Construction of persistent luminescence-plastic antibody hybrid

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Food Safety and Toxicology

Construction of persistent luminescence-plastic antibody hybrid nanoprobe for in vivo recognition and clearance of pesticide using background-free nano-bioimaging Dong-Dong Zhang, Jing-Min Liu, Shi-Ming Sun, Chang Liu, Guozhen Fang, and Shuo Wang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b02712 • Publication Date (Web): 30 May 2019 Downloaded from http://pubs.acs.org on May 30, 2019

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Journal of Agricultural and Food Chemistry

Construction of persistent luminescence-plastic antibody hybrid nanoprobe for in vivo recognition and clearance of pesticide using background-free nano-bioimaging Dong-Dong Zhang,a,c Jing-Min Liu,b Shi-Ming Sun,a Chang Liu,a Guo-Zhen Fang,a,* Shuo Wanga,b,* aState

Key Laboratory of Food Nutrition and Safety, Tianjin University of Science &

Technology, Tianjin, 300457, P.R.China. bTianjin

Key Laboratory of Food Science and Health, School of Medicine, Nankai

University, Tianjin, 300071, P.R.China. cHenan

University of Technology, Collaborative Innovation Center of Henan Grain Crops,

Henan Collaborative Innovation Center of Grain Storage and Security, Zhengzhou, 450001, P.R.China.

*Corresponding author Guozhen Fang: E-mail: [email protected], Tel: +86-22-60912493; Fax: +86 22 6091 2493, Shuo Wang: E-mail: [email protected], Tel: +86-22-85358445.

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ABSTRACT

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We prepared a specific adsorptive nanocarrier for pesticide due to its challenge to cleanup

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and low detoxification in the treatment after intake intentional or by mistake. We modified

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the plastic antibody (molecularly imprinted polymer, MIP) on the surface of persistent

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luminescence nanoparticle (La3Ga5GeO14: Cr3+, Zn2+, LGGO) as the specific adsorptive

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nanocarrier for toxic molecules, and realized the nanocarrier was widely distributed for

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absorbing pesticide and real-time in vivo bioimaging. We used LGGO as the core and

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trichlorphon as the template to prepare the plastic antibody nanocarrier. After in vivo

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bioimaging and biodistribution of mice, LGGO@MIP could be distributed evenly in the

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gastrointestinal tract, circulate in the blood for a long time, and finally excreted to achieve the

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adsorption and removal of pesticide in the body. The LGGO@MIP nanocarrier prepared in

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this study opens a new way for the treatment of poisoning.

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KEYWORDS: persistent luminescence; plastic antibody; pesticide; specific adsorption;

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bioimaging


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INTRODUCTION

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Pesticide, as an indispensable part of agricultural production, is a powerful tool for

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people to fight crop diseases and insect pests and ensure a good harvest of agriculture.1

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However, the lack of safe storage could lead to accidental ingestion or the deliberate

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swallowing by someone attempting suicide, which could cause poisoning and even death.

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This continues to be a world public health problem.2-3 According to epidemiological studies,

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up to three hundred thousand deaths in the world are caused by intentional swallowing of

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pesticides each year.4-6 The number of cases of pesticide poisoning has been increasing,

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especially in developing countries.7-8 Organophosphorus pesticides are the most common

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pesticides associated with these poisoning events. Organophosphorus pesticides inhibit the

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activity of acetylcholinesterase (AChE) and some other esterases in the body, resulting in the

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accumulation of acetylcholine in the nervous system. The excess accumulation of

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acetylchomine in the nerve cell often leads to continuous excitement of the cell, resulting in

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continuous excitement. The continuous excitement of nerve receptors can result in human

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organ dysfunction, respiratory failure, coma and death.9-10

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In the treatment of pesticide poisoning, if the poison is taken orally, the first step is to

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expel the drug that is not absorbed in the stomach from the body, it includes vomiting and

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adsorption. Vomiting and adsorbing are two commonly used effective physical therapies in

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the treatment of poisoning. Some toxic substances absorbed slowly by the body could be

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completely removed by these two methods. Vomiting, repeated decompression and gastric

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lavage are the generally methods to remove the unabsorbed pesticides directly from the

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stomach in vitro. Adsorption, there are some adsorbent, such as granular activated carbon, 3 ACS Paragon Plus Environment


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would be used to further adsorb the residual pesticides in the stomach.11-12 Second step,

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enough cholinesterase resurrection agents (such as atropine and oximes) are used

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repeatedly.13-15 However, treatment with anti-cholinesterase drugs has not improved

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significantly in the past fifty years.10, 13 In order to improve the therapeutic effect of pesticide

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poisoning, various attempts have been made, including the establishment of standardized

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procedure for gastric lavage, the establishment of standardized nursing process, the

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combination of multiple detoxification drugs and so on, and even the treatment of blood

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replacement.8,

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treatment of pesticide poisoning is limited, and the treatment using drugs is simplistic in

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many cases with a limited cure rate and often is accompanied by sequelae. The linited and

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costly development of therapeutic drugs is due to the longer drug development cycle and the

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high cost of drug research and development.6, 17 Therefore, we should start at an early stage in

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the potential exposure process to remove the exposure to pesticides as thoroughly as possible.

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As an important immune method to identify and specifically bind the invading antigen

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in vivo, antibodies have the advantages of high specificity and selectivity, which can quickly

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and accurately remove the foreign antigens and play an important role in the body

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immunity.18-19 The preparation process of natural antibodies is complicated, the preparation

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cycle is longer, and an antibody needs to be prepared in the animal body. It is expensive.

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Meanwhile, the properties of the prepared antibody may not be stable for extended periods of

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time, may require special storage conditions and the actual use conditions may be harsh.20-21

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Within a rapidly developing subgroup of polymer chemistry, the emergence of molecular

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imprinting technology is playing a more and more important role in target recognition. The

13, 16

However, the development of specific drug adsorbents used in the


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prepared molecularly imprinted polymer (MIP), known as plastic antibody, not only has

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higher specificity and selectivity than that of a natural antibody, but also can be more easily

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synthesize using simple monomers with high stability and more easily suitable to large batch

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preparation. MIP has two characteristics in adsorbing target molecules, first, the special

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configuration of imprinted template molecule in MIP; second, the chemical bonding between

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functional monomer and template molecule. It has become an ideal choice for biomimetic

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antibodies.22-23 In the process of preparation, the molecularly imprinted polymer is a polymer

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formed using the target molecule (pesticide) as the template. A functional monomer and a

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crosslinker are prepared under the action of the initiator. After eluting the template drugs

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through the eluant, the MIP has formed a template with a three-dimensional structure of the

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molecule specific hole. The prepared MIP has the specific pore structure of the target

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molecule, has the ability to adsorb the template molecule specifically, and is stable, reusable

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and biocompatible. The MIP thus formed can be used for the poison adsorption in vivo.24-27 If

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the MIP is used for the adsorption of toxic and harmful substances in the organism, it would

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further reduce the absorption of poisons by the body, and reduce the development of the toxic

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symptoms and improve the cure rate.28-29 In the construction of a nanocarrier, consideration

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may be given to the use of persistent luminescence nanopartuckes as the core. This would add

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the ability of real-time optical bioimaging of the targeted pesticide in vivo.30 Using

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biomimetic molecularly imprinted polymer as the adsorption layer and persistent

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luminescence nanoparticles as the luminescent core, we prepared a long afterglow bionic

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antibody nanocarrier LGGO@MIP.

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In this experiment, a plastic antibody was synthesized on the surface of persistent 5 ACS Paragon Plus Environment


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luminescence nanoparticles (LGGO@MIP) using as a model for the class of persticides,

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trichlorofon, as the template, methacrylic acid (MAA) as the functional monomer, ethylene

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glycol two methacrylate (EDMA) as the crosslinker, and azo two isobutanonitrile (AIBN) as

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the initiator. The prepared LGGO@MIP achieved specific adsorption of pesticides and real-

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time bioimaging. In the preparation of plastic antibodies, we optimized the proportion of

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several components on the basis of previous studies, tested the elution times of the eluant,

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and tested the physiological and biochemical characteristics of the LGGO@MIP

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nanoparticles. Finally, we used the prepared LGGO@MIP nanoparticles as a bioimaging

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agent for in vivo bioimaging after oral administration and intravenous injection of mice.

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(Figure 1)

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MATERIALS AND METHODS

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Chemicals and materials. All reagents used for synthesis were at least analytical grade. All

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the water used in the experiment was obtained through Milli-Q (Millipore, Bradford, MA,

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USA). All glassware should be soaked in aqua regia (HCl: HNO3 = 3:1, v/v) for at least 24 h

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and cleaned with ultrapure water. Trichlorofon (Product No.: 586164) was purchased from

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J&K Scientific Ltd., China. MAA (Product No.: M102640), EDMA (Product No.: E106223),

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AIBN (Product No.: A104256) were purchased from Aladdin Company, China.

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Characterization.

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characterization of the prepared materials was performed in accordance with literature

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references.31-32 The phosphorescence excitation and emission spectra, and the afterglow

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decay curves of LGGO were recorded by a Lumina spectrofluorometer (Thermo Scientific,

The

phosphorescence

properties,


structural

and

morphological

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Waltham, MA, USA) equipped with a 150 W xenon lamp. X-ray diffraction (XRD) patterns

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were acquired on a Bruker AXS D8 Advance diffractometer (Bruker, Germany) equipped

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with Cu Kα radiation (λ = 1.5418 Å). The morphology and microstructure of the prepared

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LGGO were characterized by transmission electron microscope (TEM, JEM-2010FEF,

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JEOL, Japan) operating at accelerating voltage of 200 kV, and equipped with a Phoenix 60T

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energy dispersive spectrometer (EDS).

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Preparation of LGGO@MIP. The LGGO were synthesized by one-pot method in

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combination with calcination in air.

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gallium nitrate, lanthanum nitrate, chromium nitrate, zinc nitrate, and germanium nitrate

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according to the chemical formula of La3Ga5GeO14: 0.1% Cr3+, 1% Zn2+ under vigorous

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stirring. After adjusting pH value (determined by WhatmanTM pH indicator paper) to 7.0 with

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tert-butylamine, the resulting solution was stirred for 2 h at room temperature. After that, the

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solution was mixed with oleic acid and toluene at the ratio of 15:2:15 as biphasic synthesis

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method. The mixture was transferred to Teflon-lined stainless steel autoclave and kept

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120 °C for 24 h. After cooled to room temperature, the resulting compound was precipitated

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upon addition of excess ethanol and washed with ethanol. Before annealed in air at 1000 °C

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for 1 h, the prepared precipitate was dried at 80 °C for 4 h.

31-32

The precipitated precursor was obtained by mixing

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The prepared LGGO nanoparticles as the core of LGGO@MIP. Step 1, the harvested

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LGGO was placed in fresh methanol in a polypropylene centrifugal tube, treated with

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ultrasonic bath (KQ-500B, Ningbo Scientz Biotechnology Co.,Ltd., Zhejiang, China) at room

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temperature until it was evenly dispersed. LGGO precipitation was obtained by

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centrifugation. Repeat twice. The LGGO was suspended in 20 mL fresh methanol in a glass 7 ACS Paragon Plus Environment


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flask with round bottom, treated with ultrasonic bath for 5 min. Step 2, 0.45 mmol MAA was

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added dropwise, after treated with ultrasonic bath in the glass flask for 20 min, it was keep

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40 °C for 18 h with magnetic stirring. Step 3, the resulting suspension was the LGGO-MAA

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(La3Ga5GeO14: Cr3+, Zn2+ connected to methacrylic acid), it was collected by centrifugation

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and washed with ethanol three times, and suspended in 30 mL acetonitrile solution. Step 4,

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0.6 mmol trichlorfon and 0.9 mmol MAA were added, and stirred for 2 h. Step 5, after the 2

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hours, 1.2 mmol EDMA and 10 mg AIBN were added. Step 6, the glass flask with the

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mixture treated with ultrasonic bath for 20 min and keeps stirring at 60 °C for 24 h under

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nitrogen. Step 7, the resulting compound was washed with ethanol three times, and suspended

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in 100 mL methanol/acetic acid (8:2, v/v), ultrasonic treatment for 3 h and stirring for 2 h,

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then replace the fresh solution, the same treatment, until there was no trichlorfon in the

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supernatant. The collected LGGO@MIP was washed 3 times by sterile PBS solution before

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use and finally suspended in PBS solution. As control, the LGGO modified without template

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polymer (La3Ga5GeO14: Cr3+, Zn2+@ non-molecularly imprinted polymer, LGGO@NIP) was

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prepared with the same procedures in the absence of trichlorfon.

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Adsorption of trichlorfon on LGGO@MIP. The amount of triclorfon absorbed by

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LGGO@MIP was quantified by determining the concentration of trichlorfon in the

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supernatant before and after adsorption using HPLC (1260, Agilent Technologies, Santa

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Clara, CA, USA) with UV detection, separation was achieved using a C18 column. The

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detection wavelength was 200 nm, mobile phase was water/acetonitrile = 9:1 (v/v), pH=3.0

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(regulating by phosphoric acid), and at flow rate of 1.5 mL/min. The column temperature was

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kept at 35 °C. The quantification was calculated by external reference standards. 8 ACS Paragon Plus Environment

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Cytotoxicity assay. MC 38 and HeLa cells were used to evaluate the cytotoxicity of

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LGGO@MIP in this study. The MC 38 is a tumorigenic epithelial cell line isolated from mice

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with colon adenocarcinoma, and the HeLa cell is cancer cell line isolated from malignant

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cervical tumor, there were purchased from National Infrastructure of Cell Line Resource,

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China, The MC 38 cell cultured in RPMI Medium 1640 basic (Thermo Fisher Scientific,

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Suzhou, Jiangsu, China) and the HeLa cell cultured in DMEM Medium (Thermo Fisher

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Scientific, Suzhou, Jiangsu, China), there were all added 10% fetal bovine serum (Life

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Technologies, Inc. Gaithersburg, MD, USA). After 48 hours of incubation, the cell

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concentration was adjusted to 104 mL-1, and added 100 μL/well into 96-well microplates.

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After, it was cultured in the 37 °C incubator contained 5% CO2. After mixed with various

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concentrations of sterile LGGO@MIP (Sterilization by ultraviolet radiation), the in vitro

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cytotoxicity of LGGO@MIP was assessed using the WST-1 Cell Proliferation and

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Cytotoxicity Assay Kit (Follow the product instructions, Beyotime Biotechnology, Haimen,

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JiangSu, China). At last, the absorbance of 96-well microplates at 450 wavelength was

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measured by a Varioskan LUX Spectrophotometer (Thermo Scientific, Vantaa, Finland).

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In vivo fluorescence image of LGGO@MIP in mice. All animal experiments were

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approved by the Animal Ethics Committee of Tianjin University of Science and Technology,

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and followed the guidelines of the Tianjin Committee of Use and Care of Laboratory

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Animals. This requires us to have a standard feeding environment, to monitor the quality of

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mice regularly, to purchase qualified mice, and to limit the quality of food and water for

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mice. In order to minimize the impact on mice, all experiments on mice were carried out after

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the mice were anesthetized with chloral hydrate. Five weeks old nude mice (BALB/c-nu, 9 ACS Paragon Plus Environment


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weight 20-25 g) were purchased from National Institutes for Food and Drug Control, China.

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Before animal experiments, the mice were fed in the laboratory environment for more than 7

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days to allow the animals to adapt to the new environment.

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The concentration of LGGO@MIP was adjusted to 2 mg/mL, which was excited by UV

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for 5 min. Soon, 200 μL LGGO@MIP was injected into anesthetized mice by gavage or

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caudal vein injection. The luminescence signal in mice was regularly acquired by a small

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animal imaging system (NightOWL LB983, Bethold Technologies, Bad Wildbad, Germany),

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and the LGGO@MIP in mice was repeatedly excited by a LED red lamp (650 nm) after the

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luminescence signal disappear.

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LGGO@MIP was injected into anesthetized mice at the dose of 200 μL, 2 mg/mL, after

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5 h, the mice were euthanized with an overdose of anesthetic to death. Then the main organs

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of mice were collected after anatomy and irradiated by the LED red lamp, the luminescence

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signal was acquired by the small animal imaging system (NightOWL LB983, Bethold

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Technologies, Bad Wildbad, Germany). Later, the collected organs were grinded in an agate

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mortar and the concentration of Ga3+ was determined by ICP-MS (7500CX, Agilent, Palo

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Alto, USA) after microwave digestion, and the quantity of LGGO@MIP was calculated by

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the quantity of Ga3+.

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Detoxicate test of LGGO@MIP on poisoned mice. All animal experiments were approved

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by the Animal Ethics Committee of Tianjin University of Science and Technology, and

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followed the guidelines of the Tianjin Committee of Use and Care of Laboratory Animals.

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Step 1, 20 mice were randomly selected, 50 mg/mL trichlorfon solution was injected into


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mice by gavage at a dose of 380 mg/kg body mass. Step 2, the prepared LGGO@MIP was

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injected into mice by gavage at a dose of 30 mg/kg immediately (within 1 min), which used

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as adsorbent and antidote. Step 3, the survival rate and body weight change of mice after 48 h

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were measured. Another group of mice received the same dose of trichlorfon solution

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treatment, but did not injected LGGO@MIP as control.

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RESULTS AND DISCUSSION

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Optimized preparation of LGGO@MIP. As previously reported by our group in the

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literature31, La3Ga5GeO14:Cr3+, Zn2+ (LGGO) was prepared by “one pot” combined with

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calcination, it used as the luminescence center of nanocarrier. The plastic antibody (MIP)

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acted as specific adsorption layer. Trichlorfon was used as template molecule to prepare

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LGGO@MIP, without template molecule to prepare LGGO@NIP (La3Ga5GeO14:Cr3+,

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Zn2+@non-molecularly imprinted polymer). After modified by plastic antibody MIP/NIP,

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there was no significant difference between the prepared LGGO@MIP and LGGO@NIP

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under TEM (Fig. 2A), the irregular structures of LGGO develop into regular spheres, and the

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edges were smooth, which was beneficial to the application of the nanocarrier in vivo

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bioimaging. It could reduce the possibility of phagocytosis by phagocytes and prolong the

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cycle time in vivo.33-34 In the TEM images of LGGO@MIP/NIP, the dark black LGGO core

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was clearly visible, and the outer MIP/NIP modification layer was relatively light due to it

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contains no metal ions and has a low density. The prepared nanocarriers were well dispersed,

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the thickness of MIP/NIP modified layer and morphology were uniform. It showed that the

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surface properties of LGGO nanoparticles modified by MIP/NIP were significantly

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improved, which not only changes the morphology of nanoparticles, but also solves the 11 ACS Paragon Plus Environment


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problem of aggregation of nanoparticles.

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We tried to increase the concentration of MIP/NIP components to prepare nanocarriers

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with thicker coatings. When the concentration doubled, the MIP/NIP could still be wrapped

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around LGGO (Fig. 2A/cd), the thickness of the MIP/NIP layer increased significantly, and

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some LGGO-free nanocarriers (The MIP nanocarrier does not contain LGGO core.)

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appeared. Obviously, this could increase the adsorption capacity of LGGO@MIP on target

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molecules. However, it also affects the particle size and luminescence characteristics of

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LGGO@MIP nanocarriers. As shown in Fig. 2A (cd) of TEM images, the particle size of

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nanocarries was significantly increased. Previous studies have shown that the thicker MIP-

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coated nanocarriers could be used for the development of adsorbent and antidote in the

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gastrointestinal tract. In this study, we focus on the main features of LGGO@MIP

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nanocarrier with smaller particle sizes (about 100 nm), and its application on specific

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adsorption and in vivo bioimaging.

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The effect on particle size and luminescence of LGGO. After the LGGO was modified

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with MIP, a layer of molecularly imprinted polymer was coated on the surface (Fig. 2A),

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which resulted in a slight decrease of luminous intensity (decreased by 8.46%, Fig. 2B) and a

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slight increase of nanoparticle size (Fig. 2E). The particle size of LGGO@MIP was measured

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by dynamic light scattering (DLS) method. The measured hydrate particle size was larger

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than that of TEM images, and the reason was explained by previous reports.35 The

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LGGO@MIP particle size ranged from 88.9 nm to158 nm, the average particle size was 125

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nm (PDI=0.038). According to previous reports and our study,19,

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size range could be used not only in the gastrointestinal tract of mice, but also in intravenous 12 ACS Paragon Plus Environment

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nanoparticles with this

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injection of mice. After modification, the MIP layer on the LGGO surface would inevitable

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affect the luminescence. First, the MIP layer would weaken the arrival of excitation light.

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Second, the MIP layer would weaken the emission of light. However, compared with LGGO,

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the luminescence intensity of the optimized LGGO@MIP still has 91.54% residual (Fig. 2B),

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and after UV or LED red lamp excitation, the luminescence signal of LGGO@MIP could be

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captured continuously. It has ideal NIR luminescence images and would not affect the

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application of in vivo bioimaging (Fig. 2C).

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Structural characterization of LGGO@MIP. Compared with LGGO, EDS data revealed

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the reduction of C/O ratio (The ratio of the number of carbon atoms to oxygen atoms.) in

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LGGO-MAA due to the C/O in MAA (C4H6O2), the changes of C/O ratio and the appearance

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of P/Cl (The ratio of the number of phosphorus atoms to chlorine atoms.) due to EDMA

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(C10H14O4), AIBN (C8H12N4) and trichlorfon (C4H8Cl3O4P) (Fig. 2D). Modified by MAA and

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MIP, the zeta potential of LGGO decreases continuously (Fig. 2F), which was beneficial not

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only to the stability of the nanocarrier system, but also to the adsorption of positive potential

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molecules.35-36 The pure crystallization of LGGO, LGGO@MIP and LGGO@NIP was

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confirmed by XRD (X-ray diffraction) (Fig. 2G), and after modified by MIP and NIP, the

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peak intensity decreased, but the position of the peak remains unchanged. This is consistent

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with La3Ga5GeO14 (ICSD#72-2464) and indicated that the crystal structure was stable and

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unchanged after MIP and NIP modification.37

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The pore size and specific surface area of LGGO@MIP. Through the nitrogen adsorption

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and desorption isotherms of LGGO and LGGO@MIP (Fig. 2H/I), when the pressure of

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nitrogen was low, the adsorption volume of nitrogen was small, when the pressure of nitrogen 13 ACS Paragon Plus Environment


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was high, the adsorption volume of nitrogen increases. This would indicate that the prepared

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LGGO@MIP was porous and the pore size was within the micropore range (< 2 nm). The

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average pore size was calculated by BJH (Barrett-Joyner-Halenda) method was 1.16 nm, and

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the specific surface area obtained by BET (Brunauer-Emmett-Taylor) method was 28.43

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m2/g. It was conducive to the rapid adsorption of target molecules.35, 38

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The adsorption of LGGO@MIP. The specific adsorption ability of MIP to template targets

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has been widely proved by many reports,27,

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nanocarriers was detected in this part. Fourier transform infrared spectroscopy (FT-IR) was

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used to detect the changes of each component during the preparation of LGGO@MIP

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nanocarriers and after absorbing the target trichlorphon (Fig. 3A). Compared with LGGO, the

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C-H stretching vibration peak at 2920 cm-1, the C=C stretching peak at 1654 cm-1 and the C-

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O-C symmetrical stretching vibration peak at 1050 cm-1, 1096 cm-1 on LGGO-MAA, which

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indicated the successful connection of MAA to LGGO.42-43 The strong stretching vibration

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of >C=O at 1731 cm-1, the splitting stretching vibration of C-O-C at 1255 cm-1, 1160 cm-1,

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the out-of-plane deformation of –CH3 at 755 cm-1, the stretching vibration of C-H in alkane at

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2955 cm-1, 2987 cm-1, which all indicated that the MIP on LGGO surface has complex

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covalent bonding, long chain structure and high degree of polymerization.44-46 In addition,

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when trichlorfon was embedded in LGGO@MIP, there were abundant C-Cl FT-IR peaks at

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800-600 cm-1, and when the trichlorfon was removed, the correlation FT-IR peaks

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disappeared, which also confirmed that the trichlorfon was completely removed.47-48

39-41

the adsorption curve of prepared MIP

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The prepared LGGO@MIP has a fast adsorption rate for the target trichlorphon due to

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its nano-size and large specific surface area.49-50 It has the largest adsorption rate in 1 h and 14 ACS Paragon Plus Environment

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reaches the adsorption equilibrium in 2.5 h, and the adsorption capacity was 3.83 μg/mg (Fig.

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3B). Compared with the control LGGO@NIP, the prepared LGGO@MIP has obvious

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adsorption effect on the target trichlorphon, and this could be used for rapid adsorption of

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toxic substances in the body.

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The toxicity of LGGO@MIP. The toxicity of the prepared LGGO@MIP was evaluated by

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cells and mice before it was used to adsorb toxic substances in vivo. Different concentrations

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of prepared LGGO@MIP were mixed with MC38 and HeLa cell lines for 24 hours to

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evaluate its cytotoxicity. The results showed that all the cell viability was greater than 75%,

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even in a high concentration of LGGO@MIP (400 μg/mL), and when the concentration of

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LGGO@MIP was less than 50 μg/mL, the cell activity was more than 95% (Fig. 3D). It

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indicated that the prepared LGGO@MIP has low cytotoxicity.

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The long-term toxicity of LGGO@MIP was assessed by body weight change of mice.

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After 30 days of oral administration, the mice of LGGO@MIP treatment group and control

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were in good health without obvious disease and death. The results of mouse weight

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monitoring showed that there was no significant difference in weight change between the

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treatment and control, and the weight of mice increased slightly in 30 days (Fig. 3C). It

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indicated that the mice were in good condition and LGGO@MIP had no obvious toxic and

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side effects.

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We prepared the section of MC38 or HeLa cells co-culture with LGGO@MIP for TEM

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observation. When cells were co-cultured with LGGO@MIP grew normally, there was no

297

significant difference in cell morphology under optical microscope. In the view of TEM, a

298

large number of LGGO@MIP nanoparticles adhere to the cell membrane, and some of them 15 ACS Paragon Plus Environment


299

could be free in the cytoplasm through the cell membrane (Fig. 3E/F). The ability of

300

LGGO@MIP to enter different cells indicated that it could be used to adsorb and remove

301

target molecules in cells.

302

The bioimaging of LGGO@MIP in vivo of mice. The mice were anesthetized before

303

bioimaging, and it was approved. The prepared LGGO@MIP had a long persistent

304

luminescence after excited by UV or LED red lamp, the NIR luminescence emitted by

305

LGGO@MIP has strong penetration in mice, and could be used in vivo bioimaging. In this

306

part, different bioimaging effects of different administration methods (oral administration and

307

intravenous injection) were compared, and the prepared LGGO@MIP was irradiated under

308

UV light for 5 min before oral administration or intravenous injection.

309

Oral administration. After oral administration, LGGO@MIP showed strong

310

luminescence signal in the mouth, esophagus and stomach of mice (Fig. 4). As time goes on,

311

the luminescence signal in the mouth of mice gradually weakened, and the luminescence

312

signal gradually gathered in the stomach, and continuously moved in the intestinal tract. After

313

oral administration for 40 minutes, the luminescence signals were mainly transferred from

314

stomach to small intestine, and moved along the distribution of intestinal tract, which

315

indicated that the LGGO@MIP moved continuously in the intestine of mice. After taking

316

LGGO@MIP for 5 hours, the luminescence signal of LGGO@MIP decreased continuously,

317

and the luminous intensity was weak. After the re-excitation by the LED red lamp, the

318

abdomen of the mice showed a strong luminescence signal, and the luminescence signal

319

covered the whole abdomen of the mice. This would indicate that the prepared LGGO@MIP

320

had been distributed within the gastrointestinal tract of the mice after oral administration. If 16 ACS Paragon Plus Environment

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Page 17 of 37


321

target molecules were in the gastrointestinal tract of the mice, it would be adsorbed. (Fig. 4)

322

Twenty-four hours following the ending of the dose period, no obvious luminescence signal

323

was detected in the mice, indicating that oral LGGO@MIP had been excreted from mice. The

324

collected feces of mice had strong luminescence signal after irradiated by UV. This indicated

325

that the prepared LGGO@MIP passed through the stomach and intestine of mice, and was

326

excreted from the mice after oral administration. In this process, no obvious discomfort

327

occurred in mice, and after anesthesia recovery, the diet and activities of mice were normal,

328

and no death occurred. This also showed that the prepared LGGO@MIP did not affect the

329

body of mice, nor did it threaten the life and health of mice.

330

Intravenous injection. When the LGGO@MIP was injected into the mice through the

331

tail vein, the LGGO@MIP rapidly spread throughout the entire body as evidenced by a strong

332

luminescence signal. A strong and evenly distributed luminescence signal persisted for 15

333

minutes after injection. This would indicate the uniform dispersion, small particle size, good

334

stability and abundant hydrophilic groups on the surface of the prepared LGGO@MIP. It is

335

likely that these properties reduced the probability of the LGGO@MIP being intercepted by

336

the liver and spleen, and increased the circulation time in the blood.34, 51 Thirty minutes after

337

injection, the luminous signal in the head and limbs of the mice weakened. This would tend

338

to indicate a decrease of LGGO@MIP concentration and the decay of luminous intensity

339

(Fig. 4). Therefore, we believe that the distribution of LGGO@MIP in mice is sufficient for

340

adsorption of the target molecules for up to one hour post injection. The LGGO@MIP was

341

gradually captured by liver and spleen, and only these organs showed the luminescence

342

signal. Five hours after LGGO@MIP was injected into mice, the liver and spleen still showed 17 ACS Paragon Plus Environment


343

strong luminescence signal after the re-excitation by the LED red lamp. This would indicate

344

that most of the LGGO@MIP was captured by the liver and spleen, but indicated that there

345

was sufficient material in the body to continue adsorbing the target molecules, especially the

346

target molecules gathered in the liver.

347

The luminescence signal was captured in the main organs of mice in the other group

348

after taking LGGO@MIP for 5 hours (Fig. 5). After oral administration of LGGO@MIP for

349

5 h, the whole gastrointestinal tract of mice showed strong luminescence signal (Fig. 5A/D),

350

and the concentration of LGGO@MIP in stomach and intestine had no significant difference

351

after ICP-MS detection (Fig. 5E). This indicated that LGGO@MIP was well distributed in

352

the gastrointestinal tract of mice after oral administration, and its specific adsorption ability to

353

target molecules could be used to uniformly adsorb free toxic drugs in the gastrointestinal

354

tract. In addition, there were weak luminescence signals in the lungs, liver and spleen of

355

mice, which may be due to the absorption of LGGO@MIP into the blood circulation in the

356

intestine and stomach.

357

After LGGO@MIP was injected into mice via tail vein, it mainly flowed with blood

358

circulation. After 5 hours, the luminescence signals were mainly concentrated in the liver,

359

spleen and lungs of mice, and there were also weak luminescence signals in the heart and

360

stomach, which was similar to our previous studies.19, 31-32 (Fig. 5C/D) Although the liver had

361

a strong luminescence signal due to the capture of a large number of LGGO@MIP, the

362

concentration of LGGO@MIP in the spleen was higher than that in the liver (Fig. 5E). As the

363

largest immune organs in the body, the liver and spleen identify and capture foreign

364

substances in blood. The efficiency of recognition and capture depends on the biochemical 18 ACS Paragon Plus Environment

Page 18 of 37

Page 19 of 37


365

characteristics and morphology of nanoparticles, in this study, the prepared LGGO@MIP has

366

small particle size, regular spherical shape and abundant hydrophilic groups on its surface,

367

which improves its stability, reduces the recognition probability of immune cells and

368

prolongs the circulation time in the blood. This avoids LGGO@MIP being captured by liver

369

and spleen in a short time, and is beneficial for LGGO@MIP to adsorb target molecules in

370

blood.

371

Detoxicate test of LGGO@MIP on poisoned mice. After the mice were fed in the

372

laboratory environment for more than 7 days, the average body weight of mice was

373

20.59±1.29 g. A separate control group (10 mice) was treated with trichlorphon solution by

374

gavage at 380 mg/kg according to the weight of mice. After administration of trichlorphon

375

solution, the mice suffered from acute breathing, convulsions, limb weakness and other

376

obvious poisoning symptoms. Eight out of ten mice died within 30 minutes of dosing, and the

377

remaining mice died within 1 hour. In another treatment group, the LGGO@MIP was orally

378

administered to 20 of mice at a dose rate of 30 mg/kg body weight. After oral administration

379

of trichlorphon solution, twenty mice were randomly selected for oral administration of

380

LGGO@MIP. Three mice died within 1 h, one additional mouse died shortly thereafter.

381

Within 48 h of oral administration of LGGO@MIP suspension, the total survival rate was

382

80%. In the first few hours after that, the mice had the phenomenon of breathlessness,

383

convulsions and limb weakness. After that, the mice eat freely but not as active as before in

384

cages, and 48 h later, the poisoning symptoms of mice improved significantly. In the LGGO

385

treatment group, the average body weight of the 20 mice was 20.93±1.68 g before treatment,

386

After 48 hours of oral administration of trichlorphon and LGGO@MIP, the average body 19 ACS Paragon Plus Environment


387

weight of the surviving mice was 17.74±1.79 g, indicating a slight decrease in body weight.

388

This may be due to the fact that trichlorphon poisoning has not completely disappeared,

389

affecting the normal activity and appetite of mice. When LGGO@MIP was used to adsorb

390

toxic substances in vivo, some toxic substances were absorbed by mice and could not be

391

completely adsorbed by LGGO@MIP. These results would indicate that some additional

392

antidotes will be needed to improve the survival rate and reduce the sequential effects in the

393

mice.52-53

394

In general, we have synthesized a novel nanocarrier (LGGO@MIP) for drug specific

395

adsorption and bioimaging, and validated the detoxification effect on trichlorphon in mice.

396

Firstly, long persistent luminescence nanoparticles LGGO (La3Ga5GeO14: Cr3+, Zn2+) was

397

prepared by one-pot method. Then, we modified the molecularly imprinted polymer with

398

specific adsorption function on the surface of LGGO, and constructed nanocarrier

399

(LGGO@MIP) with bright near infrared luminescence and specific adsorption function. The

400

prepared LGGO@MIP has small particle size, uniform size, good dispersion, high stability

401

and low biological toxicity. In this study, LGGO@MIP nanocarriers were constructed using

402

trichlorphon as template to achieve adsorption equilibrium for trichlorphon in a short time.

403

The advantages of fast adsorption rate have great advantages in the development and

404

application of adsorbents. Thanks to the bright near infrared luminescence of LGGO and the

405

repeated excitation by LED red lamp, LGGO@MIP could be used for in vivo long-term

406

bioimaging applications. Through in vivo bioimaging, we found that LGGO@MIP could be

407

uniformly dispersed in the intestine and stomach of mice after oral administration and

408

excreted in vitro with feces, and when LGGO@MIP was injected into the blood circulation of 20 ACS Paragon Plus Environment

Page 20 of 37

Page 21 of 37


409

mice through tail vein, it could circulate for a long time in vivo and then be captured by liver

410

and spleen. We have confirmed in the mice which took trichlorphon orally, LGGO@MIP

411

could be used to effectively adsorb trichlorphon in vivo and reduce the mortality of mice. Abbreviations Used

412 413

AIBN

azo two isobutanonitrile

414

BET method

Brunauer-Emmett-Taylor

method,

method

of

mathematical

415

description of physical adsorption based on the theory of

416

polymolecular (multilayer) adsorption.

417

BJH method

Barrett-Joyner-Halenda method, method of calculating pore size

418

distribution in a porous material using adsorption or desorption

419

isotherms.

420

C/O ratio

the ratio of the number of carbon atoms to oxygen atoms.

421

DLS

dynamic light scattering

422

EDMA

ethylene glycol two methacrylate

423

EDS

energy dispersive spectrometer

424

FT-IR

fourier transform infrared spectroscopy

425

LED

light emitting diode

426

LGGO

La3Ga5GeO14: Cr3+, Zn2+

427

LGGO@MIP

La3Ga5GeO14: Cr3+, Zn2+@ molecularly imprinted polymer

428

LGGO@NIP

La3Ga5GeO14: Cr3+, Zn2+@ non-molecularly imprinted polymer

429

LGGO-MAA

La3Ga5GeO14: Cr3+, Zn2+ connected to methacrylic acid

430

MAA

methacrylic acid 21 ACS Paragon Plus Environment


431

MIP

molecularly imprinted polymer

432

NIP

non-molecularly imprinted polymer

433

NIR

near-infrared

434

P/Cl ratio

the ratio of the number of phosphorus atoms to chlorine atoms.

435

TEM

transmission electron microscope

436

XRD

X-ray diffraction REFERENCES

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Determination of Trichlorfon in Vegetables. J Agr Food Chem 2011, 59 (24), 12745-

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12751.

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49. Ahmed, M. A.; Abdelbar, N. M.; Mohamed, A. A., Molecular imprinted chitosan-TiO2

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nanocomposite for the selective removal of Rose Bengal from wastewater. Int J Biol


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Macromol 2018, 107, 1046-1053.

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50. Urraca, J. L.; Cortes-Llanos, B.; Aroca, C.; de la Presa, P.; Perez, L.; Moreno-Bondi, M.

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C., Magnetic Field-Induced Polymerization of Molecularly Imprinted Polymers. J Phys

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Chem C 2018, 122 (18), 10189-10196.

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51. Weissleder, R.; Nahrendorf, M.; Pittet, M. J., Imaging macrophages with nanoparticles. Nat Mater 2014, 13 (2), 125-138.

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52. Lim, W.; An, Y.; Yang, C.; Bazer, F. W.; Song, G., Trichlorfon inhibits proliferation

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and promotes apoptosis of porcine trophectoderm and uterine luminal epithelial cells.

613

Environ Pollut 2018, 242, 555-564.

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53. Xiao, S. H.; Sun, J.; Chen, M. G., Pharmacological and immunological effects of

615

praziquantel against Schistosoma japonicum: a scoping review of experimental studies.

616

Infect. Dis. Poverty 2018, 7, 15.

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ACKNOWLEDGEMENTS

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This work was supported by the Open Project Program of State Key Laboratory of Food

619

Nutrition and Safety, Tianjin University of Science & Technology (No. SKLFNS-KF-

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201812).



621

FIGURE CAPTIONS

622

Figure 1. Figures illustration of the synthesis and surface modification of LGGO and their

623

application in in vivo bioimaging post oral administration and intravenous injection.

624

Figure 2. Characterization of the prepared LGGO and LGGO@MIP/NIP. (A) TEM images

625

of the LGGO@MIP (a, c) and LGGO@NIP (b, d). (B) The phosphorescence intensities of the

626

LGGO and LGGO@MIP. (C) NIR luminescence images of LGGO@MIP at different time

627

after excitation and LED reactive. (D) EDS image of the LGGO, LGGO-MAA, LGGO@MIP

628

and LGGO@NIP. (E) Size distributions of the LGGO and LGGO@MIP. (F) Zeta potential

629

changes of the LGGO, LGGO-MAA and LGGO@MIP. (G) The XRD patterns of LGGO,

630

LGGO@MIP and LGGO@NIP compared with standard PDF cards. (H) Nitrogen adsorption

631

and desorption isotherms of LGGO and LGGO@MIP. (I) Multipoint BET curve of

632

LGGO@MIP.

633

Figure 3. (A) The changes of FT-IR spectra. (B) Adsorption isotherms of LGGO@MIP and

634

LGGO@NIP toward trichlorfon. (C) In vitro viability of MC38 and HeLa cell lines incubated

635

with LGGO@MIP of various concentrations for 24 h; (D) Body weight changes of mice. The

636

mice treated with LGGO@MIP as the treatment group, and the PBS oral group as the control.

637

(E) Representative TEM images of LGGO@MIP incubated with MC38 cells. The inset

638

images are partial enlarged details. (F) Representative TEM images of LGGO@MIP

639

incubated with HeLa cells. The inset images are partial enlarged details.

640

Figure 4. In vivo NIR luminescence images of nude mice at different time after oral

641

administration and intravenous injection of LGGO@MIP.


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Figure 5. The NIR luminescence images of isolated organs of mice after oral administration

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(A) and intravenous injection (C) of LGGO@MIP for 5 h; (B) The NIR luminescence images

644

of feces after oral LGGO@MIP; (D) Quantification of LGGO@MIP in isolated organs of

645

mice detected by in vivo imaging system; (E) Quantification of LGGO@MIP concentration

646

in isolated organs of mice detected by ICP-MS.



Figure 1


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Figure 2



Figure 3


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Figure 4



Figure 5


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Table of Contents (TOC) Graphic

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Construction of persistent luminescence-plastic antibody hybrid

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