Comparison of Two Approaches for the Attachment of a Drug to Gold

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A Comparison of Two Approaches for the Attachment of a Drug to Gold Nanoparticles and their Anti-cancer Activities Yingjie Fu, Qishuai Feng, Yifan Chen, Yajing Shen, Qihang Su, Yinglei Zhang, Xiang Zhou, and Yu Cheng Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.6b00619 • Publication Date (Web): 12 Aug 2016 Downloaded from http://pubs.acs.org on August 15, 2016

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Molecular Pharmaceutics

A Comparison of Two Approaches for the Attachment of a Drug to Gold Nanoparticles and their Anti-cancer Activities Yingjie Fu1, Qishuai Feng2, Yifan Chen2, Yajing Shen2, Qihang Su2, Yinglei Zhang2, Xiang Zhou1* and Yu Cheng2* 1

College of Chemistry and Molecular Science, The Institute for Advanced Studies,

Wuhan University, Wuhan 430072, China 2

Shanghai East Hospital, The Institute for Biomedical Engineering and Nano Science,

Tongji University School of Medicine, Shanghai 200029, China Corresponding authors: [email protected]; [email protected]

ABSTRACT: Drug attachment is important in drug delivery for cancer chemotherapy. The elucidation of the release mechanism and biological behavior of a drug is essential for the design of delivery systems. Here, we used a hydrazone bond or a amide bond to attach an anti-cancer drug, doxorubicin (Dox), to gold nanoparticles (GNPs) and compared the effects of the chemical bond on the anti-cancer activities of the resulting Dox-GNPs. The drug release efficiency, cytotoxicity, subcellular distribution and cell apoptosis of hydrazone-linked HDox-GNPs and amide-linked SDox-GNPs were evaluated in several cancer cells. HDox-GNPs exhibited greater potency for drug delivery via triggered release co-mediated by acidic pH and glutathione (GSH) than SDox-GNPs triggered by GSH alone. Dox released from HDox-GNPs was released in lysosomes and exerted its drug activity by entering the

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nuclei. Dox from SDox-GNPs was mainly localized in lysosomes, significantly reducing its efficacy against cancer cells. In addition, in vivo studies in tumor-bearing mice demonstrated that HDox-GNPs and SDox-GNPs both accumulate in tumor tissue. However, only HDox-GNPs enhanced inhibition of subcutaneous tumor growth. This study demonstrates that HDox-GNPs display significant advantages in drug release and anti-tumor efficacy. KEYWORDS: gold nanoparticle, doxorubicin, drug delivery, anti-cancer activity TOC:


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INTRODUCTION Cancer is one of the most deadly threats to human health1. Chemotherapy involving local or systemic drug administration is often prescribed as a cancer treatment2. The drug molecules used in chemotherapy target cancer cells, but their effectiveness is limited by poor solubility, poor targeting efficacy, serious side effects and drug resistance3-5. Various nanoparticle-based drug delivery platforms have been developed to enhance drug distribution in tumor tissues and minimize adverse effects6-7. Moreover, these platforms can combine multiple drugs and therapeutics to boost drug efficacy and avoid drug resistance8-10. Due to the specific structures of anti-cancer drugs and nanoparticle-platforms, the drug attachment strategy has significant implications for cancer treatment. Some of the functional groups play a key role in drug efficacy and cannot be modified. Thus, it is important to optimize the attachment strategy for anti-cancer drugs on drug delivery systems11-12. The fluorescent molecule doxorubicin (Dox) exhibits highly effective anti-cancer activity and has been widely used as a model chemotherapeutic agent for the treatment of cancers, including breast, lung, brain, and other cancers13-19. However, the use of Dox is hampered by low targeting efficiency and side effects such as hair loss, nausea, myelosuppression, and cardiotoxicity20-22. Nanoparticles can be used to improve drug transport and release23-24. Chemical attachment via firm and flexible chemical bonds to achieve Dox loading is a promising approach for controlled drug release25. Two active sites on the Dox chemical structure can be exploited to create a linkage between the drug and nanoparticles. The amino group can be used to form an



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amide for functionalization26. The keto group can form a hydrazone bond, the hydrolysis of which can be catalyzed by acid. 27-30. Due to their unique properties, which include chemical inertness, non-toxicity, ease of synthesis and functionalization, gold nanoparticles (GNPs) have attracted significant attention as delivery platforms for drug release31-34. Internal and external triggers such as pH, GSH, enzymes and light, can be used for controlled and programmed drug release from GNPs35-38. Amino and keto groups are the two available sites for attaching Dox on GNPs39-42. For amino groups, a one-step coupling reaction can be utilized to load Dox on GNPs40-42. By contrast, the keto group of Dox must be functionalized via multiple reactions to form the hydrazone bond to the GNPs43-44. Due to the high GSH concentration and specific acidic micro environment in cancer cells, these strategies enable the release of the drug in targeted tissues via GSH- and pH-mediated pathways, respectively45-47. The comparison of the chemical bonds used to load Dox directly on GNPs on synthesis, stability, release efficiency, cytotoxicity and anti-cancer efficacy has not been studied. We therefore synthesized Dox-GNPs by forming a hydrazone bond (HDox-GNPs) or amide bond (SDox-GNPs) (Fig. 1). We demonstrated the efficiency of controlled release of Dox by these two systems and characterized the drug-release mechanisms. The cytotoxicities of HDox-GNPs and SDox-GNPs against cancer cells were also evaluated in vitro and in vivo.


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MATERIALS AND METHODS Materials Tetra-n-octylammonium bromide (TOAB) and reduced L-glutathione (GSH) were purchased from Sigma-Aldrich Shanghai China. Dodecylamine (DDA) and sodium borohydride (NaBH4) were purchased from Fluka Shanghai China. Chloroauric acid was purchased from Civi-Chem Suzhou China. Methyl thioglycolate (MTG) ethylenediaminetetraacetic acid disodium salt (EDTA), hydroxylamine hydrochloride, hydrazine hydrate and morpholineethanesulfonic acid (MES) were purchased from Aladdin Shanghai China. N-succinimidyl-S-acetylthioacetate (SATA) was purchased from TCI Shanghai China. Doxorubicin was purchased from Meso Suzhou China. MeO-PEG-SH (MW=5000) was purchased from SINOPEG Biotech Co. Ltd Xiamen China. The MTT kit was purchased from Roche Shanghai China. Ultrapure water was used in all experiments. All reagents were used without further purification. Preparation of m-GNPs Mono-functional GNPs (m-GNPs) were prepared according to procedures described in the literature48. Briefly, 0.25 mM TOAB and 0.6 mM DDA were dissolved in 5 mL of toluene, and 366 µL of 0.57 M HAuCl4 (17% in HCl solution) was added to obtain a dark-red solution. Then, 2 mM NaBH4 dissolved in 1 mL of ice-cold pure water was added dropwise over 2 min with vigorous stirring. After 2 h of continuous stirring, the mixture was poured into 40 mL of ethanol. The precipitate was collected by centrifugation at 4000 rpm for 10 min and redispersed in 4 mL of



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chloroform. Next, 0.04 mM of MeO-PEG-SH (MW=5000) was mixed with the GNPs in chloroform overnight. The organic phase was evaporated under vacuum and washed with water three times using centrifuge tubes (filter membrane cutoff 10 KDa). The m-GNPs were redispersed in water and centrifuged for 1 h at 12000rpm. The supernatant solution containing 5-nm m-GNPs was collected. The concentration of the m-GNPs was determined by UV-Vis spectroscopy (Cary 60 UV-Vis, Agilent Technologies) at an absorbance of 520 nm. The extinction coefficient was proved to be 1.5×107 M-1·cm-1 according to our previous work48. Preparation of SATA-Dox, SDox-GNPs and HDox-GNPs SATA-modified Dox was synthesized previously. Briefly, 0.035 mmol of Dox was dissolved in 5 mL of DMF. Then, 0.035 mmol of SATA was added, followed by stirring for 2 h at room temperature. The reaction was monitored by thin-layer chromatography. Then, the solvent was evaporated under vacuum, and the crude product was purified by column chromatography (1:7 methanol:dichloromethane as the eluent). The product was obtained as a red powder in 95% yield. Products were characterized by 1HNMR and MS (positive and negative) in supporting information. SDox-GNP: 1.6 µmol of SATA-Dox was dissolved in 1.5 mL of DMF. Then, 400 µL of PBS was added to 0.5 M hydroxylamine hydrochloride and 25 mM EDTA with stirring. Next, 1 mL of 10-5 M m-GNPs (particle number determined by UV-Vis spectra) was added and stirred for 24 h at room temperature. The solution was concentrated to 200 µL under vacuum and diluted with 10 mL of water. The SDox-GNPs were collected via centrifugation at 4000 rpm in centrifuge tubes (filter


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membrane cutoff 10 kDa) and washed with PBS (pH 7.4) until the filtrate became clear. The SDox-GNPs were collected and stored at 4°C. Fluorescence spectra (Cary Eclipse Fluorescence Spectrophotometer, Agilent Technologies) were measured to determine the attachment of Dox. HDox-GNP: 1 mL of 10-5 M m-GNPs were diluted with 5 mL of water. Then, 10-5 mol of MTG was added and stirred for 24 h at room temperature. The GNPs were collected by centrifugation at 4000 rpm in centrifuge tubes (filter membrane cutoff 10 kDa) and washed with water three times to obtain MTG-GNPs. Then, 5x10-9 mol of the MTG-GNPs were added to a mixture of 3 mL of methanol and 3 mL of hydrazine hydrate and stirred for 1 h at 60°C. The methanol and unreacted hydrazine hydrate were evaporated under vacuum, and the residue was redispersed in a mixture of 5 mL of DMF and 1.4 mmol of triethylamine. Then, 0.01 mmol of Dox was dissolved in 1 mL of DMF and added dropwise to the above mixture. After 24 h, the solvent was evaporated, and the HDox-GNPs were redispersed in 4 mL of PBS (pH 8). The HDox-GNP solution was centrifuged at 4000 rpm in centrifuge tubes (filter membrane cutoff 10 kDa) at 4°C and washed with PBS (pH 8) three times. The HDox-GNPs were diluted with PBS (pH 7.4) after the final centrifugation and stored at -20°C. The concentrations of the various GNPs were determined from UV-vis spectra. Quantification of drug loading Dox quantification was referred to literatures49-50. 10 µL SDox-GNPs were dried in vacuum and suspended in 1 mL chloroform. Then 1 mg of iodine was added to this



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solution and followed by stirring at room temperature for 3 hours. After removal of bulk gold by centrifuging, the top layer clear solution was collected and evaporated. Dox in residue was dissolved in 3 mL water and the fluorescent spectrum was collected. In addition, the concentration of Dox was also confirmed via aqua regia dissolution method. 20 µL of SDox-GNP or HDox-GNP solution was mixed with 2 µL of aqua regia in room temperature. The mixture was diluted with 2.98 mL of water immediately after the red color disappeared. Then, the fluorescence intensity at 590 nm was measured to determine the concentration of Dox in reference to the fluorescent calibration curve. Fluorescent spectrum was collected for comparison with the spectrum of the previous method . Drug release study of SDox-GNPs and HDox-GNPs Approximately 100 µL of HDox-GNPs was diluted with 1.4 mL of water, PBS (pH 7.4), PBS (pH 6.0), or MES (pH 4.7) in normal cuvettes. Then, 1.5 mL of toluene was carefully added to the aqueous phase, and the cuvettes were incubated at 37°C. Fluorescence spectra of the toluene phase were recorded after 1 min of gentle shaking. The amount of Dox release was determined from the intensity of the fluorescence at 590 nm (excitation at 488 nm). Similar releases studies of SDox-GNPs were performed in 1.5 mL of PBS (pH 4.7), GSH (10 mM) or water. A co-mediated release study of HDox-GNPs was also performed using similar methods. PBS (pH 7.4) and MES (pH 4.7) buffer containing GSH (10 mM) were employed in this study.


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Cytotoxicity of gold nanoconjugates The cytotoxicity of the gold nanoconjugates was evaluated using MTT assays. Cancer cells (U87, Hela, MCF-7, A549) were seeded into a 96-well microtiter plate overnight before treatment. The cells were then exposed to a series of concentrations (10 nM, 50 nM, 100 nM, 500 nM, 1 µM, 5 µM and 10 µM) of free Dox, SATA-Dox, HDox-GNPs or SDox-GNPs for 72 h. The dose of SATA-Dox, HDox-GNPs or SDox-GNPs was based on the Dox dose in the conjugates. Identical concentrations of free Dox were used as a control. After exposure for 72 h, the cells were incubated with MTT for 4 h. Then, 100 µL of solubilization buffer was added to dissolve the formazan crystals. The absorbance was measured at 570 nm using a microtiter plate reader (ELx808, BioTek). Cells incubated with culture medium alone were used as a reference of 100% viability. The mean percentage of cell survival relative to that of untreated cells was determined with six replicates for each condition. Cellular uptake and subcellular localization Cells were cultured on Permanox-coated chamber slides and fixed in 2% glutaraldehyde and 4% paraformaldehyde in 0.1 M sodium cacodylate buffer for 2 h. Then, the cells were washed three times with sodium cacodylate buffer and twice with serum-free medium. The cells were harvested by centrifugation and transferred to a 1.5-mL Eppendorf tube. The cells were then incubated with 1% osmium tetroxide in 0.1 M sodium cacodylate buffer for 60 min. The buffer was removed, and the cells were washed with sodium cacodylate buffer and maleate buffer and stained for 60 min in 1% uranyl acetate in maleate buffer. After dehydration and infiltration, the



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embedded cells were polymerized and spurred in a 60°C oven for 1-2 days. Then, 90-nm sections were cut using a Reichert-Jung Ultracut E and stained with uranyl acetate and lead citrate. The images were examined at 3000 kV using a FEI Tecnai F30, Gatan CCD digital micrograph. For the Dox uptake experiment, 10,000 U87 cells were placed in 6-well plates and incubated for 24 h with medium, mGNPs, HDox-GNPs, SDox-GNPs, free Dox or SATA-Dox to yield a final concentration of Dox of 1 µM. The cells were subsequently washed three times with 10% FBS DMEM and resuspended in 300 µL of FACS buffer (1% BSA and 0.01% NaN3 in PBS). Intracellular Dox fluorescence was detected by flow cytometry (Cytoflex, Beckman Coulter). Approximately 20,000 events for each condition were collected and analyzed. For the laser confocal assays, U87 cells were plated in a culture dish at a concentration of 7×104 cells per dish and incubated for 24 h before treatment. The cells were then incubated with Dox, SATA-Dox, SDox-GNPs or HDox-GNPs at equivalent concentrations of Dox of 1 µM for 24 h at 37°C. The cells were stained with 75 nM LysoTracker green for 1 h. The cells were washed with PBS twice, and the fluorescence distribution was visualized via confocal microscopy (LeicaTCS SP5). In vivo tumor inhibition activity Six-week-old male nude mice were purchased and bred at the Center of Experimental Animals at Tongji University. SH-SY5Y xenografts were established by inoculating 5×106 cells via subcutaneous injection in 100 µL of matrigel. When the


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tumor volume reached approximately 200 mm3, the mice were randomly allocated to four groups (n=6). The tumor-bearing mice were treated with free Dox, HDox-GNPs, or SDox-GNPs at a dosage of 2.5 mg Dox equiv./kg in 150 µL of saline. The drugs were administered via intraperitoneal injection every 3 days, and saline was used as a control. Animal body weight was monitored daily. Tumor size was measured daily, and the corresponding volume was calculated according to the formula V=0.5×L×W2. L represents the tumor length, and W represents the tumor width. The biodistribution studies had also been carried out. Organs including tumor, liver, spleen, heart, kidney, heart, and lung were collected from mice injected with HDox-GNPs, SDox-GNPs and saline. The paraffin-embedded tissue slices were stained with hematoxylin, eosin, and silver enhancement reagents. Images were captured at 40X magnifications via optical microscope. RESULTS AND DISCUSSION Synthesis and characterization of SDox-GNPs and HDox-GNPs The GNPs and Dox-loaded GNPs were successfully prepared (Fig. 1). For the synthesis of SDox-GNPs (Fig. 1A), SATA-Dox was easily obtained as a precursor and conveniently purified by column chromatography at room temperature under mild conditions. After the deprotection of acetyl by hydroxylamine hydrochloride, the thiol-modified Dox was mixed with the mGNPs to obtain SDox-GNPs. The process for obtaining hydrazone-modified Dox with the thiol group requires multiple chemical reactions47. Therefore, we employed a simplified method to synthesize the



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HDox-GNPs (Fig. 1B)44. The surface of the gold was first modified by MTG. After purification, hydrazine hydrate was added to obtain the acylhydrazine derivative. Then, Dox was added, and the HDox-GTPs were purified carefully at 4°C under alkaline conditions.

Figure 1. Synthetic scheme for (A) SDox-GNPs and (B) HDox-GNPs

The maximum absorbance of the HDox-GNPs and SDox-GNPs was at 520 nm (Fig. 2A), in agreement with the absorbance of 5-nm GNPs. The absorbance of Dox at 488 nm was not observed due to the higher extinction coefficient of GNPs compared with Dox. Thus, fluorescence spectra were obtained to confirm the presence of Dox. Both HDox-GNPs and SDox-GNPs exhibited Dox emission in aqueous solution, indicating that the drug was attached to the GNPs (Fig. 2B). Dox was conjugated on the surface of the GNPs via a thiol group with a short linkage, and the


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fluorescence was partially quenched due to a surface energy-transfer effect51. In addition, the presence of Dox on the GNPs was confirmed via iodine-induced decomposition method, 1HNMR and Mass Spectroscopy (Fig. S1, S2 and S3). The core diameter (based on TEM) (Fig. 2C), hydrodynamic diameter（based on DLS) and zeta potential (Table. S1) were also determined to confirm that the physical properties of the different GNPs were similar. The hydrodynamic diameters of the HDox-GNPs and SDox-GNPs (~20 nm) were similar to that of the GNPs, which were larger than the size determined by TEM (~5 nm) due to PEGylation.

Figure 2. (A) UV-visible spectra and (B) fluorescence spectra of SDox-GNP (black), HDox-GNP (red) and m-GNP (blue). Transmission electron microscopy images and size distributions of (C) SDox-GNP, (D) HDox-GNP and (E) m-GNP. In (C), (D), and (E), the bars represent 50 nm. Drug release studies of SDox-GNPs and HDox-GNPs To evaluate the drug release efficiency of the Dox conjugates synthesized by the two different methods, the environment surrounding cancer cells and normal



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conditions were simulated in a water-toluene two-phase system (Fig. 3). Low pH is a general property of most tumor microenvironments52. Here, we employed an aqueous solution with a pH of 4.7 to simulate the acidic environment in lysosomes, and PBS with a pH of 7.4 was used as the control. The Dox in HDox-GNPs is conjugated via a hydrazone bond that is sensitive to pH and can be hydrolyzed under low pH conditions. At pH 7.4, the release ratio curve of the HDox-GNPs and SDox-GNPs exhibited a rapid initial increase in fluorescence due to burst release (Fig. S4)53. At pH 4.7, more than 64.1% of the Dox was released after 48 h of incubation compared with 26.1% in the PBS buffer at pH 7.4 (Fig. 3A). Thus, low pH significantly accelerated drug release from HDox-GNPs. However, SDox-GNPs exhibited little response to low pH. Only 15.5% of the loaded drug was extracted in the organic phase, and a similar proportion (15.8%) was observed at pH 7.4 (Fig. 3B). Compared with the hydrazine groups, amide groups, which were conveniently synthesized by the amino/carboxyl coupling reaction, are more stable in acidic solution and retain their integrity at room temperature54. The acidic environment did not contribute to the release of SDox-GNPs. However, the high intracellular concentrations of GSH reduced the Au-S bond to a thiol group, resulting in drug release. The intracellular GSH concentration is 1–10 mM, substantially higher than extracellular levels in plasma46. We verified drug release from SDox-GNPs using 10 mM GSH. After 48 h of incubation in PBS with/without GSH, 68.9% of the drug was released in the presence of GSH compared with 15.8% in the absence of GSH (Fig. 3C). The Au-S bonds in the HDox-GNPs (Fig. 1B) may


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also be responsive to GSH. As expected, drug release increased significantly to 70.6% at pH 7.4 in the presence of a high concentration of GSH (Fig. 3C) compared with 26.1% in the absence of GSH. The drug was rapidly released at pH 4.7. We examined the performance of the HDox-GNPs in the presence of a high concentration of GSH under acid conditions at pH 4.7. Up to 95.6% of Dox was released after incubation for 48 h, indicating that GSH and an acidic environment can simultaneously stimulate drug release from HDox-GNPs (Fig. 3D).

Figure 3. (A) Fluorescence spectra of pH-induced release of doxorubicin from HDox-GNPs: pH 7.4 (red), pH 4.7 (black). (B) pH-mediated drug release from SDox-GNPs: pH 7.4 (red) and pH 4.7 (black) without GSH. (C) Drug release from HDox-GNPs (black) and SDox-GNPs (red) induced by GSH. HDox-GNPs (blue) and SDox-GNPs (green) in PBS at pH 7.4 were used as controls. (D) Co-mediated drug release from HDox-GNPs at pH 4.7 (red) and 7.4 (black) in the presence of 10 mM GSH. PBS buffer at pH 7.4 (blue) and MES buffer at pH 4.7 were used as the control. As shown in Fig. 3, the drug delivery vehicles were quite stable at physiological



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pH, thus allowing the drug to remain on the NPs until reaching the targeted area. The SDox-GNPs exhibited high sensitivity to GSH, and the HDox-GNPs were responsive to both GSH and low pH. Each of the individual mediators significantly accelerated the release ratio of the drug up to approximately 70% in 48 h (Fig. 3A, C). Because low pH and a high GSH concentration are inherent intracellular factors in most cancer cells, HDox-GNPs, which can respond to both pH and GSH, have greater potential as Dox-release vehicles. The drug release ratio increased to 95.6% (Fig. 3D), and both release mediators likely controlled the release process of the HDox-GNPs. GSH facilitated the escape of the prodrug from the surface of the GNPs. Without shielding of PEG, the hydrazone bonds were more easily hydrolyzed by the acidic surroundings. Thus, the co-mediated mechanism of drug release from HDox-GNPs is advantageous compared to the single mechanism of drug release from SDox-GNPs. Cytotoxicity of SDox-GNPs and HDox-GNPs To compare the cytotoxicities of the drugs or nanoparticles against cancer cells, various cell lines (A549, U87, MCF-7 and HeLa) were exposed to serial dilutions of the drug formulations (free Dox, SATA-Dox, HDox-GNPs, SDox-GNPs and m-GNPs) for 72 h. The cell viability was determined using MTT assays. As illustrated in Fig. S5, mGNPs exhibited low cytotoxicity against cells after incubation for 72 h. Therefore, mGNPs are safe and non-toxic drug delivery vehicles. Free Dox and HDox-GNPs exhibited obvious toxicity, and 50% of U87 cells were killed after incubation for 72 h with 0.19 µM and 2.45 µM Dox (Fig. 4A). However, SATA-Dox and SDox-GNPs exhibited much lower cytotoxicity against U87 cells (Fig. 4A). The


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SDox-GNPs exhibited toxicity at concentrations greater than 5 µM (Fig. S6); however, SATA-Dox exhibited no obvious toxicity, and higher doses were required to show intrinsic toxicity (Fig. 4A). Identical phenomena were observed for the other three cell lines (Fig. 4B-D). Compared to SDox-GNPs, the IC50 of HDox-GNPs was 2- to 18-fold lower, indicating that the hydrazone-bound Dox favorably maintained drug activity, whereas the amino-modified Dox lost some drug efficacy. Normally, free Dox exhibits obvious cytotoxicity at a concentration of approximately 1.5 µM 55-57. Modified or unreleased Dox requires a concentration of greater than 5 µM

25, 58

. In our study, the prodrug SATA-Dox did not exhibit

significant cytotoxicity at a high concentration compared with the original Dox, consistent with the literature. This result suggests that the amine group plays an important role in anti-cancer activity. Similarly, the 20-hydroxyl group in camptothecin is rarely modified for linking because of the resulting loss in drug efficacy59-60. The hydrazone-conjugated Dox released the original drug and maintained the medical properties of Dox. However, the amino-modified Dox and conjugated GNPs with the Au-S bond were not favorable for drug delivery, although they were convenient to synthesize and purify.



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Figure 4. Cytotoxicity assays of free Dox, SATA-Dox, HDox-GNPs and SDox-GNPs after incubation for 72 h with cancer cells: (A) U87, (B) MCF-7, (C) HeLa, and (D) A549. Cellular uptake of SDox-GNPs and HDox-GNPs To further evaluate the two conjugate systems, the drug uptake levels of Dox, HDox-GNPs and SDox-GNPs in cancer cells were evaluated by flow cytometry. The cellular uptake efficiency was assessed based on the Dox fluorescence intensity. As shown in Fig. 5A, the drug intensity peak was similar in cells which incubated with HDox-GNPs or free Dox for 3 h, indicating the conjugates were efficiently taken up into the cancer cells. By contrast, SDox-GNPs exhibited lower drug fluorescence intensity, indicating a slow intracellular process for drug release. As the incubation time increased from 3 h to 24 h, the Dox fluorescence intensity increased (Fig. 5B-D). GNPs are potent vehicles with good properties for endocytosis. However, the fluorescent signal of the SDox-GNPs was lower than that for the HDox-GNPs. According to the MS of released Dox from cells dissolution, Dox existed in cells after incubation (Fig. S3C). We inferred that the released drug would interact with GNPs


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by electrostatic interactions and hydrophobic interactions61. It took more time for drug to escape from SDox-GNPs than HDox-GNPs because of the different mechanism of drug release. The drug was nearer to the surface of the GNPs and the fluorescence was partially quenched. The fluorescence of SDox-GNPs was weaker than HDox-GNPs according to flow cytometry experiment (Fig. 5). Therefore, the co-mediated drug delivery system was more suitable for drug release because of the responses to multiple stimuli. Cellular colocalization was assessed to characterize the cellular responses of the different Dox conjugates and GNPs. After 24 h of incubation with 1 µM free Dox, Dox (in red) exhibited obvious colocalization with lysosomes (in green) (Fig. 6A-C). Drug fluorescence was also observed in the cell nuclei (Fig. 6A), where the drug intercalates DNA in cancer cells to induce apoptosis. However, the SATA-Dox and SDox-GNPs localized mainly in cell lysosomes. Neither exhibited obvious drug fluorescence signals in the nuclei (Fig. 6F, I). This result implies that the prodrug could not enter the nuclei and was trapped in the lysosomes. By contrast, the HDox-GNPs retained Dox activity and exhibited similar colocalization in the lysosomes and nuclei (Fig. 6L). The fluorescence of the HDox-GNPs in the nuclei was slightly weaker than that of free Dox, primarily due to Dox hydrolysis. This confocal study demonstrated that the HDox-GNPs preserved the drug subcellular localization, whereas the SDox-GNPs could not enter cell nuclei, explaining the anti-cancer performance of the SDox-GNPs and HDox-GNPs.



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Figure 5. Cellular uptake as investigated by flow cytometry analysis of U87 cells at (A) 3 h, (B) 6 h, (C) 12 h, and (D) 24 h of treatment with SDox-GNPs (red), free Dox (blue), HDox-GNPs (green) and control (black).


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Figure 6. Comparison of uptake, subcellular localization, and induction of nuclear damage. (A)-(C) and (D)-(F) are LSCM images of the U87 cell line incubated for 24 h with free Dox or free SATA-Dox, respectively. LSCM images of U87 cells incubated with (G)-(I) SDox-GNP and (J)-(L) HDox-GNP containing 1 µM SATA-linked Dox or hydrazone-linked Dox for 24 h are also shown. The green fluorescence (green channel) represents the location of the lysosomes stained with LysoTracker green. The Dox fluorescence (red channel) demonstrates that Dox was released in the lysosome, as indicated by the colocalization with LysoTracker green (yellow in overlay) and in the nuclei and by the uncovered red fluorescence in the center of the cells in the overlay. The bar represents 20 µm in all images.



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The endocytosis and localization of the SDox-GNPs and HDox-GNPs were evaluated via TEM. The GNPs were visualized as black dots in the lysosomes (Fig. 7), consistent with confocal images. However, no GNPs were observed in the nuclei (Fig. S7). Thus, the HDox-GNPs were taken up via endocytosis and localized in the lysosomes, and Dox entered the nuclei after its release triggered by low pH and GSH. The GNPs were primarily trapped inside the lysosomes. For the SDox-Au, both the drug and GNPs were trapped in the lysosomes.

Figure 7. TEM of U87 cells incubated with (A) HDox-GNPs and (B) SDox-GNPs at equivalent concentrations of Dox at 1 µM.

The anti-cancer activity of Dox is likely due to intercalation into DNA fragments, which may disrupt replication and transcription and lead to the death of cancer cells62-63. Therefore, the efficacy of Dox was largely increased after delivery to nuclei rather than to other parts of the cells. By contrast, the SATA-Dox and SDox-GNPs were more likely trapped in the lysosomes, with little distribution in the nuclei (Fig.


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6D-I). Thus, the prodrug could not reach the DNA and induce efficient cell death. However, the amino-modified Dox exhibited considerable toxicity after releasing free Dox under specific conditions45, 64. The irreversible modification of Dox resulted in a partial loss of endocytosis and cytotoxicity25. In contrast to SDox-GNPs, the drug on HDox-GNPs was released via hydrolysis and exhibited similar cytotoxicity and distribution as free Dox (Fig. 4-6). In vivo accumulation and anti-cancer activities To evaluate the in vivo properties of the Dox conjugates synthesized using the different attachment strategies, a subcutaneous tumor model of SH-SY5Y was constructed in nude mice. When the tumor volume reached approximate 200 mm3, the first injection was administered via intraperitoneal injection. Additional treatments were administered every three days (day 1, day 4, day 7 and day 10). At 24 h after the first injection, the HDox-GNP- and SDox-GNP-injected mice exhibited a purple color in the tumor area, indicating accumulation of the GNPs (Fig. 8A), primarily due to the enhanced permeability and retention (EPR) effect. Mice injected with HDox-GNPs and SDox-GNPs seemed darker than control group. This observation was mainly caused by the silver enhancement staining in the presence of GNPs. Particles revealed heavy color in low concentration and did not widely spread everywhere. In order to verify the distribution of GNPs, organs were collected from mice injected with HDox-GNPs, SDox-GNPs and saline. The paraffin-embedded tissue slices were stained with hematoxylin, eosin, and silver enhancement reagents. The black and brownish spots indicate Au NPs in the tissues



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(Fig. S8). Biodistribution of organs was carried out to estimate the accumulation of GNPs. As expected, obvious accumulation of GNPs was observed in liver and spleen. Consistent with this observation, a similar pattern of GNPs was observed in tumor tissues. On the contrast, heart, lung, kidney and stomach did not seem to exhibit accumulation of GNPs. Therefore, GNPs revealed relative selectivity to tumor tissue and delivered drug to tumor cells. As illustrated in Fig. 8B, the tumor volume increased rapidly without treatment. Moreover, no significant inhibition of tumor growth was observed in the SDox-GNPs group. By contrast, tumor growth was partially inhibited by free Dox and the HDox-GNPs. The efficacy of HDox-GNPs was slightly lower than that of free Dox. The body weights of the mice were also monitored post-treatment. The Dox-treated group exhibited obvious body weight loss, which was likely a side effect of Dox. The HDox-GNP and SDox-GNP groups at the same Dox dosage continued to gain weight after treatment, similar to the saline treated group (Fig. 8C). This result demonstrates that the GNPs largely eliminated the potential side effects of Dox and delivered the drug to the tumor area.


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Figure 8. (A) Images of the tumors after intraperitoneal injection of HDox-GNPs, SDox-GNPs, free Dox, and saline. (B) Tumor volume after treatment with Dox, HDox-GNPs, SDox-GNPs, or saline every three days. (C) Mice body weights as a function of time during the treatment. CONCLUSION We compared two drug chemical attachment approaches and evaluated the bioactivities of the corresponding products in vitro and in vivo. HDox-GNPs exhibited greater potency for drug delivery via co-mediated triggered release by acidic pH and glutathione (GSH) compared to SDox-GNPs triggered by GSH alone. The Dox that was bound via hydrazone on the GNPs was released in the lysosomes and maintained drug activity by entering the nuclei. Thiol-modified Dox on the GNPs was mainly localized in the lysosomes, resulting in a significant decrease in efficacy against cancer cells. Furthermore, animal studies demonstrated that HDox-GNPs are more potent as a cancer treatment than SDox-GNPs. Thus, our study demonstrated that HDox-GNPs display significant advantages as a nanoparticle delivery system for Dox



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with respect to drug release and anti-tumor efficacy. Prodrug-drug conversion should be considered during the design of drug delivery systems. ASSOCIATED CONTENT

Supporting Information: Release curves of Dox conjugates Characterization of Dox conjugates by DLS, Zeta potential and drug loading Cytotoxicity of mPEG-GNPs TEM of nuclei of U87 cells AUTHOR INFORMATION Corresponding Authors: Email: [email protected], [email protected] Notes The authors declare no competing financial interests. ACKNOWLEDGMENTS This work was sponsored by the National Science Foundation of China (No.81571803, 21432008, 91413109). Y.C. thanks the Thousand Talents Plan and Shanghai Pujiang Program (No.15PJ1407800) for support. REFERENCES 1. Boisselier, E.; Astruc, D., Gold nanoparticles in nanomedicine: preparations, imaging, diagnostics, therapies and toxicity. Chem. Soc. Rev. 2009, 38 (6), 1759-82. 2. Dreaden, E. C.; Mackey, M. A.; Huang, X.; Kang, B.; El-Sayed, M. A., Beating cancer in multiple ways using nanogold. Chem. Soc. Rev. 2011, 40 (7), 3391-404. 3. Qian Chen, X. W., Chao Wang, Liangzhu Feng, Yonggang Li, and Zhuang Liu, Drug-induced self-assembly of modified albumins as nanotheranostics for tumor-targeted combination therapy. ACS nano 2015, 9 (5), 5223–5233. 4. Chen, F.; Zhao, Y.; Pan, Y.; Xue, X.; Zhang, X.; Kumar, A.; Liang, X. J., Synergistically enhanced therapeutic effect of a carrier-free HCPT/DOX nanodrug on breast cancer cells through improved cellular drug accumulation. Mol. Pharm. 2015,


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Comparison of Two Approaches for the Attachment of a Drug to Gold

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