Keratin-Templated Synthesis of Metallic Oxide Nanoparticles as MRI

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Biological and Medical Applications of Materials and Interfaces

Keratin-templated synthesis of metallic oxide nanoparticles as MRI contrast agents and drug carriers Yan Li, Kai Song, Yu Cao, Chen Peng, and Guang Yang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b08555 • Publication Date (Web): 16 Jul 2018 Downloaded from http://pubs.acs.org on July 17, 2018

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ACS Applied Materials & Interfaces

Keratin-Templated Synthesis of Metallic Oxide Nanoparticles as MRI Contrast Agents and Drug Carriers

Yan Li2#, Kai Song3#, Yu Cao2, Chen Peng4*, Guang Yang1,2,5* 1 State Key Laboratory for Modification of Chemical Fibers and Polymer Materials，Donghua University 2 College of Chemistry, Chemical Engineering and Biotechnology, Donghua University, Shanghai 201620, China. 3 Department of Cardiac Surgery, Zhongshan Hospital, Fudan University, Shanghai, 200032, China 4 Department of Radiology, Shanghai Tenth People's Hospital, School of Medicine, Tongji University, Shanghai, 200072, PR China 5 Joint Department of Biomedical Engineering, University of North Carolina at Chapel Hill, and North Carolina State University, Raleigh, North Carolina 27695, USA.

# Yan Li and Kai Song contributed equally to this work. *Corresponding author: Prof. Guang Yang and Dr. Chen Peng Email: [email protected] (G Yang); [email protected] (C. Peng)

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ABSTRACT: Keratin is a family of cysteine-rich structural fibrous proteins abundantly present in skin and skin appendages. Inspired by the template synthesis strategy, in this work, keratin was utilized for the first time as a platform template to synthesize metallic oxide nanoparticles, such as MnO2 (MnNPs@Keratin) and Gd2O3 (GdNPs@Keratin), in a simple and environment-benign fashion. These nanoparticles possess good colloid stability and biocompatibility, high T1 relaxivity (r1 value = 6.8 mM−1s−1 for MnNPs@Keratin and 7.8 mM−1s−1 for GdNPs@Keratin) and superior in vivo MR imaging performance of tumor. Moreover, these keratin-templated nanoparticles have great potential as drug carriers with capacity of redox-responsive drug release due to the existence of disulfide crosslinking in keratin coating. These results suggest that keratin can be a promising platform template for the development of metal-based nanoparticles for cancer diagnosis and therapy.

KEYWORDS: Keratin, template synthesis, metallic oxide nanoparticles, MR imaging, redox-responsive drug delivery

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INTRODUCTION Magnetic resonance imaging (MRI) is a clinical diagnosis technique due to its high spatiotemporal resolution and good tissue penetration. The MRI performance can be improved by the MRI contrast agent1. Nowadays, the paramagnetic gadolinium (Gd III) chelates, such as Magnevist (Gd-DTPA) is a prevailing MRI contrast agent in clinical use with a longitudinal relaxivity (r1) in water of 3-5 mM−1s−1 2. Some studies have demonstrated that ultrasmall magnetic metallic oxide nanoparticles such as Gd- and Mn-based nanoparticles possess larger r1 values than gadolinium (Gd III) chelates, and the nanoparticles with a relative small particle diameter often show large r1 values 3-4. In recent decades, the template synthesis strategy has been proved effective to prepare ultrasmall metal-based nanoparticles with the assistance of different templates including proteins

5-8

, dendrimers 9, polysaccharides

10

and DNA 11. For the template

synthesis route, the metal ions are firstly sequenced onto the template molecules via binding with the thiol, carboxyl and/or nitrogen-containing groups in a mixing step; thereafter, the formation of metal-based nanoparticles is activated by adjusting the solution pH or by the inherent reductive capability of template molecules

12-13

. This

approach is mild-conditioned, environmentally benign and reproducible, and the as-obtained nanoparticles often possess superior characteristics including ultrasmall hydrodynamic diameter, good water dispersion and colloid stability 12-14.

On the other hand, inspired by the design of theranostic nanoplatforms, to combine the imaging agents with drug delivery carriers retains a focus in the field of therapy and 3



diagnostic for years

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15-18

. The common design formulation for the theranostic

nanoplatform comprises three key elements: 1) imaging agents such as fluorescent compounds, magnetic compounds or radioisotopes; 2) polymer coating to facilitate water dispersion and colloidal stability, as well as conjugation of functional units; 3) drug carriers via physical encapsulation, electrostatic interactions, or covalent cross-linking to the imaging agent or polymer coating

15

. For the drug carriers, the

responsive drug delivery systems that enable the drug release sensitive to the diseased environment are particularly expected for an enhanced therapy effect

19-21

. However,

few of the reported protein-templated metal-based nanoparticles can be applied as a responsive drug delivery system unless further chemical modification is involved 22.

Keratin is a family of structural fibrous proteins abundantly present in skin and skin appendages, such as hair, feathers, wool and nail 23. Different from general proteins, keratins feature a high concentration of cysteine residues, covering 7-20% of the total amino acids depending on the sources 24. Inspired by the template synthesis strategy, in this work, we proposed to utilize keratin as a platform template for one pot synthesis of metallic oxide nanoparticles, including manganese dioxide nanoparticles and gadolinium oxide nanoparticles. Based on the unique molecular characteristic of keratin, two main merits are expected for this template: 1) the high concentration of thiol groups could facilitate the binding of metal ions onto the protein template; 2) the formation of disulfide crosslinking in the keratin coating of the obtained nanoparticles could enable a redox-responsive drug delivery in cancer environment because of the 4


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redox potential difference between the cancer tissues and normal tissues

25-28

. To

confirm these, a detailed structural characterization, and evaluation of the stability and biocompatibility of these metallic oxide nanoparticles were investigated, as well as their potentials as the drug delivery nanoplatform in cancer therapy and their in vitro and in vivo MRI performance.

EXPERIMENTAL SECTION Detailed information about materials and experimental methods can be found in the Supporting Information.

RESULTS AND DISCUSSION In this work, keratin-mediated template synthesis approach was proposed to synthesize metallic oxide nanoparticles for MR imaging. Taking manganese dioxide nanoparticles as an example, this synthesis route was conducted by simply mixing the precursor, KMnO4 solution with the keratin solution under agitation at room temperature and neutral pH. In this process, keratin served as both the template and the reductant to impel the formation of MnO2 nanoparticles, with no extra chemical reducing agents or harsh synthesis conditions involved. The as-prepared keratin-templated manganese dioxide nanoparticles (for short, MnNPs@Keratin) exhibit a uniformly monodisperse sphere with an average size of ~ 5 nm by TEM observation (Figure 1A), and a hydrodynamic size of ~ 12 nm by DLS analysis (Figure 1B). The inset in Figure 1A is the digital picture of the lyophilized powder of MnNPs@Keratin after one batch reaction. It should be noted that this synthesis route 5



can be easily scaled up to gram-scale level. The UV-vis absorption spectrum of MnNPs@Keratin shows both the absorption bands of keratin and MnO2 at around 280 nm and 300-400 nm, respectively

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(Figure 1C). The characteristic peaks of keratin

also appear in the FT-IR and XRD spectra of MnNPs@Keratin (Figure 1D and S1). The XPS spectra of MnNPs@Keratin were collected including the main elements (Figure 1E) and the expanded pattern of Mn2p (Figure 1F). Two characteristic peaks centered at 642.4 and 653.9 eV in Figure 1F are assigned to Mn2p3/2 and Mn2p1/2 of MnO2, respectively

29

. The above results confirm the formation of MnO2 nanoparticles

mediated by the keratin template. The Mn element content in MnNPs@Keratin is 1.3% analyzed by ICP-AES.

Figure 1. (A) HR-TEM image of MnNPs@Keratin. Inset: the photograph of MnNPs@Keratin. Scale bar = 100 nm; (B) Hydrodynamic diameter of MnNPs@Keratin analyzed by DLS; (C and D) UV-vis absorption spectra and FT-IR spectra of keratin and MnNPs@Keratin; XPS spectra of MnNPs@Keratin with the 6


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main elements (E) and the expanded pattern of Mn2p (F); (G) Linear fitting of 1/T1 of MnNPs@Keratin in PBS versus Mn concentration. Inset: T1-weighted images of MnNPs@Keratin at different Mn concentrations; (H) Linear fitting of 1/T1 of MnNPs@Keratin versus Mn concentration under normal fluid and tumor microenvironment-mimicking conditions after incubation at 37 oC for 24 h.

In order to achieve high-performance MR imaging effect, we optimized the synthesis conditions of MnNPs@Keratin, especially the feeding ratio of KMnO4 to keratin (Figure S2). Under the optimum parameters, i.e., 0.05 mM KMnO4 to 25 mg/mL keratin, the T1 relaxivity (r1 value) of MnNPs@Keratin reaches up to 6.8 mM−1s−1, which approaches the reported BSA-templated MnNPs (7.9 mM−1s−1)

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(Figure 1G). The T1-weighetd MR images in the inset of Figure 1G demonstrate an evident concentration-dependent MRI performance. Like BSA-templated MnO2 nanoparticles, the high T1 relaxivity is probably ascribed to the synergistic effect of several factors, such as the shorter rotational tumbling time due to the relative large molecular weight of the particles, the high water exchange rate due to the good water retention ability of keratin molecules, and the ultrasmall size of MnO2 particles

13

.

Figure 1H exhibits the in vitro T1-weighted relaxivity of MnNPs@Keratin under varying conditions simulating the normal body fluid (pH = 7.4) and tumor microenvironment (pH = 6.5; pH = 6.5 + 10 mM GSH). The r1 value of MnNPs@Keratin realized an enhancement under the acid pH and reducing environment. This result is consistent with those from other studies, that is, MnO2 can 7



react with acids

13, 30

or GSH

31

to yield Mn2+, which can result in an enhanced

T1-weighted relaxivity. Besides, according to other reports 25, 27, we speculate that GSH could contribute to the degradation of the keratin coating via reductive cleavage of the intermolecular disulfide crosslinking between the keratin chains, thus leaving a thinner protein coating around MnO2 and facilitating the interactions between MnO2 and acids/GSH. Some previous researches have shown that the BSA-templated MnO2 could achieve an enhanced T1-weighted relaxivity under the acid pH due to the degradation of MnO2 into Mn2+

13, 30

. For our MnNPs@Keratin nanoparticles, no change in the r1

value was observed under pH 6.5 comparable to the neutral condition. We speculate that such result is mainly attributed to the thickness difference of the protein coating around MnO2 between our keratin-templated MnO2 and the reported BSA-templated MnO2

13

. According to the literature, the BSA-templated MnO2 possessed an Mn

element content of 2.8% and a hydrodynamic size of 8.7 nm13. Hence, it could be concluded that a thicker keratin coating was formed around MnO2 due to the higher hydrodynamic diameter and lower Mn element content in the MnNPs@Keratin nanoparticles, thus retarding the interaction between MnO2 and acids.

The colloidal stability of MnNPs@Keratin was investigated by measuring their hydrodynamic diameters under varying buffer pH and media (Figure S3). No obvious fluctuations of the hydrodynamic diameters were detected in the pH range of 6 to 8, or various media including water, PBS and cell medium (DMEM) for a period of 48 h, indicating a good water dispersion and colloidal stability of MnNPs@Keratin.

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Figure 2A demonstrates the relative cell viability of murine breast cancer 4T1 cells after incubated with MnNPs@Keratin for 24 and 48 h, respectively. The relative viability remained nearly 100% in the concentrations below 250 µg/mL, suggesting a low cytotoxicity of MnNPs@Keratin. A good hemocompatibility is essential for the blood-contacting biomedical materials. Hemolysis assay of MnNPs@Keratin was performed by incubating the particles with human red blood cells (HRBCs) for 2 h, using PBS and deionized water as a negative and positive control, respectively. A low hemolysis percentage of 1.16% was detected at a high concentration of 250 µg/mL, revealing a negligible hemolytic effect of MnNPs@Keratin (Figure 2B and 2C). Figure 2D shows the histological images of the main organs at 7 and 15 days post-injection of MnNPs@Keratin. No noticeable signs of histological changes or inflammation were found by comparison with the control group. These results indicated that MnNPs@Keratin possessed a good biocompatibility in vitro and in vivo.

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Figure 2. (A) Cell viability of 4T1 cells after incubation with MnNPs@Keratin for 24 and 48 h; (B, C) Hemolysis assay of MnNPs@Keratin. Inset in Figure C: the digital pictures of HRBCs after centrifugation; (D) Hematoxylin & eosin (H&E) staining images of the main organs in MnNPs@Keratin-injected mice after 7 and 15 days administration.

The metabolism of MnNPs@Keratin was investigated by measurement of the Mn element content in the main organs of mice at different time points post i.v. injection (Figure 3). After 7 days administration of MnNPs@Keratin, most of the Mn element was found in the kidney, bladder and liver, demonstrating the probable metabolic pathway of MnNPs@Keratin via the kidney and liver, likely due to the ultrasmall particle hydrodynamic diameter. At 15 days post injection, nearly no Mn element was detected in the main organs, which can avert the potential tissue damage as a result of the long-term retention in vivo.

Figure 3. In vivo biodistribution study of MnNPs@Keratin.

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The good colloidal stability and biocompatibility, as well as the impressive MR performance in vitro encouraged us to apply MnNPs@Keratin for the in vivo MR imaging of tumors. In Figure 4, a brightening effect around the tumor site of the 4T1 tumor-bearing mouse was observed at 10 min p.i. by comparison with the surrounding normal tissues. The T1-weighted MR signals (SNR) reached the summit after 2 h administration, with a high value of nearly 120%, suggesting an effective passive enrichment of MnNPs@Keratin in the tumor. Such duration offers a suitable time window for the observation via the MR scan. At 24 h p.i., the signal intensity of the tumor site gradually recovered. Similar trends in the T1-weighted MR signals of the tumor versus time were reported by the BSA-templated MnO2 nanoparticles13,

29

.

Meanwhile, a brightening effect was also observed in the kidney region, further confirming the metabolic pathway of MnNPs@Keratin via kidneys.

Figure 4. (A) T1-weighted MR images of 4T1 tumor-bearing mice after 10 min, 2 h and 24 h administrations of MnNPs@Keratin (100 µL, 33 mg/mL). Top is coronal images, and bottom is transverse images. The tumor regions are marked with red arrows; (B) T1-weighted MR signals (SNR) in the tumor calculated according to the transverse MR 11



images.

The potential of MnNPs@Keratin as the drug carrier was examined using doxorubicin hydrochloride (DOX) as the drug model via a simple mixing procedure. The UV-vis spectra of the DOX-loaded MnNPs@Keratin verified the successful loading of DOX in MnNPs@Keratin (Figure S4). The drug loading efficiency and loading capacity of DOX are 86.8% and 8.7% (100 mg of MnNPs@Keratin contains 8.7 mg DOX), respectively. Figure 5 A shows the in vitro cancer cell killing efficacy of the DOX-loaded MnNPs@Keratin by incubating them with 4 T1 cells for 24 h. Free DOX was used for comparison. It was found that at the same DOX concentration, the DOX-loaded MnNPs@Keratin exhibited higher cytotoxicity to 4T1 cells comparable to free DOX, demonstrating that MnNPs@Keratin is an effective drug delivery vehicle. It’s well known that cancer cells have multifold higher intracellular glutathione (GSH) concentration than normal cells and tissues, so the reduction-sensitive polymeric drug carriers tend to achieve an enhanced drug release in tumor tissues 19. Figure 5B exhibits the release profile of DOX from the DOX-loaded MnNPs@Keratin under the normal fluid and tumor environment-mimicking conditions, i.e. the reduced pH and reducing environment. The release rate of DOX was increased at the reduced pH by comparison with that at neutral pH within 72 h, revealing a pH-responsive release of DOX. Two factors can explain this result: one is that at acidic pH, the amino groups in DOX are prone to be protonated, which gives an enhanced solubility and hydrophilicity of DOX; the other is that the electrification change of MnNPs@Keratin (the isoelectric point of 12


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keratin is about 4.5) in the acidic environment weakens the electrostatic interaction between DOX and the particles, thus favoring the DOX release. Moreover, a significant increase in the DOX release rate was obtained in the GSH solution. This result is mainly due to the reduction sensitive characteristic of the keratin coating, that is, the disulfide crosslinking within the keratin coating can be broken under the reducing environment, thus bringing a fast release of drugs

25, 27-28

. Furthermore, the DOX

release rate attained the highest value under both the reduced pH and reducing environment, indicating a pH- and redox-dual responsive drug release behavior of the DOX-loaded MnNPs@Keratin. Next, we applied the DOX-loaded MnNPs@Keratin for in vivo therapy using the mouse 4T1 subcutaneous tumor model. Obviously, by comparison with the untreated group, injection of the DOX-loaded MnNPs@Keratin could inhibit the tumor growth (Figure 5C).

Figure 5. (A) Cell viability of 4T1 incubated with different concentrations of DOX and DOX-loaded MnNPs@Keratin by CCK-8 assay; (B) Release profiles of DOX from DOX-loaded MnNPs@Keratin under varying conditions mimicking the normal fluid and tumor environment; (C) Tumor volume growth curves of the 4T1 tumor-bearing mice after treatment with DOX-loaded MnNPs@Keratin. The mice without treatment 13



was used as control. **p< 0.01; ***p< 0.001.

The above-proposed strategy, i.e. synthesis of MnO2 nanoparticles via the keratin template can be easily extended to fabricate other metallic oxide nanoparticles, such as Gd2O3. In this synthesis process, keratin molecules first sequester the Gd ions when Gd precursor (GdNO3) and keratin solution are mixed together; thereafter, the Gd-based nanoparticles (GdNPs@Keratin) are formed in situ by adjusting the pH to 12. The whole reaction process is performed at room temperature, and no additional chemical reducing agents are involved. Figure 6A shows that GdNPs@Keratin have an average diameter of ~ 8 nm, coupled with a uniform size distribution. Figure S5A exhibits a hydrodynamic diameter of about ~ 25 nm. In Figure S5B-D, the characteristic peaks of keratin appear in the FT-IR, UV-vis and XRD spectra of the nanoparticles, confirming the involvement of keratin in the formation of GdNPs@Keratin. Figure 6B records the XPS spectra of GdNPs@Keratin. The strong peak at 143.2 eV is ascribed to Gd 4d 5/2 of Gd2O3 (Figure S5F) 10. The O 1s spectrum can be deconvoluted into three peaks at 530.9, 531.5 and 532.7 eV, originating from the O in Gd2O3 and keratin, respectively (Figure S5E)

10

. The Gd element content in GdNPs@Keratin is 3.8% analyzed by

ICP-AES. The T1 relaxivity (r1 value) of GdNPs@Keratin reached up to 7.8 mM−1s−1 under the optimum synthesis parameters, that is, 5 mM GdNO3 to 25 mg/mL keratin (Figure S6), which is much higher than that of the clinically used Gd-DTPA (Figure 6C). Meanwhile, the T1-weighetd MR images in the inset of Figure 6C revealed a concentration-dependent MRI performance. 14


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Figure 6. (A) HR-TEM image of GdNPs@Keratin. Inset: the photograph of GdNPs@Keratin. Scale bar = 20 nm; (B) XPS spectra of GdNPs@Keratin; (C) Linear fitting of 1/T1 of GdNPs@Keratin and Gd-DTPA in PBS versus Gd concentration. Inset: T1-weighted images of the samples at different Gd concentrations; (D) Cell viability of 4T1 cells towards GdNPs@Keratin and Gd-DTPA with the same Gd concentration by CCK-8 assay; (E) Cell viability of 4T1 incubated with the different concentrations of DOX and DOX-loaded GdNPs@Keratin by CCK-8 assay; (F) Release profiles of DOX from DOX-loaded GdNPs@Keratin under the normal and reducing conditions; (G) T1-weighted MR images of 4T1 tumor-bearing mice after 10 min, 2 h and 24 h administrations of GdNPs@Keratin (100 µL, 17 mg/mL). Top is coronal images, and bottom is transverse images. The tumor regions are marked with red arrows. (H) T1-weighted MR signals (SNR) in the tumor calculated according to the transverse MR images. *p < 0.05; ***p < 0.001.

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The GdNPs@Keratin nanoparticles showed a good colloidal stability in the pH range of 6-8, and different media including PBS, DMEM and water for a period of 48 h, reflected by the relatively constant values of hydrodynamic diameters (Figure S7). Figure 6D shows the cell viability of 4T1 towards GdNPs@Keratin and Gd-DTPA. GdNPs@Keratin exhibited similar or higher relative cell viabilities than Gd-DTPA, with the values above 80% at the GdNPs@Keratin concentration as high as 1000 µg/mL, suggesting a low cytotoxicity of GdNPs@Keratin. A good hemocompatibility of GdNPs@Keratin was verified by the low hemolysis percentage of less than 5% at a high concentration of 250 µg/mL (Figure S8).

Moreover, similar to MnNPs@Keratin, GdNPs@Keratin has the potential as a drug delivery system. Taking DOX as an example, an efficient loading of DOX could be achieved by a simple mixing procedure with a loading efficiency and loading capacity of 90% and 9%, respectively. The DOX-loaded GdNPs@Keratin exhibited higher in vitro cancer cell killing efficacy to 4T1 cells than free DOX (Figure 6E), as well as a redox-responsive drug release behavior (Figure 6F). The in vivo MR imaging performance indicated that GdNPs@Keratin attained a good brightening effect after 2 h administration, with a T1-weighted MR signal (SNR) of approximate 110% in the tumor. Figure S9A shows the in vivo biodistribution of GdNPs@Keratin at different time points post i.v. injection. After 7 days administration of GdNPs@Keratin, most of the Gd element was detected in the liver, and almost all the particles were gradually eliminated from the body at 15 d post-injection. No obvious signs of tissue damage were found by comparison with the control group, evidenced by the histological images of the main organs at 30 days post-injection of GdNPs@Keratin, indicating a good 16


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biocompatibility in vivo of GdNPs@Keratin (Figure S9B).

CONCLUSIONS In summary, we have demonstrated for the first time that keratin is an effective platform template for one pot synthesis of metallic oxide nanoparticles, including manganese dioxide nanoparticles and gadolinium oxide nanoparticles, with the superiority of good colloid stability, biocompatibility, as well as in vitro and in vivo MR imaging performance. Strikingly, these keratin-templated nanoparticles possess a redox-responsive drug release behavior due to the cysteine-rich characteristic of keratin. We believe that such keratin template could extend to the development of other metal nanoparticles and multifunctional applications in cancer diagnosis and therapy.

SUPPORTING INFORMATION XRD spectra of keratin and MnNPs@Keratin; Linear fitting of 1/T1 of MnNPs@Keratin versus Mn concentration under varying concentrations of KMnO4; The colloidal stability of MnNPs@Keratin towards pH values and media; UV-vis spectra of free DOX and DOX-loaded MnNPs@Keratin; Linear fitting of 1/T1 of GdNPs@Keratin versus Gd concentration under varying synthesis conditions; Hydrodynamic diameter of GdNPs@Keratin analyzed by DLS; FT-IR spectra, UV-vis absorption spectra and XRD spectra of keratin and MnNPs@Keratin; XPS spectra of GdNPs@Keratin with the expanded patterns for the elements of O 1s and Gd 4d; The stability of GdNPs@Keratin towards pH values and media; Hemolysis assay of GdNPs@Keratin; In vivo biodistribution study of GdNPs@Keratin; Hematoxylin & eosin (H&E) staining images of the main organs in GdNPs@Keratin-injected mice after 30 days administration.

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ACKNOWLEDGEMENTS This work was supported by Natural Science Foundation of Shanghai (No.15ZR1401000) and the Fundamental Research Funds for the Central Universities.

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Nanoscale‐coordination‐polymer‐shelled

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Keratin-Templated Synthesis of Metallic Oxide Nanoparticles as MRI

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