1640 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 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 Heps cell suspensisons (2.5 × 107 cells/mL) were 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 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 3 groups (n=4). Taxol®, PTX-GNPs, 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 plexus venous 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 10 ACS Paragon Plus Environment

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divided into four groups (n=5): saline, Taxol®, PTX-GNPs, and Gal/PTX-GNPs solutions containing PTX concentration of 1 mg/mL. Each treatment group was comprised of five 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. At the conclusion of the study, tumor tissues were excised, washed with saline and weighed. The therapeutic effect of the formulations was also determined in orthotopic liver cancer model. Seven days after intrahepatic tumor cell implantation (day 0), mice were divided into four groups and injected intravenously with saline, Taxol®, PTX-GNPs or Gal/PTX-GNPs 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 were determined referring 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 tumor-bearing mice were additionally stained 11 ACS Paragon Plus Environment


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with TdT-mediated dUTP nick end labeling (TUNEL) using the TUNEL apoptosis detection kit (Roche, USA).

2.15 Immunofluorescence analysis. For immunofluorescence (IF) analysis, paraffin-embedded tissue sections were deparaffinized and hydrated, then slides were incubated in 1 × citrate buffer for 10 min for antigens retrieval. Afterwards the sections were blocked with 5% bovine serum albumin in PBS for 30 min and stained with a anti-α Tublin antibody (ab80779, Abcam, Shanghai, China) at 4 °C overnight. Then a 30-minute incubation with Alexa Flour 546-labeled donkey anti-mouse 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 Niselement Basic research (Nikon) software with the magnification 400 ×.

2.16 Statistical analysis. All the results are presented as the mean ± SD. The significance of the differences between groups was evaluated using the Student t test when only two groups were compared or one-way ANOVA when there are more than two groups. P ≤ 0.05 was considered to be statistically significant.

3. Results and discussion 3.1. Preparation and Characterization


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For the fabrication of gold conjugates, citrate-protected GNPs ca. 3 nm were prepared firstly.35, 36 Two types of molecules for GNP surface modification were designed, one is GalPEG-LA, another is PTX-PEG-TA (Figure 1a). The synthesis and characterization of GalPEG-LA and PTX-PEG-TA are referred to the supporting information (Figure S1-S4, Supporting Information) and our previous report,23 respectively. After the performance evaluation of the conjugates only bearing targeting ligand or therapeutic compound, respectively, Gal-PEG400-LA showed the highest targeting ability (Figure 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 PTXPEG1000-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 PTXPEG1000-TA at the same molar ratio described above. The surface composition of gold conjugates was characterized to evaluate their receptor binding capacity, since the exposure of sugar moiety is essential for the recognition by ASGPR.37,38 The addition of galactose does not increase the hydrodynamic diameter of gold conjugates. On the contrary, dynamic light scattering (DLS) measurements (Figure 2a) show that the 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 saccharides on the surface of GNPs.39 The attachment of galactose results in a slight increase in surface charge, the zeta 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 on a X-ray photoelectron spectroscopy (XPS, Figure 2c). Gal/PTX-GNPs have higher nitrogen percentage (1.33 atom. %) than PTX-GNPs (0.42 atom. %) (Figure 2c) due to the attachment of ethylenediamine spacer connecting galactose with PEG (Figure 1a), indicating the 13 ACS Paragon Plus Environment


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exposure of galactose on the surface of the conjugates. It can be explained by the co-assembly of the rigid short PEG (Gal-PEG400-LA)40 and the flexible PTX-PEG1000-TA that possesses a coiled PEG geometry41,42 as shown in Figure 1a.

Fig. 2. Characterization and in vitro drug release of gold conjugates. (a) Hydrodynamic diameters, (b) zeta potentials, (c) XPS spectra and atomic concentrations, and (d) TEM images of PTX-GNPs and Gal/PTX-GNPs. (e) Drug release profiles of PTX from Gal/PTXGNPs 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 present the mean ± SD (n=3).

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. Transmission electron microscopy (TEM) images show that the gold cores in the conjugates have the average 14 ACS Paragon Plus Environment

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diameter of ca. 3 nm and good dispersity (Figure 2d). It can be due to the co-assembly of GalPEG400-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 for example)44,45 after the internalization of conjugates by tumor cells.

3.3. Cellular uptake and selectivity Thus, the cellular internalization and selectivity of Gal/PTX-GNPs were conducted against the human hepatic cells (L02) and human hepatocellular carcinoma cell line (HepG2). After the 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 gold content in HepG2 cells (18.03 ± 6.01 µg/L), which is 5.85 folds 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 the differences in ASGP-R expression between normal and tumor liver cells. Since HepG2 cells express ca. 2 times higher ASGP-R than L02 cells,33 Gal/PTX-GNPs show a consistent increase, ca. 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 towards gold 15 ACS Paragon Plus Environment


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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. This data demonstrates the sensitivity of HepG2 cells towards 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. Taken 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 h to 8 h.

Fig. 3. Cellular selectivity, intracellular distribution, drug release, and cytotoxicity. (a) Intracellular gold contents of PTX-GNPs and Gal/PTX-GNPs 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 16 ACS Paragon Plus Environment

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(one-sample t-test). (b) TEM images of HepG2 cells incubated with drug-free medium for 4 h (I) and Gal/PTX-GNPs for (II) 4 h and (III) 8 h; (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 t-test). (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).

3.4. Intracellular distribution, drug release, and cytotoxicity To reveal the possible treatment mechanism, the intracellular distribution of Gal/PTXGNPs was investigated by TEM (Figure 3b). Compared to HepG2 cells incubated with cell culture medium (Figure 3b, I and I’), Gal/PTX-GNPs are found in some small vesicles existed in the cytoplasm after 4 h incubation (Figure 3b, II and II’). After 8 h incubation (Figure 3b, III and III’), gold cores of the conjugates remain in the cytoplasm. However, note that a juxtanuclear movement is found at this time (Figure 3b, III and 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.48,49 After confirming the drug release site, intracellular drug concentrations of samples were detected by the reverse phase high-performance liquid chromatography (RP-HPLC) method using PTX solution as the control. Since 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 PTX-conjugated 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 to 1/3 of the PTX solution group, indicating that the galactose modification has little influence in 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 17 ACS Paragon Plus Environment


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low level (Figure 3c, columns of PTX-GNPs for HepG2 cells). However, a rapid rising of PTX is observed in Gal/PTX-GNPs 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. This 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/PTXGNPs 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. Based on the high cell selectivity and effective drug release of the designed conjugates, the cytotoxicity of PTX-conjugated 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 (from 20 to 50 µg/mL), the cell viability is significantly reduced by Gal/PTX-GNPs as compared to PTX solution and PTX-GNPs. This concentrationdependent decrease in cell viability exhibits a dosage effect on the cell killing ability of Gal/PTX-GNPs. Therefore, the accumulation of conjugates around 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 18 ACS Paragon Plus Environment

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compared with 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 Fig. S10b. 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, the 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 pass 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/PTX-GNPs, the mice with transplant Heps tumors begins to show the lowest tumor growth rate and the smallest tumor size among all test groups (Figure 4, a and b). After the 10-day treatment, the tumor weight of the mice treated by Gal/PTX-GNPs is the lightest and 0.35, 0.77, and 0.75-folds of those treated by 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 19 ACS Paragon Plus Environment


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capacities of the conjugates in tumor tissues and the subsequent cell killing effect by the release of drug. Therefore, the histological examination of H&E stained tumor tissue sections at 10-day 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.

Fig. 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 the magnification 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-day post-injection. Each point in (a, c, and e) represents the 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 between tumor and liver for PTX-GNPs and Gal/PTXGNPs is also presented in (h). *P < 0.05, **P < 0.01 (one-sample t-test).


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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 those of other groups, indicating an improved quality of life for the mice due to the low hepatotoxicity of Gal/PTX-GNPs compared to other formulations. As shown in Figure 4, f and g, Gal/PTX-GNPs show a sustained PTX concentrations 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 4, a-c. The PTX concentration ratio of Gal/PTX-GNPs in tumor to liver is in a range of 59.3% to 121.0%, which is much higher than that of PTX-GNPs (41.0%~88.0%, Figure 4h). This data is 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 both in tumor and liver (Figure 4, f and g). Its high concentrations at 0.5 h and 1 h in liver (37.1 ± 6.5 and 26.8 ± 13.5 µg/g) 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 Since 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 anti-tumor 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 line). The percentage of final weight of the 21 ACS Paragon Plus Environment


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tumor vs. liver (%, g/g) after 10-day treated with Gal/PTX-GNPs is also quite low (9.1 ± 2.5%, Figure 5b). It is 0.18-, 0.43-, and 0.37-fold for those 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 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 of Gal/PTX-GNPs, whereas the survival rate of saline- and PTX-GNPs-treated groups is 40% and no one is survival in Taxol®-treated 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-mediate dUTP nick end labeling (Figure 5f), respectively, to detect the cell killing and apoptosis degree of liver and tumor tissues in Heps orthotopic liver tumor mice. Compared to other samples, 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 5, e and 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 intra-organ target confirmed here combining with the high renal clearance of gold nanoparticles with ultrasmall size reported previously,51 Gal/PTX-GNPs would be safe in the liver cancer treatment.


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Fig. 5. In vivo antitumor efficacy in Heps orthotopic xenograft models. (a) Photos of liver tissues bearing tumors excised from mice treated by (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 the mean ± SD (n = 5). Histological observation of the liver and tumor tissue sections (e) stained with hematoxylin and eosin (the scale bar is 100 µm) and (f) stained with TdT-mediated dUTP nick end labelling using TUNEL kit, Roche, USA (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 fluorescence 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 presents mean ± SD (n=6). *p < 0.05, **p < 0.01 (one-sample t-test).



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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, un-treated and Gal/PTX-GNPs-treated Heps orthotopic liver tumor has been carried out (Figure 5g). Due to the rapid cell division of tumor cells, the expression of αTubulin 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 that of the primary antibody (α-Tubulin mAb) and competitively binds with α-Tubulin 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

4. Conclusion In the present study, we developed a PTX-conjugated GNP systme to achieve precise treatment of liver cancer via an ASGP-R mediated delivery. The designed system possesses a unique molecule structure which offers an intra-liver 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 is 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 anti-tumor therapy systems.


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Associated Content Supporting Information// The Supporting Information is available free of charge on the ACS Publications website at DOI: 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; Pharmacokinetic studies of Taxol®, PTX-GNPs, and Gal/PTX-GNPs (PDF)

Author Information Corresponding Authors *E-mail: [email protected]. Phone: +86-10-62753115. Fax: +86-10-62753115. *E-mail: [email protected]. Phone: +86-25-83271326. Fax: +86-25-83271326. ORCID Ya Ding: 0000-0001-6214-5641 Author Contributions ⊥

These authors contributed equally to this work.

Notes The authors declare no competing financial interest.

Acknowledgements This work was supported by grants from the National Natural Science Foundation of China (30900337 and 31470916), the Fundamental Research Funds for the Central Universities (2015PT036), and the Priority Academic Program Development of Jiangsu Higher Education 25 ACS Paragon Plus Environment


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Institutions, and the Open Project Program of MOE Key Laboratory of Drug Quality Control and Pharmacovigilance (DQCP2015MS01).

References [1] Langer, R. Drug Delivery and Targeting, Nature 1998, 392 (6679 Suppl), 5-10. [2] Mo R.; Jiang, T; DiSanto, R.; Tai, W.; Gu, Z. ATP-Triggered Anticancer Drug Delivery. Nat. Commun. 2014, 5 (1), 3364. [3] Bertrand, N.; Wu, J.; Xu, X.; Kamaly, N.; Farokhzad, O. C. Cancer Nanotechnology: The Impact of Passive and Active Targeting in the Era of Modern Cancer Biology. Adv. Drug Deliver. Rev. 2014, 66 (24), 2-25. [4] Poelstra, K.; Prakash, J.; Beljaars, L. Drug Targeting to the Diseased Liver. J. Control. Release 2012, 161 (2), 188-197. [5] D'Souza, A. A.; Devarajan, P. V. Asialoglycoprotein Receptor Mediated Hepatocyte Targeting - Strategies and Applications. J. Control. Release 2015, 203, 126-139. [6] Craparo, E. F.; Licciardi, M.; Conigliaro, A.; Palumbo, F. S.; Giammona, G.; Alessandro, R.; De Leo, G.; Cavallaro, G. Hepatocyte-Targeted Fluorescent Nanoparticles Based on a Polyaspartamide for Potential Theranostic Applications. Polymer 2015, 70, 257-270. [7] Ahmed, M.; Narain, R. Carbohydrate-Based Materials for Targeted Delivery of Drugs and Genes to the Liver. Nanomedicine 2015, 10 (14), 2263-2288. [8] Lee, K.; Rafi, M.; Wang, X.; Aran, K.; Feng, X.; Lo Sterzo, C.; Tang, R.;Lingampalli, N.; Kim, H. J.; Murthy, N. In Vivo Delivery of Transcription Factors with Multifunctional Oligonucleotides. Nature Mater. 2015, 14 (7), 701-706. [9] Daniel, M. C.; Astruc, D. Gold Nanoparticles: Assembly, Supramolecular Chemistry, Quantum-Size-Related Properties, and Applications toward Biology, Catalysis, and Nanotechnology. Chem. Rev. 2004, 104 (1), 293-346. [10] Sun, H.; Su, J.; Meng, Q.; Yin, Q.; Chen, L.; Gu, W.; Zhang, Z.; Yu, H.; Zhang, P.; Wang, S.; Li Y. Cancer Cell Membrane-Coated Gold Nanocages with HyperthermiaTriggered Drug Release and Homotypic Target Inhibit Growth and Metastasis of Breast Cancer. Adv. Funct. Mater. 2017, 27 (3), 1604300. [11] Li, S.-Y.; Qiu, W.-X.; Cheng, H.; Gao, F.; Cao, F.-Y.; Zhang, X.-Z. A Versatile Plasma Membrane Engineered Cell Vehicle for Contact-Cell-Enhanced Photodynamic Therapy. Adv. Funct. Mater. 2017, 27 (12), 1604916. [12] Tonga, G. Y.; Saha, K.; Rotello, V. M. Interfacing Nanoparticles and Biology: New Strategies for Biomedicine. Adv. Mater. 2014, 26 (3), 359-370. 26 ACS Paragon Plus Environment

Page 26 of 31

Page 27 of 31

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


[13] Gibson, J. D.; Khanal, B. P.; Zubarev, E. R. Paclitaxel-Functionalized Gold Nanoparticles. J. Am. Chem. Soc. 2007, 129 (37), 11653-11661. [14] Lyu, Z.; Zhou, F.; Liu, Q.; Xue, H.; Yu, Q.; Chen, H. A Universal Platform for Macromolecular Delivery into Cells Using Gold Nanoparticle Layers via the Photoporation Effect. Adv. Funct. Mater. 2016, 26 (32), 5787-5795. [15] Li, L.; Chen, C.; Liu, H.; Fu, C.; Tan, L.; Wang, S.; Fu, S.; Liu, X.; Meng, X.; Liu, H. Multifunctional Carbon–Silica Nanocapsules with Gold Core for Synergistic Photothermal and Chemo-Cancer Therapy under the Guidance of Bimodal Imaging. Adv. Funct. Mater. 2016, 26 (24), 4252-4261. [16] Vigderman, L.; Zubarev, E. R. Therapeutic Platforms Based on Gold Nanoparticles and their Covalent Conjugates with Drug Molecules. Adv. Drug Deliv. Rev. 2013, 65 (5), 663-676. [17] Ding, Y.; Jiang, Z.; Saha, K.; Kim, C. S.; Kim, S. T.; Landis, R. F.; Rotello, R. M. Gold Nanoparticles for Nucleic Acid Delivery. Mol. Ther. 2014, 22 (6), 1075-1083. [18] Liang, J.-J.; Zhou, Y.-Y.; Wu, J.; Ding, Y. Gold Nanoparticle-Based Drug Delivery Platform for Antineoplastic Chemotherapy. Curr. Drug Metab. 2014, 15 (6), 620-631. [19] Pacardo, D. B.; Neupane, B.; Rikard, S. M.; Lu, Y.; Mo, R.; Mishra, S. R.; Tracy, J. B.; Wang, G.; Ligler, F. S.; Gu, Z. Dual Wavelength-Activatable Gold Nanorod Complex for Synergistic Cancer Treatment. Nanoscale 2015, 7 (28), 12096-12103. [20] Paciotti, G. F.; Kingston, D. G. I.; Tamarkin, L. Colloidal Gold Nanoparticles: A Novel Nanoparticle Platform for Developing Multifunctional Tumor-Targeted Drug Delivery Vectors. Drug Develop. Res. 2006, 67 (1), 47-54. [21] Kingston, D. G. I.; Cao, S.; Zhao, J.; Paciotti, G. F.; Huhta, M. S. US Patent 2013, 8, 558, 019 B552. [22] Bao, Q.-Y.; Geng, D.-D.; Xue, J.-W.; Zhou, G.; Gu, S.-Y.; Ding, Y.; Zhang, C. Glutathione-Mediated Drug Release from Tiopronin-Conjugated Gold Nanoparticles for Acute Liver Injury Therapy. Int. J. Pharm. 2013, 446 (1-2), 112-118. [23] Ding, Y.; Zhou, Y.-Y.; Chen, H.; Geng, D.-D.; Wu, D.-Y.; Hong, J.; Shen, W. B.; Hang, T. J.; Zhang, C. The Performance of Thiol-Terminated PEG-Paclitaxel-Conjugated Gold Nanoparticles. Biomaterials 2013, 34 (38), 10217-10227. [24] Bao, Q.-Y.; Zhang, N.; Geng, D.-D.; Xue, J.-W.; Merritt, M.; Zhang, C.; Ding, Y. The Enhanced Longevity and Liver Targetability of Paclitaxel by Hybrid Liposomes Encapsulating Paclitaxel-Conjugated Gold Nanoparticles. Int. J. Pharm. 2014, 477 (1-2), 408415.



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

[25] Zhang, N.; Chen, H.; Liu, A.-Y.; Shen, J.-J.; Shah, V.; Zhang, C.; Hong, J.; Ding, Y. Gold Conjugate-Based Liposomes with Hybrid Cluster Bomb Structure for Liver Cancer Therapy. Biomaterials 2016, 74, 280-291. [26] Saba, T. M. Physiology and Physiopathology of Reticuloendothelial System. Arch. Intern. Med. 1970, 126 (6), 1031-1052. [27] Peer, D.; Karp, J. M.; Hong, S.; FaroKhzad, O. C.; Margalit, R.; Langer, R. Nanocarriers as an Emerging Platform for Cancer Therapy. Nat. Nanotechnol. 2007, 2 (12), 751-760. [28] Lu, Y.; Lin, Y.; Chen, Z.; Hu, Q.; Liu, Y.; Yu, S.; Gao, W.; Dickey, M. D.; Gu, Z. Enhanced Endosomal Escape by Light-Fueled Liquid-Metal Transformer. Nano Lett. 2017, 17 (4), 2138-2145. [29] Bergen, J. M.; von Recum, H. A.; Goodman, T. T.; Massey, A. P.; Pun, S. H. Gold Nanoparticles as a Versatile Platform for Optimizing Physicochemical Parameters for Targeted Drug Delivery. Macromol Biosci. 2006, 6 (7), 506-516. [30] Ojeda, R.; de Paz, J. L.; Barrientos, A. G.; Martín-Lomas, M.; Penadés, S. Preparation of Multifunctional Glyconanoparticles as a Platform for Potential Carbohydrate-Based Anticancer Vaccines. Carbohydr. Res. 2007, 342 (3), 448-459. [31] Garg, S.; De, A.; Nandi, T.; Mozumdar, S. Synthesis of a Smart Gold Nano‐Vehicle for Liver Specific Drug Delivery. AAPS PharmSciTech. 2013, 14 (3), 1219-1226. [32] Li, Y.; Huang, G.; Diakur, J.; Wiebe, L. I. Targeted Delivery of Macromolecular Drugs: Asialoglycoprotein Receptor (ASGPR) Expression by Selected Hepatoma Cell Lines Used in Antiviral Drug Development. Curr. Drug Deliv. 2008, 5 (4), 299-302. [33] Ma, Y.; Chen, H.; Su, S.; Wang, T.; Zhang, C.; Fida, G.; Cui, S.; Zhao, J.; Gu, Y. Galactose as Broad Ligand for Multiple Tumor Imaging and Therapy. J. Cancer 2015, 6 (7), 658-670. [34] Zhang, D.; Guo, Z.; Zhang, P.; Li, Y.; Su, X.; You, L.; Gao, M.; Liu, C.; Wu, H.; Zhang, X. Simplified Quantification Method for in Vivo SPECT/CT Imaging of Asialoglycoprotein Receptor with Tc-99m-p(VLA-co-VNI) to Assess and Stage Hepatic Fibrosis in Mice. Sci. Rep. 2016, 6, 25377. [35] Ding, Y.; Xia, X.-H.; Zhang, C. Synthesis of Metallic Nanoparticles Protected with N,N,N-Trimethyl Chitosan Chloride via a Relatively Weak Affinity. Nanotechnology 2006, 17 (16), 4156-4162. [36] Ding, Y.; Xia, X.-H.; Zhai, H.-S. Reversible Assembly and Disassembly of Gold Nanoparticles Directed by a Zwitterionic Polymer. Chem. Eur. J. 2007, 13 (15), 4197-4202.


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[37] Zhong, Y.; Yang, W.; Sun, H.; Cheng, R.; Meng, F.; Deng, C.; Zhong, Z. LigandDirected Reduction-Sensitive Shell-Sheddable Biodegradable Micelles Actively Deliver Doxorubicin into the Nuclei of Target Cancer Cells. Biomacromolecules 2013, 14 (10), 37233730. [38] Zou, Y.; Song, Y.; Yang, W.; Meng, F.; Liu, H.; Zhong, Z. Galactose-Installed PhotoCrosslinked pH-Sensitive Degradable Micelles for Active Targeting Chemotherapy of Hepatocellular Carcinoma in Mice. J. Control. Release 2014, 193, 154-161. [39] Ding, Y.; Liang, J.-J.; Geng, D.-D.; Wu, D.; Dong, L.; Shen, W.-B.; Xia, X. H.; Zhang, C. Development of a Liver-Targeting Gold-PEG-Galactose Nanoparticle Platform and the Structure-Function Study. Part. Part. Syst. Charact. 2014, 31 (3), 347-356. [40] Sun, J.; Zhang, L.; Wang, J.; Feng, Q.; Liu, D.; Yin, Q.; Xu, D.; Wei, Y.; Ding, B.; Shi, X.; Jiang, X. Tunable Rigidity of (Polymeric Core)-(Lipid Shell) Nanoparticles for Regulated Cellular Uptake. Adv. Mater. 2015, 27 (8), 1402-1407. [41] Kang, B.; Okwieka, P.; Schoettler, S.; Winzen, S.; Langhanki, J.; Mohr, K.; Opatz, T.; Mailänder, V.; Landfester, K.; Wurm, F. R. Carbohydrate-Based Nanocarriers Exhibiting Specific Cell Targeting with Minimum Influence from the Protein Corona. Angew Chem. Int. Ed. 2015, 54 (25), 7436-7440. [42] Pelaz, B.; Pino, P.; Maffre, P.; Hartmann, R.; Gallego, M.; Rivera-Fernandez, S.; Fuente, J. M.; Nienhaus, G. U.; Parak, W. J. Surface Functionalization of Nanoparticles with Polyethylene Glycol: Effects on Protein Adsorption and Cellular Uptake. ACS Nano 2015, 9 (7), 6996-7008. [43] Hong, R.; Han, G.; Fernandez, J. M.; Kim, B. J.; Forbes, N. S.; Rotello, V. M. Glutathione-Mediated Delivery and Release Using Monolayer Protected Nanoparticle Carriers. J. Am. Chem. Soc. 2006, 128 (4), 1078-1079. [44] Lu, X.; Howard, M. D.; Talbert, D. R.; Rinehart, J. J.; Potter, P. M.; Jay, M.; Leggas, M. Nanoparticles Containing Anti-Inflammatory Agents as Chemotherapy Adjuvants II: Role of Plasma Esterases in Drug Release. AAPS. J. 2009, 11 (1), 120-122. [45] Qiu, N.; Liu, X.; Zhong, Y.; Zhou, Z.; Piao, Y.; Miao, L.; Zhang, Q.; Tang, J.; Huang, L.; Shen, Y. Esterase-Activated Charge-Reversal Polymer for Fibroblast-Exempt Cancer Gene Therapy. Adv. Mater. 2016, 28 (48), 10613-10622. [46] Shukla, R.; Bansal, V.; Chaudhary, M.; Basu, A.; Bhonde, R. R.; Sastry, M. Biocompatibility of Gold Nanoparticles and Their Endocytotic Fate Inside the Cellular Compartment: A Microscopic Overview. Langmuir 2005, 21 (23), 10644-10654.



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[47] Heller, B.; Adu-Gyamfi, E.; Smith-Kinnaman, W.; Babbey, C.; Vora, M.; Xue, Y.; Bittman, R.; Stahelin, R. V.; Wells, C. D. Amot Recognizes a Juxtanuclear Endocytic Recycling Compartment via a Novel Lipid Binding Domain. J. Biol. Chem. 2010, 285 (16), 12308-12320. [48] Snyder, J. P.; Nettles, J. H.; Cornett, B.; Downing, K. H.; Nogales, E. The Binding Conformation of Taxol in Beta-Tubulin: A Model Based on Electron Crystallographic Density. Proc. Natl. Acad. Sci. USA 2001, 98 (9), 5312-5316. [49] Jordan, M. A.; Wilson, L. Microtubules as a Target for Anticancer Drugs, Nat. Rev. Cancer 2004, 4 (4), 253-265. [50] Namgung, R.; Lee, Y. M.; Kim, J.; Jang, Y.; Lee, B.-H.; Kim, I.-S.; Sokkar, P.; Rhee, Y. M.; Hoffman, A. S.; Kim, W. J. Poly-Cyclodextrin and Poly-Paclitaxel Nano-Assembly for Anticancer Therapy. Nat. Commun. 2014, 5 (6183), 3702. [51] Zhou, C.; Long, M.; Qin, Y.; Sun, X.; Zheng, J. Luminescent Gold Nanoparticles with Efficient Renal Clearance. Angew Chem. Int. Ed. 2011, 50 (14), 3168-3172. [52] Sun, Q.; Sun, X.; Ma, X.; Zhou, Z.; Jin, E.; Zhang, B.; Shen, Y.; Van Kirk, E. A.; Murdoch, W. J.; Lott, J. R.; Lodge, T. P.; Radosz, M.; Zhao, Y. Integration of Nanoassembly Functions for an Effective Delivery Cascade for Cancer Drugs. Adv. Mater. 2014, 26 (45), 7615-7621. [53] Giannakakou, P.; Sackett, D. L.; Kang, Y. K.; Zhan, Z. R.; Buters, J. T. M.; Fojo, T.; Poruchynsky, M. S. Paclitaxel-Resistant Human Ovarian Cancer Cells Have Mutant BetaTubulins that Exhibit Impaired Paclitaxel-Driven Polymerization. J. Biol. Chem. 1997, 272 (27), 17118-17125. [54] Kowalski, R. J.; Giannakakou, P.; Hamel, E. Activities of the Microtubule-Stabilizing Agents Epothilones A and B with Purified Tubulin and in Cells Resistant to Paclitaxel (Taxol®). J. Biol. Chem. 1997, 272 (4), 2534-2541.


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Graphic for manuscript


Intraorgan Targeting of Gold Conjugates for Precise Liver Cancer

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