Preliminary Quality Criteria of Citrate-Protected Gold Nanoparticles for

Subscriber access provided by UNIV OF NEW ENGLAND ARMIDALE

Preliminary quality criteria of citrate-protected gold nanoparticles for medicinal applications Jia-Jia Shen, Pinghu Zhang, Feng Zheng, Huan Chen, Wei Chen, Ya Ding, and Xing-Hua Xia ACS Appl. Nano Mater., Just Accepted Manuscript • DOI: 10.1021/acsanm.8b00192 • Publication Date (Web): 18 Apr 2018 Downloaded from http://pubs.acs.org on April 23, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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

ACS Applied Nano Materials

Preliminary quality criteria of citrate-protected gold nanoparticles for medicinal applications Jia-Jia Shen †, Ping-Hu Zhang ‡, Feng Zheng †, Huan Chen ⊥, Wei Chen ║, Ya Ding†,*, Xing-Hua Xia §

†

Key Laboratory of Drug Quality Control and Pharmacovigilance, China Pharmaceutical

University, Ministry of Education, Nanjing 210009, China ‡

Jiangsu Key Laboratory of New Drug Screening & Jiangsu Center for Pharmacodynamics

Research and Evaluation, China Pharmaceutical University, Nanjing, 210009, China ⊥

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

University, Nanjing 210009, China ║

Higher Educational Key Laboratory for Nano Biomedical Technology of Fujian Province,

Department of Pharmaceutical Analysis, Fujian Medical University, Fuzhou 350004, China §

State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and

Chemical Engineering, Nanjing University, Nanjing 210093, China * Corresponding author, email: [email protected]

ACS Paragon Plus Environment

ACS Applied Nano Materials 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

ABSTRACT. Gold nanoparticles (GNPs) are one of the most widely studied and utilized materials in nanomedicine, but there is no business or national quality standard for their medicinal applications. This has become the bottleneck in the translation of clinical applications of gold-based nanomaterials. To solve this issue, the most frequently used citrate-reduced GNPs were selected as demonstration in this study to conduct quality standard research. The different molar ratios of chloroauric acid to trisodium citrate in preparation were changed, from 1:1 to 1:5, to adjust the particle size and main ingredients in gold colloids. The composition of reduced Au0 and residual Au3+ were determined by X-ray photoelectron spectroscopy (XPS), inductively coupled plasma-atomic emission spectroscopy (ICP-AES), and photodynamic catalysis, while the residual citrate and its oxidation products in supernatants were detected by nuclear magnetic resonance (1H NMR) spectroscopy and nonaqueous titration. Cell viability of human normal liver (L02) and hepatoblastoma (HepG2) cells was detected to evaluate the cytotoxicity of main ingredients (Au0) and impurities (Au3+ and citrate salt) in gold colloids. The results showed that the main impurities in gold colloids did not show significant effect on the cell viability and the cytotoxicity was mainly caused by GNPs. Furthermore, in the experiments of the cell apoptosis, cycle, autophagy, and hemolysis assays, GNPs were not completely inert when the gold concentration exceeded 1.5 mg/L. The smallest GNPs showed the greatest degree of apoptosis, autophagy, and G2/M phases retention against L02 and HepG2 cells when compared with the other two larger gold colloids. Based on the above results, a preliminary quality criterion was drafted to assure the safety of GNPs for their medicinal application.

KEYWORDS. gold nanoparticles, quality criteria, particle size, impurities, biological effect


Page 2 of 28

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


INTRODUCTION Nanomaterials utilized in the field of medicine have proven to improve the therapeutic efficiency and biosafety and are promising for precise medical treatment.1 Up to now, numerous nanomaterials, such as iron oxide nanoparticles, carbon nanotubes, silica nanoparticles, and gold nanoparticles (GNPs), have been designed in the construction of various nanosystems for the diagnosis and therapy of cancer.2 It is noted that, all drugs, excipients, and medical probes applied to patients require quality criteria to ensure their clinical controllability, safety, and effectiveness.3 Establishment and refinement of the methods for characterizing organic compounds have been almost a century, and this requirement has a legal effect and is authoritative.4 However, different from the small molecule with a clear chemical structure, nanomaterials are polyatomic aggregates and the structure is much more complex. The standardization of characterization procedures and requirements for nanomaterials progressed much slower than the chemical molecules, which has become the bottleneck of nanomedicine. So far, the quality standards of only two nanomaterials, Ferumoxides (injection and oral suspension)5 and Colloidal Silicon Dioxide, are included in American Pharmacopoeia.6 Colloidal chromium phosphate [32P] injection, a kind of nanocolloid, for the tumor adjuvant therapy is also included in China Pharmacopoeia.7 GNPs are undoubtedly one of the most extensively studied nanomaterials in the fields of biosensing, molecular imaging, and nanomedicine.8,9 GNPs are considered to be good radiosensitizers for cancer therapies based on their high X-ray absorption.10 Owing to their low toxicity in associated with the anti-inflammatory effect, GNPs are also utilized as effective therapeutic agents for the treatment of some inflammatory diseases such as rheumatoid arthritis.11 In addition, it is found that GNPs is effective in suppressing inflammation and stimulating re-epithelialization during the healing process. This indicates that the amelioration of excision



wounds using GNPs can be a novel therapeutic way to improve wound healing in clinical practice.12 Their advantages in biological inert,13 optical and photothermal properties,14 and shape-, size-, and surface chemistry-adjusted performance in vivo15,16 made GNPs the promising material in the future as has been approved by the Food and Drug Administration (FDA).17,18 However, there is no any business or national standards for the medicinal application of GNPs yet. As early as 1952, Peck and Gale suggested that the accepted requirements for full characterization of an organic compound place emphasis on the analysis of sample purity, physical properties, elementary composition, functional groups, and spatial relationships based on structural formula.19 On the contrary, the criteria of nanomaterials would focus on the analytical methods of main ingredients and limits of impurities, as well as their possible influence on the biosafety. Herein, to discuss the quality control of gold-based nanomaterials, we chose the simplest and most widely utilized citrate-reduced GNPs as a demonstration to establish the analytical methods for main ingredients and impurities. The possible effects of the main ingredients and impurities on the medicinal application of diagnosis and therapy were also investigated. To evaluate the influence of the diversity for the preparation of citrate-reduced GNPs, different molar ratios of chlorine acid to trisodium citrate (1:1, 1:2, and 1:5) were used to obtain different gold colloids, denoted as GNPs1:1, GNPs1:2, and GNPs1:5, respectively. Assay of Au0, Au3+, and trisodium citrate in gold colloids was carried out using inductively coupled plasma-atomic emission spectrometry (ICP-AES), photocatalysis, nonaqueous titration, and X-ray photoelectron spectroscopy (XPS) methods. Oxidation products of trisodium citrate formed in the GNPs fabrication were characterized using 1H NMR spectra. Compared with existing nanomaterial quality standards we mentioned above, the limits of impurities have been set according to in vitro cellular properties (e.g. cytotoxicity, hemolysis, cell apoptosis, cell cycle, and cell autophagy). Finally, a preliminary quality


Page 4 of 28

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


criterion for the citrate-reduced GNPs has been drafted as a reference to control the safety and effectiveness of gold nanomaterials.

MATERIALS AND METHODS Materials. Hydrogen tetrachloroaurate hydrate (HAuCl4·4H2O, Au% ≥ 47.8), trisodium citrate dehydrate (Na3C6H5O7·2H2O, ≥ 99.0%), sulfuric acid (H2SO4, 98.08%), glacial acetic acid (C2H4O2, ≥ 99.5%), perchloric acid (HClO4, 70.0-72.0%) and potassium acid phthalate (C8H5KO4, ≥ 99.8%) were purchased from Nanjing Chemical Reagent Co., Ltd (Nanjing, China). Sodium periodate (NaIO4, ≥ 99.5%) and rhodamine B (C28H31ClN2O3) were obtained from Aladdin Reagent Co., Ltd (Shanghai, China). Standard storage solution of gold (Au3+, 1.000 g/L) was purchased from National testing center of nonferrous metals and electronic materials analysis (Beijing, China). Acetic anhydride (C4H6O3, ≥ 98.5%) was provided by Ling Feng Chemical Reagent Co., Ltd (Shanghai, China). Crystal violet was obtained from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). Dulbecco’s Modified Eagle Media (DMEM) was obtained from Thermo Fisher Scientific Inc. (China) and fetal bovine serum (FBS) was purchased from Tianhang biological Polytron Technologies Inc. (Hangzhou, China). Tetrazolium bromide (MTT), Annexin V-FITC/PI Apoptosis Detection Kit, and Cell Cycle and Apoptosis Analysis Kit were supplied by Yisheng Biotechnology Co., Ltd (Shanghai, China). Primary antibodies against LC3B (3868), SQSTM/p62 (8025), and ACTIN were purchased from Cell Signaling Technology (Danvers, MA, USA). All aqueous solutions were prepared using deionized water (>18 MΩ, Purelab Classic Corp., USA). Instruments. Zeta potential was determined using Zetasizer (90plus, Brookhaven, USA). UV-Vis spectroscopic measurements were carried out using a UV-2401 PC UV/Vis spectrophotometer (Shimadzu, Japan). Morphological observation of GNPs was visualized by a



Page 6 of 28

transmission electron microscope (TEM, JEM-200CX, JEOL, Japan). XPS spectra of GNPs were measured on a PHI 5000 Versaprobe X-ray photoelectron spectroscope (UlVAC-PHI, Japan). Gold content was detected using ICP-AES (Leeman Prodigy, USA). The chemical structure of oxidation products of trisodium citrate was detected using lH NMR spectroscopy. 1H NMR spectra were recorded on a Bruker (AVACE) AV-500 spectrometer. Chemical shifts (δ) are listed in parts per million (ppm), using tetramethylsilane as an internal reference. Preparation of GNPs. GNPs were prepared using a citrate reduction method.20 Volumes of 49.63, 49.26, and 48.14 mL aqueous solutions containing HAuCl4·4H2O (14.65 µmol) were heated to boiling for 15 min, respectively. Volumes of 0.37, 0.74, and 1.86 mL Na3C6H5O7·2H2O (39.20 mM) were added into the boiling HAuCl4 solutions, respectively, reacting for another 15 min. The mixture solutions changed into red color and were cooled to room temperature. The as-prepared GNP solutions were stored in brown vials at 4 oC. ICP-AES assay. To separate GNPs from three gold colloids rapidly, the colloids were frozen at -20 oC overnight and warmed to liquid. After centrifugation at 5,000 rpm for 5 min at 4 oC, the black precipitates of GNPs and colorless supernatants were collected separately. The gold atoms in the precipitates (M) and supernatants were detected by ICP-AES. The number of gold atoms fitting into each volume of gold nanoparticles (U) was calculated on the basis of the equation (1).21

(1) where, D is the diameter of GNPs and ɑ is a fixed value of 4.0786 Å. The number of GNPs in three gold colloids (N) was calculated in terms of equation (2).

N=

M U

(2)

Photodynamic catalysis assay. After separating the supernatant and precipitate of the gold


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


colloids, the precipitate was digested with aqua regia. The concentration of Au3+ both in the supernatants and precipitates of three gold colloids was detected using photodynamic catalysis method. RhB solution (0.5 g/L, 0.3 mL), NaIO4 solution (0.005 M, 0.25 mL), and H2SO4 solution (6 M, 1 mL) were added into a 50 mL volumetric flask and mixed well. The solutions containing Au3+ from the supernatant and precipitate in the same volume but different concentrations were added into the mixture. After reaction at 70 oC for 15 min, the solution was cooled to room temperature to terminate the reaction, and then UV-vis spectra were measured. Nonaqueous titration of trisodium citrate. Quantitative analysis of trisodium citrate in three gold colloids was performed using the nonaqueous titration method. Firstly, an accurate concentration of 0.1040 mol/L of perchloric acid solution was calibrated using pharmacopoeia method. The lyophilized residue of the supernatant from gold colloids was dissolved in 5 mL of glacial acetic acid. Then, 10 mL of acetic anhydride and a drop of crystal violet were added. The mixture solution was titrated with perchloric acid solution until the color of solution became a bluish green, e.g. the end point. In addition, a blank test was performed to make a necessary correction. Cell culture. Human normal hepatocytes (L02) and human liver hepatocellular carcinoma (HepG2) cells was purchased from the China Center for Type Culture Collection (Shanghai, China). For cell tests, cells were seeded in cell culture plates using Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum (FBS) and incubated at 37 °C under a humidified atmosphere with 5% CO2 for 24 h to reach 80% confluence. MTT assay. For cytotoxicity assay, L02 and HepG2 cells were seeded in 96-well plates at a density of 1×104 cells per well, respectively. After 24 h, the cells were incubated with different concentrations of gold colloids, Au3+ standard solutions, and trisodium citrate solutions for another



24

h.

The

cells

were

subsequently

washed

with

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazoliumbromide

Page 8 of 28

PBS

twice

(MTT,

and

0.5

Shanghai

mg/mL YeSen

Biotechnology Co. Ltd, China) in growth medium was added for an additional incubation of 4 h at 37 oC. After removing the medium, 150 µL dimethyl sulfoxide (DMSO) was added to each well and the absorbance of formazan product was measured at 490 nm using a microplate reader (Bio-rad, USA). The cell viability (%) was determined by comparing the absorbance at 490 nm with control wells containing only cell culture medium. Data are presented as the means ± SD (n=5). Hemolysis assay. For removal of plasma and leukocytes, whole blood from human was suspended in PBS and centrifuged at 5,000 rpm for 5 min. Supernatant was removed and pellet consisting of red blood cells (RBCs) was resuspended and rinsed with PBS three times. A 2% RBCs solution in PBS (V/V) was then prepared for the hemolysis studies. Different concentrations (100 µL) of gold colloids (50, 25, 12.5, 6.25, 3.15 µg/mL) were respectively mixed with 100 µL 2% RBCs solution. RBCs mixed with distilled water and PBS were used as positive and negative controls, respectively. After incubation at room temperature for 3 h, the mixtures were centrifuged at 10,000 rpm for 3 min, and 100 µL of supernatant from each group was transferred to a 96-well plate. The absorbance of the supernatant solution was measured at 570 nm with a microplate reader (Multiskan FC, Thermo). The hemolysis rate was calculated using the formula: (sample absorbance - negative control absorbance) / (positive control absorbance - negative control absorbance) ×100%. Cell apoptosis assay. For assessment of apoptosis, HepG2 and L02 cells were respectively seeded in 12-well plates at a concentration of 4.5×105 cells per well. After culturing for 24 h, cells were treated with gold colloids and incubated for another 24 h. Subsequently, cells were harvested after trypsinization and stained with Annexin V-FITC and propidium iodide (PI) using the Annexin


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


V-FITC Apoptosis Detection Kit (Shanghai YeSen Biotechnology Co. Ltd, China), according to manufacturer's instruction. The percentage of cells in each quadrant was analyzed by flow cytometry using BD Accuri C6 software (n=3). Cell cycle assay. The effect of GNPs on cell cycle distribution and apoptosis were further analyzed by Cell Cycle and Apoptosis Kit using propidium iodide (PI) staining. Briefly, HepG2 and L02 cells were respectively cultured in 6-well plates at a concentration of 1.1×106cells /well and treated with gold colloids (5739 µg/L) after culturing for 24 h. After that, both the floating and attached cells were collected, washed with ice-cold PBS and fixed with 70% ethanol at 4 oC for 12 h. Subsequently, cells were stained with PI in the dark at 37 oC for 30 min. The cell cycle distribution was detected by flow cytometry (Shanghai YeSen Biotechnology Co. Ltd, China) analyzed using the ModFit software. Cell autophagy assay. Based on the results from cell viability and apoptosis assays, HepG2 cells were more relatively sensitive to GNPs than L02 cells, and thus were further selected to evaluate the autophagy-related activity of GNPs. Briefly, HepG2 cells were seeded into 6-well plates for overnight, and then were incubated with three gold colloids at a concentration of 1500.0 µg/L for 24 h, chloroquine (CQ) as positive control. The expression of autophagy-related marker, SQSTM1/p62 (p62) and LC3B were evaluated by immunoblotting. Relative fold-increased LC3B-II and p62 normalized to ACTB were presented. Cellular uptake Assay. L02 and HepG2 cells were seeded in cell culture dishes for 24 h to reach a confluence of 80%. The media were removed, and cells were washed with PBS twice. Cells were further incubated with GNPs1:1, GNPs1:2, GNPs1:5 at gold dose of 5 µg/mL for 4 h and 12 h. The excess medium was removed, and cells were washed with PBS, trypsinized, and centrifuged. Then samples were separately nitrified in 5 mL of aqua regia. The residual aqua regia was



evaporated by heating. The residue was then redissolved in 1 mL 30% H2O2 and evaporated until less than 0.5 mL. The resultant samples diluted with distilled water to 3 mL and were analyzed via ICP-AES. The results were represented as the mean ± SD (n=3).

RESULTS AND DISCUSSION Preparation, Characterization, and Stability. GNPs were prepared using the citrate reduction method.22 By adjusting the molar ratio of HAuCl4: citrate salt to be 1:1, 1:2, and 1:5, transparent and odorless colloids were obtained with the colors of purple, fuchsia, and salmon pink, respectively (Figure 1A). UV-vis spectra of the gold colloids are presented in Figure 1B. All test solutions showed characteristic localized surface plasmon resonance (LSPR) bands of GNPs. With the increasing proportion of trisodium citrate in the feeding solutions, the maximum absorption wavelength blue-shifted from 551.5 nm to 528.3 nm and then to 518.6 nm for GNPs1:1, GNPs1:2, and GNPs1:5, demonstrating a decrease in particle size. In addition, the zeta potentials of GNPs1:1 (-33.13 ± 1.9 mV), GNPs1:2 (-17.60 ± 1.9 mV), and GNPs1:5 (-13.98 ± 3.1 mV) showed that the prepared GNPs were negatively charged owing to the surface-capped citrate salts (Figure 1C). From Figure 1D, spherical shape of GNPs was found in TEM images and smaller particles exhibited more regular shape and better dispersity. The average particle size of 100 GNPs was measured in TEM images and calculated to be 56.2 ± 4.2 nm, 33.3 ± 1.7 nm, and 15.5 ± 0.5 nm for GNPs1:1, GNPs1:2, and GNPs1:5, respectively, which are consistent with the results demonstrated in UV-vis spectra. In addition to usual translation diffusion, nonspherical particles would provide a contribution from the rotational diffusion to the results detected by DLS, therefore, we adopted TEM method to measure the particle sizes of gold nanoparticles.23-25


Page 10 of 28

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


Figure 1. (A) photograph, (B) UV-vis spectra, (C) zeta potentials, and (D) TEM images of (a) GNPs1:1, (b) GNPs1:2, and (c) GNPs1:5, respectively. The data are presented as the mean ± SD (n=3). *, P < 0.05; **, P < 0.01.

In addition, we investigated the short-term stability (not more than 72 h) in different media and long-term storage stability (three months) of prepared gold colloids in the darkness and at 4 oC. The method and results of the short-term stability were included and showed in Figure S1 (Supporting Information). According to the changes of particle size and the maximum absorption wavelength of solutions, all samples didn’t show significant difference. They were not stable when diluted by only phosphate buffered solution (PBS), while in the presence of fetal bovine serum (FBS) the particles are stable at least for 36 h without the addition of DMEM. This means citrate-protected GNPs are ACS Paragon Plus Environment


sensitive to high salts but FBS can stable GNPs and keep their dispersity. Moreover, after three months storage of gold colloids at 4 oC, GNPs1:2 and GNPs1:5 maintain better long-term stability than GNPs1:1. The maximum absorption wavelength remain unchanged at ca. 528 nm and 518 nm for GNPs1:2 and GNPs1:5 solutions, respectively, while black precipitation appeared in GNPs1:1 after three weeks’ preservation. Assay of gold nanoparticles and salts. To detect the different element valence of gold, XPS spectra of three samples were showed in Figure S2. A strong and sharp peak at ca. 84 eV represents Au 4f7/2 component, and a relatively week peak at ca. 88 eV is corresponded to the Au 4f5/2 components. XPS data clearly showed the existence of Au0 at 84.0 eV, while other valences of gold were not seen by this method. It might be the content of gold ions was too low to be detected. Therefore, ICP-AES and photodynamic catalysis were further employed to quantitatively analyze GNPs (Au0) and soluble gold salt (Au3+) in the prepared gold colloids, respectively. Gold contents in the precipitate and supernatant detected using ICP-AES are shown in Figure S3, A and B. From the data in Figure S3B, the reduction rate of gold salt by trisodium citrate can be calculated (Figure S3C). More than 99.9% of the chloroauric acid was reduced to zero-valent gold and three types of formed GNPs did not show significant difference. Dozens of micrograms of gold in one liter (< 100 µg L-1) were detected in the supernatants, indicating that the remaining of small amount of gold salt was an impurity component of GNPs. Although three gold colloids had similar Au0 concentration, they showed significant differences in nanoparticle numbers that were negatively correlated with the diameters of GNPs (Figure S3D).26 GNPs1:1, GNPs1:2, and GNPs1:5 with the average diameter of 56.2, 33.3, and 15.5 nm had 3.2 × 1010, 1.6 × 1011, and 2.4 × 1012 particles per mililiter in their solutions, respectively. Photodynamic catalysis of Rhodamine B (RhB) oxidation27 using NaIO4 needs the participation


Page 12 of 28

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


of Au3+. We found that the absorbance of RhB displayed a linearly negative correlation with Au3+ concentrations (Figure S4A). The catalytic reaction equation is as follows:28 Au3+

RhB

CO2 + H2O + NO3- + NH4+

NaIO4

(Colorless)

(Peachblow)

(3)

Gold contents in precipitate and supernatant detected using this catalytic reaction are shown in Figure S4, B and C. Based on these data, the reduction yields of chloroauric acid were high (> 99.9%, Figure S4D), similar to the results as determined by ICP-AES (Figure S3C). To compare these two methods, we list all parameters in Table 1. The data demonstrated that ICP-AES method displayed higher accuracy and precision while photodynamic catalysis method was relatively simpler in the sample processing and the instrument. The reduction efficiency of gold salt by trisodium citrate reduction was close to 100% when the molar ratio of trisodium citrate: chloroauric acid was not less than 1:1.

Table 1. Assay of gold contents by ICP-AES and photodynamic catalysis (mean ± SD, n=3). Total amount of gold / mg 0

-1

3+

-1

Methods

Samples

Au / mg L

Au / µg L

ICP-AES

GNPs1:1 GNPs1:2 GNPs1:5

51.2 ± 7.7 53.0 ± 2.3 52.7 ± 9.1

GNPs1:1 GNPs1:2 GNPs1:5

53.4 ± 12.2 60.7 ± 12.3 47.2 ± 12.8

Photodynamic catalysis

Reduction yield /%

Theoretical value

Measured value

97.2 ± 13.8 26.3 ± 2.1 57.2 ± 11.2

2.87

2.57 ± 0.39 2.65 ± 0.12 2.64 ± 0.45

99.8 ± 0.00 99.9 ± 0.00 99.9 ± 0.02

23.2 ± 7.2** 31.6 ± 6.9 19.2 ± 5.7**

2.87

2.67 ± 0.61 3.04 ± 0.62 2.36 ± 0.64

99.9 ± 0.01 99.9 ± 0.01 99.9 ± 0.01

*, P < 0.05; **, P < 0.01.

Assay of citrate salt and its oxidation products. After citrate reduction reaction, the surplus citrate and its oxidation products remained in the gold colloids. Hydrogen nuclear magnetic resonance spectroscopy (1H NMR) and nonaqueous titration method were employed to characterize the chemical structure of the oxidation products and determine the citrate content. As shown in ACS Paragon Plus Environment


Page 14 of 28

Figure 2A, the signals of methylene protons in trisodium citrate were found to be a pair of doublets in all spectra at ca. 2.6 ppm and 2.5 ppm (signals 1 and 2). New peaks appeared at ca. 2.3 and 3.9 ppm (signals 3 and 4), indicating the formation of decomposition product of trisodium citrate, e.g. acetoacetic acid (Figure 2B). In addition, signal for formic acid (signal 5) at 7.9 ppm was found, which may be generated through an intermediate product, acetone dicarboxylic acid that was not observed in 1H NMR spectra because it is unstable at an elevated temperature such as the boiling condition of GNP preparation.29 Thus, the possible decomposition pathways and oxidation products of trisodium citrate are shown in Figure 2B. All these intermediates are not stable enough. They would finally decarboxylate and release carbon dioxide. Therefore, the equation for the reduction of chloroauric acid by trisodium citrate can be written as:30 HAuCl4 + Na3C6H5O7

Boiling

(Ketoglutarate sodium)

(Trisodium citrate)

Na2C5H4O5 + HCl (Ketoglutarate sodium) Na2C4H5O3 + HCl (Acetoacetate sodium)

Au0 + Na2C5H4O5 + NaCl + CO2 + 3HCl

Boiling

NaC4H5O3 + CO2 + NaCl

(Acetoacetate sodium) Boiling

C3H6O + CO2 + NaCl (Acetone)


(4)

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


Figure 2. (A) 1H NMR spectra of trisodium citrate and its oxidation products in the supernatants of GNPs1:1, GNPs1:2, and GNPs1:5, respectively. (B) Decomposition procedure of trisodium citrate in gold colloid preparation. (C) Remained and (D) consumed amount of trisodium citrate in the supernatants of gold colloids detected using the nonaqueous titration method (mean ± SD, n=3).

Hydrogen integrals in 1H NMR spectra demonstrated that unreacted trisodium citrate was the main component in the supernatant while the decomposition impurities had very little proportion. Therefore, we only determined the content of trisodium citrate by nonaqueous titrations (Figure 2C). Nonaqueous titration is the most commonly used titrimetric procedure included in pharmacopoeia. It is an effective method for the analysis of weak acids, such as citric in the supernatant of gold colloids. Subtracted by the amount of trisodium citrate added in the feeding solutions, the consumed trisodium citrate in GNPs1:1, GNPs1:2, and GNPs1:5 was calculated to be 2.72 ± 0.10 mg, 5.37 ± 0.16 mg, and 11.99 ± 0.47 mg, respectively (Figure 2D). Here, GNPs1:5 consumed the most trisodium citrate in amount even though all the GNPs showed similar reduction yield in Table 1. It can be explained by the most citrate adsorption on the largest specific surface area of GNPs1:5,



which was removed with the precipitate in the separation procedure.

Cytotoxicity and limit control of impurities. Based on the above analysis, besides the main ingredient of Au0 in gold colloids, there existed important impurities mainly including Au3+ and trisodium citrate. The existence of these impurities would influence the quality and biosafety of GNPs. Therefore, the impurity limit should be controlled to eliminate cytotoxicity. There are large school of literatures reported the liver accumulation and relative side effects of GNP-based nanosystems in vivo,31,32 herein, the human normal liver (L02) and hepatoblastoma (HepG2) cells were thus employed to validate the cellular uptake and cytotoxicity of GNPs. As shown in Figure S5, GNPs1:5 showed the highest uptake of gold in both L02 and HepG2 cells, while at the same time the cellular uptake of GNPs1:1 and GNPs1:2 displayed no significant difference. The intracellular gold content of GNPs1:5 was 1.7 and 1.8 times higher than that of GNPs1:1 against L02 and HepG2 cells, respectively. It indicated that citrate-protected GNPs can be internalized by cells and smaller particles have higher uptake efficiency. The cell viability incubated with gold colloids, HAuCl4, and trisodium citrate solutions in different concentrations was detected using MTT assay (Figure 3). Gold colloids exhibited very low cytotoxicity in both L02 and HepG2 cells. From Figure 3, A and B, Gold colloids did not show inhibition effect on both cells at the gold concentration lower than 1.5 mg/L (Au0 plus Au3+). However, when the gold concentrations were higher than 1.5 mg/L, cell growth was slightly inhibited and HepG2 cells (Figure 3B) were more sensitive than L02 cells (Figure 3A). Among three colloids, GNPs1:1 having less trisodium citrate showed higher cytotoxicity than the other two colloids (Figure 3B), indicating that the cytotoxicity was mainly caused by GNPs rather than trisodium citrate. Combining the cellular uptake results, the fact that GNPs1:1 has the lowest uptake


Page 16 of 28

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


efficiency but exhibits the lowest cell viability suggests that the bigger particles would lead to higher cytotoxicity.

Figure 3. MTT assay of (A) L02 and (B) HepG2 cells incubated with different concentrations of gold colloids for 24 h. The cytotoxicity of (C) Au3+ and (D) trisodium citrate at different concentrations against L02 and HepG2 cells. *P < 0.05, **P < 0.01, ***P < 0.001 vs. the cell viability at the lowest concentration. Each data represents mean ± SD, n=3.

To determine the limit of major impurities, cell viability of Au3+ and trisodium citrate is presented in Figure 3, C and D. With the increase of Au3+ and trisodium citrate concentrations, there was no obvious toxicity in both cells below 1.0 mg/L and 1.0 g/L, respectively. While above these two concentrations, cell viabilities gradually decreased and HepG2 cells showed more sensitivity towards both Au3+ and trisodium citrate. In addition, the maximum concentration of Au3+ and trisodium citrate in these three gold colloids were 0.1 mg/L and 0.2 g/L. Combining with the solution composition and cell viability results, the limits of Au3+ and trisodium citrate are suggested ACS Paragon Plus Environment


at 0.2 mg/L and 0.4 g/L, respectively. It was obvious that the impurity limits were 2 folds of the impurity content in gold colloids and at the same time far below the concentration that induced obvious cytotoxicity. Under the control of this limit, they can be considered as biosafe at their preparation concentration, although the as-prepared gold colloids were not completely inert and nontoxic. Hemolysis assay and other biological effects. Hemolytic rate (< 5%) is a commonly used index for the biocompatibility evaluation of intravenous materials according to the national standard of China, GB/T14233.2. In this case, after the incubation of gold colloids with human red blood cells (RBCs) for 3 h, all samples showed no obvious hemolysis (less than 5%, Figure 4), indicating the favorable biocompatibility of citrate-protected GNPs.

Figure 4. Hemolysis quantification of RBCs at concentrations of gold at 3.15, 6.25, 12.5, 25, and 50 µg/mL, respectively. RBCs incubated with PBS and water were used as negative and positive controls, respectively. ***, P < 0.001 vs. hemolytic rates of GNPs-incubated samples. Each data represents mean ± SD (n=3).

Other biological effects of citrate-protected GNPs on cell apoptosis, cell cycle, and cell autophagy were also investigated to evaluate the possible toxicity. Annexin V-FITC/PI apoptosis detection kit was used to detect the apoptosis of HepG2 and L02 cells after the incubation with gold ACS Paragon Plus Environment

Page 18 of 28

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


colloids at gold concentration of 5.7 mg/L (Figure S6), the maximum concentration in MTT assay. At this relatively high concentration, cell apoptosis was observed in both L02 and HepG2 cells. However, the total apoptosis rate in L02 was no more than 18%, indicating that gold colloids did not have obvious pro-apoptotic effect. This phenomenon was also observed in GNPs1:1 and GNPs1:2 groups against HepG2 cells. It is interesting that GNPs1:5 showed the highest apoptosis in HepG2 cells (28.4%), which indicated that the liver tumor cell may be sensitive to smaller-sized GNPs. The similar phenomenon was also found in cell cycle assay (Figure 5A). Comparing with the control group, the percentage of both L02 and HepG2 cells in the G2/M phases increased with the decrease of size of GNPs. The data changed from 11.34%, 12.29%, to 17.99% for the groups of GNPs1:1, GNPs1:2, and GNPs1:5, respectively (Figure 5A, a-d). HepG2 cells were more sensitive to the gold colloids. The percentage of G2/M phases was higher than those of L02 cells, e.g. 14.01%, 14.84%, and 20.75% corresponding to the GNPs1:1, GNPs1:2, GNPs1:5, respectively (Figure 5A, e-h). In the quantitative analysis of the respective cell cycle statuses by flow cytometry (Figure 5B), HepG2 cells showed more cells staying in G2/M phases after an incubation for 24 h, especially for GNPs1:5. This stay indicated that the cell proliferation rate was reduced and HepG2 cells were more sensitive to gold colloids than L02 cells.18 Since G2/M phase was the most sensitive phase to radiotherapy, we could use GNPs to induce G2/M phase statues of tumor cells and enhance radiotherapy effect.



Figure 5. Cell cycle assay of L02 cells (a-d) and HepG2 cells (e-h). These two cell lines were exposed to 5739.0 µg/L gold colloids for 24 h and cell cycle was analyzed by flow cytometry. (A) The representative images of L02 cells (control (a), GNPs1:1 (b), GNPs1:2 (c), GNPs1:5 (d)) and HpeG2 cells (control (e), GNPs1:1 (f), GNPs1:2 (g), GNPs1:5 (h)) were shown. (B) The relative cell numbers of different cell cycles (G1/G0 phases (red), S phase (green), and G2/M phases (blue) were analyzed statistically. (C) Cell autophagy assay of GNPs. HepG2 cells were treated with 1.5 mg/L gold colloids for 24 h, chloroquine (CQ) as positive control and ddH2O as negative control. Proteins LC3B-I, LC3B-II, p62, and ACTIN were evaluated by immunoblotting and the relative expression levels normalized (norm) to negative control were quantified by Quantity One.

Moreover, autophagy is a lysosome-based degradative pathway which plays an essential role in maintaining cellular homeostasis. During autophagy, autophagosomes engulf the intracellular ACS Paragon Plus Environment

Page 20 of 28

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


contents and fuse with lysosomes to form hybrid organelles named autolysosomes.33 LC3B, an important autophagy protein exists as two forms, LC3B-I and the autophagosome membrane-bound LC3B-II. In the process of autophagy, the cytoplasmic form LC3B-I can be converted to LC3B-II. Therefore, LC3B-II is widely used as an autophagic biomarker to monitor autophagic activity.34 p62 as an autophagy substrate is preferentially degraded in the process of autophagic degradation. As shown in Figure 5C, all three GNPs induced the conversion of LC3B-II and increased the accumulation of p62, suggesting blocking the autophagic degradation. Compared with GNPs1:1 and GNPs1:2, GNPs1:5 with the smaller particle size induced stronger autophagy activity in HepG2 cells. However, the detailed mechanism of GNPs-induced autophagy is needed to further clarify. Based on the above biological effects induced by gold colloids, GNPs1:5 demonstrated stronger cell apoptosis, G2/M phase retention, and autophagic degradation suppression, especially in tumor cells. Therefore, referring to the relevant provisions of Ferumoxides Injection and Colloidal Silicon Dioxide in the United States Pharmacopoeia, the preliminary quality criteria of citrate-protected GNPs (Colloidal Gold) were drafted on the basis of above results as follows.

Preliminary quality criteria. Definition Colloidal Gold is a colloidal suspension associated with trisodium citrate in water for injection. It contains no less than 95 percent and no more than 105 percent of the labeled amount of gold. It contains in each mL no less than 0.03 mg and no more than 0.4 mg of trisodium citrate. It contains no antimicrobial agents. The GNPs particle size is between 15 and 60 nm detected using transmission electron microscope (JEM-200CX, JEOL, Japan). Characters GNPs colloids show transparent purple, fuchsia, salmon pink colors according to different sizes.



Identification Add 3 mL gold colloids to 1 cm ultraviolet quartz dish and determine the ultraviolet characteristic absorption spectrum by wavelength scanning for GNPs. The characteristic maximum absorption wavelength is in the range of ca. 510-560 nm. pH Between 3.0 to 6.0. Colloidal particle size Apparatus−Use a transmission electron microscope. Procedure−Transfer 8 µL of gold colloids to copper network. Place in the transmission electron microscope and then measure the particle sizes. The particle size is between 15 and 60 nm. Impurities Au3+, Methods 1 Apparatus−Use an inductively coupled plasma atomic emission spectrometry instrument. Procedure−Freeze the gold colloid 50 mL at -20 oC overnight and separate the precipitation by freezing centrifugation at 5000 rpm for 5 min at 4 oC. Obtain the supernatants and determine gold content by ICP-AES. Acceptance criteria: NMT 0.2 ppm according to the limit. Au3+, Methods 2 Apparatus−Use an ultraviolet spectrophotometer instrument. Procedure−Freeze the gold colloid 50 mL at -20 oC overnight and separate the precipitation by freezing centrifugation at 5000 rpm for 5 min. Obtain the supernatant and determine gold content by photodynamic catalysis assay. Acceptance criteria: NMT 0.2 ppm according to the limit. Trisodium citrate Standardized titration solution−Use a standardized perchloric acid solution (0.1 mol/L)


Page 22 of 28

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


Procedure−Dissolve the lyophilized residue in the supernatant in 5 mL of glacial acetic acid. Add 10 mL of acetic anhydride and a drop of crystal violet. The mixture solution is titrated with perchloric acid solution until the color of solution becomes a bluish green. Acceptance criteria: NMT 400 ppm according to the limit. Assay for gold nanoparticles Apparatus−Use an inductively coupled plasma atomic emission spectrometry instrument. Procedure−Freeze the gold colloid 50 mL at -20 oC overnight and separate the precipitation by freezing centrifugation at 5000 rpm for 5 min at 4 oC. Obtain the precipitate and digest with 10 mL aqua regia. Determine the gold content by ICP-AES. The number of gold atoms in precipitates (M) is calculated from ICP-AES data. Containers and storage Preserve in well-closed and shading containers at 4 oC.

CONCLUSION In this study, three types of citrate-reduced and protected GNPs were prepared and analyzed to establish the preliminarily quality criteria of gold colloids. The colloid solutions were characterized in detail. The main ingredient and impurities, Au0, Au3+, and citrate salt, were qualitatively and quantitatively analyzed using ICP-AES, photocatalytic, 1H NMR, and nonaqueous titration methods. Based on the cytotoxicity test, the impurity limits have been determined. Moreover, other cytological properties including cell apoptosis, cell cycle, and autophagy have been investigated. The gold colloids did not show significant influence on normal liver cells, while they demonstrated potential inhibition effects on liver tumor cells through cell apoptosis, G2/M phase retention, and autophagic degradation suppression, especially for the colloids containing smaller size particle (e.g. GNPs1:5). Finally, a preliminary quality criterion of gold colloid has been drafted. This criterion



provides a reference for the further medicinal application and clinical transformation of gold-based as well as other nanomaterials.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: . ICP-AES assay of GNPs and unreduced gold salts in the gold colloids; Determination of gold in supernatants and precipitates by photodynamic catalysis assay; Cell apoptosis assay of L02 and HepG2 cells exposed to 5.7 mg/L of gold colloids for 24 h (PDF) AUTHOR INFORMATION Corresponding Author * E-mail: [email protected] ORCID Wei Chen: 0000-0003-3233-8877 Xing-Hua Xia: 0000-0001-9831-4048 Ya Ding: 0000-0001-6214-5641 Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT


Page 24 of 28

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


This work was supported by grants from the National Natural Science Foundation of China (31470916), 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).

REFERENCES (1) Liu, J.; Zheng, X. P.; Yan, L.; Zhou, L. J.; Tian, G.; Yin, W. Y.; Wang, L. M.; Liu, Y.; Hu, Z. B.; Gu, Z. J.; Chen, C. Y.; Zhao, Y. L. Bismuth Sulfide Nanorods as a Precision Nanomedicine for in vivo Multimodal Imaging-guided Photothermal Therapy of Tumor. ACS Nano 2016, 12, 696-70. (2) Yezhelyev, M. V.; Gao, X. H.; Xing, Y.; Ahmad, A. H.; Nie, S. M.; O’Regan, R. M. Emerging Use of Nanoparticles in Diagnosis and Treatment of Breast Cancer. Lancet Oncol. 2006, 7, 657-667. (3) Li, J. X.; Chang, X. L.; Chen, X. X.; Gu, Z. J.; Zhao, F.; Chai, Z. F.; Zhao, Y. L. Toxicity of Inorganic Nanomaterials in Biomedical Imaging. Biotechnol. Adv. 2014, 32, 727-743. (4) Hermann, R.; Cnota, P.; Maus, J. Minimum Quality Criteria are Needed in the Assessment and Communication of Unexpected Drug Safety Findings of Marketed Products from Randomized Controlled Trials. Brit. J. Clin. Pharmaco. 2012, 74, 207-208. (5) National Pharmacopoeia Committee. Pharmacopoeia of the United States of America. USP35-NF30, 2012: Text 1949. (6) National Pharmacopoeia Committee. Pharmacopoeia of the United States of America. USP35-NF30, 2012: Text 3183-3185. (7) National Pharmacopoeia Committee. Pharmacopoeia of People’s Republic of China. Part 2. Beijing: Chemical Industry Press, 2015: Text 1600. (8) Akthakul, A.; Hochbaum, A. I.; Stellacci, F.; Mayes, A. M. Size Fractionation of Metal Nanoparticles by Membrane Filtration. Adv. Mater. 2005, 17, 532-535. (9) Yohan, D.; Cruje, C.; Lu, X. F.; Chithrani, D. B. Size-Dependent Gold Nanoparticle Interaction at Nano-Micro Interface Using Both Monolayer and Multilayer (Tissue-Like) Cell Models. Nano-Micro Lett. 2016, 8, 44-53. (10) Her, S.; Jaffray, D. A.; Allen, C. Gold Nanoparticles for Applications in Cancer Radiotherapy: Mechanisms and Recent Advancements. Adv. Drug Deliver. Rev. 2017, 109, 84-101. (11) Hornos Carneiro, M. F.; Jr, B. F. Gold Nanoparticles: A Critical Review of Therapeutic Applications and Toxicological Aspects. J. Toxicol. Env. Heal. B 2016, 19, 129-148. (12) Naraginti, S.; Kumari, P. L.; Das, R. K.; Sivakumar, A.; Patil, S. H.; Andhalkar, V. V. Amelioration of Excision Wounds by Topical Application of Green Synthesized, Formulated Silver and Gold Nanoparticles in Albino Wistar Rats. Mat. Sci. Eng. C 2016, 62, 293-300. (13) Zhang, X. Gold Nanoparticles: Recent Advances in the Biomedical Applications. Cell Biochem. Biophys.



2015, 72, 771-775. (14) Huang, X.; El-Sayed, M. A. Gold nanoparticles: Optical Properties and Implementations in Cancer Diagnosis and Photothermal Therapy. J. Adv. Res. 2010, 1, 13-28. (15) Arnida.; Janátamsbury, M. M.; Ray, A.; Peterson, C. M.; Ghandehari, H. Geometry and Surface Characteristics of Gold Nanoparticles Influence their Biodistribution and Uptake by Macrophages. Eur. J. Pharm. Biopharm. 2011, 77, 417-423. (16) Carnovale, C.; Bryant, G.; Shukla, R.; Bansal, V. Size, Shape and Surface Chemistry of Nano-gold Dictate its Cellular Interactions, Uptake and Toxicity. Prog. Mater. Sci. 2016, 83, 152-190. (17) Iqbal, M.; Usanase, G.; Oulmi, K.; Aberkane, F.; Bendaikha, T.; Fessi, H.; Zine, N.; Agustia, G.; Errachid, E. S.; Elaissari, A. Preparation of Gold Nanoparticles and Determination of Their Particles Size via Different Methods. Mater. Res. Bull. 2015, 79, 97-104. (18) Wang, P. Y.; Wang, X.; Wang, L. M.; Hou, X. Y.; Liu, W.; Chen, C. Y. Interaction of Gold Nanoparticles with Proteins and Cells. Sci. Technol. Adv. Mater. 2015, 16, 1-15. (19) Peck, R. L.; Gale, P. H. Characterization of Organic Compounds. Anal. Chem. 1952, 24, 116-120. (20) Frens, G.; Kolloid, Z. Controlled Nucleation for the Regulation of the Particle Size in Monodisperse Gold Suspensions. Nat. Phys.Sci. 1973, 241, 20-22. (21) Chithrani, B. D.; Ghazani, A. A.; Chan, W. C. Size and Shape Dependence of Nanoparticles on Cellular Uptake. Nano Lett. 2006, 6, 662-668. (22) Frens, G.; Kolloid, Z. Controlled Nucleation for the Regulation of the Particle Size in Monodisperse Gold Suspensions. Nat. Phys. Sci. 1973, 241, 20-22. (23) Mingalyov, P. G.; Kolyagin, Y. G.; Lisichkin, G. V. Solid-state NMR Spectroscopic Study of the Structure of Grafted Layers of Phosphonic Acid Ester-modified Hydroxyapatite. Colloid J. 2011, 73, 83-87. (24) Haiss, W.; Thanh, N. T. K.; Aveard, J.; Fernig, D. G. Determination of Size and Concentration of Gold Nanoparticles from UV-vis Spectra. Anal. Chem. 2007, 79, 4215-4221. (25) Njoki, P. N.; Lim, I. S.; Mott, D.; Park, H. Y.; Khan, B.; Mishra, S.; Sujakumar, R.; Luo, J.; Zhong, C. J. Size Correlation of Optical and Spectroscopic Properties for Gold Nanoparticles. J. Phys. Chem. C 2007, 111, 14664-14669. (26) Piella, Jordi.; Bastús, Neus. G.; Puntes, V. Size-Controlled Synthesis of Sub-10-nanometer Citrate-Stabilized Gold Nanoparticles and Related Optical Properties. Chem. Mater. 2016, 28, 1066-1075. (27) Zhu, X. S.; Zhang, Y. Q. Inhibition Kinetic Determination of Trace Amount of Iodide by the Spectrophotometric Method with Rhodamine B. Spectrochim. Spectrochim. Acta A 2008, 70, 510-513. (28) Hou, M. F.; Liao, L.; Zhang, W. D.; Tang, X. Y.; Wan, H. F.; Yin, G. C. Degradation of Rhodamine B by Fe(0)-based Fenton Process with H2O2. Chemosphere 2011, 83, 1279-1283. (29) Munro, C. H.; Smith, W. E.; Garner, M.; Clarkson, J.; White, P. C. Characterization of the Surface of a Citrate-Reduced Colloid Optimized for Use as a Substrate for Surface-Enhanced Resonance Raman Scattering. Langmuir 1995,11, 3712-3720. (30) Ojeajiménez, I.; Campanera, J. M. Molecular Modeling of the Reduction Mechanism in the Citrate-Mediated Synthesis of Gold Nanoparticles. J. Phys. Chem. C 2012, 116, 23682-23691. (31) Glazer, E. S.; Zhu. C.; Hamir, A. N.; Borne, A.; Thompson, C. S.; Curley, S. A. Biodistribution and Acute Toxicity of Naked Gold Nanoparticles in a Rabbit Hepatic Tumor Model. Nanotoxicology 2011, 5, 459-468.


Page 26 of 28

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


(32) Balasubramanian, S. K.; Jittiwat, J.; Manikandan, J.; Ong, C. N.; Yu, L. E.; Ong, W. Y. Biodistribution of Gold Nanoparticles and Gene Expression Changes in the Liver and Spleen after Intravenous Administration in Rats. Biomaterials 2010, 31, 2034-2042. (33) Zhou, Y. F.; Wang, Q.; Song, B.; Wu, S. C.; Su, Y. Y.; Zhang, H. M.; He, Y. A Real-time Documentation and Mechanistic Investigation of Quantum Dots-induced Autophagy in Live Caenorhabditis Elegans. Biomaterials 2015, 72, 38-48. (34) Zhang, P. H.; Zheng, Z. G.; Ling, Li.; Yang, X. H.; Zhang, N.; Wang, X.; Hu, M. Z.; Xia, Y.; Ma, Y. W.; Yang, H. R.; Wang, Y. Y.; Liu. H. Q. A Novel Autophagy Enhancer, Induces Autophagy-dependent Cell Apoptosis via Activation of EGFR-mediated RAS-RAF1-MAP2K-MAPK1/3 pathway. Autophagy 2017, 13, 1093-1112.



Graphic for manuscript


Page 28 of 28

Preliminary Quality Criteria of Citrate-Protected Gold Nanoparticles for

Recommend Documents