Persistent Luminescence Nanophosphor Involved Near-Infrared

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Persistent Luminescence Nanophosphor Involved Near-Infrared Optical Bioimaging for Investigation of Foodborne Probiotics Biodistribution in Vivo: A Proof-of-Concept Study Yaoyao Liu,†,∥ Jing-Min Liu,‡,∥ Dongdong Zhang,† Kun Ge,† Peihua Wang,† Huilin Liu,§ Guozhen Fang,*,† and Shuo Wang*,‡ †

Key Laboratory of Food Nutrition and Safety, Ministry of Education, Tianjin University of Science and Technology, Tianjin, 300457, China ‡ Research Center of Food Science and Human Health, School of Medicine, Nankai University, Tianjin 300071, China § Beijing Advanced Innovation Center for Food Nutrition and Human Health, Beijing Technology & Business University (BTBU), Beijing, 100048, China ABSTRACT: Probiotics has attracted great attention in food nutrition and safety research field, but thus far there are limited analytical techniques for visualized and real-time monitoring of the probiotics when they are ingested in vivo. Herein, the optical bioimaging technique has been introduced for investigation of foodborne probiotics biodistribution in vivo, employing the near-infrared (NIR) emitting persistent luminescence nanophosphors (PLNPs) of Cr3+-doped zinc gallogermanate (ZGGO) as the contrast nanoprobes. The ultrabrightness, super long afterglow, polydispersed size, low toxicity, and excellent photostability and biocompatibility of PLNPs were demonstrated to be qualified as a tracer for labeling probiotics via antibody (anti-Gram positive bacteria LTA antibody) recognition as well as contrast agent for long-term bioimaging the probiotics. In vivo optical bioimaging assay showed that the LTA antibody functionalized ZGGO nanoprobes that could be efficiently tagged to the probiobics were successfully applied for real-time monitoring and nondamaged probing of the biodistribution of probiotics inside the living body after oral administration. This work presents a proof-of-concept that exploited the bioimaging methodology for real-time and nondamaged researching the foodborne probiotics behaviors in vivo, which would open up a novel way of food safety detection and nutrition investigation. KEYWORDS: persistent luminescence, probiotics, antibody, near infrared, bioimaging

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INTRODUCTION A large number of bacteria in the human intestine constitute one of the most complex ecosystems, providing a significant contribution to human biology and development.1 In this microecosystem, the host and its microbiota develop a direct symbiotic and mutually beneficial relationship that affects the physical function of the host.2,3 Intestinal microbiota could enhance fermentation of the diet that the host is unable to digest, synthesize low weight molecules to assist host nutrition, facilitate maturation of the epithelial barrier, influence intestinal immune development, and enhance the host defense mechanisms.4−7 In past decades, the importance of the gut microbiota to human health and disease has been discussed a lot, which reached the conclusion that intestinal microflora not only contribute to creature innate and adaptive immune systems but also to the immune homeostasis and metabolism.8 Probiotics bacteria, an important group of the gut microbiota, confer the major health benefit to the consumers when administered in adequate amounts.9,10 The action mechanisms were reported that probiotic bacteria could regulate and be conducive to host health.11,12 Probiotics not only improve the competitive exclusion of the pathogenic bacteria and regulate the activity of the endogenous microorganisms but also strengthen epithelial barrier function via an immunoregulation signaling pattern13,14 or generate immunomodulatory proteins to prevent apoptosis.15 Simultaneously, probiotics can motivate © 2017 American Chemical Society

the immune system behavior of the host through transient predominance in the small intestine region that possesses the vast majority of the immune responses.16,17 In the patient with inflammatory bowel disease (IBD), it is found that the disease of IBD is closely related to microbial dysbiosis and the probiotics have the ability to restore this imbalance to normal.18 In addition to the innate factors, the acquired dietary pattern has a great influence of the intestinal flora colonization pattern, therefore, there is a proposal that offering the possibility of a new route for ingestion nutrition and intervention therapy, such as orally up-taking food containing live bacteria (such as yoghurt, cultured buttermilk, fermented vegetable juice), of which the probiotics are supposed to partly colonize in the gastrointestinal tract to exert function.19 In spite of the well-studied benefits and special functions to human health, the metabolism, distribution, and related immunomodulation of probiotics in vivo is still currently poorly understood and the function knowledge and general action of most new species remain unidentified and need to be assessed to acquire more information. With advances of modern molecular-based biotechnology, various methods, such as Received: Revised: Accepted: Published: 8229

June 23, 2017 August 23, 2017 August 24, 2017 August 24, 2017 DOI: 10.1021/acs.jafc.7b02870 J. Agric. Food Chem. 2017, 65, 8229−8240

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Journal of Agricultural and Food Chemistry

via hydrothermal method combination with solid phase calcination in the air have demonstrated remarkable advantages, including ultrabrightness, super long afterglow, polydispersed size, low toxicity, and excellent photostability and biocompatibility, which ensured the success of high-sensitivity and longterm bioimaging in living systems. For the first time, PLNPs were utilized as targeted optical probes for selective tagging of the food probiotics (Lactobacillus as the representative) with the assistance of anti-Gram positive bacteria LTA antibody bioconjugated on the PLNPs surface. After oral administration, we can trace labeled bacteria to supervise their progress though the gastrointestinal tract in vivo after excitation and acquaint their biodistribution by animal optical detection. Consequently, this work has proposed a new perspective for in vivo investigation of food probiotics biodistribution with the advantageous real-time monitoring and nondamage detection ability and broadened the methodology development for food safety detection and nutrition investigation based on the advanced functional nanomaterials.

polymerase chain reaction combined with denaturing gradient gel electrophoresis (PCR-DGGE), real-time PCR, DNA microarray, pyrosequencing, and high-throughput sequencing, have been proposed to determine the DNA sequences of biological samples to reveal the types, distribution, and metabolism of probiotics and their relative abundance in gut.20−22 However, the above-mentioned techniques usually need to harvest the samples (faeces or vivo tissue) and design probes/primers and time-consuming statistical analysis. Moreover, these methods do not allow in situ detection, which can hardly provide the real scene of probiotics behaviors inside the living body with nondestructive and real-time in situ monitoring. Karimi et al. have constructed the plasmids carrying reporter genes expressing fluorescent and luminescent proteins then cloned it into two Lactobacillus reuteri strains, resulting in the bacteria emitting bright signals of fluorescence and luminescence to reveal the localization and distribution of the probiotics in the gut by in vivo and in vitro imaging.23 Fluorescein isothiocyanate (FITC) has been applied to label probiotics by Xing et al. combined with fluorescence imaging to investigate modulation of gut microbiota and the distribution and colonization of Lactobacillus kefiranofaciens in the mouse intestinal tract.24 In vivo optical bioimaging with the ability of noninvasive conception of structural and functional processes at the cellular and molecular level appears as the ideal methodology for investigation of probiotic behaviors after being taken up in the living body.25 Bioimaging has become the indispensable tool for establishing a means of real-time monitoring and nondamaged detection within the living body for diagnosis and therapy in biomedical fields25−28 and monitoring the change of biological environment in vivo.29,30 The fluorescence imaging basically relied on luminescence biolabels, and the current research focused on the development of highly efficient luminescent materials as fluorescence imaging probes such as quantum dots (QDs),31,32 organic dyes,33−35 fluorescent proteins,36,37 noble metal nanomaterials,38−40 carbon dots,41,42 and upconversion nanoparticles.43 However, most of them still have shortages of high photobleaching rate, poor signal-to-noise ratio, short luminescence lifetimes, and poor biocompatibility.25 Persistent luminescence nanophosphors (PLNPs) possess the remarkable optical phenomenon of the continuous afterglow in the nearinfrared (NIR) spectral regions (650−1000 nm) for minutes, hours, or even days after stoppage of the excitation that makes them qualified as a new generation of in vivo optical bioimaging nanoprobe.44−47 The typical PLNPs, zinc gallogermanate (ZGGO) and zinc gallate (ZGO), have demonstrated several distinctive features: (i) the ability to store the excitation energy and lentamente release by a photonic emission after removal of the excitation source, (ii) no need of external excitation that leads to complete overcome of the tissue autofluorescence and significant enhancement of signal-to-noise ratio and sensitivity, (iii) the adjustable red-NIR luminescence located in the region of tissue transparency window that further increases the detection depth, (iv) the red-light re-excitable luminescence that favored the in vivo long-term bioimaging.25,48,49 Therefore, in past few years, near-infrared persistent luminescence nanoparticles continuously arouse the extensive exploration interest of making them as biological imaging probes.50−54 Herein, with the proof-of-concept, the optical bioimaging technique has been introduced for investigation of foodborne probiotic biodistribution in vivo by employing the Cr3+-doped ZGGO PLNPs as the contrast nanoprobes. The PLNPs prepared

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MATERIALS AND METHODS

Materials and Instruments. All reagents were of the highest available purity and at least of analytical grade. Ultrapure water (MilliQ quality water system, 18.2 MΩ cm, Millipore, USA) was used throughout all experiments. Zn(NO3)2·6H2O (99.99%), Ga2O3 (99.999%), GeO2(99.999%), Cr(NO3)3·6H2O (99.99%), 1-ethyl-3(3-(dimethylamino)propyl)carbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), (3-aminopropyl) triethoxysilane (APTES), and dimethylformamide (DMF) were all purchased from Aladdin (Shanghai, China). tert-Butylamine, ammonium hydroxide, concentrated nitric acid, and hydrochloride, NaCl, KCl, MgCl2, CaCl2, FeCl2, AgNO3, Cu(NO3)2, Zn(NO3)2, Al(NO3)3, amino acids (Cys, Phe, Ala, Gly, Glu, Gln, Met, Arg, Lys, Tyr, Leu, Pro, Trp, Ser, Thr, Asp, Asn, Val, Ile, His), de Man, Rogosa, and Sharpe culture medium (MRS) were all purchased from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China), and bovine serum albumin (BSA), bovine hemoglobin (BHB), glutathione (GSH), ovalbumin (OVA), peroxidase (POD), casein, phosphoeptide, thrombin, lysozyme, pepsase, pancreatin, and cytochrome C (cyt-C) were purchased from Sigma-Aldrich (St Louis, MO, USA). Gram-positive bacteria monoclonal antibody (BDI380) was obtained from Amyjet Scientific Inc. (Wuhan, China). All glassware used in the experiment was soaked in a bath of freshly prepared aqua regia (HCl:HNO3 = 3:1, v/v) and rinsed thoroughly with H2O before use (caution: aqua regia is dangerous and should be handled with care). The photoluminescence excitation, emission spectra, and afterglow decay curves of ZGO and ZGGO were measured by a Lumina spectrofluorometer (Thermo Fisher Scientific, Waltham, MA, USA) equipped with continuous (150 W) and pulsed xenon lamps as an excitation source, and the phosphorescence signals were recorded using a standard photomultiplier tube (Hamamatsu, R928) detectors. The crystal microstructure, size, and morphology of the prepared nanoparticles were observed and recorded by high resolution transmission electron microscopy (HRTEM) using on a JEM-2100F field emission transmission electron microscope (JEOL, Japan) operated at a 200 kV accelerating voltage. The biological section sample TEM images were obtained on HT7700 Hitachi (Japan) operating at a 100 kV accelerating voltage. X-ray diffraction (XRD) patterns were carried out by using a D/max-2500 diffractometer (Rigaku, Japan) equipped with Cu Kα radiation (λ = 1.5418 Å). The elemental analysis was performed by inductively coupled plasma mass spectrometry (ICP-MS, Agilent 7500CX). The UV−vis absorption spectra were recorded on a UV-3600 UV−vis−NIR spectrophotometer (Shimadzu, Japan) with 1 cm path-length. All the measurements were executed at room temperature. Fourier transform infrared (FT-IR) spectra (4000−400 cm−1) in KBr were recorded on a Magna560 spectrometer (Nicolet, Madison, WI). 8230

DOI: 10.1021/acs.jafc.7b02870 J. Agric. Food Chem. 2017, 65, 8229−8240

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Journal of Agricultural and Food Chemistry Synthesis of ZGO and ZGGO PLNPs. The ZnGa1.995O4:0.5%Cr3+ and Zn1.25Ga1.5Ge0.25O4:0.5%Cr3+ nanoparticles were both synthesized by hydrothermal method combination with calcinations in air according to the reported procedures.55,56 ZGO PLNPs. First, 0.013 mmol chromium nitrate and 3.01 mmol zinc nitrate dissolved in 10 mL ultrapure water were mixed with 10 mL of gallium nitrate (0.6 M) solution under vigorous stirring. Subsequently, the white precipitated precursor was obtained by adding the tert-butylamine to adjust the pH to 7.5. After 3 h of stirring and ultrasonic treatment at room temperature, the reaction solution was transferred into a 50 mL Teflon-lined stainless steel autoclave and treated under 120 °C for 24 h. After being cooled to room temperature, the resulting compound was washed three times with water and ethanol, respectively, and finally annealed in air at 750 °C for 5 h. The obtained PLNPs powder was ground after a freeze-drying process and stored in dark. ZGGO PLNPs. The synthesis procedure of Zn1.25Ga1.5Ge0.25O4:0.5% Cr3+ was similar to that of ZGO with some modifications. In brief, zinc nitrate (5 mmol), chromium nitrate (0.3 mmol), and 10 mL of ammonium germinate (0.1 M) were mixed with 10 mL of gallium nitrate (0.6 M) aqueous solution under vigorous stirring. The tertbutylamine was rapidly added to adjust the pH to 8, and the turbid liquid was kept stirring for 1 h, followed by an ultrasonic treatment at room temperature. The obtained mixture was then transferred into a 50 mL Teflon-lined stainless steel autoclave for a 15 h heat treatment at 120 °C. The resulting precipitate was washed three times with water and ethanol, respectively, and finally annealed in air at 1000 °C for 1 h. The precipitate powder was ground with a mortar and pestle and stored in the dark after freeze-drying. Surface Functionalization of PLNPs. The surface amino functionalization of PLNPs was performed via the Stöber procedure with APTES as silane coupling agent according to the previous literature.44,57 The ZGGO powder was wet ground with alcohol three times. Subsequently, the sample was suspended in the NaOH (5 mM) solution under ultrasonic treatment for 1 h and vigorously stirred overnight at room temperature. The white colloidal suspension solution was first selected by centrifugation at 4500 rpm for 10 min to remove the large sized particles. Then the supernatants were gathered and centrifuged at 3500 rpm for 15 min to collect the nanoparticles with 20−50 nm diameter. The obtained freeze-drying precipitate (5 mg) was resuspended in 2 mL of DMF by sonication, followed by dropwise addition of 20 μL of APTES to initiate the formation of the NH2-ZGGO. The reaction was kept under vigorous stirring at 80 °C for 24 h. Finally, the resulted NH2-ZGGO nanoparticles were washed with DMF to remove the unreacted APTES and gathered by centrifugation. Preparation of Antibody-ZGGO Conjugates. Anti-Gram positive bacteria LTA [3801] monoclonal antibody was immobilized on ZGGO nanoparticle surfaces according to the literature method with some modification.58,59 Typically, 1 mg of antibody was dissolved in 10 mL of PBS (0.1 M, pH 7.4), followed by addition of NHS (15 mg) and EDC (15 mg). The mixture was incubated for 30 min at room temperature to fully activate the carboxyl group of the antibody. NH2-ZGGO suspension solution was obtained by adding 10 mg of NH2-ZGGO power into 10 mL of PBS (0.1 M, pH 7.4) buffer and supersonic treatment for 30 min. The above-mentioned two kinds of solution were mixed together thoroughly for another 4 h reaction at room temperature under gently stirred. The resulting product antibody-ZGGO was centrifuged (4 °C, 7000 rpm, 5 min) and washed with PBS buffer three times to obtain the conjugation of antibodyZGG, freeze-dried, and stored at 4 °C for standby. Strain and Microbial Cultures Conditions. A bacterial strain ZW-128 of Lactobacillus reuteri [“Gram-positive group”] in this study was available from the animal Resources Development and Functional Food Lab at the College of Food Engineering and Biotechnology, Tianjin University of Science and Technology, Tianjin, China, which was used as target and bioimaging in all experiments. Pure Lactobacillus reuteri cultures were prepared by activating twice from −80 °C frozen inventories in de Man, Rogosa, and Sharpe (MRS) broth at 37 °C under shake-flask culturing for 12 h to achieve

logarithmic growth phases. In experiments, 3 mL of the bacterial culture were added to 100 mL of MRS broth medium and incubated overnight at 37 °C. Cell pellets was harvested by centrifugation (6000g for 10 min) at 4 °C from the overnight grown cultural suspension, the supernatant was removed and the pellet subsequently washed twice with sterilized PBS (0.01 M, pH 7.4) buffer and adjusted the bacterial concentrations by determining the optical density at 600 nm (OD600). Preparation of L. reuteri Competent Cells. The competent cell was prepared according to the previously method with some modification.60 First, 25 mL of preculture L. reuteri cell in logarithmic growth phase at 37 °C was inoculated into 100 mL of sterilized MRS medium supplemented with 1% (w/v) glycine. The culture was incubated at 37 °C until the cell was at exponential phase (OD600 = 0.6), then the cells were cooled on an ice-bath for 10 min and harvested by centrifugation at 4 °C, 3000g for 5 min and the supernatant was removed softly (the parameters depend on various strains). The pellet of cells was washed with 100 mL of precooling 10 mM MgCl2 and centrifuged at 4 °C, 3000g for 5 min. Afterward, cells were resuspended charily in 100 mL of precold SM solution (925 mM sucrose and 3.5 mM MgCl2), and the pellet of thallus was collected by centrifugation (5000g, 10 min) at 4 °C. After repeating this step twice, cells were resuspended gently in 1 mL of precooling SM solution and stored in an ice-bath before further treatment, of which process should be implemented as quickly as possible. Interaction of ZGGO with L. reuteri. Three different incubation ways between L. reuteri cell suspension (109 cfu mL−1) and ZGGO nanoparticles have been compared. (i) Competent cells (60 μL) prepared as described above were mixed with 40 μL of ZGGO nanoparticles dispersion in PBS (1 mg mL−1) and suffered from an electric pulse in a 0.1 cm cuvette by using a Gene Pulser and a Pulse Controller apparatus (Gene Pulser Xcell, Bio-Rad, USA). Immediately, the cells was gained by centrifuging the above mixture at 4 °C, 1500g for 5 min, and decanted the supernatant. (ii) The competent cells (1 mL) were treated with ZGGO nanoparticles (500 μL, 1 mg mL−1) under soft shaking at 37 °C for 6 h and washed with PBS twice (0.01 M, pH 7.4). (iii) Meanwhile, bacteria (L. reuteri) from logarithmic phase were obtained by rinsing with PBS (0.01 M, pH 7.4) twice and resuspending them in PBS, which was mixed with antibody-ZGGO nanoparticles and incubated for 1 h at 37 °C. As the control, suspended bacterial cells in PBS without any treatment were interacted directly with ZGGO nanoparticles under soft shaking at 37 °C for 6 h. After treating, all above bacterial cells were fixed with 2.5% glutaraldehyde in PBS solution right away to the pellet in the tube and stored at 4 °C overnight for TEM samples preparation. The interaction of ZGGO with L. reuteri was evaluated via TEM. In a typical assay, the slice sample was prepared as follows: The abovementioned pellet was washed with PBS for three times to remove the glutaraldehyde, then incubated with 2% osmium tetraoxide (OsO4) in PBS at 4 °C for 30 min. The superfluous OsO4 was eliminated by washing with PBS, and the fixed pellet was dehydrated through different levels of ethanol (30%, 50%, 70%, 90%), respectively, followed by series washing using 90% ethanol and 90% acetone (1:1, v/v), 90% acetone, and 100% acetone (3 times) for 20 min at the room temperature. The pellet sample was prepared by immersing into Araldite resin medium overnight and embedding into the resin. The sample was cured at 60 °C overnight in a vacuum oven until it thoroughly infiltrated in the resin. Using an ultramicrotome (Power Tome-PC, RMC, USA), the resin-embedded pellets were cut into 60−70 nm ultrathin slices, which were then put on the Cu grids for TEM examination. Evaluation of ZGO and ZGGO Stability. First, 20 mg of prepared ZGO and ZGGO nanoparticles were infiltrated into 20 mL of PBS, HEPES, HBSS, Tris-HCl, artificial gastric juice, artificial intestinal juice, and ultrapure water, respectively. Then the element content of Zn, Ga, and Cr in the supernate of materials suspension was monitored by ICP-MS analysis at the time of the 0, 1, 2, 3, 4, 5, 6,12, 24, 48 h. In addition, prepared nanoprobe solid solutions were separately incubated with nine species of metal salts, 20 kinds of amino acids, 12 common biomolecules, and ultrapure water to record the change of phosphorescence intensity (excitation 295 nm, emission 8231


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In Vivo Luminescence Imaging. The adult athymic BALB/c mice (15−20 g) were obtained from Beijing HFK Bioscience Co., Ltd. (Beijing, China). All animal experiments were implemented in accordance with guidelines of Tianjin Committee of Use and Care of Laboratory Animals, and all project protocols were approved by the Animal Ethics Committee of Nankai University. In vivo experiments were performed on anesthetized mice with chloral hydrate (200 μL, 4%). The prepared L. reuteri-ZGGO (2 mL, 1 × 109 cfu mL−1) or ZGGO (2 mL, 1 mg mL−1) dispersed in 10 mM sterile PBS solution was given to the mice through oral administration, respectively. The nude mouse was fasted for 36 h before oral administration of materials. After 120 min of gavage, the mice were excited with a LED light for 60 s to reactivate the persistent luminescence of ZGGO and the signal acquisition was resumed under the optima camera.

698 nm) to evaluate the photostability and biocompatibility of ZGO and ZGGO. Evaluation of the ZGGO Toxicity. The methyl thiazolyl tetrazolium (MTT) assays were performed to determine the in vitro cytotoxicity of the ZGGO nanoprobes. Mouse embryo fibroblast cell lines (Balb/3T3), human cervical carcinoma cell lines (Hela), and human breast carcinoma cell lines (MCF-7) were obtained from China Center for Type Culture Collection (Wuhan, China). Briefly, the three cell lines were plated at a density of 4 × 104 cells per well in 96-well plates and grown for 24 h at 37 °C in 5% CO2. The NH2-ZGGO nanoparticles dispersed in 10 mM PBS solution with a wide concentration range from 50 to 1000 μg mL−1 were subsequently added into the cell and incubated for another 24 h under the same conditions as above. MTT (10 μL, 3 mg mL−1) was added to each well and incubated for another 4 h at 37 °C. Then, 150 μL of DMSO was added to each well, and the plate was kept at room temperature for 10 min. OD570 (Abs. value) of each well was measured by the Multiskan Spectrum multifunction microplate reader (Labsystems, Thermo, USA).

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RESULTS AND DISCUSSION Principle of in Vivo Investigation of Probiotics Biodistribution. As illustrated in Figure 1, to directly track the biodistribution of probiotics inside the living body, lactic acid bacillus was labeled by antibody-ZGGO nanoprobe with ultrabright long afterglow luminescence and the bioimaging assay was performed post oral administration. First of all, the surface of PLNPs was activated with considerable amount of hydroxyl groups through 5 mM NaOH treatment (OH-PLNPs). Subsequently, amination of the PLNPs was formed by Stöber method via the reaction of the silanol groups of APTES with the surface hydroxyl groups of PLNPs, making the amino group decorated onto the ZGGO PLNPs surface (NH2-PLNPs). Afterward, with the aid of NHS-EDC catalysis, the LTA antibody could react with the −NH2 groups on the surface of NH2-PLNPs via carbodiimide method to obtain binding sites for the probiotics and then produced the antibody-PLNPs nanoprobes, which were used to combine with the lipoteichoic

Figure 1. Schematic illustration of the utilization of the antibodyZGGO nanoprobes for orally administrated in vivo bioimaging.

Figure 2. Structural characterization and persistent luminescence properties of the ZGO and ZGGO. (A) Excitation and emission spectra of ZGGO and ZGO. (B) NIR afterglow decay curve of ZGGO and ZGO powder after 120 s irradiation with a 254 nm UV lamp. (C) XRD patterns of ZGO and ZGGO PLNPs powder. (D) Photographs of the ZGO (left) and ZGGO (right) nanomaterials solid powder under sunlight (up) and UV 254 nm (down). 8232


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Figure 3. HRTEM images of ZGGO (A) and ZGO (E). EDX analysis of the ZGGO (B) and ZGO (F). Size distribution of ZGGO and NH2-ZGGO (C) and ZGO (G) nanoparticles. Decay curves of ZGGO (D) and ZGO (H).

Figure 4. Evaluation of the photostability of ZGO and ZGGO powder in the different media: common biomolecules (A,B), amino acids (C,D), and metal ions (E,F).

form of white powder by a hydrothermal method combined with calcination in air according to the reported methods with slight modifications.55,56 In the ZGO and ZGGO PLNPs nanostructures, zinc gallate acts as the host, while the Cr3+ and Ge4+ are the doped ions, as the luminescent centers and defect centers (vacancies), respectively. Depending on the crystal-field environment of the host lattices, Cr3+ ions act as a favorable

acid (LTA) antigen on surface of probiotics. Finally, the antibody-PLNPs nanoprobes were employed as luminescence tagging for in vivo bioimaging of the labeled probiotics after oral administration. Preparation and Characterization of the ZGO and ZGGO PLNPs. The Cr3+-doped ZGO and ZGGO persistent luminescence nanoparticles were successfully synthesized in the 8233


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Figure 5. Evaluation of the structural stability of ZGGO (A,C,E) and ZGO (B,D,F) power in different simulated media.

phase of ZnGa2O4 (JCPDS no. 38-1240) and Zn2GeO4 (JCPDS no. 25-1018), respectively (Figure 2C). HRTEM characterization revealed that both ZGO and ZGGO PLNPs were generated with the diameter ranging from 30 to 80 nm and good dispersity. Compared with the traditional high temperature solid calcination method, the hydrothermal method could effectively control the nanoparticle size and achieve improved dispersity of PLNPs. Hydrodynamic diameter of the nanoparticles measured by dynamic light scattering was 45 ± 5 nm (ZGGO) and 48 ± 6 nm (ZGO), respectively (Figure 3C,G), The elemental composition of ZGO and ZGGO PLNPs was further confirmed by the EDX analysis, which showed the presence of Ge elements in the ZGGO nanostructure (Figure 3B,F). Biocompatibility, Stability, and Toxicity of PLNPs. Before application of the synthesized PLNPs for in vivo bioimaging, the biocompatibility, stability, and toxicity of the nanoprobes have been integrally evaluated. The biocompatibility of PLNPs was evaluated via determining the photoluminescence intensity changes of ZGO and ZGGO nanomaterials in the presence of various substances inside the living body, including metal ions, amino acids, and proteins. As shown in Figure 4, the tested metal ions, common amino acids, proteins, and enzymes produced negligible effect to the luminescence of

emitter, which have a broadband NIR emission from 650 to 950 nm, overlapping with the biologically transparent window (650−1100 nm).61,62 As the defect center, Ge4+ is responsible to extend the afterglow time in the persistent luminescence nanostructures. Figure 2 shows the excitation, emission spectra, and NIR persistent luminescence of the ZGO and ZGGO powder at the room temperature. After excitation at 260 nm, the prepared ZGO and ZGGO nanophosphors both showed NIR emissions centered at 694 nm with a broad emission band from 650 to 900 nm. The excitation spectra of ZGO and ZGGO monitored at 694 nm corresponded to three excitation broad bands due to the inner transitions of Cr3+, which possessed a broad excitation range from 200 to 600 nm. In addition to the intense and broad NIR photoluminescence, the as-prepared ZGO and ZGGO both demonstrated remarkable long afterglow features, with the persistent luminescence lifetime over 90 and 150 h, respectively, which formed the basis of long-term bioimaging application (Figure 3D,H). Through the comparison of their afterglow decay curves, ZGGO showed better afterglow properties than ZGO due to the presence of Ge4+ ion in the lattices. The nanocrystal phase was confirmed by XRD analysis, which indicated the XRD pattern of ZGO and ZGGO were consistent with the spinel 8234


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Figure 6. (A) In vitro viability of 3T3, MCF-7, and HeLa cell lines incubated with ZGGO of various concentrations for 24 h. (B) Weight change of the normal mice with or without gavage by the ZGGO imaging probes (1 mg mL−1, 0.5 mL). (C) FT-IR spectra of ZGGO, NH2-ZGGO, and antibody-ZGGO. (D) PL intensity of ZGGO, antibody-ZGGO, and L. reuteri-ZGGO incubation with PBS (10 mM, pH 7.4). The inset was the photograph of the dispersion of ZGGO, antibody-ZGGO, and L. reuteri-ZGGO incubation with ultrapure water under 254 nm UV excitation. (E) Zeta potential of ZGGO, NH2-ZGGO, and antibody-ZGGO. (F) Fluorescence intensity change of the ZGGO against time immersed in ultrapure water.

3T3 normal cell, MCF-7 cancer cell, and Hela cancer cell. As shown in Figure 6A, after exposure to the concentration as high as 1000 μg mL−1 of ZGGO nanoprobes for 24 h, the viability of three type cells was still higher than 80%, which proved no significant toxicity of the ZGGO PLNPs to three types of cells. Furthermore, ZGGO PLNPs were fed to mice and the body weight change of the mice was continuously recorded to evaluate the long-term in vivo toxicity. After 30 days of ZGGO PLNP oral administration, the mice viability was still 100% and there was no significant difference of the body weights between control and treated groups (Figure 6B). On the basis of above phenomenon, the ZGGO PLNPs were proved to be of low toxicity and suitable for in vivo imaging. Surface Functionalization of ZGGO PLNPs. To reinforce the targeted specificity, anti-Gram positive bacteria LTA antibody was grafted onto the surface of the ZGGO PLNPs via the NHS-EDC assisted amidation reaction. The surface functionalization with APTES and antibody to ZGGO PLNPs were confirmed by FT-IR analysis (Figure 6C) and ζ potential (Figure 6E). FT-IR spectra showed that there appeared to be a strong absorption band at 3437 cm−1 (stretching vibrations of O−H), manifesting the successful modification of hydroxyl groups.

PLNPs nanomaterials (ZGO and ZGGO), which proved that ZGGO and ZGO PLNPs both possessed excellent biocompatibility in vivo. For the investigation of the chemical stability of PLNPs, the ZGO and ZGGO powders were separately incubated with different media, including ultrapure water, PBS (10 mM, pH 7.4), Tris-HCl buffer (10 mM, pH 7.4), HEPES buffer (10 mM, pH 7.5), HBSS buffer, simulated intestinal fluid, and simulated gastric juice (Figure 5). Then the element contents of the supernatants of the solid solutions were measured by ICP-MS elemental analysis. After a 48 h continuous monitoring, the results of the metal element contents in the supernatant revealed there were few Ga3+, Zn2+, and Cr3+ ion leakages from the nanocrystals when treated by the seven media. Therefore, it was proved that the PLNPs nanoparticles can maintain long-time structure stability existing in various biological fluids, which significantly favored the following in vivo bioimaging assay via oral administration. In consideration of the better afterglow performance and comparable biocompatibility and chemical stability, ZGGO PLNPs were finally chosen as the optical centers for the following biolabeling and bioimaging assays. The in vitro cytotoxicity of the ZGGO nanoprobes was assessed via cell counting assay performing on three types of cell lines, 8235


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Journal of Agricultural and Food Chemistry The strong FT-IR band at 1011 and 1115 cm−1 (stretching vibrations of O−Si−O), 2938 cm−1 (−CH2 stretching bands), and 3421 cm−1 (N−H stretching bands) indicated the successful modification with APTES. The FT-IR absorption bands of antibody-ZGGO at 1716 cm−1 (stretching vibration of CO) suggested the antibody successfully grafted onto the surface of the NH2-ZGGO. With the different surface modification process, the surface ζ potentials change of the ZGGO nanoparticles were used to verify the state of the materials surface functional group. After treatment with NaOH, the ZGGO revealed a negative ζ potential of −30.9 mV due to the presence of hydroxyl groups on surface. Then the ζ potential changed to +12.9 mV after the APTES silane coupling reaction, proving the decoration of −NH2 on particle surfaces. Subsequently, binding of antibody with the NH2-ZGGO produced a negatively charged surface of −14.4 mV. Allof the above evidence confirmed the successful preparation of the bioconjugate of antibody-ZGGO nanoprobes. In addition, the luminescence intensity of NH2-PLNPs maintained over 80% after soaking in ultrapure water for 30 days, indicating its excellent photostability (Figure 6F). Meanwhile, there was not much loss of luminescence intensity of PLNPs after being modified with antibody or binding with L. reuteri (Figure 6D). Interaction of PLNPs Nanoprobes with Probiotics. To realize the optimal labeling of antibody-ZGGO nanoprobes with the targeted Lactobacillus, two interaction approaches have been explored, internalization and antibody−antigen specific binding. It is well-studied that nanoparticles can be internalized via receptor-mediated endocytosis by some special mammalian cells such as macrophages, hemameba, and neutrophils.63,64 However, it is generally agreed that bacterial cells hardly support endocytosis, pinocytosis, or exocytosis due to the presence of the thick peptidoglycan cell wall.65,66 TEM analysis of sample thin sections can be able to directly visualize the states of the microorganisms upon incubation with ZGGO (1 mg mL−1) in sterilized PBS (Figure 7). The L. reuteri

Gram-positive cell with the typical tubular shape demonstrated complete cell structure and membrane layer. After incubation, most of the nanoparticles existed away from the cells and failed to label the cells of interest owing to the thick cell wall of bacteria. Therefore, electroporation treatment was carried out to facilitate the bacterial cells to uptake the nanoparticles. Primitively, the competent L. reuteri cells with better penetrability than the normal cells were homogeneously mixed with ZGGO nanoprobes, followed by the electroporation treatment immediately, and incubated at 37 °C for 6 h. The same process was performed on the competent cells without the following electroporation treatment as a control. However, similar results were obtained that a limited amount of nanoparticles can penetrate the cell membrane into the cells, of which the amount was insufficient to realize the luminescence tagging for the following bioimaging assay (Figure 8A−D).

Figure 8. Representative TEM images of L. reuteri competent cell after incubating for 6 h with ZGGO nanoparticles (A,B), L. reuteri competent cell incubating with ZGGO by electroporation treatment (C,D), and L. reuteri after incubating for 1 h with the antibody-ZGGO nanoparticles (E,F).

In the following assay, the specific immunoreaction was introduced for biolabeling probiotics. The antibody against the LTA antigen on the surface of L. reuteri Gram-positive cells was modified to the NH2-ZGGO nanoparticles to make the antibody-ZGGO nanoprobes that can interact with L. reuteri Gram-positive cells via surface antigen−antibody binding. TEM results strongly support the idea that considerable amount of

Figure 7. Representative TEM images of normal L. reuteri (A,B) and normal L. reuteri after incubating for 6 h with ZGGO nanoparticles (C,D). 8236


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Journal of Agricultural and Food Chemistry nanoparticles were decorated onto the bacteria surface via the immunoreaction of antibody with the LTA antigen epitopes on the cell wall of the L. reuteri bacteria, leading to the nanoparticles embraced around the cells as clearly shown in Figure 8E,F. Consequently, the ZGGO PLNPs have been successfully labeled to the targeted probiotics via the immunoreaction to form the L. reuteri-ZGGO bioconjugates for the following bioimaging application. To assess the activity of Lactobacillus cells after labeling, the bacterial activity also has been investigated after modification of the antibody. The activity of the bacterial after labeling and untreated bacterial were measured by Bacterial Activity Kit-8 (CCK-8) assay (Bestbio Biotech, Shanghai, China), respectively. After 12 or 24 h

of treatment, the viability of the activity of the bacteria was still higher than 80%, which proved there is no significant influence of nanoprobes to the bacterial activity. (Figure 9) To understand the adhesion performance of probiotics after labeling with nanoprobes, the MC38 cells were used to make a simple contrast test to investigate the changes of the adhesion performance of probiotics before and after modification through in vitro culture by the method of counting viable bacteria.67 The results showed that the adhesion performance of the ZGGO-Lactobacillus has declined 34% compared with the L. reuteri without modification. This indicates that although parts of the LTA on the surface of bacteria are occupied by antibody-ZGGO conjugates, the bacteria maintained significant adhesion ability to the surface of cells in gut. (MC38 cells were purchased from National Infrastructure of Cell Line Resource, China, and cultured in RPMI medium 1640 basic with 10% fetal bovine serum. Cells were cultured in the 37 °C incubator contained 5% CO2.) The above results supported our principle of nanoimaging guided in vivo investigation study of bacteria. Biodistribution and Orally Administrated in Vivo Bioimaging. To intuitively monitor the biodistribution of the probiotics L. reuteri inside the living body, the prepared L. reuteri-ZGGO was orally administrated into mouse by gavage. As shown in Figure 10B, NIR light emitting persistent luminescence of L. reuteri-ZGGO appeared in the stomach 1 min after oral administration, subsequently arrived at the intestinal tract, and spread around the whole digestive tract region of the mouse 6 min later and became obvious in the intestine region at 30 min. After 60 min, L. reuteri-ZGGO still remained in the stomach and intestines sites, probably due to the gradual adhesion of L. reuteri cells in the gastrointestinal

Figure 9. In vitro viability of L. reuteri cell incubated with antibodyZGGO of various concentrations for 12 and 24 h.

Figure 10. Biodistribution of NH2-ZGGO and L. reuteri-ZGGO inside the mice body after oral administration. (A) In vivo NIR luminescence images of PBS dispersion of NH2-ZGGO (1 mg mL−1, 0.5 mL) were excited for 10 min using a LED lamp before gavage. (B) In vivo NIR luminescence images of L. reuteri-ZGGO (109 cfu mL−1 in PBS, 0.5 mL) were excited for 10 min using a LED lamp before gavage. The mice were reactivated with a LED lamp at 120 min post administration. (C) FL of isolated organs of mice after 5 min irradiation with a LED lamp recorded by CCD camera. (D) Ga concentration of ZGGO in isolated organs of mice measured by ICP-MS elemental analysis. The acquisition was performed 24 h after the application of ZGGO. 8237



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tract. Although the persistent luminescence intensity of ZGGO gradually decreased without in situ excitation, the signal-tonoise ratio was still >20 at 120 min post oral administration. NH2-ZGGO nanoparticles were orally administrated via the same procedure as a control. The persistent luminescence appeared in intestinal tract, spread around the whole body within 6 min, and gradually disappeared 120 min post oral administration without noteworthy accumulation in the gastrointestinal tract. After 120 min of oral administration, the mice were reirradiated by a LED light to reactivate ZGGO (Figure 10). It was observed that NH2-ZGGO nanoparticles in the control group spread over the whole mouse body and mainly accumulated in the liver and spleen, whereas the biodistribution of the L. reuteri-ZGGO remained in the digestive tract areas. Furthermore, the in vivo distribution of antibody-ZGGO labeled Lactobacillus and NH2-ZGGO has been further evaluated by anatomy experiments followed by fluorescence and ICP-MS measurements of organs, of which results were consistent with those of bioimaging assay (Figure 10C,D). The above results demonstrated that the L. reuteri-ZGGO bioconjugates could effectively track the biodistribution of probiotics inside the living body, and as a proof-of-concept, the PLNP-based long-term optical bioimaging was able to be utilized for probing the bacteria behaviors in vivo. In summary, the optical bioimaging technique has been introduced for investigation of foodborne probiotics biodistribution in vivo, employing the ZGGO PLNPs as the contrast nanoprobes. The ultrabrightness, super long afterglow, polydispersed size, low toxicity, and excellent photostability and biocompatibility of PLNPs were demonstrated to be qualified as tracers for labeling probiotics via antibody recognition as well as contrast agent for long-term bioimaging of the probiotics. This work has proposed a new perspective for in vivo investigation of food probiotics biodistribution with advantageous real-time monitoring and nondamage detection ability and broadened the methodology development for food safety detection and nutrition investigation based on the NIR PLNPs assisted in vivo bioimaging assay.

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AUTHOR INFORMATION

Corresponding Authors

*For G.F.: phone, +86-22-60912493; fax, +86 22 6091 2493; E-mail, [email protected]; address, No. 29, 13th Avenue, Tianjin Economic and Developmental Area (TEDA), Tianjin 300457, China. *For S.W.: E-mail, [email protected]; address, No. 94 Weijin Road, Tianjin, 300071, China. ORCID

Guozhen Fang: 0000-0001-5346-953X Author Contributions ∥

Article

Y.L. and J.-M.L. contributed equally to the work.

Funding

This study was funded by the Ministry of Science and Technology of China (no. 2012AA101602). This work was supported by International Science and Technology Cooperation Program of China (no. 2014DFR30350), National Key Research and Development Program of China (no. 2016YFD0401202), and Youth Innovation Fund of Tianjin University of Science & Technology (no. 2016LG01). Notes

The authors declare no competing financial interest. 8238


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