Nano-corona formation between foodborne nanoparticles exacted

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Food Safety and Toxicology

Nano-corona formation between foodborne nanoparticles exacted from roast squid and human serum albumin Ronggang Liu, Kangjing Liu, and Mingqian Tan J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b04425 • Publication Date (Web): 30 Aug 2019 Downloaded from pubs.acs.org on August 30, 2019

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Journal of Agricultural and Food Chemistry

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Nano-corona formation between foodborne nanoparticles exacted from roast

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squid and human serum albumin

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Ronggang Liu1,2.3 Kangjing Liu1,2.3 and Mingqian Tan1,2,3*

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1School

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Dalian116034, Liaoning, People’s Republic of China

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2 National Engineering Research Center of Seafood, Dalian116034, Liaoning, People’s

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Republic of China

of Food Science and Technology, Dalian Polytechnic University,

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3Engineering

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116034, Liaoning, People’s Republic of China

Research Center of Seafood of Ministry of Education of China, Dalian

12 13 14 15 16 17

*Corresponding author (Tel& Fax: +86-411-86318657, E-mail: [email protected],

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ORCID:

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Dalian116034, Liaoning, People’s Republic of China

0000000275350035).

Address:

Qinggongyuan1,

20 21 22

ACS Paragon Plus Environment

Ganjingzi

District,


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Abstract:

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Foodborne nanoparticles (FNPs) produced by roasting have attracted people’s

25

attention owing to their safety risk to body health. Herein, we reported the formation,

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physicochemical properties, elemental composition, bio-distribution and binding with

27

human serum albumin (HSA) of FNPs extracted from roast squid. The results showed

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that the FNP size gradually decreased from 4.1 to 2.3 nm as the roasting temperature

29

changed from 190 to 250 °C. The main component elements of FNPs are carbon, oxygen,

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and nitrogen, and the carbon and nitrogen contents of FNPs increased with the roasting

31

temperature rising. The surface of FNPs contained hydroxyl, amino and carboxyl

32

functional groups. The FNPs can emit fluorescence in ultraviolet light and show

33

excitation-dependent emission behavior. Furthermore, it was found that the FNPs

34

derived from roast squid could be accumulated in stomach, intestine and brain of

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BALB/c mice after oral feeding. Static fluorescence quenching of HSA was found by

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Stern-Volmer equation and ultraviolet−visible (UV-vis) spectrum analysis after

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interaction with the FNPs. After the addition of FNPs, the α-helix content of HSA

38

decreased, and the morphological height of HSA increased, which indicated that the

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FNPs could cause structural changes in HSA. The AFM characterization showed the

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formation of nano-corona between FNPs and HSA.

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Keywords: Foodborne nanoparticles, Roast squid, Human serum albumin, Interaction,

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Binding mechanism

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Introduction:

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Nanoparticles (NPs) possess extremely small size, ultra-high surface to volume

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ratio, and good permeability, which have received growing public concerns about

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potential safety risk to human health.1-3 Many studies have shown that the artificially

49

synthesized NPs may induce toxicity for organisms because of their unique properties.

50

For example, Silver NPs were observed in the main organs of mice after oral feeding,

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and have adverse effects on the liver and kidneys4 and carbon nanotubes can cause

52

dose-dependent pulmonary granuloma formation.5 Chitosan NPs can induced damage

53

of cell membrane integrity of human hepatocytes, leading to necrosis or autophagic cell

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death.6-7 Certain NPs led cells to produce ROS (reactive oxygen species), and resulted

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in oxidative stress and promote apoptosis.8-9

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In addition to the adverse effects caused by exogenous engineered NPs, the food-

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borne NPs (FNPs) have been found recently in nutrition matrices during food thermal

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processing.10-18 High temperature caused complicated physical and chemical reactions

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of nutrients in foods such as water loss, protein denaturation and lipid oxidation,19

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resulting in formation of nano-structures with unique fluorescent property. Sk et al.20

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discovered the fluorescent FNPs in some carbohydrate-rich foods by heating raw

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materials and caramelization reactions. Recently, our group have reported the universal

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presence of FNPs from vinegar, grilled fish, roast chicken, roast duck and baked lamb.9-

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15

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deserve more attention owing to their ubiquitous presence in processed foods.

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Since the endogenous FNPs can enter human body with food, their potential risks

Squid is a kind of marine food rich in protein, taurine, phosphorus, vitamin B1 and



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other nutrients.21 Roast squid is a very popular seafood in the world. However, to the

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best of our knowledge, there is no research about the discovery and assessment of FNPs

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in roast squid. No information is available about roasting temperature effects on the

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morphology, bio-distribution of the FNPs derived from squid and potential biological

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interaction with biomacromolecules. Human serum albumin (HSA), the most abundant

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protein in plasma, has been found with many unique physiological activities and

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functions, including transportation and delivery of fatty acids, bilirubin, steroids etc.22-

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23

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encounter HSA. Studies have shown that the binding of HSA to exogenous

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nanoparticles changed HSA conformation.24-25 For example, Huang et al.25 found

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quantum dots significantly competed with warfarin by binding with HSA in site I. Song

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et al15 reported that that the FNPs from roast chicken can cause fluorescence quenching

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of HSA by static interaction, and the interaction-induced changes were studied by

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spectroscopy and thermodynamics. All of these changes affected the physiologic

81

function of the HSA. Hence, HSA was chosen as a representative sample to assess the

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FNP’s interaction with serum proteins.

After oral uptake with roast squid, the FNPs might enter the blood stream and

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The aims of this work are to study the formation and physicochemical

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characteristics including particle sizes, elemental composition, optical properties,

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surface chemical groups and fluorescent stability of FNPs extracted from squid at

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different roasting temperatures (190, 220, 250 °C). The ex vivo bio-distribution in mice

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fed with the squid FNPs was investigated. Additionally, the molecular interaction

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between the FNPs from roast squid and HSA was explored the nano-corona formation


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by spectroscopy and atomic force microscopy (AFM). The results of this study may

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provide useful information about the generation and biological effects of FNPs for roast

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squid and other seafoods.

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MATERIALS AND METHODS:

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Materials:

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Squids were bought at a seafood store in Dalian, China. The human serum albumin

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(HSA) was supplied by Beijing Solarbio Science & Technology Co., Ltd. In Beijing,

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China. All other chemicals and reagents were of analytical grade bought from

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commercial companies unless otherwise stated.

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Extraction of FNPs from roast squid

99

Viscera (inedible portion) were removed from fresh squids (515g), and then the

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squids were rinsed with distilled water and cut into pieces of 4 cm × 4 cm × 0.5 cm.

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The squid samples were placed in an electric roasting oven (EP2PT, Foshan, China)

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and roast at 190, 220, and 250 °C, respectively, for 30 min. The charred portion was

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peeled off from roast squid and stirred with 1.2 L of 100% ethanol for 48 h. The solid

104

precipitate was filtered, and the filtrate was concentrated by rotary evaporation. The

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raw product was then dispersed in 120 mL water and washed three times with

106

chloroform (250 mL). The water fraction was collected and passed through a 0.22 µm

107

filter and dialyzed with distilled water for 72 h by a membrane bag with molecular

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weight cut-off 500 Da. The FNP powder was obtained after dialysis and lyophilizing.

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The yields of the FNPs from squid after roasting at 190, 220, and 250 °C were 0.009%,

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0.014% and 0.017% (calculation based on the wet weight of squid), respectively, which



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they were named as FNPs-190, FNPs-220 and FNPs-250.

112

Instrumentation:

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Transmission electron microscopy (TEM) was used to analyze FNPs under 200

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kV voltage by using a JEM-2100 model instrument from JEOL company in Tokyo,

115

Japan. X-ray diffraction (XRD), Fourier transform infrared (FTIR), ultraviolet-visible

116

(UV-vis) absorption, fluorescence, fluorescence lifetime (τ), X-ray photoelectron

117

spectrometry (XPS) and Circular dichroism (CD) spectra were measured according to

118

the previous methods.16 The fluorescence intensity of the roast squid sample was

119

measured using XFP-BIX multi-functional in vivo imaging system (MIIS) (Molecular

120

Device Corporation, Sunnyvale, CA, USA) with excitation wavelength at 365 nm,

121

emission wavelength of 515 nm long pass. The average molecular weight of FNPs from

122

roast squid was measured with matrix-assisted laser desorption ionization time-of-flight

123

mass spectrometry (MALDI-TOF-MS; Auto flex, Bruker Co., Germany). The method

124

to measure quantum yield (QY) of squid FNPs was similar to previous study.16 Three-

125

dimensional (3-D) topography, two-dimensional (2-D) and height of the squid sample

126

surface were measured using atomic force microscopy (AFM) (AFM-5500M Hitachi,

127

Tokyo, Japan).26

128

Fluorescence Analysis of Interaction

129

The method of fluorescence analysis was similar to previous work.15 HSA

130

concentration was kept at 2 × 10−6 mol L−1 and the squid FNP concentration was 0, 0.3,

131

0.9, 1.5, 2.1, 2.7 × 10−4 mol L−1. The fluorescence emission spectra within 290-450 nm

132

were measured under the excitation of 280 nm (excitation of tryptophan and tyrosine


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residues).

134

Nuclear Magnetic Resonance (NMR) and Magnetic Resonance Imaging (MRI)

135

Characterization

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NMR and MRI measurement of squid samples weighed 5.1 ± 0.5 g was performed by

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heating the samples at various temperature (190, 220 and 250 °C) for 30 min. Squid

138

samples were placed in a benchtop MiniMR-Rat low field-NMR analyzer produced by

139

Suzhou Newmag Analytical Instruments Ltd. Co. (Suzhou, China) equipped with a 0.5

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T permanent magnet using a coil in 60 mm diameter at 32 °C. Carr-Purcell-Meiboom-

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Gill (CPMG) sequence was used for the measurement of transverse relaxation T2

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attenuation using the following parameters: 180° pulse = 22 μs, 90° pulse = 11 μs, echo

143

number of 2000, and duration time between consecutive scans of 3000 milli seconds.

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T1-weighted imaging was conducted using a spin echo sequence with the following

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acquisition parameters: echo time 60 ms, repetition time 1900 ms, average 2, slice gap

146

4 mm, sheet width 6.7 mm, and phase size 192.26

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Biodistribution of the FNPs in mice

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Ex vivo imaging of the FNPs-250 in major mice organs was carried out with male

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adult BALB/c mice supplied by Experimental Animal Center of Dalian Medical

150

University. The BALB/c mice were kept in standard cages for 2 weeks, then they were

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intragastrically gavaged with FNPs-250 at a dose of 2 g kg-1 (n = 5). After oral

152

administration of FNPs-250 at 1.5, 4, 6, and 24 h, the mice were sacrificed, and the

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main organs of the mice were imaged with an XFP-BIX MIIS analyzer (Molecular

154

Device Corporation, Sunnyvale, CA, USA) under the excitation of 365 nm by collecting



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the emission wavelength of 400 nm long-pass, and acquisition time 180 ms. The control

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mice were intragastrically gavaged with saline solution. The mice were maintained at

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Animal Core Facility at Dalian Polytechnic University in line with the animal protocol

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(No. 2017-0005) approved by the Animal Ethics Committee.

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Statistical Analysis

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All the diagrams in this work were plotted by the Origin 8.5 software (OriginLab

161

Corporation, MA, USA). Significant analysis was performed using the software of

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Statistical Product and Service Solutions (SPSS Inc., Chicago, IL, USA).

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Results and Discussion

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Characterization of FNPs derived from roast squid

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Recent studies showed that food thermal process can resulted in the formation of

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FNPs.27 To determine the existence and formation of FNPs in the roasting squid, the

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fluorescent fraction of ethanol extractant from roast squid was characterized by TEM.

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Figure 1 clearly revealed the presence of FNPs after the squid roast at 190, 220 and 250

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°C

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1d, 1g). Lattice-like structure was also found in the insets of high-resolution TEM

172

images of the FNPs. The size of FNPs-190, FNPs-220 and FNPs-250 was 4.1 ± 1.3, 3.0

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± 0.6 and 2.3 ± 0.8 nm, respectively (Figure 1b, 1e, 1h). It noteworthy that the size of

174

FNPs decreased when the roasting temperature went up, and the FNPs were smaller

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than the exogenously synthesized nanoparticles.28 The result of MALDI-TOF-MS

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showed that the average molecular weight of FNPs-250 was 837 Da (Figure S1). The

for 30 min, and the FNPs have spherical shape and good dispersibility (Figure 1a,


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XRD patterns of FNPs-190, FNPs-220 and FNPs-250 exhibited a broad peak around 2

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θ = 22° (Figure 1c, 1f, 1i), suggesting an amorphous structure for FNPs. In a word, the

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FNPs were universally formed in the roasting squid as we hypothesized (Figure 1j),

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which was similar to those FNPs exacted from other foods in previous studies.10

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(Figure 1)

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The surface groups of FNPs were determined by FTIR spectroscopy. Strong

183

absorption peak around 3424-3269 cm-1 was ascribed to O-H stretching or NH2

184

vibration, and the peak at 2962-2925 cm-1 was attributed to C-H vibrations of methylene

185

(Figure 2a). the band at 1655-1545 cm−1 was assigned to the amide bond in the -CONH-

186

or carbonyl groups. Moreover, the peak at 1400 and 1048 cm−1 corresponded to C−N

187

and aromatic alkoxy (C-O-C) bonds, respectively. All the signals indicted the existence

188

of hydroxyl, amino and carboxyl groups on the surface of FNPs. XPS spectra showed

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three peaks around 285, 399.8 and 532 eV for FNPs-190, FNPs-220 and FNPs-250

190

(Figure 2b, 2c and 2d). The detailed elemental contents of various FNPs were

191

summarized in Table 1. These results demonstrated that the FNPs contained carbon,

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nitrogen and oxygen, and carbon was the most abundant element. As the roasting

193

temperature rose, the carbon and nitrogen content of FNPs increased, and the oxygen

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content decreased, indicating that the squid proteins were decomposed at higher

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temperature and more nitrogen elements were doped into the resulting FNPs.26

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Table 1. Elemental content of FNPs derived roast squid Sample

C (%)

N (%)

O (%)

N/C (%)

O/C (%)

FNPs-190

60.54

5.49

30.22

9.07

49.92

FNPs-220

62.56

9.49

25.36

15.17

40.54

FNPs-250

67.61

12.60

17.30

18.64

25.59

200 201

(Figure 2)

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The functional groups on FNPs were further identified by the high-resolution XPS

203

spectra. The C1s peak of FNPs could be divided three sub-peaks with binding energies

204

(BEs) at 284.5, 286.4, 288.6 and 285.5 eV that belonged to the C=C, C−O, C=O, and

205

C-N, respectively (Figure 3a, 3b, 3c).14 The O1s peak of FNPs could be divided into

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three peaks with BEs at 531.4, 532.3, and 533.2 eV, which could be assigned to the

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*O=C−O,

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of FNPs could be divided into two peaks with BEs at 399.3 and 400.0 eV, which were

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assigned to the amino N and pyrrolic-like N, respectively (Figure 3g, 3h, 3i).30

210

Therefore, these findings provided evidence for the existence of hydroxyl, amide and

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carboxyl groups on the surface of the FNPs from roast squid. All these results revealed

212

that the squid FNPs have semblable characteristics to the synthesized carbon dots

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derived from small molecules.31

C−O, and O=C−O*, respectively (Figure 3d, 3e, 3f).29 The N1s level spectrum

214

(Figure 3)

215 216

Optical property analysis of FNPs


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The optical properties of FNPs were analyzed by UV-vis absorption and

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fluorescence spectroscopy. First, the UV-vis absorption spectra of FNPs showed only

219

a peak at about 250 nm due to the π-π* transition of conjugated biomolecules on the

220

surface of FNPs (Figure 4a, 4c, 4e). Second, all the FNPs exhibited strong fluorescence

221

and excitation-dependent emission behavior. The maximum excitation wavelength of

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the FNPs-190, FNPs-220 and FNPs-250 (Table 2) was 350 nm, and the maximum

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emission wavelength was in the range of 449 - 426 nm. The FWHM for the FNPs-190,

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FNPs-220 and FNPs-250 was 71, 66 and 94 nm, respectively. The fluorescence spectra

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of FNPs showed red shift as the excitation wavelength increased (Figure 4a, 4c, 4e),

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which was quite similar to the previous results.9,11 This fluorescence bathochromic-shift

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behavior may be caused by the existence of different emission centers formed by

228

various surface groups on the surface of FNPs, and the extensive size distribution of

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FNPs. Moreover, at room temperature, the fluorescence attenuation spectra of FNPs in

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aqueous solution could be fitted by a double exponential function, and the fluorescence

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lifetime of FNPs-190, FNPs-220 and FNPs-250 were 7.57, 7.63 and 8.14 ns,

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respectively (Figure 4b, 4d, 4f). The QY of the FNPs-190, FNPs-220 and FNPs-250

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were determined to be 2.21%, 3.07% and 7.33%, respectively (Table 2). As the roasting

234

temperature went up, the QY of FNPs increased as well. The reason for the higher QY

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value was probably due to the greater nitrogen-doping content of in FNPs, thus forming

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more trap excitons under excitation.10

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Table 2. Fluorescence properties of the FNPs from roast squid. Sample

Max Ex

Max Em

FWHM

Lifetime

QY

(nm)

(nm)

(nm)

(ns)

(%)

FNPs-190

350

449

71

7.57

2.21

FNPs-220

350

447

66

7.63

3.07

FNPs-250

330

426

94

8.14

7.33

240

Max: maximum, Ex: excitation, Em: emission, FWHM: full width at half maximum,

241

QY: quantum yield.

242 243

(Figure 4) Stability of the FNP fluorescence

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Effects of various metal ions, pH values, and NaCl concentrations on FNP

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fluorescence were studied to evaluate the stability of FNPs from roast squid. The

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addition of Cu2+ exhibited different quenching effect on the fluorescence of FNPs by

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contrast to other metal ions (Figure 5a, 5d, 5g). The possible reason might be owing to

248

the blockage of nonradiative electron-transfer from the squid FNP excited state to the

249

electric orbital of Cu2+. No obvious fluorescence intensity change was observed for

250

FNPs-190 in different pH solution (Figure 5b), whereas the fluorescence intensity of

251

FNPs-220 and FNPs-250 increased in the range of pH 2-6 and decreased gradually in

252

the range of pH 8-11 (Figure 5e, 5h). Under extreme pH conditions, the fluorescence

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intensity of FNPs only decreased by about 19%. The pH influence may be related to

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the protonation and deprotonation of various functional groups on the surface of FNPs,

255

which affected electron transition process.32 The fluorescence intensity of FNPs-190


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decreased with the increase of NaCl concentration, however, only 23% fluorescence

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decreased at the maximum concentration (Figure 5c). NaCl concentrations exhibited

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slight influence on the fluorescence intensity of FNPs-220 and FNPs-250 (Figure 5f,

259

5i). Stability studies showed that the FNPs exacted from roast squid were relatively

260

stable and might retain their unique properties if they were orally uptake. Therefore, it

261

was necessary to study the molecular interaction between FNPs and HSA.

262

(Figure 5)

263 264

Formation of FNPs exacted from squid during roasting

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Multiple imaging techniques were used to study the effects of different roasting

266

temperatures on FNPs formation, including bright field imaging, MRI imaging, AFM,

267

and fluorescent imaging. The color of raw squid meat was pinkish white (Figure 6a).

268

Ass the roasting temperature rose, the color of roast squid became yellow (Figure 6b),

269

then changed to brownish yellow, finally turned black brown (Figure 6c, 6d).

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Meanwhile, the size of the sample shrunk due to the loss of water. The CPMG

271

attenuation curve of the squid sample at different roasting temperature in Figure S2a

272

showed that the squid sample with prolonged roasting time decayed faster than those

273

with short roasting time. This was probably owing to the mobility of protons decreased

274

as roasting time increased. Moreover, the dynamics and mobility of internal water in

275

roasting squid samples were examined using a transverse relaxation (T2) spectroscopy

276

(Figure S2b). Three water populations were identified: 1) T21 of binding water in the

277

range of 0.49-2.10 ms was closely connected with macromolecules located in water-



278

plasticized structures; 2) T22 of entrapped water in the range of 31.44-126.04 ms, and

279

3) T23 of free water in the range of 821.43-249.51 ms was related to high-order protein

280

structures such as myofibrillar protein.33-34 T21, T22 and T23 showed a downward trend

281

when the roasting temperature went up, demonstrating a decrease water mobility.

282

Generally speaking, the peak area of the transverse relaxation time was proportional to

283

the total amount of protons in the squid sample. When the roasting temperature went

284

up, the total amount of protons decreased significantly. Hydrodynamic and internal

285

structural changed of squid samples at different roasting temperatures were also

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monitored by MRI.35 The contrast enhancement showed a downward trend for the T1

287

weighted MRI images of roast squid (Figure 6e, 6f, 6g and 6h) with the increase of the

288

temperature, indicating a moisture loss in roast squid sample. In addition, the formation

289

of FNPs from roast squid was monitored using the XFP-BIX MIIS system for optical

290

fluorescence imaging. The results showed that fluorescence intensity gradually

291

increased as the roasting temperature rose (Figure 6i, 6j, 6k and 6l). No fluorescence

292

was observed in the raw meat (Figure 6i), which was consistent with the AFM result

293

(Figure 6m). The AFM result (Figure 6n, 6o, 6p) clearly showed the formation of FNPs,

294

and the aqueous solutions of FNPs-190, FNPs-220 and FNPs-250 exhibited bright blue

295

fluorescence under 365 nm UV light (Figure 6q, 6r, 6s, and 6t). The higher the roasting

296

temperature, the stronger the aqueous solution. No fluorescence was found for the

297

extractant from the raw squid without roasting. All the results indicated that the

298

formation of the FNPs in squid was temperature-dependent.

299

(Figure 6)


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Bio-distribution of FNPs

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To explore where the FNPs were distributed after oral uptake, and whether the

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FNPs are accumulated in organism, the biodistribution of FNPs in major organs of

303

BALB/c mice were investigated after 1.5, 4, 6, and 24 h oral administration with FNPs-

304

250 at a dose of 2g kg-1 body weight. Figure 7 showed the bio-distribution of FNPs in

305

major organs of mice. The fluorescence signal detected in the intestine of control mice

306

may be from mouse fodder. The fluorescence signal increased with prolonged time and

307

reached the maximum after 4h. After 24 h, the fluorescence intensity decreased

308

eventually.

309

Compared with the control group, the FNPs-250 accumulated in small intestine

310

for 1.5 to 6 h, then were excreted at 24 h (Figure 7a). Similarly, fluorescence of FNPs

311

in other organs including brain, heart, lung, kidney, and stomach showed a time-

312

dependent accumulation (Figure 7b-7f, Figure S3). Strikingly, the fluorescence

313

intensity of the brain increased after 1.5 h oral administration and reached its maximum

314

value at 6h (Figure 7e), indicating that the FNPs could enter the mice brain through the

315

blood-brain barrier.26 After oral administration for 24 h, the fluorescence intensity of

316

major organs decreased to the level similar to that before administration, suggesting

317

that the FNPs were finally excreted from mice. The results indicated that the FNPs

318

derived from squid appeared in the main organs of mice after ingestion. They

319

accumulated in the main organs of organism through metabolic absorption in a short

320

time, and were then gradually excreted out. Therefore, potential health risk is unknown

321

and much effort is required to assess the effects of the FNPs from squid, such as non-



322

specific binding between the FNPs and important bioactive macro molecules.

323 324

(Figure 7) Interaction Between HSA and FNPs

325

HSA can act as carriers for the transportation of fatty acids, amino acids, steroid

326

hormones, metal ions and various bioactive molecules in human body fluids and

327

maintain normal osmotic pressure in the blood.36 Thus it seems to us that the FNPs are

328

probably transported into blood stream and will meet the HSA after oral uptake.

329

Therefore, it is necessary to investigate the interaction between the HSA and FNPs. It

330

is known that the tryptophan residue of HSA has strong fluorescence, which can reflect

331

the changes after encountering with FNPs. Figure 8a shows the fluorescence spectra of

332

HSA in the absence and presence of FNPs-250. Upon adding FNPs-250, the

333

fluorescence intensity of HSA obviously reduced, indicating that the addition of FNPs-

334

250 led to the decrease of intrinsic fluorescence of HSA molecules. With the increase

335

of FNPs-250 concentration, the endogenous HSA fluorescence reduced gradually,

336

suggesting that the interaction between HSA and FNPs-250 in a concentration-

337

dependent manner. No red-shift in the maximum emission peak was found because the

338

interaction did not cause a dramatic change in the conformation near the tryptophan

339

residue. The results showed that there was a linear relationship between F0/F-1 value

340

with the concentration of FNPs-250, which was consistent with the Stern-Volmer

341

equation (Figure 8b). As we all known that the mechanism of fluorescence quenching

342

included dynamic and static quenching.37 If the excited state of the HSA was changed,

343

the fluorescence of HSA would be dynamically quenched, whereas static fluorescence


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344

quenching, in most cases, was assigned to the non-luminescent complex formation.38

345

The UV-vis absorption spectroscopy has been proved to be an effective method to

346

elucidate the fluorescence quenching mechanism of HSA with other complexes39. The

347

UV-vis absorption spectra of HSA, FNPs, HSA-FNPs and [HSA-FNPs] -FNPs

348

(otherness of absorption spectra between HSA-FNPs and FNPs) were displayed in

349

Figure 8c. The peak pattern of [HSA-FNPs] -FNPs was inconsistent with that of HSA

350

at 210-300 nm, which manifested that the FNPs caused changes of HSA UV-vis

351

absorption spectrum, resulted in a static quenching of HSA fluorescence.

352

Time-resolved fluorescence spectroscopy also can provide useful information in

353

clarifying the fluorescence quenching mechanism through measurements of lifetime for

354

HSA-FNPs complexes. Figure 8d shows the average fluorescence lifetimes from time-

355

resolved fluorescence measurements, and the lifetime of HSA-FNPs and HSA alone

356

was about 4.97 and 4.63 ns, respectively, which varied by 7.34%. The amplitude change

357

was quite less than that of dynamic quenching,37 revealing that the addition of FNPs

358

resulted in a fluorescence quenching of HSA via a static quenching mechanism between

359

HSA and FNPs.

360 361

(Figure 8) Conformational change analysis

362

FTIR spectrometry is considered as an effective method in detecting

363

conformational change in protein secondary structure upon encountering NPs.40 Two

364

absorption bands, the amide II band (C–N stretch and N–H bending) at 1,537cm-1 and

365

the amide I band (C=O stretching) at 1,649 cm-1 (Figure 9a), were observed in HSA.41-42



366

When the FNPs-250 were added, the peak of the amide II band changed from 1,537 to

367

1,542 cm-1. At the same time, the peak of the amide I band, moved from 1,649 to 1,659

368

cm-1. This revealed that the FNPs not only affected the vibration of C=O groups but

369

also affected C–N stretching and N–H bending. The interaction of FNPs with HSA

370

induced a change in the secondary structure of HSA. Hence, the CD spectroscopy was

371

further used to examine specific changes in the secondary structure of HSA. The CD

372

spectra collected in a far ultraviolet range of 195-250 nm demonstrated the absorption

373

of the protein amide band.43 With the addition of various concentration of FNPs, two

374

negative bands at 208 and 222 nm from α-helix showed a decrease trend (curves 1 to 5

375

in Figure 9), indicating an apparent conformational change of protein accompanied by

376

the loss of α-helix content. The greater the concentration of FNPs was, the more the

377

content of α-helix decreased. When the FNPs content reached 2.7 × 10-4 mol L-1, the α-

378

helix content of HSA decreased from 39.4 to 29.9% (Table 3). It can be seen that the

379

FNPs caused significant change of the original secondary structure of HSA. Therefore,

380

the existence of FNPs might lead to HSA skeleton loose and fold, and the structural

381

change might affect the biological activity and protein function.

382

Table 3. α-helix changes of HSA with the addition of different concentrations of FNPs. FNPs concentration (mol L−1) 0 0.3×10-4 0.9×10-4 1.5×10-4 2.1×10-4 2.7×10-4

383

α–helix content (%) 39.4 37.4 36.7 34.2 32.8 29.9 (Figure 9)


Page 18 of 39

Page 19 of 39

384


Surface topography investigation

385

AFM is an effective biophysical technique in determining the surface topography

386

of certain NPs.44 The 3-D (Figure 10a) and 2-D image (Figure 10b) of HSA alone show

387

that the HSA exhibited good dispersion, and the main height was measured to be 20.23-

388

21.40 nm (Figure 10c). The AFM image of FNPs-250 showed that the FNPs were

389

evenly dispersed on the mica sheet. (Figure 10d, 10e). The surface morphology of FNPs

390

was sharper than that of HSA, and the main height of FNPs-250 was around 18.53-

391

22.32 nm (Figure 10f), probably due to the FNP aggregation after being dried on the

392

mica sheet. The AFM images of HSA-FNPs system (Figure 10g, 10h) showed that the

393

size of HSA-FNP was relatively uniform, and the morphology was quite different from

394

HSA and FNPs alone. The main height of the HSA-FNPs system was 28.54-37.25 nm

395

(Figure 10i), exhibiting an increase trend as compared with that of HSA. This further

396

revealed that the HSA-FNPs might form nano-corona, thus affecting the function of

397

HSA.

398

(Figure 10)

399

In summary, this work explored the effect of different roasting temperatures on the

400

formation of FNPs from roast squid. The FNPs were mainly composed of carbon,

401

oxygen and nitrogen, which exhibited temperature-dependent particle sizes and

402

fluorescence properties. The FNPs could be distributed into the main organs after oral

403

uptake and pass through the blood–brain barrier to enter the brain of BALB/c mice. In

404

addition, the FNPs interacted with bio-macromolecular HSA and altered the structure

405

and morphology of HSA through forming nano-corona. These results provided useful



406

information in understanding the properties of FNPs from roast squid, and much effort

407

is needed in assessing their biological effects.

408

Acknowledgements

409 410

This work was supported by the National Natural Science Foundation of China (31872915).

411 412

Conflicts of Interest The authors declare that they have no conflict of interest.

413 414 415

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Lumin. 2006, 121 (1), 179-186.

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serum albumin and its effect on protein conformation stability. Food Chem. 2016, 192,

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Biochem. 1981, 114 (2), 199-227.

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39. Zhenxing, C.; Rutao, L. Phenotypic characterization of the binding of tetracycline

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to human serum albumin. Biomacromolecules 2011, 12 (1), 203-209.

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secondary structure. Anal. Biochem. 2000, 277 (2), 167-76.

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41. Zhang, G.; Wang, L.; Pan, J. Probing the binding of the flavonoid diosmetin to

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human serum albumin by multispectroscopic techniques. J. Agric. Food Chem. 2012,

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44. Best, R. B.; Brockwell, D. J.; Toca-Herrera, J. L.; Blake, A. W.; Smith, D. A.;

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Radford, S. E.; Clarke, J. Force mode atomic force microscopy as a tool for protein

540

folding studies. Anal. Chim. Acta. 2003, 479 (1), 87-105.

541 542 543 544 545 546 547 548 549 550 551

Figure caption:

552

Figure 1. TEM images of (a) FNPs-190, (d) FNPs-220 and (g) FNPs-250, respectively.

553

Size distribution of (b) FNPs-190, (e) FNPs-220, (h) FNPs-250. XRD pattern of FNPs-

554

190 (c) FNPs-220, (f) FNPs-250. (j) Schematic illustration of fluorescent FNPs derived

555

from roast squid.

556

Figure 2. (a) FTIR spectra of FNPs-190, FNPs-220 and FNPs-250, corresponding to 1,

557

2 and 3 in the figure, respectively. XPS spectrum of (b) FNPs-190, (c) FNPs-220 and

558

(d) FNPs-250.

559

Figure 3. High-resolution C1s spectra of (a) FNPs-190, (b) FNPs-220, and (c) FNPs-


Page 26 of 39

Page 27 of 39


560

250. High-resolution O1s spectra of FNPs of (d) FNPs-190, (e) FNPs-220 (f) FNPs-250.

561

High-resolution N1s spectra of (g) FNPs-190, (h) FNPs-220 and (i) FNPs-250.

562

Figure 4. UV-vis absorption and fluorescence spectra of (a) FNPs-190, (c) FNPs-220

563

and (e) FNPs-250. Fluorescence decay curve of (b) FNPs-190 (d) FNPs-220 and (f)

564

FNPs-250.

565

Figure 5. Effects of metal ions on the fluorescence intensity of (a) FNPs-190, (b) FNPs-

566

220 (c) FNPs-250. Effects of pH on the fluorescence intensity of (d) FNPs-190 (e)

567

FNPs-220, (f) FNPs-250. a, b, c, d, e, f and g referred the values with significant

568

difference.

569

Figure 6. Bright-field photographs (a–d), MRI images (e–h), surface fluorescence

570

images under 365 nm excitation (i–l), AFM images (m–p) and photographs of the FNPs

571

aqueous solution under a 365 nm UV light (q–t) of the squid samples before and after

572

roasting at 190, 220 and 250 °C, respectively.

573

Figure 7. Ex vivo fluorescence images of major organs in mice under the excitation of

574

365 nm, (a) intestine, (b) heart, (c) lung, (d) brain, (e) kidney, (f) stomach, after oral

575

administration of 0.9% NaCl solution (control) and FNPs-250 at a dose of 2 g kg-1 body

576

weight at 1.5, 4, 6, and 24 h, respectively.

577

Figure 8. (a) Fluorescence emission spectra of HSA with increase of FNP concentration.

578

c (HSA) = 2 × 10−6 mol L−1, c (FNPs), a−f: 0, 0.3, 0.9, 1.5, 2.1, 2.7 × 10−4 mol L−1,

579

pH=7.4. (b) Stern-Volmer plot of HSA fluorescence quenched by FNPs at 298 K. (c)

580

UV-vis absorption spectra of HSA, HSA with FNPs and FNPs alone. c (HSA) = 2 ×

581

10−6 mol L−1, c (FNPs) = 2 × 10−4 mol L−1. (d) Fluorescence decay traces of HSA



582

existence and nonexistence FNPs, c (HSA) = 2 × 10−6 mol L−1, c (FNPs) = 2 × 10−4 mol

583

L−1, pH = 7.4, T = 298 K.

584

Figure 9. (a) FT-IR spectra of HSA and HSA in the presence of FNPs, c (HSA) = 2 ×

585

10−6 mol L−1, c (FNPs) = 2 × 10−4 mol L−1. (b) CD spectra of HSA in the presence of

586

FNPs at different concentrations of 0, 0.3, 0.9, 1.5, 2.1 and 2.7 × 10−4 mol L−1 (splines

587

from 1-5); c (HSA) = 2 × 10−6 mol L−1; T = 298 K; pH = 7.4.

588

Figure 10. (a) 3-D AFM images of HSA, (d) FNPs-250 and (g) HSA-FNPs system. (b)

589

2-D AFM photographs of HSA, (e) FNPs-250 and (h) HSA-FNPs system. The height

590

profiles of (c), (f), (i) corresponding to the area circled in (b) (e) (h), respectively.

591 592 593


Page 28 of 39

Page 29 of 39


594 595

Figure 1.



a 2

3 C-H -OH or -NH2

C-O-C

N1s

C1s N1s

400

600

0

d

200

800

Bingding Energy (e.V)

400

600

800

Bingding Energy (e.V) O1s

C1s

Intensity (a.u.)

Intensity (a.u.)

O1s

200

C1s

CONH2

3000 2000 1000 Wavenumber (cm-1)

0 597

C-N

O1s

Intensity (a.u.)

Transmittance (a.u.)

b 1

c

596

Page 30 of 39

N1s

0

200

400

600

800


Figure 2.

598 599 600



Intensity (a.u.)

g

284

602

288

290

C-O

O-C=O*

*O-C=O

530

532

534


Amiono N

396

601

286


Pymolic N

400

404

408


C-O C=O

282

e

284

286

288

Intensity (a.u.)

C=O

C-N

290

f

Bingding Energy (e.V) O-C=O*

C-O *O-C=O

528

530

532

h Amiono N

Pymolic N

396

400

404

C-O C=O

284

408


Figure 3.

603 604 605 606


286

i

288

290

Bingding Energy (e.V) O-C=O*

C-O

530

534


392

C-N C=C

Intensity (a.u.)

C-O

Intensity (a.u.)

C-N

C=C

Intensity (a.u.)

Intensity (a.u.)

d

c

b C=C

Intensity (a.u.)

Intensity (a.u.)

a

Intensity (a.u.)

Page 31 of 39

532

*O-C=O

534

Bingding Energy (e.V) Amiono N Pymolic N

392

400

408



a

600

300

607 608

0 300 310 320 330 340 350 360 370 380 390 400

400

500

Wavelength (nm)

f

600

80

χ2 =1.02

40

Time (ns)

Counts (a.u.)

500

40

Time (ns)

d

Wavelength (nm)

Absorbance (a.u.)

e

0

300 310 320 330 340 350 360 370 380 390 400

400

Counts (a.u.)

600

FL Intensity (a.u.)

300

χ2 =1.141

Counts (a.u.)

450

Wavelength (nm)

FL Intensity (a.u.)

300

FL Intensity (a.u.)

Absorbance (a.u.)

b 300 310 320 330 340 350 360 370 380 390 400

Absorbance (a.u.)

c

Page 32 of 39

80

χ2 =1.078

0

Figure 4.

609 610 611 612 613 614


40

Time (ns)

80


C

on

tr C ol u 2+ Zn 2 M + g 2+ C a2 M + n 2+ N 2 i + C o 2+ Fe 2 +

a

FL Intensity(a.u.)

d

e

cd cd e

e bc

e b

on

tr o C l u 2+ Zn 2 M + g 2+ C a2 M + n 2+ N 2 i C + o 2+ Fe 2 +

a

FL Intensity(a.u.)

C

g

616

e

e cd de b bc de

on

tr C ol u 2+ Zn M 2+ g2 C + a 2+ M n 2+ N 2 i C + o 2+ Fe 2 +

a

f

e d d c

b a

Control 2 3 4 5 6 8 9 11 12

Control 10 25 50 100 250 500

e

f

pH

bc bc bcbc c

c

b b b a

Control 2 3 4 5 6 8 9 10 11

pH

h

fg g a e f f d c b a

Control 2 3 4 5 6 8 9 10 11

pH

C

615

f

FL Intensity(a.u.)

b

c

Figure 5.

617 618 619 620 621 622 623 624 625


NaCl (mmol/L)

FL Intensity(a.u.)

g f

e

bc bcde e d d b bc c a

b b b b

a a

a

Control 10 25 50 100 250 500

NaCl (mmol/L)

i

FL Intensity(a.u.)

c c d

b

FL Intensity(a.u.)

FL Intensity(a.u.)

h

FL Intensity(a.u.)

a

FL Intensity(a.u.)

Page 33 of 39

d d c c

b a b

Control 10 25 50 100 250 500

NaCl (mmol/L)


626 627

Figure 6.

628 629 630 631 632 633 634 635 636 637 638


Page 34 of 39

Page 35 of 39


639 640

Figure 7.

641 642 643 644 645 646 647 648 649 650



Page 36 of 39

651

a

b

FL Intensity(a.u.)

a

F0 / F-1

f

0.08

400

440

b

c a d

b c a

220

240

300

280nm

240

260

280

Wavelengh (nm)

360

1

Wavelengh (nm)

300

2

420

3

104 FNPs (mol L-1)

d

a HSA b [HSA-FNPs] c FNPs d [HSA-FNPs]-[FNPs]

d

0

HSA+FNPs

Counts (a.u.)

360

Wavelength (nm) Absorance (a.u.)

Absorance (a.u.)

0.12

F0 /F-1=0.0527×104[FNPs]+0.0102

c

653

298K R2=0.9989

0.04

320

652

0.16

HSA alone

HSA alone

0

Figure 8.

654 655 656 657 658 659 660 661 662 663


100

Time (ns)

200

Page 37 of 39


664

b 60 HSA HSA+FNPs 1537

666

1649

C-N/N-H 1541

1500

665

40

CD (mdeg)

Transmittance (a.u.)

a

1662

1600

C=O

1700

Wavenumber (cm )

20 0

-20

6

-40

1

200

-1

HSA alone

210

220

230

240

Wavelength (nm)

Figure 9.

667 668 669 670 671 672 673 674 675 676 677 678 679 680 681


250


682

683 684

Figure 10.

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Nano-corona formation between foodborne nanoparticles exacted

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