Subscriber access provided by University of South Dakota
Article
Gold Nanoparticles Cure Bacterial Infection with Benefit to Intestinal Microflora Juanjuan Li, Ruitao Cha, Xiaohui Zhao, Hongbo Guo, Huize Luo, Mingzheng Wang, Fengshan Zhou, and Xingyu Jiang ACS Nano, Just Accepted Manuscript • Publication Date (Web): 27 Mar 2019 Downloaded from http://pubs.acs.org on March 27, 2019
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 48 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 Nano
Gold Nanoparticles Cure Bacterial Infection with Benefit to Intestinal Microflora Juanjuan Li1, 2, Ruitao Cha2, *, Xiaohui Zhao2, Hongbo Guo2, Huize Luo1, 2, Mingzheng Wang1, 2, Fengshan Zhou1,*, Xingyu Jiang2, 3, 4* Beijing Key Laboratory of Materials Utilization of Nonmetallic Minerals and
1
Solid Wastes, National Laboratory of Mineral Materials, School of Materials Science and Technology, China University of Geosciences (Beijing), No. 29 Xueyuan Road, Beijing 100083, P. R. China; 2
Beijing Engineering Research Center for BioNanotechnology and CAS Key
Laboratory for Biomedical Effects of Nanomaterials and Nanosafety, CAS Center for Excellence in Nanoscience, National Center for NanoScience and Technology, No. 11 Zhongguancun Beiyitiao, Beijing 100190, P. R. China; 3
Department of Biomedical Engineering, Southern University of Science and
Technology, No. 1088 Xueyuan Road, Nanshan District, Shenzhen, Guangdong 518055, P. R. China; 4
University of Chinese Academy of Sciences, 19 A Yuquan Road, Shijingshan District, Beijing 100049, P. R. China.
1 ACS Paragon Plus Environment
ACS Nano 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
Page 2 of 48
ABSTRACT: Antibiotics which are most used to cure bacterial infections in the clinic would result in the imbalance of intestinal microflora, destroy the intestinal barrier, and induce bacterial resistance. Therapy for bacterial infections without destroying intestinal microflora suggests an urgent need for antibacterial agents. Herein,
we
applied
4,
6-diamino-2-pyrimidinethiol
(DAPT)-coated
Au
nanoparticles (D-Au NPs) for therapy of bacterial infection induced by
Escherichia coli (E. coli) in gut. We cultured D-Au NPs and E. coli in anaerobic atmosphere to evaluate their bactericidal effect. We studied the microflora, distribution of Au, and biomarkers in mice after a 28-day oral administration to analyze the effect of Au NPs on mice. D-Au NPs cured bacterial infections more effectively than levofloxacin without harming intestinal microflora. D-Au NPs showed great potential as alternatives of oral antibiotics.
KEYWORDS: Au nanoparticles, bacterial infections, Escherichia coli, intestinal microflora, intestinal epithelial cells
2 ACS Paragon Plus Environment
Page 3 of 48 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 Nano
Infections caused by Escherichia coli (E. coli) in gut can induce peritonitis, cholecystitis, and appendicitis.1 Serious infection induced by E. coli would cause blood infections and life-threatening septicaemia, which affects thousands of patients every year.2 E. coli could enter the brain of infants through blood stream, resulting in neonatal meningitis, which inhibits the intelligence development of infants.3 The most used treatment for E. coli is antibiotic in the clinic, such as levofloxacin (LFX), ampicillin, clindamycin, and gentamicin.4 Administration of antibiotic for E. coli treatment causes several side effects, especially diarrhea.5 The world suffers from serious overuse of antibiotics over the last decades.6-8 Antibiotic abuse would cause liver and kidney dysfunction, which may lead to serious infection, and even antibiotic resistance.9, 10 Oral antibiotics would disrupt the balance of intestinal microflora, especially the broad-spectrum antibiotics.11, 12 They killed both probiotic and pathogenic bacteria, and destroyed intestinal epithelial cells. Prenatal and postnatal antibiotic administration may affect the composition of early intestinal microflora in preterm infants.13 Oral administration of penicillin destroyed the intestinal microflora of mice, and enhanced the effect of high-fat diet induced obesity. Ampicillin, streptomycin, and clindamycin induced the disorder of intestinal microflora, which would result in depressive behaviors and hinder social cognition.14 In order to fight against the crisis of antibiotic abuse, the development of antibacterial agent which could cure infections with specificity has been 3 ACS Paragon Plus Environment
ACS Nano 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
proposed.15 As early as the Tang Dynasty (A.D. 618-907), Chinese realized that gold was beneficial for improving physical fitness.16 Compendium of Materia Medica (A.D. 1552-1587, Shizhen Li), which is the most complete and comprehensive medical book in ancient China, mentions that gold foil (thickness ~120 nm) has a tranquilizing effect.17 Gold is one of components in Tsothel that is a representative of Tibetan medicine and can protect liver and cure indigestion.18, 19
Drinking-gold, consisting of the gold nanoparticles (Au NPs), cured most diverse
mental illnesses in 16th century Europe and acted as therapeutic agents for intemperance in America.20, 21 Drinking gold could treat arthritis by strengthening bone and reliving the pain of patients.22, 23 In 1890s, researchers found that Au NPs could kill Mycobacterium tuberculosis.24 In 1939, Au NPs acted as antibacterial agents to cure infections in the clinic.25 Au-based NPs showed good biocompatibility,26-28 and there were many Au-based antibacterial agents under development.29-31 4-Dimethyl aminopyridinium propylthioacetate coated Au NPs showed antibacterial activity toward E. coli.32 Au NPs modified by extract from
Galenia Africana and Hypoxis hemerocallidea were antimicrobial to Pseudomonas aeruginosa.33 We synthesized many Au NPs-based antibacterial materials to fight bacteria, such as 4, 6-diamino-2-pyrimidinethiol (DAPT), D-alanyl-D-alanine, 5Amino-1, 3, 4-thiadiazole-2-thiol coated Au NPs.34-40 It is a great advantage to replace the current antibiotics with Au NPs.41-43
4 ACS Paragon Plus Environment
Page 4 of 48
Page 5 of 48 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 Nano
Therapies containing metals have recently undergone rapid development, such as treatments for cancer and microbial infection.44,
45
Rhodium-containing
complex showed antitumor activity against triple-negative breast cancer.44 Platinum-containing nanoparticles were efficient for eradicating the tumor burden on a high-fidelity patient-derived lung cancer model.45 Nanoparticles could act as antimicrobial agents of high-performance with security and durability.28, 41 However, several nanoparticles, such as zinc oxide (ZnO) and silver NPs, have adverse effects on intestinal microflora.46 ZnO NPs decreased the number of total species and biodiversity (α-diversity) of intestinal microflora.47 Silver NPs reduced the abundance of Bacteroidetes to 40% and increased the abundance of Firmicutes to 58%, which was harmful to health.48 Intestinal microflora maintains the physiological activities of hosts, such as anti-infection and stimulating immune response.45, 46 Imbalance of intestinal microflora is closely related to the chronic metabolic diseases, including diabetes, hypoglycemia, gout, and protein energy malnutrition.49,
50
The α-diversity of intestinal microflora is
closely associated with the health of host, containing richness and evenness. Abundance-based coverage estimator (ACE) and Chao1 reflect the total number of species in samples, and Shannon and Simpson are quantitative indicators of biodiversity in a region.51,
52
The decrease of α-diversity may induce the
occurrence of obesity or enteritis, and the increase of α-diversity promotes the metabolic process.53 5 ACS Paragon Plus Environment
ACS Nano 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
Au NPs showed antibacterial effect with high efficiency through injection, which could act as alternatives to antibiotics.54-59 Oral administration is more efficient for curing bacterial infections in gut (diarrhea and intestinal bleeding) than intravenous and intramuscular injection.60 Herein, we studied the therapeutic effect of DAPT coated Au (D-Au) NPs on mice models, and the interaction between E. coli and D-Au NPs by incubating them in anaerobic atmosphere. We evaluated the effects of Au NPs on intestinal microflora after a 28-day administration, such as the diversity of microflora, and the abundance of
Firmicutes, Bacteroidetes, Verrucomicrobia, and Actinobacteria in microflora. Additionally, we assessed the toxicity of Au NPs in mice by detecting the amounts of Au NPs in tissue and the biomarkers in blood. RESULTS AND DISCUSSION We observed the Au NPs with TEM (Figure 1). The N-Au and D-Au NPs dispersed uniformly, and the sizes of N-Au and D-Au NPs were around 5 nm (Figure 1a and c). The average hydrodynamic sizes of N-Au and D-Au NPs were 12.90 and 16.57 nm (Figure 1b and d), and the zeta potentials of N-Au and D-Au NPs were -2.3 and 14.7 mV. The absorption peaks of Au NPs were around 520 nm in the UV spectra due to the surface plasma resonance of Au NPs (Figure 1e).38 The peaks of C, O, and Au displayed in the XPS spectrum of N-Au (Figure 1f), and the peaks of S and N appeared on the spectra of D-Au NPs,38 indicating that DAPT was capped on Au NPs. 6 ACS Paragon Plus Environment
Page 6 of 48
Page 7 of 48 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 Nano
Figure 1. Structure of Au NPs. a) TEM image of N-Au NPs; the figure inserted in (a) was the scheme of N-Au NPs. b) Size distribution of N-Au NPs. c) TEM image of D-Au NPs; the figure inserted in (c) was the scheme of D-Au NPs. d) Size distribution of D-Au NPs. e) UV spectra of Au NPs. f) XPS spectra of Au NPs. We administered D-Au NPs, N-Au NPs, and LFX to the model mice infected by
E. coli at dose of 1700 g/kg to evaluate their therapeutic effect on mice model, since the dosage of LFX used in clinic is 1700 g/kg (Figure 2). We obtained the bacteria from feces of mice after they were treated with agents at 0, 12, 24, 48, and 7 ACS Paragon Plus Environment
ACS Nano 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
72 h, and spread the bacteria on eosin-methylene blue (EMB) medium, in which the green color in the plate indicates the growth of E. coli (Figure 2a). The amount of E. coli in PBS group was 1.21107 CFU/g before treatment, and there were no E.
coli in mice of control group (Figure 2b). The amount of E. coli in PBS, D-Au 1700, N-Au 1700, and LFX 1700 group decreased to 2.67106, 3.76105, 2.47106, and 5.23105 CFU/g at 12 h after infection. There was no E. coli in mice of D-Au 1700 group, and the number of E. coli in mice of LFX 1700 group decreased to 3.76105 CFU/g in 24 hours. Until 48 hours, there was no E. coli in mice of LFX 1700 group. LFX that is one kind of quinolones kill the bacteria by inducing faulty copies of gene and may lead to bacterial resistance by chromosomal mutation.61, 62 D-Au NPs are more efficient in the treatment of gut infection than LFX.
8 ACS Paragon Plus Environment
Page 8 of 48
Page 9 of 48 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 Nano
Figure 2 Treatment of N-Au NPs, D-Au NPs, and LFX on mice model infected by
E. coli. a) The photograph of eosin-methylene blue (EMB) gel plate after a 24hour-cultivation. We spread the bacteria from feces of mice on EMB plate at different time points after treatment, and the green color in the plate indicates the growth of E. coli. b) The amounts of E. coli in the feces of mice before and after treatment, which were counted from (a). c) The level of CRP in serum, which indicated the level of inflammation d) The level of IL-4 that is an inflammatory
9 ACS Paragon Plus Environment
ACS Nano 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
cytokine in serum. e) The level of INF-β that is an inflammatory cytokine in serum. f) HE staining of small intestines. The scale bar is 100 m. We detected the concentration of CRP, IL-4, and INF-β in serum by ELISA kits to evaluate the inflammation in mice at different time point before and after treatment (Figure 2c-e). The CRP (Figure 2c), IL-4 (Figure 2d), and INF-β (Figure 2e) of mice in PBS and N-Au 1700 group still maintain high level until 48 h after infection, and those of mice in D-Au 1700 group decreased after 12 h. We examined the pathology of small intestines in mice to assess the curative effect of D-Au NPs (Figure 2f). Muscular detachment happened on the small intestines of mice in PBS and N-Au 1700 groups, while that did not appear in D-Au 1700 and LFX 1700 group. The D-Au NPs were superior to LFX in relieving the inflammation in body and the damage on small intestines by killing the E. coli in gut. Since infections caused by E. coli in gut and antibiotics decreased the diversity of microflora, maintaining the diversity of microflora is matter of great concern in the treatment of infections in gut.4 The diversity of microflora in patients decreased by 25% after they took LFX.63 We analyzed the operational taxonomic units (OTUs, an operational definition used to classify groups of closely related individuals) and richness (ACE and Chao1 index) to study the diversity of microflora (Figure S1a-b). The OTUs, ACE and Chao1 index of microflora in mice of D-Au 1700 group showed no obvious difference from normal group. Compared 10 ACS Paragon Plus Environment
Page 10 of 48
Page 11 of 48 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 Nano
with normal group, the OTUs, ACE and Chao1 index of microflora in mice of LFX 1700 group decreased 16.2%, 9.1%, and 8.6%. D-Au could maintain the richness of microflora effectively.
Firmicutes and Bacteroidetes are the main phyla, which formed 90% of intestinal microflora in animals. Their relative abundance is closely related to the health of host.5 We calculated the ratio between Firmicutes and Bacteroidetes of microflora (F/B). Compared with the control group, the F/B value decreased after oral administration of norfloxacin and ciprofloxacin.64 Infections in gut would increase the F/B value.65 We studied the effect of N-Au 1700, D-Au 1700, and LFX 1700 on F/B value (Figure S1c). The F/B values of D-Au 1700 and normal mice groups showed no obvious difference, and F/B values of LFX 1700 group were 13.3% higher than that in normal mice groups. LFX cured infections while the microflora showed difference from normal mice. D-Au NPs would treat the bacterial infections and restore the microflora. We cultured E. coli and Au NPs (34 g/mL) in anaerobic incubator to simulate their interaction in small intestine. We observed the growth and morphology of E.
coli after they were treated with Au NPs (Figure 3). We measured the OD600 of samples after incubation for 6, 12, 24, 48, and 72 h, and low OD600 of D-Au and LFX indicated the death of E. coli in these two groups (Figure 3a). We observed the morphology of E. coli after a 12 h-incubation with Au NPs and LFX under confocal microscope and SEM (Figure 3b). We counted the average bacterial 11 ACS Paragon Plus Environment
ACS Nano 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
numbers in confocal images by Image J. In one counted field, the numbers of live bacteria (green florescent) in of D-Au and LFX groups were 91.2% and 85.2% less than N-Au and control groups, indicating that the D-Au and LFX were effective for killing E. coli. The cell membrane of E. coli was broken in SEM images of DAu groups, indicating that the D-Au NPs destroyed the cell membrane in anaerobic conditions, which would happen with the presence of oxygen.38
12 ACS Paragon Plus Environment
Page 12 of 48
Page 13 of 48 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 Nano
Figure 3 Growth and morphology of E. coli after treated by Au NPs (34 g/mL). a) OD600 of E. coli after treated by N-Au, D-Au, and LFX at 0, 6, 12, 24, 48, and 72 h. b) SEM images, confocal images, and average bacterial number of E. coli (107 CFU/mL) after 12 h-incubation with Au NPs and LFX. Green color in confocal 13 ACS Paragon Plus Environment
ACS Nano 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
images indicates live bacteria, and red color indicates dead bacteria. Average bacterial numbers in one counted field of confocal images (40000 m2). We evaluated the effects of long-term administration of Au NPs on mice. We administered the mice with D-Au and N-Au in different dosage, containing 170, 1700, and 17000 g/kg for 28 days, and detected the microflora, bodyweight, biomarkers of mice and the distribution of Au NPs in mice. We observed the small intestine of mice under TEM to study the distribution of Au NPs in small intestine and the effect of Au NPs on small intestine (Figure 4a). After a 28-day administration, Au NPs could penetrate intestinal wall, and distributed in a clustered manner or individually in the intestinal epithelial cells. We studied the morphology of mitochondria in small intestine since mitochondria could produce energy for peristalsis of small intestine and assist food digestion. DAu NPs did not damage the mitochondria of the intestinal epithelial cell, whereas N-Au NPs destroyed the mitochondrial membrane (images inserted in Figure 4a). N-Au NPs could affect the normal physiological function of the small intestine. We further studied the interaction between Au NPs and intestinal epithelial cells (MODE-K) by incubation for 24 h, including the morphological change of cells and cell uptake. We stained and observed the MODE-K cells which were treated with Au NPs under confocal microscope. The cells after treated by Au NPs for 24 h were well-stacked, which were plumper than control group (Figure 4b). Compared with control group, cell number in D-Au group increased by 24%, and 14 ACS Paragon Plus Environment
Page 14 of 48
Page 15 of 48 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 Nano
cell number in N-Au NPs decreased by 40%. D-Au NPs would promote the proliferation of cells. In TEM image of MODE-K cells, the Au NPs could enter the cells after they were incubated with MODE-K cells (Figure 4c).
Figure 4 Morphology of small intestines and MODE-K cells. a) TEM images of small intestine in mice which were orally administered N-Au 17000 and D-Au 17000 NPs for 28 days. Blue and red arrows indicate N-Au NPs and D-Au NPs, and white arrows indicate mitochondria. Images inserted are the morphology of mitochondria in small intestines (intestinal epithelial cells). b) Confocal images of MODE-K cells after they were treated by Au NPs (340 g/mL) for 24 h, in which 15 ACS Paragon Plus Environment
ACS Nano 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
the green color is cytoskeleton and the blue color is nucleus. c) TEM images of MODE-K cells after they were treated by Au NPs (340 g/mL) for 24 h. Blue and red arrows indicate N-Au NPs and D-Au NPs. We studied the biocompatibility of D-Au and N-Au NPs towards MODE-K cells by CCK8 and Live-Dead cell staining kits (Figure 5). We calculated the cell viability of MODE-K cells after they were treated with D-Au and N-Au NPs by measuring OD450. When the Au concentration was 500 g/mL, the viability of cells in D-Au group was 16.5% higher than that in N-Au NPs (Figure 5a). We observed the live and dead cells after treated by Au NPs under confocal microscope (Figure 5b). The number of dead cells (in red color) in D-Au group was 60% less than that in N-Au group, and the number of live cells (green color) in DAu group was 200% more than that in N-Au groups. The D-Au NPs are thus more biocompatible to MODE-K cells than N-Au NPs. We studied the α-diversity of intestinal microflora in mice after a 28-day oral administration of D-Au and N-Au NPs, containing the richness (ACE and Chao1 index) and evenness (Shannon and Simpson index) (Figure 6). Low α-diversity of the intestinal microflora would induce obesity, insulin resistance, lipid metabolism disorders, and chronic inflammation.66 The diversity of intestinal microflora in DAu NPs groups was higher than that in N-Au groups as more OTUs in the rankabundance curve of D-Au NPs (Figure 6a). The D-Au NPs would not affect the richness and evenness of microflora in mice. ACE, Chao1, Shannon, and Simpson 16 ACS Paragon Plus Environment
Page 16 of 48
Page 17 of 48 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 Nano
index showed no obvious difference between D-Au NPs treated groups and control group (Figure 6b-c). In contrast, the richness of microflora decreased after administration of N-Au NPs. ACE index decreased from 335.61 to 272.68 and Chao1 index decreased from 339.86 to 279.02. Shannon and Simpson index showed no difference, indicating that the N-Au NPs decreased the richness of intestinal microflora. We did principal component analysis (PCA) to evaluate the difference between microflora of mice in different groups (Figure 6d), in which the distance between each point reflect the difference between samples. PCA based on bacterial genera composition showed that the microflora from mice in DAu and control groups differed from mice in N-Au NPs. Long-term administration of antibiotic would decrease the diversity of microflora,63 whereas D-Au NPs seldom influence the diversity of microflora after a 28-day administration.
17 ACS Paragon Plus Environment
ACS Nano 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 5 Biocompatibility of Au NPs with MODE-K cells. a) Cell viability of MODE-K cells after treated by different concentration of D-Au and N-Au NPs. b) Confocal images and average number of MODE-K cells after treated by D-Au and
18 ACS Paragon Plus Environment
Page 18 of 48
Page 19 of 48 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 Nano
N-Au NPs. Green color in confocal images indicates live cell, and red color indicates dead cell.
Figure 6 Diversity of intestinal microflora in mice after oral administration of Au NPs for 28 days. a) Rank abundance of OTUs in different groups; more OTUs indicates higher richness. b) ACE and Chao1 index of different groups; higher value indicates higher richness. c) Shannon and Simpson index of different groups; higher value indicates more evenness. d) PCA ordination plot of intestinal microflora in different groups; the distance between each point reflect the difference between different samples. ** denotes extremely significant difference (P
