Plant Protein-Directed Synthesis of Luminescent Gold Nanocluster

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Plant Protein-Directed Synthesis of Luminescent Gold Nanoclusters Hybrids for Tumor Imaging Zhao Li, Haibao Peng, Jialin Liu, Ye Tian, Wuli Yang, Jinrong Yao, Zhengzhong Shao, and Xin Chen ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b13088 • Publication Date (Web): 08 Dec 2017 Downloaded from http://pubs.acs.org on December 10, 2017

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ACS Applied Materials & Interfaces

Plant Protein-Directed Synthesis of Luminescent Gold Nanoclusters Hybrids for Tumor Imaging id Zhao Li†,‡, Haibao Peng†, Jialin Liu†,‡, Ye Tian†, Wuli Yang*,† ○ , Jinrong Yao†,‡, Zhengzhong

id Shao†,‡, Xin Chen*,†,‡ ○

†

State Key Laboratory of Molecular Engineering of Polymers, Department of Macromolecular Science, Fudan University, Shanghai 200433, P. R. China. ‡

Laboratory of Advanced Materials, Fudan University, Shanghai 200433, P. R. China.

KEYWORDS: gold nanomaterials, pea protein isolate, composites, red blood cell, biocompatibility, bioimaging

ABSTRACT: Nowadays, fluorescence detection has emerged as one of the most frequently used non-invasive biosensing method to selectively monitor biological processes within living systems. Among fluorescent nanoparticles, gold nanoclusters (AuNCs) have been intensively studied because of their intrinsic fluorescence and their endowed biocompatible surface. Herein, we selected an abundant, low-cost and sustainable plant protein, the pea protein isolate (PPI) for its excellent biocompatibility, biodegradability, and non-allergenic character, to be employed as both a reducing and stabilizing agent to facilely produce AuNCs exhibiting a strong red fluorescence. Afterwards, the formed AuNCs-PPI mixture was able to self-assemble into nanoparticles (AuNCs/PPI NPs) with the size of about 100 nm simply through a dialyzing

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process. Taking advantage from the protein nature of PPI, AuNCs/PPI NPs demonstrate both excellent biocompatibility and colloidal stability. Moreover, AuNCs/PPI NPs showed a great capability when employed as a bioimaging probe for both in vitro and in vivo imaging. Finally, AuNCs/PPI NPs were coated with red blood cell (RBC) membranes to improve their blood circulation property and enhance their tumor enrichment ability in order to meet the requirement for practical use. Results convincingly show that such super nanoparticles (RBC-coated AuNCs/PPI NPs) were able to successfully locate tumor in vivo with an excellent imaging capability, which provides a new strategy for bioimaging with fluorescent nanoparticles.

INTRODUCTION Noble metal quantum clusters, also known as nanoclusters, exhibit interesting molecule-like properties (e.g. strong fluorescence in the red to near-infrared region) due to the discrete and size-tunable electronic transitions resulted from the strong spatial confinement of free electrons in the particles.1-3 Among noble metals, gold nanoclusters (AuNCs) stand out thanks to their easy synthesis, intensive fluorescence, good water solubility, excellent biocompatibility, extraordinary photostablity, and low cytotoxicity. AuNCs are thus expected to be the perfect alternative for semiconductor quantum dots and organic dyes in the area of sensitive bioimaging in vitro as well as in vivo.4-9 For instance, Liu et al. found that glutathione-coated luminescent gold nanoparticles, with rapid clearance in normal tissue, long retention in tumors, and high tumor targeting specificity, have great potential in cancer diagnosis.10 Another example is that Zhao et al. have developed 64Cu doped AuNCs with AMD3100 targeting ligand for CXCR4 detection in tumor, achieving accurate and sensitive cancer imaging.11 Inspired by the outstanding properties and potential applications of AuNCs, many synthesis methods have been reported using different


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capping molecules, such as mercaptopropionic acid,12 meso-2,3-dimercaptosuccinic acid,13 dendrimers,14 DNA,15 amino acids,6 peptides,16, 17 and proteins.1 Protein-directed synthesis, in which proteins act as reducing and stabilizing agents, is particularly appealing for developing sustainable synthesis methods,18 owing to the obvious advantages of low environmental impact, mild reaction conditions, good water solubility, and natural biocompatibility.19 Up to now, several proteins have been applied in the synthesis of AuNCs, including bovine serum albumin (BSA),1,

7, 20, 21

lysozyme type VI,22,

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tryspin,24

pepsin,25 insulin,26 human transferrin,27 lactoferrin28 and horseradish peroxidase.29 Despite many discussions showing the efficient fabrication of AuNCs, very few reports have focused on the self-assembly of proteins with AuNCs to form nanoparticles.30 Another restriction in the application of the synthetic noble metal nanomaterials is that they are easily detected as intruders by the immune system and cleared out from the body by the reticuloendothelial system (RES; e.g., liver and spleen); this leads to a shortened lifetime in the systemic circulation and a weak accumulation in tumor tissues.31, 32 Even though poly(ethylene glycol) (PEG) grafting could prolong the blood circulation time,33 more and more reports have evidenced that PEG-modified nanoparticles may stimulate immune responses after multiple injections.34 Apart from PEG, another surface modification strategy relies on lipid membrane coatings, red blood cell (RBC) membranes in particular, which can realize both surface shielding and functional group incorporation.35 Being long-circulating carriers, RBCs have inspired numerous investigations on artificial biomaterials and delivery systems.36 RBCs are recognized as self by the body due to their natural surface “make-up”, comprising a series of proteins residing on the membranes, such as the CD47, which acts as a self-marker to prevent the uptake of macrophages, and various membrane proteins, which avoid the attack from the complement


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system.35, 37 For example, Zhang et al. have successfully enclosed gold nanoparticles (AuNPs) in continuous RBC membranes, conferring immunosuppressive capabilities to AuNPs to fend off the macrophage uptake.35 In addition, RBC membrane coatings resulted in a prolonged blood half-life and reduced blood clearance compared to the PEGylation technique.32 However, to our best knowledge, there was no previous report on coating the AuNCs and proteins together with RBC membranes for bioimaging. Herein, we report a simple, one-step route to synthesize AuNCs using an abundant, low-cost, and sustainable plant protein (pea protein) as the reducing and stabilizing agent. Inspired by our previous results where silk fibroin nanofibrils and soy protein isolate (SPI) were employed to control the shape and dimension of gold nanomaterials,38, 39 pea protein isolate (PPI), another precious gift from nature with inherent biocompatibility, tunable biodegradability, and more importantly, non-allergenic character,40-42 was chosen as the reductant and stabilizer of AuNCs. PPI contains two major components, one is pea legumin that represents the 11S globulin fraction (molar mass between 350 and 400 kDa), and another is vicilin and convicilin that represent the 7S globulin fraction (molar mass of about 150 kDa).43, 44 The isoelectric point of PPI is about 4.3.45 In order to obtain AuNCs showing strong red fluorescence, the synthesis conditions were thoroughly investigated through the adjustment of material concentrations and reaction time. Hybrid nanoparticles (AuNCs/PPI NPs) were successively fabricated through the selfassembling of PPI and AuNCs through a simple dialysis process (Scheme 1). Finally, RBC membranes were used to coat hybrid AuNCs/PPI NPs in order to prolong their blood circulation time and to concentrate in tumor sites: imaging properties of the RBC-coated AuNCs/PPI NPs were evaluated on a tumor-bearing mice in vivo.


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Scheme 1. Schematic illustration of the preparation process of AuNCs/PPI@RBC and its bioimaging application in a tumor-bearing mouse. EXPERIMENTAL SECTION Materials. The PPI powder was purchased from Staerkle & Nagler AG, Switzerland. Chloroauric acid was purchased from Sigma-Aldrich. The other chemicals were purchased from Sigma-Aldrich and used as received. The water used in all experiments was deionized by Millipore purification apparatus (resistivity >18.2 MΩ·cm). Preparation of the PPI Aqueous Solution. The PPI aqueous solution was prepared on the basis of a well-established procedure reported in our previous work on SPI solution.46, 47 In brief, the raw PPI powder was dissolved in 6 mol/L guanidine hydrochloride aqueous solution with continuous stirring for 3 h under room temperature, followed by the addition of 25 mmol/L dithiothreitol solution. After dialysis against NaOH solution (pH=10) for two days and deionized water for one more day, the obtained solution was centrifuged (9000 r/min, 10 min) to remove the possible insoluble components and finally lyophilized to get the pure PPI powder. The asprepared pure PPI powder was then dissolved in deionized water to get PPI solutions with controlled concentrations.


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Preparation of AuNCs and AuNCs/PPI NPs. AuNCs were prepared in aqueous medium at different PPI and chloroauric acid (HAuCl4) solution concentrations and different reaction times. The pH of the reaction system was adjusted to 13 by adding 1 mol/L NaOH solution. Under optimal conditions, 10 mmol/L HAuCl4 solution was added into 10.0 wt% PPI solution with equal volume and reacted at 60 °C for 30 min. To form AuNCs/PPI NPs, the as-prepared AuNCs-PPI mixture was dialyzed against deionized water in a Visking dialysis tube (MWCO: 100 Da) for 3 days at room temperature, slowly re-adjusting the pH to the neutral value. AuNCs were also synthesized by replacing PPI with SPI, corn zein protein (CZP), BSA, and histidine (His). The AuNCs synthesis method using SPI and CZP was the same as for PPI, while the preparation using BSA1 and His6 followed typical protocols described in the literature. The four AuNCs-protein mixtures were dialyzed against deionized water for 3 days in controlled pH. Preparation of AuNCs/PPI@RBC. RBC membranes were obtained following a wellestablished protocol of hypotonic treatment and extrusion.35 1 mL of mouse eye blood was added into 5 mL PBS with some heparin to prevent blood coagulation, followed by centrifugation at 3000 r/min for 5 min at 4 °C to collect the erythrocytes. The packed RBCs were washed with PBS before the application of the hypotonic treatment, which was applied by adding 0.25×PBS (pH 7.4, 1.675 mmol/L PO43-) into the washed erythrocytes so as to release the intracellular components. The mixture was vibrated, centrifuged at 16,000 r/min for 10 min and washed with PBS twice to collect the RBC membranes. The as-prepared RBC membranes were stored in PBS at −80 °C for subsequent use. The AuNCs/PPI NPs were mixed with 200 µL RBC membranes to get a final Au concentration of 0.2 mg/mL and then sonicated for 60 s (53 kHz, 100 W). Subsequently, the mixture was extruded through 200 nm porous membranes for 20 times and


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centrifuged (3500 r/min, 5 min, 4 °C) to remove excessive RBC membranes. Finally, the AuNCs/PPI@RBC at the bottom of the centrifuge tube was re-dispersed for next experiments. Characterization of the Different AuNCs-Related Materials. The digital photos of different reaction solutions were taken under daylight and the fluorescent ones under 365 nm UV light. The morphology of the AuNCs-PPI mixture, AuNCs/PPI NPs and AuNCs/PPI@RBC was observed with an FEI Tecnai G2 20 TWIN transmission electron microscope (TEM) operated at 200 kV. The absorption spectra of the AuNCs-PPI aqueous medium were acquired with a Hitachi UV 2910 UV-vis spectrophotometer in the wavelength range between 325 and 825 nm. Fluorescence spectra were collected with a FLS 920 fluorescence spectrometer. The hydrodynamic diameter and the zeta potential of AuNCs/PPI NPs and AuNCs/PPI@RBC and the hydrodynamic diameter of AuNCs-SPI, AuNCs-CZP and AuNCs-BSA systems after dialysis were measured by dynamic light scattering (Zetasizer Nano ZS90). Confocal Fluorescence Imaging. Human breast adenocarcinoma cells (MCF-7 cells) were used to verify the cell accumulation of AuNCs/PPI NPs and AuNCs/PPI@RBC. The cells were seeded in a 35 mm glass bottom culture dish at a seeding density of 2×105 cells/well (2 mL DMEM media) for 24 h and then treated with AuNCs/PPI NPs or AuNCs/PPI@RBC dispersions for 4 h respectively. After washing three times with PBS, cells were immobilized using a 4% glutaraldehyde solution for 15 min and stained by 4’, 6-diamidino-2-phenylindole (DAPI) for 15 min. Then the media was removed and the cells were washed again with PBS. After washing, another 2 mL PBS were added to the well to keep the cells hydrated. Fluorescence images were recorded by an Olympus Fluoview FV 1000 laser scanning confocal microscope (LSCM). Cell Viability. The cytotoxicity of the AuNCs/PPI NPs was measured using the Cell Counting Kit-8 (CCK-8) assay on MCF-7 cells. The cells were seeded in a 96-well tissue culture plate at a


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seeding density of 1×104 cells/well (100 µL DMEM media) for 24 h and then treated with AuNCs/PPI NPs dispersion with a concentration ranging from 6.25 to 100 µmol/L for 24 h and 72 h. After incubation, PBS was used to wash the cells three times before measuring the cell metabolic viability using a CCK-8 assay. In vivo Tumor Imaging. Female nude mice, 4 weeks old, were obtained from Fudan University Experimental Animal Center (Shanghai, China) and carefully handled under the guidelines approved and supervised by the ethics committee of Fudan University. They were used as tumor-bearing models by subcutaneous injection of 2×106 MCF-7 cells to the left shoulder. After about 7 days, the tumor size reached 100-120 mm3, making it adapt for the following tumor imaging studies. For the in tumor imaging study, 200 µL of 10 mg/mL AuNCs/PPI NPs dispersion was injected into the tumor site, followed by immediate imaging using an optical and X-ray small animal imaging system (In Vivo Xtreme, Bruker, USA) with a central excitation wavelength at 480 nm. For the in vivo imaging study, 200 µL 10 mg/mL AuNC-His mixture, AuNCs/PPI NPs, and AuNCs/PPI@RBC dispersion were injected into the tail vein of the tumor-bearing mice respectively, followed by imaging using the same instrument under the same condition after 1 h, 2 h, 8 h, 12 h and 24 h post-injection. Then, mice were euthanized and dissected 24 h post-injection and the fluorescence images of the major organs (i.e., liver, heart, tumor, kidney, spleen and lung) were collected and analysed by ex vivo fluorescence imaging under an excitation wavelength of 480 nm. RESULTS AND DISCUSSION Synthesis of AuNCs by PPI. A facile, one-step routine is proposed to synthesize highly fluorescent AuNCs using PPI in aqueous medium. As illustrated in Scheme 1, the PPI solution, with the desired pH, was mixed with same volume of HAuCl4 aqueous solution under stirring.


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After thoroughly mixing, the pH of the reaction solution was adjusted to 13 using 1 mol/L NaOH solution. To activate the reducing capacity of PPI, the mixture was incubated at 60 °C for a certain time to form AuNCs. In order to optimize the synthesis conditions, the influence of the PPI content, the HAuCl4 concentration, and the incubation time were investigated (Figure 1 and Figure S1). Figure 1a, b and c show the appearance and properties of samples made by varying the PPI content (0, 0.2, 1.0, 2.5, and 5.0 wt%) while keeping fixed HAuCl4 concentration (5 mmol/L) and reaction time (30 min). As can be seen from UV-vis spectra (Figure 1a), the absence of peak at 520 nm indicates that no Au nanoparticles (AuNPs) were formed to stimulate surface plasmon resonance under these conditions.48 Fluorescence emission spectra (Figure 1b) show that, under an excitation wavelength of 480 nm, an emission peak at 609 nm starts to emerge when the PPI content reaches 1.0 wt%. By increasing the PPI content, the intensity of this emission peak increases and the maximum intensity occurs for a PPI content of 5.0 wt%. At the same time, the position of the maximum emission peak exhibited red shift, which can be explained by the increase of the AuNCs size.49 A more intuitive perception was obtained from the digital images shown in Figure 1c. As the PPI content increases from 0 to 5.0 wt%, the solution color turned from pale yellow to brownish yellow under daylight (Figure 1c, upper panel). When samples with a PPI content of 1.0, 2.5, and 5.0 wt% were exposed to 365 nm UV light, red fluorescence occurred; the strongest fluorescence appeared in sample with the highest PPI content (5 wt%, Figure 1c, lower panel). Notably, the further increase of the PPI concentration (e.g., to 7.5 wt%) resulted in a protein aggregation and the formation of a precipitate when adding the HAuCl4 solution into the PPI solution.


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Figure 1. (a) UV-vis absorbance spectra of AuNCs-PPI solutions with different PPI contents; (b) Fluorescence emission spectra of AuNCs-PPI solutions with different PPI contents; (c) Digital images of AuNCs-PPI solutions with different PPI contents under visible (upper line) and 365 nm UV light (lower line); (d) Digital images of AuNCs-PPI solutions with different HAuCl4 concentrations under visible (upper line) and 365 nm UV light (lower line). Subsequently, we studied the effect of the HAuCl4 concentration (0, 2.5, 5.0, and 10 mmol/L) for a fixed PPI content (5 wt%) and reaction time (30 min). The pure PPI solution displays a light yellow color in daylight and a light blue fluorescence under UV light (Figure 1d). After the addition of the HAuCl4 solutions, the color turned to a darker tone and red fluorescence occurred. Figure 1d shows clearly that the 5 mmol/L HAuCl4 concentration resulted in the brightest red fluorescence with no measurable formation of large AuNPs; this was further confirmed by the UV-vis and fluorescence spectra (Figure S1a and b) indicating that the sample with 5 wt% PPI and 5 mmol/L HAuCl4 resulted in the strongest fluorescence. Therefore, these conditions were chosen to investigate the impact of incubation time on the synthesis of AuNCs.


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During the first 3 h (Figure S1c), no peak was observed at 520 nm, meaning that no sufficiently large AuNPs were formed. Nevertheless, with the progress of the reaction, a broad peak emerges at 520 nm, indicating the formation of AuNPs. Regarding fluorescence spectra (Figure S1d), a weak peak was detected at 630 nm after 15 min incubation; this peak reached a maximum intensity at 30 min then slowly decreased. A similar phenomenon was previously observed in the AuNCs-SPI system.39 We attributed this phenomenon to the degradation of PPI under strong alkaline conditions. The protection of the AuNCs by PPI chains is weakened due to the protein degradation; this allows Au3+ to be reduced on the AuNCs surface, which results in an increase of the AuNCs size. When the AuNCs size exceeds a certain value, fluorescence vanishes and a surface plasmon resonance related absorbance peak appears. According to the above observations, the optimal conditions for the synthesis of AuNCs is a mixture containing 5 wt% of PPI and 5 mmol/L HAuCl4, reacting at pH=13 at 60 °C for 30 min. Preparation and Characterization of AuNCs/PPI NPs. The microstructure of as-prepared AuNCs was characterized by transmission electron microscopy (TEM). As shown in Figure 2a, AuNCs exhibit a good dispersity and uniform size distribution, as well as a clear crystalline structure. After dialysis post-treatment to remove NaOH and unreacted HAuCl4, and after having readjusted the pH to neutral, self-assembled nanoparticles were formed spontaneously with the PPI wrapping AuNCs (Figure 2b). There are no additional steps needed to get the AuNCs/PPI NPs. This phenomenon was ascribed to the change of surface charging of the PPI during the dialysis process. When the protein solution is subjected to an extreme alkaline pH value, the PPI is negatively charged, which promotes the intramolecular repulsion and partial unfolding. After dialysing to neutral environment, the electric repulsion is reduced so that protein molecules refold and assemble into globular structures.50, 51


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Figure 2. (a) TEM image of as-prepared AuNCs; (b) TEM image of AuNCs/PPI NPs; (c) Fluorescence emission spectra of AuNCs/PPI NPs under different conditions; (d) NIR fluorescence image in vivo after subcutaneous injection of AuNCs/PPI NPs into the tumor site. Since the environment in vivo is rather complex, we further investigated the fluorescent and structural stability of the AuNCs/PPI NPs. Figure 2c shows that the incubation of AuNCs/PPI NPs with PBS at pH 7.4 or 5% FBS at 37 °C for 24 h weakly impacted on their fluorescent properties, while a longer storage time (at 4 °C for 1 month) and with PBS at low pH (5.5) slightly decreased the fluorescence intensity, without affecting the spectral line shape. Moreover, after the incubation, the AuNCs/PPI NPs size did not show significant changes (Figure S2a). Especially for the 5% FBS sample, the hydrodynamic diameter for AuNCs/PPI NPs barely changed after the incubation, proving that PPI is an ideal surface ligand for AuNCs. This is due to its inertness to serum protein, since surface ligands with a purely positive or negative charge


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have high affinity with the serum protein, leading to a markedly enlarged hydrodynamic diameter.52 In addition, with the PPI protection, AuNCs/PPI NPs dispersion could be lyophilized and stored in a solid form for several months without losing its fluorescent property (Figure S3). These studies clearly indicate that the PPI protection provided for a great fluorescence properties and structural stability of AuNCs under a biologically relevant environment. We further investigated the universality of this phenomenon by replacing PPI with other proteins. Although SPI, CZP and BSA proteins allowed to produce AuNCs under certain conditions (Figure S4a), after the dialysis process, the behaviour was fairly different. Self-assembling was only observed with SPI and CZP but resulting in a larger hydrodynamic diameter with respect to PPI NPs. However, the self-assembling phenomenon was not observed with BSA (Figure S4b). This could be explained by the nature of the different proteins. For SPI, the composition of amino acid residues, as well as the content of polar amino acids, is very close to PPI, resulting in a similar self-assembling performance. CZP is also a non-physiologically active protein as PPI and SPI, and possesses a similar content of polar amino acids. Therefore, as PPI, SPI and CZP have the potential to form spherical structures in the aqueous phase. BSA is an animal protein which is quite different from the three plant proteins; its physiological activity and amino acids composition are different, which partially explains its different behaviour. Of course, a more profound understanding of the mechanism would require a dedicated work. After comprehensively understanding the structure-property relations, we evaluated the biocompatibility and biofunctionality of AuNCs/PPI NPs. Firstly, the cytotoxicity of AuNCs/PPI NPs was measured using MCF-7 cells by CCK-8 assay for 24 h and 72 h (Figure S2b). Interestingly, the MCF-7 proliferation remained at a relatively constant level even at high AuNCs/PPI NPs concentration (100 µmol/L) for 72 h, indicating an excellent biocompatibility.


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The biocompatibility and fluorescence properties of AuNCs/PPI NPs motivated their use as bioimaging agent in MCF-7 tumor xenograft bearing-mice. As clearly shown in Figure 2d, the subcutaneous injection of the AuNCs/PPI NPs dispersion into the xenograft tumor site resulted in a strong fluorescence signal, indicating a great potential for in tumor imaging applications in vivo. Preparation and Characterization of AuNCs/PPI@RBC. Despite the encouraging results achieved with the subcutaneous injection method, the accumulation and imaging results obtained with the intravenous injection of AuNCs/PPI NPs were still unsatisfactory (Figure S2c). In order to prolong the blood circulation and avoid the macrophages uptake, as-prepared AuNCs/PPI NPs were encapsulated into RBC membrane-derived vesicles. TEM was used to monitor the changes before and after the coating process (Figure 3a, b and Figure S5), which shows the diameters of AuNCs/PPI NPs after membrane fusion were a little bit larger than those before the fusion (all around 100 nm). This phenomenon accords with previous reports,32, 35, 53 showing an increase of the particle diameter after RBC coating. However, the DLS result showed a decrease in the average diameter of AuNCs/PPI NPs after membrane fusion (188 ± 4 nm) than before (152 ± 2 nm) (Figure 3c). This can be explained by the fact that the TEM was observed in dry state while DLS test was performed in aqueous environment. It is easy to understand that, it is easier for the molecular chains in AuNCs/PPI NPs to extend in an aqueous medium than those in AuNCs/PPI@RBC, as the latter is restricted within a membrane. The surface zeta potential of AuNCs/PPI NPs also changes from -11.2 ± 0.8 mV to -7.1 ± 0.5 mV upon fusion with RBC (Figure 3c): this drop was ascribed to the charge screening effect of RBC membranes. Size and potential changes both indicate the successful fusion of RBC membranes and AuNCs/PPI NPs. We also determined the stability of the as-prepared AuNCs/PPI@RBC by comparing the particle


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size before and after incubation in FBS for 24 h. As shown in Figure S6, negligible change in particle sizes was found, which suggest the excellent stability of AuNCs/PPI@RBC in serum. In this work, we wish to establish an effective fluorescent probe for tumor imaging based on AuNCs, so the affinity of AuNCs to tumor cells is of vital significance. Since the fusion of RBC membranes with AuNCs/PPI NPs barely had any influence on the fluorescence intensity (inset of Figure 3a and b), we compared the affinity between MCF-7 cells and AuNCs/PPI NPs, as well as AuNCs/PPI@RBC. As clearly shown in Figure 3d, after 4 h of incubation, fluorescent signals can be detected from the MCF-7 cells for both the coated and uncoated nanoparticles, indicating that the fusion with RBC membranes have a weak effect on the interactions between AuNCs/PPI NPs and MCF-7 cells. The z-stack confocal images indicated that both AuNCs/PPI NPs and AuNCs/PPI@RBC were localized inside of the cells (Figure S7), suggesting the uptake of both AuNCs/PPI NPs and AuNCs/PPI@RBC by MCF-7 cells. Thus, we conclude that AuNCs/PPI@RBC can be applied as a bioimaging probe in vivo. In vivo Tumor Imaging with AuNCs/PPI@RBC. Considering the good biocompatibility and in vitro imaging capability of AuNCs/PPI@RBC, we further assessed its fluorescent imaging property in vivo on MCF-7 tumor-bearing nude mice: results have been compared with AuNCs/PPI NPs and AuNCs reduced by histidine. The synthesis of AuNCs by histidine followed an established protocol6 and its morphology and fluorescent property are displayed in Figure S8. As illustrated in Figure 4a (lower panel), fluorescence emission is observed at the tumor site just 1 h after the injection of the AuNCs/PPI@RBC. Within the next 8 h, the intensity of the fluorescence signal gradually increases and the tumor area becomes better defined. Despite the decrease of fluorescence intensity in the subsequent 16 h, the tumor was still clearly displayed 24 h after the injection. We assume the decrease in fluorescence intensity mainly due to the


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urination, as the fluorescence in the bladder of the mice is quite intense in first 8 h. In comparison, without the RBC coating or AuNCs reduced by His, the shorter blood circulation resulted in an insufficient accumulation at the tumor site (Figure 4a, upper and middle panels). 24 h after the injection, mice were euthanized and dissected respectively to harvest major organs for ex vivo imaging and analyse the biodistribution of the RBC-coated and uncoated AuNCs/PPI NPs (Figure 4b and Figure S9). Results demonstrate RBC coating allows the AuNCs/PP NPs to preferentially accumulate at the tumor site due to longer blood circulation and enhanced permeability and retention (EPR) effect,35, 54 testifying outstanding bioimaging capability for in vivo tumor imaging.


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Figure 3. (a) TEM image of AuNCs/PPI NPs; (b) TEM image of AuNCs/PPI@RBC; (c) Hydrodynamic diameter and zeta potential of AuNCs/PPI NPs before and after the RBC membrane coating. (d) Confocal images of MCF-7 cells incubated with AuNCs/PPI NPs and AuNCs/PPI@RBC for 4 h.

Figure 4. (a) Real-time NIR fluorescence images in vivo after intravenous injection of AuNCs reduced by His (upper panel), AuNCs/PPI NPs (middle panel) and AuNCs/PPI@RBC (lower panel) in tumor-bearing mice for 1 h, 2 h, 8 h, 12 h and 24 h; (b) Ex vivo tissue images of tumor bearing-mouse injected with AuNCs/PPI@RBC (from left to right: liver, heart, tumor, kidney, spleen, and lung).


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CONCLUSIONS In this work, we present a facile, one-step method to produce AuNCs using abundant, cheap, sustainable, and most importantly, non-immunogenic plant protein (PPI) acting as both the reductant and the stabilizing agent. After thoroughly investigating the synthesis procedure, we obtained the optimal reacting condition to synthesize the AuNCs showing strong red fluorescence, that is to add HAuCl4 solution into PPI solution with equal volumes, resulting in a final concentration of 5.0 mmol/L HAuCl4 and 5.0 wt% PPI, and to perform the reaction at 60 °C for 30 min. Interestingly, we found that AuNCs/PPI NPs were readily formed through the selfassembling of PPI simply by dialyzing the AuNCs-PPI mixture solution. The AuNCs/PPI NPs displayed an outstanding stability under different conditions, including long time storage (1 month), storage in PBS (both pH = 5.5 and 7.4), and in 5% FBS. The simple method to produce protein embedded AuNCs NPs resulted to be almost universal as we could successfully use other plant proteins having similar polar amino acid residues content to PPI (e.g., SPI and CZP). Furthermore, thanks to the excellent biocompatibility of PPI, the AuNCs/PPI NPs were successfully applied as bioimaging probe in vitro and in tumor. In order to make the AuNCs/PPI NPs more practical in clinical use, we successfully fused them with RBC membranes. As expected, AuNCs/PPI@RBC exhibited a prolonged blood circulating life, thus making them promising in applications such as in vivo tumor imaging. Therefore, we believe that such a highly biocompatible noble metal/plant protein hybrid nanoparticles possesses a bright future in biomedical area for tumor imaging. ASSOCIATED CONTENT Supporting Information. UV-vis absorbance spectra and fluorescence emission spectra of AuNCs-PPI solutions with different HAuCl4 concentration and reaction time. Hydrodynamic


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diameters of AuNCs/PPI NPs under different conditions. Cell viability of MCF-7 cells incubated with AuNCs/PPI NPs for 24 h and 72 h under different concentration. NIR fluorescence image in vivo 12 h after tail vein injection of AuNCs/PPI NPs. Digital images of the lyophilized AuNCs/PPI NPs under visible and 365 nm UV light. Fluorescence emission spectra and hydrodynamic diameters of AuNCs-PPI, AuNCs-SPI, AuNCs-CZP and AuNCs-BSA. TEM image of AuNCs/PPI NPs and AuNCs/PPI@RBC. Hydrodynamic diameters of AuNCs/PPI NPs and AuNCs/PPI@RBC in 5% and 10% FBS. Z-stack confocal fluorescence images of MCF-7 cells incubated with AuNCs/PPI NPs and AuNCs/PPI@RBC for 4 h. TEM image, fluorescence excitation and emission spectra of AuNCs-His. Ex vivo tissue images of tumor bearing-mouse injected with AuNCs/PPI NPs. These materials information are available free of charge via the Internet at http://pubs.acs.org/. AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] (X. C.); [email protected] (W. Y.). ORCID Xin Chen: 0000-0001-7706-4166 Wuli Yang: 0000-0003-0384-9213 Author Contributions The manuscript was written through contributions of all authors. Z. L. and H. P. contributed equally to this work. Notes


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The authors declare no competing financial interest. ACKNOWLEDGEMENTS X. C. thanks the National Natural Science Foundation of China (No. 21274028, 21574023 and 21574024) for financial support. W. Y. thanks the National Key R&D Program of China (No. 2016YFC1100300) and the National Natural Science Foundation of China (No. 51473037) for financial support. We also thank Prof. Ping Yao for providing pea protein isolate and Dr. Yuhong Yang for her valuable discussion and comments.


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Table of Contents Graphic


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Plant Protein-Directed Synthesis of Luminescent Gold Nanocluster

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