Albumin-Mediated Biomineralization of Shape-Controllable and

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Albumin-Mediated Biomineralization of ShapeControllable and Biocompatible Ceria Nanomaterials Zhangyou Yang, Shenglin Luo, Yiping Zeng, Chunmeng Shi, and Rong Li ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b15442 • Publication Date (Web): 02 Feb 2017 Downloaded from http://pubs.acs.org on February 4, 2017

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ACS Applied Materials & Interfaces

Albumin-Mediated Biomineralization of Shape-Controllable and Biocompatible Ceria Nanomaterials Zhangyou Yang†,§, Shenglin Luo†,§, Yiping Zeng†,§, Chunmeng Shi†,* and Rong Li†,* †Institute of Combined Injury, State Key Laboratory of Trauma, Burns and Combined Injury, Chongqing Engineering Research Center for Nanomedicine, Department of Preventive Medicine, Third Military Medical University, Chongqing, 400038, China.

KEYWORDS: ceria nanostructure, green synthesis, metal-protein complex, BSA, biocompatibility ABSTRACT Although ceria-based nanostructures have emerged as fascinating materials with diverse biological activities, developing a facile, rapid and biocompatible method of their preparation remains a major challenge. Herein we describe bovine serum albumin (BSA) protein-directed synthesis of ceria-based nanostructures, including ceria nanoclusters (CNLs), nanoparticles (CNPs) and nanochains (CNHs). Their preparation is simple, one-pot, and performed in a mild reaction condition with “green” synthetic approach. Most importantly, these three kinds of ceria-based nanostructures can be synthesized in a shape and size controllable manner by tuning the reaction time, temperature and molar ratio. The formation mechanism shows that growth of these ceria nanostructures is mediated by Ce3+/Ce4+ switchable redox system, reducible disulfide bonds and unique spatial structures in albumin proteins. More importantly, these albumin-based ceria nanostructures remain exhibit high superoxide dismutase (SOD) mimetic activity as well as good biocompatibility, providing a promising prospect in biomedical application.

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Introduction Ceria-based nanostructures, such as ceria nanoparticles, nanorods, nanowires and nanotubes,1-5 have emerged as fascinating materials with diverse biological activities, including antiinflammation, antiaging, anticancer, radiation protection, or as potential bioscaffoldings for regenerative medicine.6-14 Especially with co-existence of two oxide states (Ce3+/Ce4+) on their surfaces, the charge redistribution results in oxygen vacancies in the lattice of nanostructures, allowing them to switch redox system reversibly.15 This interchangeable property makes ceria nanomaterials have the regenerative antioxidant property, further promising the potential of biomedical application. There are many methods developed for the preparation of ceria-based nanostructures,

such

as

microemulsion,

hydrothermal,

microwave-assisted, thermal decomposition et. al.

16-21

reversed

micelles,

Different methods are usually

utilized to obtain different shapes of ceria-based nanostructures with varied physicochemical and biological activities. Although many methods are becoming sophisticated and efficient, most of them need multi-step, complicated chemical reactions or harsh conditions (high temperature or organic solvents). Besides, in order to improve the stability and biocompatibility for biological application, various surface modifications of ceria nanomaterials are usually indispensable, such as polyethylene

glycol

(PEG),

dextran,

poly(acrylic

acid)

(PAA),

chitosan,

N-acetylglucosamine and polyvinylpyrrolidone (PVP).22-27 However, extra surface modification with chemical molecules mean additional synthetic steps, costs, and more convoluted behavior in vivo, which would confront more difficulties in applying for clinic trial and real application. Therefore, it remains a major challenge to develop a facile, rapid and biocompatible method to prepare ceria nanomaterials especially for biomedical application. Bovine serum albumin (BSA), composed of 583 amino acid residues with a molecular weight of 66.4 kDa, is abundant as an important blood protein. It is predominantly α-helical with three homologous domains (I, II, and III) and some reducible disulfide bonds,28 providing an excellent platform to deliver lipophilic drug as a surfactant or generate metal-protein conjugates as a reductant. Generally, BSA


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can display as an expansive hollow nanocage in basic environment owing to its unique spatial structure and molecular chain flexibility,29,30 which could be explored as the nanoreactor to mediate the growth of metal ions toward metal nanoclusters (e.g., Au, Mn, Cu, Cd clusters).31-36 The protein-mediated approach exhibits a green process to synthesize nanomaterials, such as room temperature, short reaction time and aqueous solution.37-39 Furthermore, nanomaterials conjugated with BSA as surface modification, not only exhibit good dispersibility and biocompatibility, but also facilitate

post-surface

modification

with

functional

ligands

for

further

applications.40,41 With these advantages, it is highly attractive to modify nanostructures with BSA for biomedical application. Here we aimed to explore BSA-directed synthesis of ceria nanomaterials. With two main oxide stations (Ce3+ and Ce4+) of nanoceria, BSA with reducible disulfide bonds would have potential as the nanoreactor to direct growth of ceria nanoclusters by forming Ce-BSA complexes. Owing to its switchable redox system, the recovered reduction potential would keep directing the protein-mediated synthesis to grow different shapes of ceria-based nanostructures. Based on this design, in this work we not only obtained size-uniform BSA-coated ceria nanoparticles (CNPs), but also obtained ceria nanoclusters (CNLs) and ceria nanochains (CNHs) for the first time. Three kinds of ceria-based nanomaterials could be synthesized in a controllable manner by adjusting reaction time, temperature or molar ratio (Figure 1a). More importantly, the BSA-coated ceria nanomaterials were demonstrated with high superoxide

dismutase

(SOD)

mimetic

activity

meanwhile

with

excellent

biocompatibility based on in vitro and in vivo evaluation. Results and Discussion A protein-mediated approach to synthesize different shapes of ceria nanostructures was explored initially by tuning reaction time. The experimental details can be found in the experimental section and Table S1-S3 in the Supporting Information. Briefly, BSA protein in water was mixed with cerium nitrate under vigorous vortexing. After dissolved well, the mixture at 37 °C was rapidly added KOH to modify pH to 13. It



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has revealed that there are 17 disulfide bridges protected in BSA at pH between 5−7. However, when pH is increased to 10, some disulfide bonds become solvent-accessible and can be easily cleaved by a reducing agent.42,43 The basic environment possibly triggered the reaction Ce(NO3)3 + 3KOH = Ce(OH)3 + 3KNO3 and simultaneously transformed BSA into unfolded status, which may facilitate the subsequent the reaction 4Ce(OH)3 + O2 = 4CeO2 + 6H2O. The reaction was stirred for 15 min, 1 h, 4 h, 12 h, respectively. After purified with dialysis and centrifugation filtration (molecular weight cutoff: 100 Kda), the residual mixture was characterized with transmission electron microscopy (TEM). As shown in Figure 1b, within 15 min reaction, CNLs with the diameter of 1.7 ± 0.5 nm and lattice fringe of d111 = 0.31 Å (inset in Figure 1b) were formed, which is in agreement with the reported ceria nanostructures.44 After 12 h reaction, CNHs with an average length about 100 nm and width about 2 nm were prepared (Figure 1c-d). The results suggested that the CNHs would be formed from the growth of CNLs. Time-dependent growth of ceria nanostructures from CNLs to CNHs were further validated by TEM images (Figure

Figure 1. a) The schematic diagram of albumin-mediated tunable synthesis of ceria-based nanomaterials, including ceria nanoclusters, nanoparticles, and nanochains; TEM images measurements of the prepared b) CNLs (bar, 50 nm) and c-d) CNHs (bar, 100, 10 nm respectively).


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S1a-d), which exhibited to produce ceria nanomaterials with increasing length (from about 2 nm to 10, 50, 100 nm) after the reaction time was extended from 15 min, to 1 h, 4 h or 12 h, respectively. Dynamic light scattering (DLS) measurement showed good polydispersity of BSA-coated CNLs, and also demonstrated the growing trend of ceria nanomaterials along with the increasing reaction time (Figure S1e-f). The physicochemical properties of CNLs and CNHs were characterized. UV–vis absorbance spectra showed that both CNLs and CNHs have the characteristic peak of BSA at 260 nm, and the wide absorbance of ceria-based nanomaterials between 250-400 nm (Figure 2a). Thermal gravity analysis (TGA) under nitrogen atmosphere showed that the main weight lost (from 300-500℃) of CNLs and CNHs was contributed by their surface coated BSA (Figure 2b). The CNLs and CNHs could be freeze-dried, and stored in the solid form (Figure 2b, insert) stably for at least 3 months. According to X-ray photoelectron spectroscopy (XPS) analysis by mean of argon sputtering (Figure 2c), the sulfur (S2p) signals can be detected in both two ceria nanostructures, suggesting successful conjugation of BSA to ceria nanomaterials. Selected XPS segments related to the valence state of cerium ions with corresponding binding energy peaks for Ce3+ (880.20, 885.00, 899.50 and 903.50 ev) and Ce4+ (882.10, 888.10, 898.00, 900.90, 906.40 and 916.35 ev) were also confirmed in both two ceria nanostructures (Figure 2c, inset).45 The XPS analysis further demonstrated that the Ce3+ percentage in CNLs and CNHs were calculated high up to 45.7% and 43.8%, respectively (Figure S2). It is well known that ceria nanomaterials with a higher Ce3+ to Ce4+ ratio, are more effective against diseases associated with oxidative stress or inflammation, owing to higher oxygen vacancy and SOD mimetic activity.46,47 FT-IR spectrum (Figure 2d) revealed the common characteristic peaks (483, 1650 and 3300 ) of BSA in CNLs and CNHs, further verifying the successful conjugation.



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Figure 2. Characterization of CNLs and CNHs. a) UV-vis absorbance spectra (inset: aqueous solution of samples). b) TGA analysis (inset: freeze-dried samples). c) The full XPS analysis (inset: selected segmentrelated to valence state of cerium element). d) FT-IR analysis of BSA, CNLs and CNHs. To demonstrate the effects of molar ratio and pH value on BSA-mediated synthesis of ceria nanomaterials, different control experiments were performed (Table S1). After addition of KOH into the mixture of BSA and cerium nitrate (BSA:Ce mole ratio ~ 1:13:150), a lot of precipitates generated immediately but subsequently redissolved in this reaction system within several seconds. However, without BSA, yellow precipitates (probably cerium hydroxide, Figure S3a) generated. Without KOH, BSA could disperse cerium nitrate but it was not stable in aqueous solution (Figure S3a). Without cerium nitrate, BSA mixed with KOH, could form a light yellow solution. However, TEM images and DLS (Figure S3b-c) showed a large range of sizes (5 nm-150 nm) of spherical nanoparticles prepared, which would be self-assembled BSA


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according to the previous reports.48,49 Modifying pH from acid to basic condition, it showed that yellow precipitates were formed in acid and neutral environment (Table S1). In addition, when Lysozyme

50

(a 14.3 kDa protein only with 5 disulfide bonds)

or Hemoglobin (HGB which has the same molecular weight but different protein conformation as compared to BSA) was selected as control protein to replace BSA, massive precipitates generated and ceria nanomaterials failed to synthesis (Figure S4 and 5). It seems that both the number of activatable disulfide bonds and specific

Figure 3. Photographs and TEM images of BSA-mediated synthesis of CNPs under different temperatures. a) Images of mixture with cerium nitrate and BSA before addition of KOH; b) Images of mixture 2 h after addition of KOH; c-f) TEM images of BSA-coated CNHs and CNPs by elevating reaction temperature from 4 °C, 37 °C, 55 °C to 80 °C, respectively. (bar, 50 nm)



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three-dimensional structure of BSA play important roles in the preparation of these ceria nanostructures. To demonstrate whether the morphology of BSA-coated ceria nanomaterials could be modified by thermal control, different reaction temperatures were investigated. Because temperature is closely related to the number of disulfide bonds activable in BSA,51,52 we varied the reaction temperature from 4 °C to 80 °C in above reaction system, in which 37 °C and 2 h were used for preparation of CNHs. Figure 3a showed that when the temperature was high up to 55 °C, opacification was observed before KOH addition. All the reaction mixtures under different temperatures finally became a clear yellow solution after KOH addition and 2 h stirring (Figure 3b). TEM images exhibited that the average length of CNHs gradually decreased along with the raising reaction temperature, from about 40 nm (4 °C, Figure 3c), to 30 nm (37 °C, Figure 3d), and 10 nm mixed with a small part of CNPs (55 °C, Figure 3e). In particular, when the temperature was high up to 80 ℃, spherical CNPs with the average diameter of 3.7± 0.7 nm were obtained (Figure 3f). To characterize BSA-coated CNPs, TEM image (Figure 4a) demonstrated the nanoparticles with uniform size and ceria lattice fringe of d111 = 0.32 Å. Their average hydrodynamic diameter tested by DLS (Figure 4b) also showed about 25 nm with the polydispersity index of 0.095, indicating a monodispersive nanostructure. The energy-dispersive spectrometry (EDS) and elemental mapping image analysis of BSA-coated CNPs bulk indicated the existence and the homogeneous distribution of O, S, and Ce elements (Figure S6). FT-IR spectrum, TGA, and XPS tests (Figure S7a-d) confirmed the successful synthesis of BSA-contained CNPs and co-existence of two oxide states (Ce3+/Ce4+) with high Ce3+ percentage about 49.7%. According to X-Ray diffraction (XRD, Figure 4c) analysis, more characteristic peaks of ceria-based crystallographic structures were clearly found in CNPs (80 °C) than that in CNHs (37 °C), e.g. 111, 200, 220, 311, 400, 331. This result could explain the reason why clearer TEM images were seen in CNPs than other two nanostructures. Circular dichroism (CD) is a widely used technique to quantify partial conformational changes of proteins. The results of CD analysis (Figure 4d) revealed that all kinds of our newly


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prepared ceria nanomaterials induced significant conformational changes of BSA. They were found with high percentage of Beta (β) fold, which does not exist in BSA protein. In contrast, the α-Helix level which was calculated up to 77.8% in BSA protein, decreased obviously in these BSA-mediated synthesized ceria nanomaterials. Particularly, CNPs prepared under high temperature showed the highest percentage of β fold (65.1%) but the lowest α-Helix level (6.1%), suggesting the formation of CNPs would be actively mediated by BSA rather than simply physical attachment. In addition, CNHs prepared with short branches exhibited the highest percentage of Random rotation (34.9%), showing the linear anisotropic constructs. The result indicated that BSA would actively participate in the growth of CNHs.

Figure 4. a) TEM images (bar, 10 nm) and b) DLS analysis of CNPs; c) XRD analysis of BSA, BSA-coated CNHs (37°C, 2 h) and CNPs (80 °C, 2 h). d) Circular dichroism spectrum of BSA, BSA-coated CNLs, CNPs and CNHs (inset: the main data of conformational changes).



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It has been proved that CNPs (8-9 nm) can interact to metallothionein, and the possible reaction mechanism is the formation of a disulfide bridge/Ce3+ complex between Ce4+ and the thiol groups.53 Hence, the formation mechanism of CNHs would possibly be illustrated as shown in Figure 5a. In a strong basic environment, BSA protein may unfold and open its disulfide bonds to thiol groups. The active thiol groups have the potential to direct the growth of CNLs by forming disulfide bridge/Ce3+complexs. With lengthening the reaction time, CNLs could be gradually growing to CNHs. In order to verify this proposed mechanism, different experiments were performed (Table S4). First, we replaced cerium nitrate by HAuCl4 to investigate whether Au nanochains could be synthesized. In fact, with the same condition for CNHs as described before, BSA-conjugated Au nanoparticles (~25 nm) were obtained (Figure 5b). When extending the reaction time to 8 h, CNHs were prepared as shown by TEM image (Figure 5c) while the preparation of Au nanochains failed. The different results indicated that the growth of CNHs would be also relevant to their own switchable redox system between Ce3+ and Ce4+. Second, dithiothreitol (DTT), a well-known thiol-based protein disulfide reducing agent, 51 was used to investigate the effect of disulfide-bond cleavage on the formation of ceria nanochains. The results showed that DTT inhibited the growth of CNHs from CNLs (Figure 5d), and also inhibited the formation of long CNHs from short CNHs (Figure 5e). In addition, we performed the reaction started with 37 °C for 2 h to produce short CNHs and kept 80 °C for another 6 h to investigate whether the growth of CNHs could be inhibited, since high temperature can inhibit disulfide-bond formation. The result showed that the growth of CNHs was inhibited by high temperature, and a part of CNPs were produced (Figure 5f). When the reaction started with 80°C and kept stirring for 8 h, the size of CNPs was almost same to that prepared with 80°C for 2 h (Figure 5g). There is also no trend to form CNHs even increasing the reaction time. However, KMnO4, as a strong oxidation agent, was added in the synthesis system of CNPs (Mn: Ce=1:10). Interestingly, the results showed that KMnO4 could promote the growth of CNHs even under 80℃ for 2 h. In contrast, if there was just KMnO4 in this synthesis system, only Mn nanosheet was gained (Figure S8a, b). Therefore, above results


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demonstrated the importance of disulfide bridge/Ce3+ complex on the growth of CNHs.

Figure 5. The possible growth mechanism of CNHs and the results of verification tests. a) The proposed formation mechanism of the CNHs. b) TEM image of Au nanoparticles, when replaced Ce(NO3)3 by HAuCl4, at 37°C for 8 h. c) TEM image of CNHs, when used Ce(NO3)3 at 37°C for 8 h. d) TEM image of the inhibited formation of CNHs from CNLs, when prepared at 37°C for 15 min (0.25h), then added DTT for another 7.75 h. e) TEM image of the inhibited growth of CNHs, when prepared at 37°C for 2 h, then added DTT for another 6 h. f) TEM image of the inhibited growth of CNHs, when prepared at 37°C for 2 h, followed by 80 °C for another 6 h. g) TEM image of CNPs, when prepared at 80°C for 8 h. (bar, 100 nm) The enzyme mimetic activity of ceria nanomaterials is critical for most of their biological effects. In order to evaluate enzyme mimetic activity of BSA-coated ceria nanomaterials, their SOD activity was determined by inhibiting the generation of superoxide free radicals, which were produced from riboflavin under light illumination. The results demonstrated that all the newly developed BSA-coated ceria



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nanomaterials (CNLs, CNPs and CNHs) exhibited the significant SOD activity as compared to the blank PBS or BSA aqueous solution (Figure 6a). Among them, CNLs showed the highest SOD activity, and CNHs showed higher SOD activity than CNPs at the same ceria concentration. High specific surface area of CNLs may be ascribed for its high enzyme mimetic activity. The results also indicated that CNHs with short branches, likely prolonged from CNLs, facilitated the improvement of SOD activity. Additionally, the SOD mimetic activity and stability analysis of CNLs in PBS and PBS+10% FBS at different day showed the ceria nanomaterials could maintain high SOD mimetic activity and stability at least 7days (Figure S9a, b). The XPS (Figure S9c, d) analysis of CNLs at 7 day also showed no obvious Ce3+ percentage change (46.5%) compared newly developed BSA-coated CNLs (45.7%).

Figure 6. SOD activity, biodistribution and cytotoxicity of BSA-coated ceria nanomaterials. a) The SOD activity of BSA (10 µM), CNLs, CHPs and CNHs in different concentration. b) Distribution of CNLs, CHPs and CNHs in organs. c) The cell viability of CNLs, CHPs and CNHs (cerium, 0.01~10 mg/mL) at 24, 48, 72h incubation with L-02 cells. d) ICP-MS analysis the cellular uptake behavior for CNLs, CHPs and CNHs at 0.5, 4, 24h incubation with L-02 cells.


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To investigate the biodistribution of BSA-coated ceria nanomaterials in main organs, the MALB/c mice were sacrificed one week after tail vein injection. The amount of ceria accumulated in heart, liver, spleen, lung and kidney was determined by ICP-MS. The results showed that they as well as other nanomaterials mainly accumulated in tissues (Figure 6b). CNHs with average 100 nm size also exhibited higher accumulation in lung than CNLs and CNPs. Due to high accumulation in liver, the cytotoxicity of these newly prepared ceria nanomaterials on L-02 liver cells was further evaluated (Figure 6c). As well as PBS and BSA (1, 10 µM), incubation of CNLs, CNPs and CNHs with 0.01~10 mg/L Ce concentration showed no damage on cell viability. Otherwise, the cytotoxicity of these ceria nanomaterials to other cells, including normal (HK-2) and tumor (HeLa) cells were also evaluated with above Ce concentrations. The results exhibited no damage on cell viability (Figure S10a, b). Hence, the above results verified that BSA modification ceria nanomaterials exhibit good biocompatibility. It is well known that different morphologies and sizes of nanomaterials would exhibit different physicochemical and biological activities.54 Generally, many metal ions only allow the formation of very small metal clusters with BSA-directed synthetic strategy.31,

55-57

However, when used Ce ions, BSA-mediated synthesis of ceria

nanostructures can be achieved in a controllable manner from nanoclusters to nanoparticles and nanochains in this work. The synthetic yields of CNLs, CNHs, and CNPs, calculated by ICP-MS analysis of the total amount of Ce element, were about 73%, 75%, 78%, respectively. The three kinds of new ceria nanostructures could be uptake by cells efficiently based on ICP-MS analysis (Figure 6d). In addition, we used human serum albumin (HSA), a clinically approved protein, 48, 58 to replace BSA. We found HSA protein could mediate the synthesis of different shapes of ceria nanostructures as well as BSA (Figure S11a, b, c). Therefore, all the above findings indicate that this albumin-based strategy to prepare ceria nanostructures would be feasible and attractive, exhibiting a promising potential of their practical application in biomedicine.



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Conclusion In this report, BSA or HSA coated CNLs, CNPs and CNHs can be synthesized in a controllable manner. Their preparation is simple, one-pot, high-yield, and performed in a mild reaction condition with “green” synthetic approach. The formation mechanism shows that growth of these ceria nanostructures is mediated by Ce3+/Ce4+ switchable redox system, reducible disulfide bonds and unique spatial structures in albumin proteins. These albumin-based ceria nanostructures remain high superoxide dismutase (SOD) mimetic activity while exhibit good biosafety. Different from the traditional surface modification (such as PEG, PAA, PVP), albumin-coated CNLs, CNPs and CNHs would have advantages of good dispersibility, biocompatibility and easily post-surface modification, providing a bright future for their biomedical application.

Experimental Section Materials. All chemicals were purchased from Sigma-Aldrich and used as-received. Ultrapure Millipore water (18.2 M Ω) was used. All glassware was washed with chromic acid lotion and then rinsed with ultrapure water. (Caution: chromic acid lotion is a very corrosive oxidizing agent, which should be handled with great care.) Synthesis of CNLs. In a typical experiment, 0.25 mL 0.1 M Ce(NO3)3.6H2O aqueous solution was added to 4.5 mL 25 mg/mL BSA (or HSA) solution under vigorous stirring at 37 °C for 15 min. 0.3 mL 1 M KOH solution was quickly added into above mixture, and the reaction was kept stirring for 15 min. Then, the obtained CNLs were dialyzed in ultrapure water with dialysis bag (Thermo, 10 kd) for 24 h (notice: change the ultrapure water every 6 hours). For further purification, the CNLs were placed in sleeve tube (Millipore, 100 kd) with 4500 r/min by centrifugation and rinsed with ultrapure water repeatedly. Finally, the obtained CNLs were dispersed in water and


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stored at 4 °C before used. Synthesis of CNPs. In a typical experiment, 0.25 mL 0.1 M Ce(NO3)3.6H2O aqueous solution was added to 4.5 mL 25 mg/mL BSA (or HSA) solution under vigorous stirring at 80 °C for 15 min.0.3 mL 1 MKOH solution was quickly added into above mixture, and the reaction was kept stirring for 2h. The purification procedure of CNPs was same to that for CNLs synthesis and stored at 4 °C before used. Synthesis of CNHs. In a typical experiment, 0.25 mL 0.1 M Ce(NO3)3.6H2O aqueous solution was added to 4.5 mL 25 mg/mL BSA (or HSA) solution under vigorous stirring at 37 °C for 15min. 1 M KOH solution (0.3 mL) was quickly added into above mixture, and the reaction was kept under this condition for different time phase (1~12 h). The purification procedure of CNHs was same to that for CNLs synthesis and stored at 4 °C before used. Conditions for synthesis of different ceria nanomaterials and mechanism research. As seen in Table S1-S4 (Supporting Information), different conditions were investigated for synthesis of ceria nanomaterials and mechanism study. In all cases 0.25 mL of Ce(NO3)3.6H2O (0.1 M) was added into a 4.5 mL BSA solution while varying the reaction time, temperature or the concentration of KOH, DTT, et al. For control experiments, 0.25 mL of HAuCl4 was added into a 4.5 mL 25 mg/mL BSA to replace the Ce(NO3)3.6H2O. Lysozyme solution (25 mg/mL, 4.5 mL) was used to replace BSA solution. Nanoparticle Stability. The CNLs (25 mg/L) was incubated with PBS and PBS+10% FBS for 0, 0.5, 1, 3, 7 days, then the stability of nanoparticles was tested by DLS using a Brookheaven Zeta PALS analyzer after. TEM Characterization and XRD measurement. TEM analysis were conducted using Tecnai G2 F20 S-TWIN(200KV). Samples were prepared by casting a drop of nanomaterials dispersion onto a carbon-coated copper grid. The ceria nanomaterialswere dispersed in water. Element mapping images and EDS results were obtained using a field-emission scanning electron microscope (FESEM, Ultra55,



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Zeiss, Germany). The DLS of ceria nanomaterials were analysis by Brookheaven Zeta PALS analyzer. XRD patterns were acquired from dried nanomaterials samples with a Persee XD3 X-ray diffractometer using Cu NF radiation at 36 kV and 20 mA. Structural Characterization. UV-Vis spectra weremeasured at 25 °C with a Shimadzu UV-3600 UV/Vis spectrophotometerequipped with a 10 mm quartz cell. FT-IR spectrum was measured from 500 to 4000 cm−1 using a Thermo scientific nicolet iS10 infrared spectrometer. The nanomaterials were dried on EYELA FDU-2100 freeze drier for 24 h. Samples were milled with dried KBr and the mixture was pressed into a pellet for analysis. TGA experiments were performed on TA Q50 undernitrogen atmosphere. Samples of the nanomaterials were dried on EYELA FDU-2100 for 24 hbefore analysis. The dried nanomaterials were tested with temperature increase rate of 30 ºC/min. XPS measurement. XPS experiments were performed using a multipurpose surface analysis system (SCIENTIFIC ESCALAB 250, Thermo, UK) by the way of argon sputtering.The photoelectron spectra were excited by an Al Ka (1486.6 eV) anode operating at 100 W. The base pressure during XPS analysis was maintained at less than10-9 mbar, and the binding energy scale was calibrated from the C1s peak at 284.8 eV. The 3d peak positions of cerium were fitted using XPSPEAK41 software with binding energy from 870 to 925 ev. CD spectra measurement. CD spectra were recorded on a Jasco 715.The parameters used were as follows: bandwidth, 1.0 nm;measurement range, 190–250 nm;response time, 4 sec;sensitivity, standard; date pitch, 0.1 nm; scan speed, 50 nm min-1. The cerium concentration of samples were tested by ICP-MS (Agilent 7500ce) before digested in concentrated sulfuric acid at 80 ºC for 3 h. Superoxide Dismutase Mimetic Activity. The superoxide dismutase mimetic activity of ceria nanomaterials was tested by inhibiting the superoxide free radical generated by riboflavin under light illumination.59100 µL riboflavin (1.2 mmol/L) was added into the solution which containing 150 µL nitrotetrazolium blue chloride (2


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mmol/L), 400 µL EDTA (0.1 mol/L) and 5.8 mL sodium phosphate buffer (pH 7.8, 10.0 mmol/L), and the mixture was used as detection solution. The different concentration of CNLs, CNPs, CNPs and BSA in 50 µL water were miexed with 100 µL detection solution (n=5). The mixture was shook at 37 ºC for 5 min followed by illumination for 2 min using a 27 W light tube. The excessive superoxide free radical which could not be inhibited by CNPs reacted with nitrotetrazolium blue chloride to generate blue product which could be detected at 560 nm. The inhibition was calculated by the decrease of absorbance at 560 nm. Cell cuture and uptake. The liver L-02 cells were cultured in a humidified atmosphere (5% CO2) at 37 ºC, and grown in RPMI-1640 medium supplemented with 10% FBS. For cell viability assay, the cells were seeded in 96 well plate at 4000 cells in 0.1 mL medium 24 h prior to the experiment. The CNLs, CNPs, CNHs (with cerium concentration of 0.01, 0.1, 1.0, 10 mg/L) and BSA (1,10 µM) were added to the cell culture medium (n=5). The cells were cultured for another 24, 48 and 72 h followed by adding cell viability detection reagent CCK-8 (10 µL/well).

After

incubation for 2 h, the absorbance at 450 nm was detected for viability analysis. The experiment procedures of cytotoxicity evaluation for CNLs, CNPs, CNHs to HK-2 and HeLa cells were the same as above mentioned except using different cell lines. For cell uptake, the L-02 cells (20×104 cells/well, seeded in 6-well plates) were plated 24 h prior to incubation with CNLs, CNPs, CNHs (Ce 5 mg/L) for different time periods (0.5, 4 and 24 h) with three wells at each time point. The cells were then lightly washed three times with PBS and digested in sulfuric acid (500 µL/well) at 75 °C for 4 h. The intracellular cerium concentrations were determined by ICP-MS Agilent 7500ce. Organ distribution. Male MALB/c mice were purchased from the Third Military Medical University Animal Center, Chongqing, China. Animals were housed under controlled laboratory conditions with a 12: 12 h light: dark cycle at 20 ± 5 °C and 40-70% humidity. All animal protocols were reviewed and approved by the Animal Ethics Committee of the Third Military Medical University. The ceria nanomaterials



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were injected via the tail ven with Ce dose of 10 mg/kg (n=3). The animals were sacrificed at day 7. The organs including heart, liver, spleen, lung and kidney were collected and weighted. The tissues were rinsed with ultrapure water to remove any superficial cerium. After that the tissues were added 3.0 mL concentrated nitric acid and digested with a microwave reaction system (CEM MARS 6) at 150 °C for 20 min. Finally, the Cerium concentration in the tissues were determined by ICP-MS (n=3). ASSOCIATED CONTENT Supporting Information The Supporting Information file contains conditions synthesis, mechanism research, characterization of ceria nanomaterials, TEM analysis of HSA-mediated synthesis ceria nanomaterials. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Authors * Email: [email protected] * Email: [email protected] Author Contributions

§The manuscript was jointly written by all authors. Z.Y., S.L., and Y. Z. contributed equally. Notes The authors declare no competing financial interests. ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (No. 81472914 and 81402784) and the National Basic Research Program of China (973 Program, No. 2012CB518103).

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Graphical Abstract


Albumin-Mediated Biomineralization of Shape-Controllable and

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