Cell-Permeable, MMP-2 Activatable, Nickel Ferrite and His-Tagged

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Cell-Permeable, MMP-2 Activatable, Nickel Ferrite and His-tagged Fusion Protein Self-Assembled Fluorescent Nanoprobe for Tumor Magnetic Targeting and Imaging Lu Sun, Shuping Xie, Jing Qi, Ergang Liu, Di Liu, Quan Liu, Sunhui Chen, Huining He, and Victor C. Yang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b12918 • Publication Date (Web): 23 Oct 2017 Downloaded from http://pubs.acs.org on October 24, 2017

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ACS Applied Materials & Interfaces

Cell-Permeable, MMP-2 Activatable, Nickel Ferrite and His-tagged Fusion Protein Self-Assembled Fluorescent Nanoprobe for Tumor Magnetic Targeting and Imaging Lu Sun a, Shuping Xie a, Jing Qi a, Ergang Liu b, Di Liu a, Quan Liu b, Sunhui Chen a, Huining He a, *, Victor C. Yang a,c, * a. Tianjin Key Laboratory on Technologies Enabling Development of Clinical Therapeutics and Diagnostics (Theranostics), School of Pharmacy, Tianjin Medical University, Tianjin 300070, PR China b. Collaborative Innovation Center of Chemical Science and Engineering, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, PR China c. Department of Pharmaceutical Sciences, College of Pharmacy, University of Michigan, Ann Arbor 48109-1065, USA

KEYWORDS: MMP-2 activatable, His-tagged fusion protein, magnetic targeting, cellpenetrating peptides (CPP), low molecular weight protamine (LMWP), tumor imaging, intracellular protein delivery.

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ABSTRACT: Matrix metalloproteinases (MMP) activatagble imaging probe has been explored for tumor detection. However, activation of the probe is mainly occurred in the extracellular space without intracellular uptake of the probe for more sensitivity. Whilst cell penetrating peptides (CPPs) have been demonstrated to enable intracellular delivery of the imaging probe, they nevertheless encounter off-target delivery of the cargos to normal tissues. Herein, we developed a dual MMP-2-activatable and tumor cell-permeable magnetic nanoprobe to simultaneously achieve selective and intracellular tumor imaging. This novel imaging probe was constructed by self-assembling of a His-tagged fluorescent fusion protein chimera and nickel ferrite nanoparticles via a chelation mechanism. The His-tagged fluorescent protein chimera consisted of a red fluorescent protein mCherry that acted as the fluorophore, the LMWP (Low Molecular Weight Protamine) peptide as a CPP, and the MMP-2 cleavage sequence fused with the hexa-histidine tag, whereas the nickel ferrite nanoparticles functioned as the His-tagged protein binder and also the fluorescent quencher. Both in vitro and in vivo results revealed that this imaging probe would not only remain non-permeable to normal tissues thereby offsetting the non-selective cellular uptake but was also suppressed of fluorescent signals during magnetic tumor targeting in the circulation, primarily due to masking of the CPP activity and quenching of the fluorophore, respectively, by the associated NiFe2O4 nanoparticles. However, these properties were recovered or “turned on” by the action of tumor-associated MMP-2 stimuli, leading to cell penetration of the nanoprobes as well as fluorescence restoration and visualization within the tumor cells. To this regard, the presented tumor-activatable and cell-permeable system deems to be an appealing platform to achieve selective tumor imaging and intracellular protein delivery. Its impact is therefore significant, far-reaching and wide-spread.


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1. INTRODUCTION Tumor imaging techniques have made great progress in modern diagnostics and surgical guidance in the past decades.1-2 However, conventional imaging techniques still suffer from insufficient resolution due to poor sensitivity and specificity of the imaging agents 1. Stimuliresponsive imaging probes, which benefit from signal enhancement in response to stimuli from either internal conditions, such as altered pH or differentially expressed enzyme concentrations in tumor tissues due to physiological or pathological changes, or external triggers including heat, light, magnetic and electrical fields, provide a logical strategy to overcome these problems.3-5 As a subtype of these stimuli-responsive tumor imaging systems, enzyme-activatable imaging systems enhance the specificity by activation and enhancement of the local imaging agent concentration in the tumors via enzymatic reaction.6-8 Among a variety of enzymes, matrix metalloproteinases (MMPs) deem to be valuable candidates for tumor detection and disease controlling. MMPs are zinc-dependent endopeptidases that are reported to be frequently overexpressed in the majority of solid tumors such as breast, colon and prostate cancers.9-11 As a key member of the MMP family, MMP-2 plays a key role in the progression and modulation of tumor-associated angiogenesis, rendering it an important target for tumor diagnosis and therapy.12-15 However, for a system that was responsive to proteases expressed outside the cells or on the cell surface, such as MMP-2, the imaging signal would be restricted outside or around the tumor region but not inside the tumor cells. To enhance the imaging payloads inside the cells, a more powerful cell penetrating helper, such as the cell-penetrating peptides (CPPs) hotly pursued by investigators in recent years, could be utilized to augment their tumor imaging sensitivity.


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CPPs are effective in promoting membrane transportation of the attached cargos including peptides, proteins, ribonucleic acids, mimics of oligonucleotides, quantum dots, or nano-carriers into live cells, as well as the ability for deeper tumor penetration to further enhance the imaging or therapeutic efficacy of these cargos.16-19 However, the lack of tumor selectivity of CPPs hinders them from wider applications, rendering the need of a more safely designed system to avoid the off-targets effects in their future development.20-21 Therefore, a combination of the enzyme responsive system with a CPP-mediated imaging agent delivery could, in principle, overcome the cell membrane barrier of the conventional non-permeable imaging agents as well as the selectivity hurdle of the unmodified CPP-based delivery approaches.8 Magnetic iron oxide nanoparticles (MION) are widely used in imaging and drug delivery systems due to their magnetic-responsive behavior.22 This property allows MION to be guided and held in a desired location by a magnetic field, such as those applied in tumor targeting. Recently, MION was reported to be a promising and highly efficient fluorescence quencher by either covalent or noncovalent fluorophores-loaded strategies in fluorescence assays, and proven to function as an activatable probe scaffold.23-24 even though the mechanism of fluorescence quenching by NiFe2O4 nanoparticle has not been thoroughly investigated yet. Application of fluorescence quenching and magnetic property of MION nanoprobes would thus be a promising combination that used for imaging, especially for more precise detection of the tumor margin for surgical guidance, as well as the residual tumor tissues after surgical excision. Nevertheless, the magnetic nanoparticles scaffold as fluorescence protein quenchers in the imaging system has till now rarely reported. Herein, we reported the development of a readily fabricated, cell-permeable, enzymeactivatable, fluorescent protein-modified and magnetic nanoparticle-based imaging probe to


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enhance tumor targeting efficiency and selectivity (Figure 1). Briefly, this imaging probe was made up of two components. The fluorophore component contained a red fluorescent fusion protein that acted as the signal donor, whereas the other component consisted of the (Ni)modified magnetic nanoparticles that acted as the signal quencher. The protein compartment was genetically engineered to contain a His-tagged fusion protein chimera consisting of the red fluorescent protein mCherry, the LMWP peptide (Low molecular weight protamine, a potent CPP that was developed in our own laboratories), and a MMP-2 (Matrix Metalloproteinases-2)cleavable specific peptide linker. The magnetic component, on the other hand, was synthesized by using nickel (Ni)-modified MION (i.e. nickel ferrite nanoparticles). These two components were self-assembled, via the automatic chelating effect between the nickel ions and hexahistidine tags, into the final imaging probe. It should be noted that the fluorescence signal of mCherry would be quenched by the nickel ferrite nanoparticles, and then amplified when the cleavable linker was hydrolyzed by the endogenous MMP-2 which was specifically overexpressed in the matrix of intracellular space of the tumor tissues. During application, the imaging probe would be guided to and then retained at the tumor target by the influence of an externally applied magnetic field. Owing to the masking effect by the nickel ferrite nanoparticles, LMWP would be deprived of its cell-penetrating ability and remained inactive during circulation for tumor targeting; thereby aborting the off-target internalization of the imaging probes to normal cells. Once reaching the vicinity of the tumor, the protection on CPP would be uncovered due to detachment of the nickel ferrite nanoparticles over cleavage of the specific peptide linker by MMP-2, rendering CPP to be fully exposed thereby mediating a potent yet selective intracellular delivery of the fluorescent probe. In addition, without quenching by the chelated nickel ferrite nanoparticles, the cytosol-delivered mCherry protein would restore its


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red fluorescent activity, yielding intracellular tumor imaging. To this regard, the MMP-2 cleavable linker could be regarded as a switch to automatically turn on the fluorescent probe inside the cells. Both in vitro and in vivo “proof-of-concept” investigations were carried out to confirm the feasibility and success of this innovative tumor imaging system.

Figure 1. Schematic illustration of the imaging process of the activatable magnetic nanoprobes.


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2. EXPERIMENTAL SECTION 2.1 Materials. Restriction enzymes were purchased from New England BioLabs (Ipswich, MA). pET-21b vector was obtained from Merck Millipore (Darmstadt, Germany). Isopropyl-βD-1-thiogalactopyranoside (IPTG), yeast extract, and tryptone were provided by Sigma-Aldrich (Saint Louis, MO). Competent E. coli BL21(DE3)pLysS cells were obtained from CWBIO (Beijing, China). The nickel affinity column was purchased from QIAGEN (Hilden, Germany). PageRuler-prestained protein ladder and RPMI1640 Medium were provided by Thermo Fisher Scientific (Waltham, MA). Phalloidin-iFluor™ 488 Conjugate was purchased from AAT Bioquest (Sunnyvale, CA). DAPI was purchased from Beyotime Biotechnology (Shanghai, China). All the other chemicals were of the highest grade commercially available. 2.2 Cell lines and animals. HT-1080 (human fibrosarcoma) cell lines were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). BALB/c nude mice (4 weeks old) and KM mice (18-22 g) were obtained from Chinese Academy of Military Medical Sciences (Beijing, China). All animal experiments were carried out according to the Animal Management Rules of the Ministry of Health of the People’s Republic of China. 2.3 Plasmid DNA construction. A fusion protein composed of mCherry, LMWP (VSRRRRRRGGRRRR) and MMP-2 cleavable site (PLGVR) was designed. The DNA fragment encoding this fusion protein was produced via gene synthesis by GENEWIZ. Inc. (Jiangsu, China), then cloned into pET-21b vector through BamH I and Hind III sites. The insert was confirmed by sequencing. 2.4 Expression and purification of fusion proteins. The plasmids with target sequence were transformed into E. coli expression strain BL21(DE3)pLysS. For expression of fusion proteins,


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cells were cultured with 2×YT media with 100 µg/mL ampicillin at 37°C. When the optical density at 600 nm reached 0.6-0.8, cells were induced for 6 h at 24°C by adding IPTG to a final concentration of 0.5 mM. After induction, cells were harvested. Purification of protein was carried out as flowing steps: First, cell pellet was suspended in lysis buffer (20 mM Tris-Cl, 500 mM NaCl, 5 mM imidazole, pH 8.0) at 1 mL per gram wet weight, and then was lysed by sonication in ice bath. After that, the cell lysate was centrifuged at 12,000 rpm for 1h and the supernatant was loaded onto Ni-NTA resin pre-equilibrated with lysis buffer. The impurities were washed with 20 mM Tris-Cl, 500 mM NaCl, 80 mM imidazole, pH 8.0, and the target protein was eluted with 20 mM Tris-Cl, 500 mM NaCl, 250 mM imidazole, pH 8.0. The eluted protein was concentrated and exchanged into buffer (20 mM Tris-Cl, 150 mM NaCl, pH 8.0) with Microsep-centrifugal device, MWCO at 3 kDa. For animal studies, the target protein was further purified by HisTrap HP affinity resin according to the manufacturer’s protocol. 2.5 Preparation of NiFe2O4 magnetic nanoparticles. NiFe2O4 magnetic nanoparticles were synthesized by a hydrothermal process using nickel chloride hexahydrate (NiCl26H2O) and iron chloride hexahydrate (FeCl36H2O) according to the reported procedure.25-26 Briefly, 1.08 g (1.5 mmol) of NiCl26H2O and 2.43 g (3 mmol) of FeCl36H2O were dissolved in 90 mL of ethylene glycol and stirred to form a clear solution. Then, 2.16 g of sodium acetate was added to the mixture, and was stirred vigorously for 30 min. After that, 0.9 g of KNO3 was added and stirred for another 10 min. Finally, the solution was sealed in a teflonlined stainless steel autoclave (100 mL capacity). The autoclave was heated at 200°C for 16 h and then naturally cooled to room temperature. The final products were then washed with ethanol for several times and dried at 60°C for 10 h.


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Fe3O4 magnetic nanoparticles used as a control in the experiment were synthesized following the same procedure without NiCl26H2O. 2.6 Characterization of NiFe2O4 magnetic nanoparticles. X-ray diffraction (XRD) of powder samples was made using a Riguka D/MAX-2500 equipped with copper anodes. The Xray source operated at 40 kV and 100 mA, and the 2θ scan was performed in the 10° < 2θ < 80° range each 0.02° with scan step speed of 0.15 s. TEM and energy-dispersive X-ray spectrometry (EDX) were obtained using a FEI Tecnai G2 F20 microscope at an acceleration voltage of 200 kV and equipped with a Genesis system EDAX spectrometer (EDAX, USA). For preparing the samples, NiFe2O4 magnetic nanoparticles dispersed in pure water were dropped-cast onto copper grid. Size analysis of NiFe2O4 magnetic nanoparticles were performed using Zetasizer NanoZS90 (Malvern, UK). A magnetic property measurement was carried out using a superconducting quantum interference device (SQUID) magnetometer (Quantum Design, MPMS XL-7). 2.7 MMP-2 Assay. Recombinant human MMP-2 was expressed and purified according to the reported procedure.27 Equal amount of fusion protein modified NiFe2O4 magnetic nanoparticles were incubated with recombinant human MMP-2 separately in the assay buffer (50 mM TrisHCl pH 8.0, 150 mM NaCl, 10 mM CaCl2, 0.1 mM ZnCl2, 0.05% Brij35), at 37°C. Fusion protein modified NiFe2O4 magnetic nanoparticles incubated without recombinant human MMP-2 were used as controls. The fluorescence intensity of supernatant was recorded from 30 min to overnight. 2.8 Cell culture and tumor-bearing mice. The human fibrosarcoma cells (HT-1080) cultured in 10% FBS RPMI1640 Medium in a well-humidified incubator with 5% CO2 and 95% air at


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37°C. The suspensions of HT-1080 cells (1×106 cells in PBS) were subcutaneously transplanted into the right flank of nude mice. Tumor volume was calculated as 0.5×length×width2. 2.9 Ex vivo tumor imaging. The nanoprobes/control were administered to tumor-xenograft mice via tail vein injection. As described previously, four mice with similar size of tumor were divided into two experimental groups: the magnet treatment group and the one without treatment. In each group, one mouse was injected with rCCMH-bound imaging probes whereas the other mouse was injected with the rCCMH-N-bound nanoprobes to serve as the control. Twenty four hours post-injection, the tumors were collected for cryosection. Then the slides were stained with DAPI and Phalloidin-iFluor 488 Conjugate. The fluorescence was observed with a confocal microscopy (Olympus, Japan). 2.10 Optical in vivo imaging. When the average tumor volume reached approximately 0.51cm3, four mice with similar size of tumor were chose and divided into two experimental groups: the magnet treatment group and the one without treatment. In each group, one mouse was injected with rCCMH-bound imaging probes whereas the other mouse was injected with the rCCMH-N-bound nanoprobes to serve as the control. The tumor-bearing mice were intratumoral injected with 10 nmol of probe/control and an IVIS spectrum imaging system (Perkin Elmer, Massachusetts, USA) was used to detect the mCherry fluorescence (Ex/Em = 570/620 nm). During the imaging, the mice were anesthetized with 2.5% isoflurane gas in the oxygen flow (1.5 L/min). Images were analyzed using Living Image ® 4.3.1 software (Xenogen). 2.11 In vitro and in vivo biosafety study of the nanoprobes. The cytotoxicity was studied in non-tumoral cell lines of normal human umbilical vein endothelial cell (HUVEC). The cell viability was measured by a standard MTT assay.


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Thirty two KM mice (18-22 g) were divided into four groups - the blank, NiFe2O4 nanoparticles, His-tagged fusion protein and nanoprobe groups - for preliminary safety evaluation. The body weight changes, organ coefficient were recorded, and major organs were processed by histological examination.

3. RESULTS AND DISCUSSION 3.1 Production and Characterization of the Fluorescent His-tagged Fusion Proteins. Two hexa-histidine tagged (His-tag) fusion proteins, both possessed a calculated molecular weight of 32.3 kDa and termed rCCMH and rCCMH-N, respectively, were produced via recombinant technology and purified using a Ni-NTA column. Briefly, rCCMH is acronyms of the recombinant, mCherry, Cell penetrating peptide, MMP-2 cleavable sequence and His-tagged protein chimera, whereas rCCMH-N is acronyms of the recombinant, mCherry, Cell penetrating peptide, mutated MMP-2 cleavable sequence, His-tagged but non-cleavable (by MMP-2) protein chimera. The rCCMH protein chimera was expressed to contain the red fluorescent protein mCherry, the LMWP peptide (a CPP), and the specific cleavage site for MMP-2. On the other hand, the rCCMH-N, where the MMP-2 cleavage site (PLGVR) on rCCMH was mutated to PLGKL, was also produced as a control protein. It was demonstrated via kinetic analyses that the PLGKL sequence was not recognized by MMP-2.28 Both of these fusion proteins contained a His-tag at the C-terminal to allow for their chelation with the NiFe2O4 magnetic nanoparticles. Successful expression of these two fusion proteins and their purification via a NI-NTA column was validated by SDS-PAGE and western blot assay (Figure 2). A dense protein band with molecular weight of about 32kDa, representing the rCCMH and rCCMH-N fusion protein (Lane 1 & 2 in Fig. 2, respectively) was observed on the SDS-PAGE. It was reported in the literature


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that the C=N bond formed by Phe70 and Met71 in mCherry would be hydrolyzed upon boiling.29-30 In agreement with findings by other investigators,31 our fusion mCherry protein displayed two fragment bands; one was at about 22 kDa whereas the other fragment was at about 10 kDa. The presence of a His-tag at the C-terminus for these fusion proteins was also confirmed by western blot assay using an anti-His tag antibody (Lane 3 & 4; Figure 2).

Figure 2. Expression and purification of fusion proteins. SDS-PAGE and western blot assay results for rCCMH and rCCMH-N. Lane M: markers of the protein molecular weight standard. Lane 1 and 2 represented proteins purified using a Ni-NTA column, whereas Lane 3 and 4 denoted the western blot results for rCCMH (1 & 3)and rCCMH-N (2 & 4), respectively. 3.2 Characterization of the Nickel Ferrite (NiFe2O4) Magnetic Nanoparticles. Characterization of the Nickel Ferrite (NiFe2O4) Magnetic Nanoparticles was shown in Figure 3. The crystalline structures of NiFe2O4 magnetic nanoparticles were examined using XRD, as shown in Figure 3A. The x-ray diffraction pattern was consistent with that of the standard nickel ferrite indexed in JCPDS 86–2267. The EDX spectrum (Figure 3B) also displayed the presence of both iron and nickel elements in the sample. These results confirmed that synthesis of the


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NiFe2O4 magnetic nanoparticles were successfully, with Ni ions being available on the surface of these particles to allow for chelation with the His-tagged proteins. The size and shape of the nanoparticles were examined by DLS assay and TEM (Figure 3C & 3D). The hydrodynamic diameter of NiFe2O4 magnetic nanoparticles was 131.2 ± 0.9 nm in aqueous solution based on the DLS assay. The TEM images revealed that the average size of the NiFe2O4 magnetic nanoparticles was about 80.3 ± 3.0 nm. The magnetic properties were examined using the superconducting quantum interference device (i.e. the SQUID magnetometer) at room temperature. As shown in Figure 3E, the magnetic hysteresis loop of the NiFe2O4 magnetic nanoparticles yielded a relatively high magnetic saturation value (47.51 emu/g) and superparamagnetic behavior. These properties confirmed that the NiFe2O4 magnetic nanoparticles were suitable for magnetic targeting.


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Figure 3. Characterization of the NiFe2O4 magnetic nanoparticles. (A) XRD patterns and (B) EDX spectrum of the synthesized NiFe2O4 magnetic nanoparticle; (C) Size distribution and (D) TEM micrograph of the NiFe2O4 magnetic nanoparticles; (E) The magnetic hysteresis loop displayed by the synthesized NiFe2O4 magnetic nanoparticles. 3.3 Binding Capacity of His-tagged Protein on NiFe2O4 Magnetic Nanoparticles. The binding property of synthesized NiFe2O4 magnetic nanoparticles was examined by incubating 500 µg of such nanoparticles with various amounts, ranging from 10 to 300 µg of the His-tagged rCCMH protein. After 2 h of incubation at room temperature, the NiFe2O4 nanoparticles were separated from the supernatant by applying an external magnet, according to the procedures described by Lee and co-workers.25 The binding capacity of the magnetic nanoparticles was then calculated by measuring the reduction in fluorescence intensity in the supernatant. Results in Figure 4 showed that the amount of bound rCCMH reached a plateau at an input protein dose about 200 µg, yielding a binding capacity of approximately 155 µg of the His-tagged rCCMH would bind to 500 µg of the synthesized NiFe2O4 magnetic nanoparticles.


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Figure 4. Binding capacity of the His-tagged rCCMH protein on the NiFe2O4 magnetic nanoparticles under an identical dose of 500 µg of the synthesized nanoparticles. 3.4 Binding Specificity of His-tagged Protein on NiFe2O4 Magnetic Nanoparticles. The specific binding affinity between His-tagged rCCMH and NiFe2O4-based magnetic nanoparticles was characterized and compared with the control consisting of Fe3O4-based nanoparticles and normal BSA protein without the His-tag modification. Equal amount of NiFe2O4- and Fe3O4based nanoparticles were separately incubated with the same amount of His-tagged rCCMH at room temperature, and the supernatants from these two reaction mixtures were isolated following incubation by using an external magnet. Experimental results in Figure 5A demonstrated that the NiFe2O4-based nanoparticles yielded a significantly enhanced binding capacity to the His-tagged rCCMH than that by the Fe3O4-based magnetic nanoparticles, presumably due to the chelating effect between the Ni2+ ions on NiFe2O4 and the His-tags on rCCMH. It should be pointed out


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that the reduced fluorescent intensity of rCCMH incubated with Fe3O4-based magnetic nanoparticles was probably caused by the non-specific binding of proteins to the nanoparticles. A measurement of the fluorescent intensity of rCCMH incubated with Fe3O4-based magnetic nanoparticles was also conducted, and result was included in Figure S1 in the Supporting Information. These findings suggested that the chelating binding was far superior to the physical adsorption between the nanoparticles and rCCMH. In addition, the bound His-tagged protein on NiFe2O4-based nanoparticles could be eluted using a 1.5 M imidazole solution, as Figure 5B displayed an incubation time-dependent, continuous increase in fluorescence intensity in the imidazole eluents. Furthermore, SDS-PAGE results showed while both the His-tagged rCCMH and rCCMH-N proteins were completely pulled down by the NiFe2O4 nanoparticles thereby leaving nothing in the supernatants (Lane 4 & 5 in Figure 5C, respectively). On the other hand, native BSA without His-tag modification, which served as the control protein, was hardly bound to the NiFe2O4 nanoparticles and thus remained in the supernatant following incubation (Lane 6, Figure 5C). Overall, all of these findings confirmed that NiFe2O4 magnetic nanoparticles could be used as a carrier for the His-tag proteins via the chelation effect between Ni2+ ions on NiFe2O4 and the His-tags on rCCMH and rCCMH-N. It should be noted that the coordination between Ni2+ and His-tag has been widely and conventionally applied for affinity purification of His-tagged recombinant proteins, especially in the area of immobilized metal affinity chromatography (IMAC) involving nickel ion charged nitriletriacetic acid adsorbent (Ni-NTA) to greatly facilitate the purification process. Whilst application of Ni-bound iron oxide nanoparticles on separation of His-tagged proteins have been reported in the literature,25, 32 our system offers a unique and innovative utilization of this coordination science on the design and assembly of


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protein-based delivery systems and imaging probes. To this regard, the impact of the presented technology is far-reaching and wide-spread.

Figure 5. Specific binding affinity between NiFe2O4 magnetic nanoparticles and His-tagged rCCMH and/or rCCMH-N proteins. (A) Fluorescent images of His-tagged rCCMH in the supernatants before (Lane 1) and after treatment with Fe3O4 nanoparticles (Lane 2) or NiFe2O4 magnetic nanoparticles (Lane 3), respectively; (B) Fluorescence spectra of solutions after eluting the protein-bound NiFe2O4 nanoparticles with 1.5 M imidazole solution for various incubation


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times of 1 min (red line) and 30 min (blue line). Dark line represented the emission intensity of 1.5 M imidazole solution; (C) SDS-PAGE results of control solutions (i.e. without treatment with NiFe2O4 nanoparticles) containing His-tagged rCCMH (Lane 1), rCCMH-N (Lane 2) and native BSA without His-tag modification (Lane 3), as well as the supernatants of rCCMH (Lane 4), rCCMH (Lane 5) and BSA (Lane 6), respectively, after treatment with NiFe2O4 nanoparticles. 3.5 Fluorescence Quenching of mCherry Induced by NiFe2O4 Magnetic Nanoparticles. Magnetic iron oxide nanoparticles (MION) were widely used in imaging and drug delivery systems for their magnetic behavior which could be guided and retained in a desired location under the influence of an external magnetic field. Recently, MION was also being shown to function as a highly efficient quencher towards a variety of fluorophores 23-24. Hence, application of magnetic nanoparticles in our imaging system would render the probe to be capable for both tumor targeting based on their magnetic property and on/off signal switching in accordance to their quenching ability. The mechanism of fluorescence quenching by NiFe2O4 nanoparticle has not yet been thoroughly investigated. However, it does not appear to be a FRET-based quenching effect. Thus far, a variety of models have been explored to explain the quenching mechanism when nano-sized metal is being used as the acceptor. Most of these studies have been mainly focused on gold nanoparticles (AuNP), which possess a high fluorescence quenching efficiency. At this moment, the nano-metal surface energy transfer (NSET) model is most widely accepted to explain AuNP quenching over a broad spectrum of fluorophores and over a wide range of distances between the dye and AuNP.33-34 Yet, this model still needs rigorous testing to further validate the fluorescence energy transfer mechanism involving NiFe2O4 nanoparticles. Since the NiFe2O4 magnetic nanoparticle were utilized as a quencher in our designed probe, their fluorescence quenching efficiency were examined by titrating a fixed concentration of


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fluorescent rCCMH with various amounts of the NiFe2O4 nanoparticle suspension. Based on results shown in Figure 6, the fluorescence intensity of rCCMH (10µg/mL) was markedly reduced when increasing the dose of the NiFe2O4 nanoparticles from 0 to 0.107 mg/mL, and then dropped to the minimum level of being almost completely quenched when concentration of the nanoparticles reached 0.083 mg/mL. Hence, the optimal concentration ratio of mCherry/NiFe2O4 for quenching was 1.0:8.3, and this concentration ratio of 1.0:8.3 was selected as the optimal ratio for subsequent tumor imaging studies.

Figure 6. Fluorescence intensity of rCCMH in response to titration with various concentrations of NiFe2O4 nanoparticle suspensions. The rCCMH concentration was fixed at 10 µg/mL, whereas the doses of the NiFe2O4 nanoparticle suspensions was gradually increased to: (a) 0; (b)


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0.0067; (c) 0.013; (d) 0.019; (e) 0.026; (f ) 0.033; (g) 0.039; (h) 0.046; (i) 0.052; (j) 0.058; (k) 0.071; (l) 0.083; (m) 0.095; (n) 0.107 mg/mL. The inset displayed the fluorescence emission spectra of rCCMH under titration with various concentrations of the NiFe2O4 suspensions ranging from (a) to (n). 3.6 Characterization of the Fabricated Fluorescent Imaging Probe. Based on results from the fluorescence quenching experiments, the rCCMH:NiFe2O4 concentration ratio of 1.0:8.3 was adopted to fabricate our imaging probe via the aforementioned chelation-mediated self-assembly process. Morphology of this imaging probe was examined by both DLS assay and transmission electron microscopy (TEM). As shown in Figure 7A & 7B, The hydrodynamic diameter of the fabricated probes were about 192.8 ± 5.3 nm in size in aqueous solution based on the DLS assay, while TEM images revealed that the average size of the fabricated probes were about 86.5±5.6 nm, slightly larger than the bare NiFe2O4 magnetic nanoparticles without rCCMH attachment. The display of nitrogen element in the EDX spectrum in Figure 7C confirmed the presence of rCCMH in the imaging probe. These results were in agreement with previous findings, and further confirmed that His-tagged rCCMH was chelated to the surface of the NiFe2O4 magnetic nanoparticles through Ni-mediated coordination binding. It should be pointed out that presence of the Cu peaks in the EDX spectrum were from the copper grids that were used to support the samples for TEM examination. On the other hand, the presence of small Si peaks in the spectrum was not from our sample specimens, since the same peak existed in the EDX spectrum of brand new copper grids without applying any sample specimen, as shown in the EDM spectrum (data not included). To this regard, this Si peak might be attributed to the internal fluorescence peak from the Si dead layer of the Si-Li detector. As reported in the literature,35 such a Si peak would appear in the spectra when a Si K X-ray was


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generated in the Si dead layer at the front of the detector. Consequently, Si would enter the active region and was then being detected.

Figure 7. Characterization of the fabricated fluorescent imaging probe. (A) Size distribution and (B) TEM micrograph of rCCMH-attached NiFe2O4 nanoparticles; (C) EDX spectrum displayed the presence of nitrogen element in the sample.


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3.7 In Vitro Activation of the Fluorescent Imaging Probe by Recombinant MMP-2 Protease. In our designed imaging system, the fluorescence signal of mCherry would be quenched by the nickel ferrite nanoparticles, and subsequently be amplified when the cleavable linker was hydrolyzed by the MMP-2 protease which was specifically overexpressed in the matrix of the intracellular space of tumor tissues. To examine whether our fabricated probe could be activated by MMP-2, the nanoprobes were evaluated in vitro via the use of human recombinant MMP-2. Briefly, equal amount of rCCMH and rCCMH-N-bound NiFe2O4 nanoparticles, whereas the later served as the control, were incubated with recombinant human MMP-2 in the assay buffer. Fluorescence intensity in the supernatant was then measured at various time points ranging from 30 min to overnight. The fluorescence intensity in the samples without the addition of MMP-2 was used as the background intensity. As shown in Figure 8, supernatant from the control (i.e. rCCMH-N-bound nanoparticles) exhibited a statistically insignificant change in fluorescence intensity following incubation with MMP-2 at various time periods; presumably due to mutation of the MMP-2 cleavable sequence in the probes, rendering them non-cleavable to MMP-2. In sharp contrast, the rCCMH-bound probes displayed a far more significant yet time-dependent increase in fluorescence intensity in the supernatant or, in other words, in the release of the fluorescent protein following incubation with MMP-2. These results confirmed our hypothesis that the designed fluorescent was responsive to the enzymatic activity of MMP-2, and release of the fluorescence signal could be “turned on” by the MMP-based protease switch.


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Figure 8. Fluorescence intensity in the supernatant following incubation of the recombinant human MMP-2 with rCCMH-bound NiFe2O4 nanoparticles (red color) or rCCMH-N-bound NiFe2O4 nanoparticles (blue color). Nanoparticles were incubated with MMP-2 in the assay buffer at 37°C, and at various incubation times fluorescence intensity in the supernatant was recorded. The assays were repeated three times, and data are shown as the mean ± SD, n.s.: not significant, asterisks (*) represented statistically significant (*p < 0.05, **p < 0.01, ***p < 0.001). Flow cytometric analysis was carried out to demonstrate the cell-penetrating ability of the synthesized nanoprobes. In brief, the cells were exposed to the medium containing the nanoprobes pre-activated by the recombinant human MMP-2. After thorough wash with PBS, the


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cells were collected and subjected to flow cytometric analysis. As shown in the Figure S2 in the Supporting Information, the flow cytometric analysis results clearly indicated that cellular uptake of nanoprobes was significantly higher than the control nanoprobes, due to the aid of the cell penetrating peptide LMWP. 3.8 In Vivo Activation of the Nanoprobe by MMP-2 Leads to Tumor Cell Penetration of mCherry. According to principle of our designed imaging probe, the fluorescence signal of mCherry would be quenched by the nickel ferrite nanoparticles, and then amplified when the cleavable linker was hydrolyzed by the endogenous MMP-2 which was specifically overexpressed in the tumor tissues. Meanwhile, during application, the imaging probe would be guided to and then retained at the tumor target by the influence of an externally applied magnetic field. As described previously in Figure 1, owing to the masking effect by the NiFe2O4 nanoparticles, LMWP was deprived of its cell-penetrating ability and remained inactive during circulation for tumor targeting; thereby aborting the off-target internalization of the nanoprobes into normal cells. Once reaching the tumor target, however, protection on LMWP would be uncovered due to detachment of the NiFe2O4 nanoparticles by cleavage of the peptide linker via MMP-2, rendering CPP on the mCherry protein to be fully exposed, thereby mediating a potent intracellular delivery of the fluorescent protein. Furthermore, without quenching by the NiFe2O4 nanoparticles, the cytosol-delivered mCherry protein would restore its red fluorescent activity, yielding cellular tumor imaging. To verify feasibility and success of our tumor imaging system, the nanoprobes were administered to tumor-xenograft mice via tail vein injection. Four mice were divided into two groups: one group was being applied with an external magnetic field around the tumor region, whereas the other group was without magnetic treatment. In each group, one mouse was injected


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with the rCCMH-bound imaging probes (20nmol of His-tagged protein was used for probe fabrication) whereas the other mouse was injected with the rCCMH-N-bound nanoprobes to serve as the control. Twenty four hours post-injection, mice were sacrificed, and tumor tissues were collected for cryosection process. The tissue slices were stained for F-actin and cell nuclei and then observed using fluorescence microscopy. It should be pointed out that although flow cytometric analysis was presumably the most conventional method to demonstrate the cellpenetrating ability of the synthesized nanoprobes, in our case, however, confocal immunofluorescence microscopy deemed to be a better choice to validate this behavior of the nanoprobes. As noted, cationic CPPs such as LMWP used in our experiments are known to bind to the outside surface of the cell membrane, and can thus lead to a false positive conclusion; as fluorescence analysis is not fully capable of discriminating the cell-internalized signals from those coming from the surface-bound CPP-mCherry conjugates.36-37 As shown in Figure 9A, rCCMH-bound imaging probes were activated by the overexpressed MMP-2 of the tumor, leading to the restoration of mCherry fluorescence in the tumor. In addition, accumulation of rCCMH-bound imaging probes in the tumor was significantly enhanced under the influence of external magnetic field in the magnet treatment group. On the other hand, the fluorescent activity of rCCMH-N-bound probes was relatively inactive due to the mutation of MMP-2 cleavage sites, rendering it non-cleavable to MMP-2. Once the rCCMH-bound probes were activated, demasking of CPP occurred, leading to intracellular delivery of the fluorescent protein into the cells. High-magnification confocal immunofluorescence images of the tumor tissues obtained from the magnet-treated, rCCMH-bound probes showed that the mCherry fluorescence signal (red) was overlapped with iFluor 488 signal (green) which indicated the localization of cytoskeleton,


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and partially with DAPI signal which indicated the localization of nucleus (Figure 9B). Since the LMWP was inserted at the C-terminal of the mCherry protein during recombinant conjugation, the cell-penetrating functions of LMWP in the recombinant protein chimera would not affected. As shown from results in Figure 9B, the LMWP-conjugated mCherry chimera was localized in the cytosol and nucleus, indicating a similar localization pattern as FITC-labeled LMWP; a finding that was reported previously by our research group.38 Overall, these findings revealed that the rCCMH-bound nanoprobes could be activated by tumor-secreted MMP-2, releasing mCherry which was subsequently delivered into the cells due to the aid of CPP. In addition, restoration and retention of the red fluorescent mCherry at or inside the tumor occurred due to influence of the external magnetic field, yielding intracellular tumor imaging. Taken together, these findings suggested the fundamental plausibility of tumor imaging and drug delivery by our nanoprobe-based platform. As noted, the effect of the external magnetic field was for both targeting and retention of the nanoprobes at the tumor target. Unfortunately, because of the exceedingly rapid clearance of both the mCherry protein39 and the iron oxide magnetic nanoparticles,40 the events of tumorspecific targeting and activation of the nanoprobes, as well as intracellular delivery of these fluorescent nanoprobes could not be visually identified from the “whole body” fluorescence images of the mice, whether the experiments were carried out with or without applying an external magnetic field. This was primarily due to an exceedingly low accumulation of the nanoprobes at the tumor target, because the large majority of the administered nanoprobes were cleared from the circulation shortly following their tail vein injection. To visually verify these important events, intratumoral injection of the nanoprobes combined with a magnetic-induced


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nanoprobe retention at the tumor region, as discussed in the next section below, became a worthwhile alternative to significant magnify tumor accumulation of the nanoprobes.


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Figure 9. Subcellular distribution of probe in HT-1080 cells after tail vein injection. (A) The spatial distribution of fluorescence for MMP-2 activatable probe in frozen tissue slices 24 hours after injection. The slides were stained for F-actin and cell nuclei with Phalloidin-iFluor 488 Conjugate (green) and DAPI (blue), mCherry fluorescence was shown in red. (B) Highmagnification confocal immunofluorescence microscopy photographs of magnet treatment and rCCMH-bound probes injected mouse tumor tissue. Original magnification: 1000×. 3.9 In Vivo Tumor-Activated Fluorescent Imaging by the Designed Probe. Although preliminary proof-of-concept in vivo studies have been carried out to assess the feasibility of the probe in tumor magnetic targeting and imaging, tumor imaging need to go through a cryosection process, this may due to the unperfect accumulation of probes in tumor site. In vivo tumoractivated fluorescent imaging of the nanoprobes was further confirmed via intratumoral injection to improve the concentration of probes accumulated in the tumor. Briefly, tumor-bearing mice were subjected to the IVIS imaging system following intratumoral injection of the nanoprobes (10nmol of His-tagged protein was used for probe fabrication), and fluorescent images were acquired 0, 1, 4, 6, 24 h post-injection. Four mice were divided into two groups: one group was being applied with an external magnetic field around the tumor region, whereas the other group was without magnetic treatment. In addition, in each group, one mouse was injected with the


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rCCMH-bound imaging probes whereas the other mouse was injected with the rCCMH-N-bound nanoprobes to serve as the control, of which the fluorescent protein contained a mutated site that was non-cleavable to MMP-2; as described previously. As shown in Figure 10A & 10B, whilst the fluorescent intensity was consistently increased over the incubation time, it nevertheless reached the maximum level after 24 hours of incubation; suggesting that nanoprobes were continuously accumulated at the tumor region in a time-dependent manner, and were then rapidly activated by the overexpressed MMP-2 of the tumor. Furthermore, activation of the probes deemed to be more effective in the presence of an external magnetic field, suggesting an enhanced retention and accumulation of the magnetic nanoparticles due to the aid from magnetic attraction. These findings further confirmed the in vivo feasibility of our designed nanoprobes for both magnetic targeting and tumor-activated fluorescent imaging. The presence of fluorescence signals in the animal group with intratumoral injection of the non-activatable(-)rCCMH-N-bound nanoprobes seen in Figure 10A could be accounted for in terms of two reasons. First, the control nanoprobes might be activated non-specifically by other proteases from the MMP family. It was reported that MMPs displayed overlapping substrate cleavage preference when there were high sequence similarity or comparable three-dimensional structures among such substrates.41-43 Although it was reported that MMP-2 was overexpressed in HT-1080, and the mutated MMP-2 cleavage site of the control nanoprobes was presumably non-cleavable by MMP-2, the rCCMH-N-bound control nanoprobes could still be hydrolyzed by other MMP family members expressed in the HT-1080 cells.44 Secondly, our results indicated that the chelating effect between the Ni2+ ions on NiFe2O4 and the His-tags on the fusion protein was superior to the physical adsorption between the mCherry protein chimera and the magnetic nanoparticles, and thus the latter was relatively unstable, resulting in an unexpected release of the


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fluorescent mCherry at the tumor target following administration of the rCCMH-N-bound nanoprobes. All of these events would lead to a relatively high background in the imaging.


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Figure 10. In vivo imaging of HT-1080 tumor-bearing nude mice after intratumoral injection of the nanoprobes. (A) Fluorescence images at various time points after intratumoral injection. Fluorescent images were acquired 0, 1, 4, 6, 24 h post-injection. One group was being applied with an external magnetic field (+), whereas the other group was without magnetic field (-). In addition, in each group, one mouse was injected with MMP-2 activatable (+) rCCMH-bound imaging probes, whereas the other mouse was injected with the non-activatable (-) rCCMH-Nbound nanoprobes to serve as the control. (B) The radiant efficiency in the tumor sites. 3.10 Preliminary Safety Evaluation of the Nanoprobe. Since the cytotoxicity of nanoprobe is a priority consideration for biomedical applications. The biocompatibility of the activatable magnetic nanoprobes was investigated both in vitro and in vivo. The biocompatibility of the nanoprobe was examined with non-tumoral HUVEC cell line in vitro. The results indicated that the fabricated nanoprobes exhibited no significant inhibitory effect on cell growth, and cell viability at 200 µg/mL was all above 70% (Figure 11A). The biocompatibility in vivo was further performed on KM mice. Dosage used in the each injection was 13.6 mg/kg (protein), and the animals were given two injections in 2 weeks. As shown in Figure 11B, the nanoprobe had no significant effect on body weight compared to the blank group. At the endpoint, the organ coefficients indicated no significant changes in the heart, liver, lung and kidney between the NiFe2O4 nanoparticles treated group, protein treated group and nanoprobe group, compared with the blank group (Figure 11C). And being processed by the histopathological examination, the H&E stained images of the heart, lung, kidney, liver and spleen demonstrated that tissue structures of main organs (heart, liver, spleen, lung and kidney) of mice in experimental groups displayed no distinguishable changes comparison with the


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control group after intravenous injection of nanoprobes (Figure 11D). All these results confirmed the good biocompatibility of fabricated nanoprobe for application in vivo.


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Figure 11. Preliminary toxicity assessment of the nanoprobes. Cell viability studies with HUVEC cells. (B) The change of the body weight over the regimen. (C) The organ coefficients after treatment. (D) Histological examination of heart, liver, spleen, lung, and kidney.

4. CONCLUSIONS The MMP-2 protease plays a pivotal role in the multistep processes of tumor growth, invasion and metastasis, including proteolytic degradation of the extracellular matrix (ECM), alteration of the cell-cell and cell-ECM interactions, migration and angiogenesis. Therefore, development of a MMP-2-activatable fluorescent probe undoubtedly would significantly augment the sensitivity, selectivity and specificity of the probe for tumor detection and imaging. Normally, tumoractivatable probes could accumulate at the tumor target by the passive enhanced permeability and retention (EPR) effect via the leaky vasculatures of the growing tumor.45-46 However, most of the nanoprobes would be restricted outside or at the interstitial space of the tumor cell (i.e. extracellular accumulation), which could be rapidly cleared from the bloodstream after protease activation. To this regard, incorporation of the so-called cell penetrating peptides, such as the LMWP peptide used in this paper, to the imaging probes would provide them with unique capability to cross the cell membrane, rendering these probes suitable for specific and selective intracellular tumor imaging. In this paper, we presented an innovative, magnetic-targeted, cell-permeable, tumor activatable and revivable fluorescent imaging probe. This nanoprobe was constructed by self-assembling, via a chelation mechanism, of His-tagged fluorescent fusion protein chimera and NiFe2O4-based magnetic nanoparticles. The fusion protein chimera consisted of a red fluorescent protein mCherry, a CPP (i.e. LMWP) and the specific cleavage site for MMP-2. Both in vitro and in vivo


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results revealed that this imaging probe would not only remain non-permeable to normal tissues thereby offsetting the non-selective cellular uptake but was also suppressed of fluorescent signals during magnetic tumor targeting in the circulation, primarily due to masking of the CPP activity and quenching of the fluorophore, respectively, by the associated NiFe2O4 nanoparticles. However, these properties were recovered or “turned on” by the action of tumor-associated MMP-2 stimuli, leading to cell penetration of the nanoprobes as well as fluorescence restoration and visualization within the tumor cells. In the in vivo studies, when the nanoprobes were administered to tumor-xenograft mice via tail vein injection, tumor imaging need to go through a cryosection process, this may due to the unperfect accumulation of probes in tumor site that would be improved by prolong the circulation time via PEGylation.47-48 We are now optimizing of the probe for further application. Nevertheless, this target-specific on-off property can be a useful tool for cancer imaging and drug delivery. Since poor cell permeability and lack of targeting selectivity are two primary hurdles encountered when using protein-based therapeutics, our tumor-activatable and CPPmediated cell-entry system deems to be an appealing platform to achieve selective tumor imaging and intracellular protein delivery. To this regard, the impact of this technology is significant, far-reaching and wide-spread.

ASSOCIATED CONTENT Supporting Information.


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Fluorescence intensity of rCCMH in response to titration with various concentrations of Fe3O4 nanoparticle suspensions and flow cytometric analysis of cell uptake (PDF).

AUTHOR INFORMATION Corresponding Author E-mail address: [email protected] (Victor C. Yang) E-mail address: [email protected] (Huining He)

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT This work was supported in part by the National Natural Science Foundation of China (21402143, 81402856 and 81361140344). This research was also partially sponsored by Tianjin Municipal Science and Technology Commission (15JCQNJC13600 and 15JCYBJC28700) and National Key Research and Development Plan (2016YFE0119200).

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Cell-Permeable, MMP-2 Activatable, Nickel Ferrite and His-Tagged

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