Activatable T1 Relaxivity Recovery Nanoconjugates for Kinetic and

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Activatable T1 Relaxivity Recovery Nanoconjugates for Kinetic and Sensitive Analysis of Matrix Metalloprotease 2 Xianglong Zhu, Hongyu Lin, Lirong Wang, Xiaoxue Tang, Lengceng Ma, Zhong Chen, and Jinhao Gao ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b05389 • Publication Date (Web): 12 Jun 2017 Downloaded from http://pubs.acs.org on June 14, 2017

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ACS Applied Materials & Interfaces

Activatable T1 Relaxivity Recovery Nanoconjugates for Kinetic and Sensitive Analysis of Matrix Metalloprotease 2

Xianglong Zhu,†,‡,# Hongyu Lin,†,# Lirong Wang,†,# Xiaoxue Tang,† Lengceng Ma,§ Zhong Chen,§ and Jinhao Gao*,†

†

State Key Laboratory of Physical Chemistry of Solid Surfaces, The MOE Key Laboratory of

Spectrochemical Analysis & Instrumentation, The Key Laboratory for Chemical Biology of Fujian Province, and Department of Chemical Biology, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen, Fujian 361005, China. ‡

College of Chemistry and Chemical Engineering, Xinyang Normal University, Xinyang, Henan

464000, China. §

Department of Electronic Science and Fujian Key Laboratory of Plasma and Magnetic Resonance,

Xiamen University, Xiamen 361005, China.

*Email address: [email protected]

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ABSTRACT Sensitive detection of Matrix metalloproteinase 2 (MMP-2, an important cancer marker associated with tumor invasion and metastasis) activity in vitro and at cellular level is of great significance to clinical diagnosis and medical treatment. With unique physical properties, nanoparticles are emerging as a platform to construct conjugates of various biological molecules, which can be expected to generate new types of biosensors. In this work, Fe3O4 NPs were modified with Gd chelates via linking peptides to construct NP-substrate (Fe3O4-pep-Gd) conjugates for kinetic MMP-2 activity assessment in vitro, at cellular level and in vivo. Superparamagnetic Fe3O4 quenched the longitudinal relaxation effect (T1 relaxivity) of attached Gd chelates by perturbing proton relaxation process under an external magnetic field. MMP-2 cleaved the peptide substrates and released Gd chelates from the local magnetic fields accompanied with T1 relaxivity recovery and T1 contrast enhancement. Benefiting from signal amplification through binding multiple Gd chelates to one linking peptide, Fe3O4-pep-Gd conjugates exhibited high sensitivity for detection of MMP-2 (as low as 0.5 nM). Enzymatic processes were in good agreement with the integrated Michaelis-Menten model, revealing an unexpected activity enhancement in the initial stage. Fe3O4-pep-Gd conjugates could also probe MMP-2 at cellular level and in vivo, that indicates a great promise in in vitro diagnosis (IVD) and disease monitoring.

Keywords: matrix metalloproteinase; enzymatic activity; T1 relaxivity recovery; signal amplification; in vitro diagnosis; in vivo sensing

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INTRODUCTION Metastasis, as one of three hallmarks of malignancy, spreads cancer cells to lung, liver, brain or other key organs, which significantly increases difficulty in treatment and results in elevated cancer mortality.1 Tumor metastasis involves multiple processes including escaping from the primary tumor, invading surrounding tissues, entering the vasculature, and eventually reaching secondary sites.2-4 During these processes, matrix metalloproteinases (MMPs) play a key role in regulating the tumor microenvironment.5 There is a considerable amount of evidences that enhanced MMP activity is associated with malignant state.6, 7 Among MMPs, MMP-2 is an important player for metastasis because of its ability to degrade the extracellular matrix (ECM), which is the main barrier preventing cancer cells from invading.8,

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Therefore, sensitive detection of MMP-2 activity is of great urgency to medical

diagnosis. Traditional assays, including gelatin-PAGE,10, 11 ELISA12, 13 and colorimetry,14, 15 suffer from cumbersome pretreatment and poor detection sensitivity. Recently great progress has been made in the development of bioassay strategies owing to immense application of modern technologies, such as surface plasmon resonance (SPR),16 fluorescence,17 fluorescence resonance energy transfer (FRET)18 and electrochemistry19, with simple operation and fantastic sensitivity. For example, the electrochemical biosensor for MMP-2 showed a low detection limit with 0.078 pg/mL.20 However, it remains a challenge to develop a sensitive and in vivo biosensor for in situ detection of MMP-2. In the past decade, nanoparticle (NP)-bioconjugates have found applications in a wide range of diagnosis and bioanalysis.21, 22 In particular, using NP-substrates conjugates for in vitro detection and kinetic analysis of enzyme has attracted more and more research interest.23 Various nanomaterials, such as carbon nanotubes,24 quantum dots (QDs),25-27 magnetic NPs,28,

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upconversion NPs30,

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and Au

NPs,32-34 were utilized to construct NP-substrates. For example, Zhu and coworkers constructed a biosensor of tyrosine-conjugated QD for kinetic analysis of Tyrosinase.35 Nowadays, activatable

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magnetic resonance imaging (MRI) agents, which respond to biological makers associated with diseases greatly promote the diagnostic magnetic resonance (DMR), a new concept referring to the in vitro MR analysis with high sensitivity, use of small samples, low cost and convenience.36, 37 However, activatable T1 or T2 contrast agents, which result in modulation of either longitudinal (T1) or transverse (T2) relaxation, always suffer from weak sensitivity because of low signal-to-noise ratios (SNR). Recently, it was reported that T1 relaxivity (r1 value) of Gd chelates would be quenched upon conjugation to T2 contrast agents. Cheon and coworkers supposed that the local magnetic field generated by superparamagnetic T2 contrast materials perturbed the relaxation process of paramagnetic T1 contrast agents in close proximity.38 Theoretically, conjunction of T1 agents with superparamagnetic T2 materials would cause the former spin fluctuation slowed such that it is ineffective for water proton relaxation, resulting in a low T1 MRI signal. Although the quenching theory is still unclear and requires more detailed investigation,39, 40 it has been in part confirmed and employed to promote the sensitivity by repressing the noise.41, 42 Herein, we demonstrate an activatable T1 relaxivity strategy by using Fe3O4-pep-Gd as NPsubstrates for kinetic analysis of MMP-2 in vitro and at cellular level. As shown in Figure 1, MMP-2 cleaves the peptide that links Gd chelates to Fe3O4 NPs, releasing the former from the local magnetic fields. Since the local magnetic field generated by the central Fe3O4 is known to be dependent on 1/d3 (d is the distance away from the magnetic core),43 its quenching effect on T1 relaxivity decays quickly as Gd chelates move away. Recovery of T1 relaxivity confirms that the central superparamagnetic Fe3O4 nanocrystal could act as a magnetic quencher only when Gd chelates confined within its local magnetic field range. In addition, we introduce signal amplification into the sensing system by using the peptides with multiple amino residues and G2 PAMAM (polyamidoamine) dendrimers to provide more conjugating sites for Gd chelates. We envision that the analytical sensitivity would be improved, not

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only because of more Gd chelates on each peptide, but also due to the prolonged rotational correlation time (τR) of Gd chelates with bigger molecular weight.44, 45 Benefiting from the activatable MRI strategy and signal amplification, analysis sensitivity of Fe3O4-pep-Gd NP-substrates to MMP-2 was dramatically raised with detection limit as low as 0.5 nM. This enzyme responding system based on T1 relaxivity recovery was also explored on MMP overexpressing HT-1080 human fibrosarcoma cells, both at cellular level and in xenograft tumor, to evaluate its cellular and in vivo sensing capacity. Fe3O4-pepGd conjugates with kinetic, sensitive, and cellular detection capacity of MMP-2 will hold great promises for in vitro diagnostics (IVD) and in vivo diseases monitoring applications.

EXPERIMENTAL SECTION Chemicals. Peptide KSKSQSGPLGLAGGSQPC, KSKSQSGplglagGSQPC (lowercase letters for D-type amino acid) and Ac-KSGSQSGPLGLAGGSQPC were purchased from GL Biochem (Shanghai), p-SCN-Bn-DTPA was purchased from Macrocyclics, Recombinant human MMP-2 protein (active) was purchased from Abcam, and other reagents were purchased from Sigma-Aldrich. All reagents were used without further purification. HT-1080 cell line was purchased from Cell Bank of Chinese Academy of Sciences (Shanghai, China). Characterizations. TEM images were taken with JEM-2100 microscope (JEOL) at an accelerating voltage of 200 kV. The element analysis of Fe and Gd in the samples was measured by inductively coupled plasma mass spectroscopy (ICP-MS). The T1-relaxation time measurements and magnetic resonance imaging (MRI) phantoms were performed on a 0.5 T MRI scanner (NMI20-Analyst NMR Analyzing & Imaging system from Niumag). In vivo MRI imaging were performed on a 7 T MRI scanner (Varian 7 T micro MRI System), equipped with 10 cm bore imaging gradients (40G/cm). Synthesis of Dendrimer N3-PAMAM-G2 (N3-G2). Dendrimer N3-G1.5 solution (0.53 mmol in 8 mL

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of methanol) was added dropwise into ethylenediamine solution under N2 over 10 h at room temperature and reacted for 4 days. After methanol was evaporated, excess ethylenediamine was removed azeotropically with a mixture of toluene and methanol (9:1) (bath temperature less than 55 °C) to give crude product, which was further purified using semi-preparative HPLC with gradient elution (H2O and acetonitrile) to afford pure product (25%): 1H NMR (400 MHz, CDCl3) 2.27 (t, J = 6.3 Hz, 4H), 2.32 (t, J = 6.3 Hz, 8H), 2.43 (m, 12H), 2.57 (t, J = 6.0 Hz, 2H), 2.64 (t, J = 6.3 Hz, 8H), 2.72 (t, J = 6.3 Hz, 4H), 3.21 (m, 14H), 6.84 (t, J = 5.0 Hz, 6H); MS (ESI, m/z) calcd for [M]+ 770.5, found 770.8. Assembly of Fe3O4-pepA-Gd Conjugates. Multi-step conjugations were conducted on the surface of Fe3O4 NPs to install Gd chelates on the central nanoparticle via cleavable linkers. Firstly, Fe3O4 NPs were coated with SiO2 shell with amino groups modification on the surface, which were then conjugated to

heterobifunctional

linkers,

4-(N-maleimidomethyl)cyclohexane-1-carboxylic

acid

3-sulfo-N-

hydroxysuccinimide ester sodium salt, via amide formation. Secondly, Peptide A (sequence: KSKSQSGPLGLAGGSQPC) was conjugated to the nanoparticles through the reaction between maleimide and thiol of cysteine residues, then alkynyl groups were introduced by the reaction between the amino group on the peptide and propargyl-N-hydroxysuccinimidyl ester. After that, dendrimer azidePAMAM (N3-G2) was introduced via standard Sharpless click conditions using ascorbic acid and CuSO4. Thirdly, Gd chelates were conjugated by modified nanoparticles with p-SCN-DTPA, and Gd3+ chelation with GdCl3·6H2O. Ultrafiltration was executed after each modification step to remove excess reactants from the NPs. We also assembled Fe3O4-pepB-Gd (without signal amplification) and Fe3O4pepA(D)-Gd (uncleavable peptide) conjugates for negative control, see Supporting information for details. Kinetic analysis. Fe3O4-pepA-Gd in PBS buffer (1×, pH 7.4) with different iron concentrations (0.1, 0.2, 0.4 and 0.8 mM) were prepared. For each concentration, active MMP-2 were added to reach

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final enzyme concentrations of 1.5, 3, 6 and 12 nM, respectively. All reaction mixtures were incubated at 37 °C. During the incubation, samples were collected every hour within 8 h. T1 and T1-weighted MRI phantoms were acquired accordingly. Cellular Sensing. HT-1080 cells were seeded in 3.5 cm culture dish at a density of 1×105 cells and incubated for 24 h. Serum free media replaced the original media and cells were further incubated for 4 h. The concentrated conjugates (Fe3O4-pepA-Gd, Fe3O4-pepA(D)-Gd, and Fe3O4-pepB-Gd) solution was diluted with serum free media until the final concentration of iron was 0.2 mM. For enzyme inhibition experiments, the media were pretreated with 10 mM Batimastat for 10 min before addition of Fe3O4pepA-Gd conjugates. During the incubation, media were collected at 0, 2, 4, and 8 h, then T1 relaxation time and T1-weighted phantom imaging of the collected solutions were acquired accordingly. In vivo sensing. BALB/c nude mice were obtained from Laboratory Animal Center of Xiamen University and operated in compliance with the guideline for the care and use of Laboratory Animal Center of Xiamen University. To induce a solid tumor, HT-1080 cells (5×106 in 100 µL PBS) were injected subcutaneously into the right rear flank areas of the mice. The mice were used when the tumor grew to ~5 mm in diameter, which is visible from skin. After intraperitoneal injection of chloral hydrate, the mice were then intratumorally injected with Fe3O4-pepA-Gd and Fe3O4-pepA(D)-Gd solutions (50 µL, [Fe] = 0.2 mM). T1-weighted MR images were acquired pre- and post-injection at 2, 4 h with the same slices. All the images were acquired using fSEMS sequence under the following parameters: TR/TE = 300/2.4 ms, 256 × 256 matrices, slices = 5, thickness = 2 mm, averages = 8, FOV = 80 × 80 mm2.

RESULTS AND DISCUSSION Design and construction of Fe3O4-pep-Gd conjugates as NP-substrate. It has been found that T1

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relaxivity of Gd chelates would be quenched in the local magnetic field generated by superparamagnetic materials.41, 46 Activatable MRI probes based on this phenomenon have been developed for biosensing.47, 48

However, to the best of our knowledge, there is no report on enzyme activity sensing, which may be

ascribed to low SNR. We chose Fe3O4 NPs with an average diameter of 12 nm as cores, each of which was coated with a SiO2 shell (~6 nm) through reverse microemulsion (Figure S1a). The silica shell kept ambient H2O molecules away and blocked their chemical exchange with magnetic Fe3O4 surface, thereby shielded the T1 shortening ability of Fe3O4 NPs, which was the major source of noise. To further improve SNR, we introduced multiple Gd chelates to one NP as a means of signal amplification. Detailed construction procedures were demonstrated in Figure S2. Briefly, MMP-2 cleavable peptides (peptide A, sequence: KSKSQSGPLGLAGGSQPC) were conjugated onto Fe3O4@SiO2 through the cysteine residue at C-terminal and linked to G2 dendrimers via the three amino groups at N-terminal. Since every G2 dendrimer carries 4 amino groups, at maximum, there would be 12 amino groups at the end of each peptide for introducing Gd chelates. Then Gd chelates were attached onto the peptide through the coupling between amino group and isothiocyanate of chelating ligand. Chemical reactions in each step were fast, efficient, selective and RT reactive. Ultrafiltration was performed after each step to remove excess reactants. NPs maintained their morphology and monodispersity after a series of surface modification (Figure S1b). Inductively coupled plasma atomic emission spectroscopy (ICP-AES) analysis indicated the Gd/Fe ratio reached as high as 0.17:1. By similar steps, we also synthesized Fe3O4-pepA(D)-Gd as negative control by using a cleavable-resistant peptide, in which L-amino acids were converted to D-isomers at MMP-2 hydrolysis site (peptide, KSKSQSGplglagGSQPC, lowercase letters represented D-type amino acids). Fe3O4-pepA(D)-Gd had the same Gd/Fe ratio as high as 0.17:1. To demonstrate the significance of signal amplification, we also constructed Fe3O4-pepB-Gd conjugates by using peptide B (sequence: Ac-KSGSQSGPLGLAGGSQPC), which could also be cleaved by MMP-

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2 but contained only one binding site for Gd chelates (dendrimers were not used). Without the strategy of signal amplification, Gd/Fe ratio of Fe3O4-pepB-Gd dropped to 0.04:1. Therefore, once enzymatic cleavage of peptide A would release 4.5 times more Gd chelates than peptide B, which should lead to a significant improvement in sensitivity. Static relaxivity measurement to evaluate the signal amplification. Signal amplification was achieved through introducing multiple Gd chelates to the N-terminal of peptide A via PAMAM dendrimers (termed as multi-Gd-pepA, i.e., pepA-PAMAM-Gd complex). We reasoned that compared to single-Gd-pepB complex (one peptide attach only one Gd chelates), the enlarged molecular size of multi-Gd-pepA complex would lead to slower rotations in bulk solution upon released from Fe3O4 surface, therefore increase its T1 relaxivitiy (r1). So we compared the T1 relaxivities of multi-Gd-pepA and single-Gd-pepB complexes on a 0.47 T MRI scanner. As expected, multi-Gd-pepA complex showed almost 1-fold increase in r1 value (15.59 ± 0.14 mM-1 s-1, Figure 2a) over single-Gd-pepB (7.89 ± 0.11 mM-1 s-1). The tumbling of multi-Gd-pepA complex was slowed down by their large molecular size, prolonging the rotational correlation time (τR), which is one of the key contributors to T1 relaxivity. To evaluate the effect of signal amplification, we prepared simulated solutions after enzymatic hydrolysis. Multi-Gd-pepA and single-Gd-pepB complexes were added into the solutions containing superparamagnetic Fe3O4 NPs, according to the corresponding Gd/Fe ratios of Fe3O4-pep-Gd (i.e. 0.17 for multi-Gd-pepA, 0.04 for single-Gd-pepB, respectively). Then we measured T1 relaxivities of the following solutions: Fe3O4 (control), Fe3O4-pepA-Gd, Fe3O4-pepB-Gd (NP substrates solutions before enzymatic hydrolysis), and Fe3O4 + multi-Gd-pepA, Fe3O4 + single-Gd-pepB (simulating the solutions after enzymatic hydrolysis). As shown in Figure 2b, Fe3O4, Fe3O4-pepA-Gd and Fe3O4-pepB-Gd showed similar r1 values (2.95, 3.02, 3.03 mM-1 s-1, respectively). These results did not comply with the equation:49

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1 = 1 + Fe + Gd

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(1)

where the observed solvent relaxation rate, 1 , was the sum of the intrinsic diamagnetic solvent relaxation rate in the absence of contrast agents, 1 , and the additional relaxivity contribution from Fe3O4 ( Fe) and Gd chelates ( Gd). This equation describes the linear relationship between T1 relaxivity and concentration of paramagnetic species, without solute-solute interaction.50 The disagreement between experiment data and Eq. (1) indicated strong T1 relaxivity quenching of Gd chelates in close proximity to Fe3O4 core. In contrast, in the simulation experiments in which multi-GdpepA and single-Gd-pepB conjugates were simply added to Fe3O4 solutions, the T1 relaxivities (5.52 and 3.29 mM-1 s-1, respectively) complied with Eq. (1), suggesting no magnetic quenching by Fe3O4. The distinct r1 increase of Fe3O4 + multi-Gd-pepA also validated the effective signal amplification, and inspired us to employ Fe3O4-pepA-Gd conjugates for kinetic analysis and sensitive detection of MMP-2. Kinetic Analysis of MMP-2. Fe3O4-pepA-Gd conjugate solutions with iron concentrations at 0.8, 0.4, 0.2 and 0.1 mM (quantitative NPs concentrations as 21.9, 11.0, 5.48 and 2.74 nM, respectively) were prepared for kinetic analysis of MMP-2 (12, 6, 3, 1.5 nM). During the enzymatic process, we collected samples at 60 min intervals, then performed T1 measurement and T1-weighted phantom imaging on a 0.47 T MRI scanner. It contained a permanent magnet with low magnetic strength, conforming to device miniaturization of DMR, as well as low-cost and convenience. Time-dependent T1 reduction (Figure 3a) reflected the relaxivity recovery, corresponding to the release of multi-Gd-pepA by enzymatic cleavage. The quick declining curves suggested that Fe3O4-pepA-Gd conjugates could respond to MMP-2 with the concentration as low as 0.5 nM. Although this detection limit was much

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higher than a few recent reported assays, it was still sufficient in practical cases, such as at cellular level and in vivo. The classic MM equation, with Briggs-Haldane steady-state treatment, can be transformed into its integrated form to involve the time-dependent substrate concentration, which can be extracted from T1 relaxation contribution of multi-Gd-pepA complex:51

ln

+ − = !"# $ %

(2)

where [S]0 and [S]t are the initial and time-dependent substrate concentrations. We used [E]0t (enzymetime) as an independent variable instead of time in the fitting for convenience. As shown in Figure 3b, the time-dependent enzymatic process curves were presented in enzyme-time courses, and the released Gd-pepA concentrations were extracted from their T1 relaxation contribution. The well overlapped curves indicated the same nature of Fe3O4-pepA-Gd conjugates as substrates, which was irrelevant to MMP-2 concentrations.52 To better understand the interaction between NP-substrate and enzyme, the comparison between our experimental curves and curves fitted with MM model was necessary. To facilitate the comparison, we made two assumptions: (1) in the initial stage of the enzymatic reaction, all enzymes were saturated by excess NP-substrates, and the formation of enzyme-substrate complexes was not affected by NP in this period; (2) the subsequent step, in which enzyme-substrate complex broke down to yield free enzyme and product, was fast and identical. With these two assumptions, the initial rate of the reactions, designated as V0, could be expressed as a function of [S]0:53

& =

'()* + ,- .

(3)

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Here, we retrieved V0 from tangents to the initial stage of T1 curves (Figure 4). The kinetic parameter kcat was calculated to be 42.39±6.05 min-1 after fitting with Eq. (3) (curves displayed as dashed lines). Then we obtained the best fit for the released multi-Gd-pepA conjugates in enzyme-time course with the integrated MM model (Figure 3b, cyan dashed lines), and calculated the apparent Km, Km,app by assuming kcat unchanged (Table S1). The experimental curves agreed with the trend of fitting curves well, although positive and negative deviation could be observed in the initial and final stage, respectively. We attributed the negative deviation in the final stage to the cleaved multi-Gd-pepA that still adhered to NPs non-covalently. Recently, Algar et al. described a “hopping” model for enhanced trypsin proteolysis by virtue of high local substrate concentration at QD interface.54 In this light, we reasoned that the enhanced MMP-2 activity in the initial stage came from the relatively high peptide A concentration confined in the nanometer-scale space around NP surface. Substrate confinement induced more frequent collisions and faster enzyme-substrate complex formation, especially at the beginning of reaction when most of multi-Gd-pepA remained. The increasing Km,app with the rising of Fe3O4-pepAGd concentration was against the MM model and the “hopping” hypothesis, indicating factors that suppressed enzyme-substrate collisions and complex formation. This was probably caused by useless collisions between enzymes and NPs that had been hydrolyzed, preserving no or few substrate. We also acquired the corresponding T1-weighted phantom images, which showed brightness gradients over time (Figure 5). To quantify the imaging contrast, we calculated the SNR by finely analyzing the regions of phantoms and utilized the values of SNRpost/SNRpre to represent the signal changes (Figure S3). The SNR values were obtained according to SNRROIs = SIROIs/SDnoise (SI stands for signal intensity and SD stands for standard deviation). For each iron concentration, SNR increased along with the brightness gradient, in accordance with decreasing T1. It should be noticed that the T2 shortening effect of superparamagnetic Fe3O4 would oppress T1 signal during MRI, reducing the overall

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SNR, especially at high iron concentrations. Hence, phantoms of the group with the highest iron concentration (0.8 mM, Figure 5d) displayed the darkest appearance and the lowest SNR, whereas the group with the suitable iron concentration (0.2 mM, Figure 5b) possessed the best brightness distinction among all groups. We also performed MMP-2 analysis by Fe3O4-pepA(D)-Gd, which had similar construction with Fe3O4-pepA-Gd but containing the cleavage-resistant Peptide a. By contrary, Fe3O4-pepA(D)-Gd illustrated no T1 relaxivity recovery with MMP-2, neither T1 relaxation time nor T1-weighted MRI phantoms (Figure S4). The control results indicated the specificity of Fe3O4-pepA-Gd for MMP-2 analysis. Sensing at cellular level. The success of in vitro MMP-2 analysis using NP-substrate conjugates encouraged us to use them to detect MMP-2 at cellular level. We chose HT-1080 cells as an experimental model due to their high MMP-2 secretion level and have been used to investigate other activatable sensing agents.55,

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Firstly, the biocompatibility of Fe3O4-pepA-Gd, Fe3O4-pepB-Gd and

Fe3O4-pepA(D)-Gd conjugates was assessed by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT) assays, and no apparent cytotoxicity was observed on HT-1080 cell lines (Figure S5). HT-1080 cells were seeded in the 3.5 cm culture dish at a density of 1×105 cells, and cultured for 4 h. After that, the concentrated conjugates (Fe3O4-pepA-Gd, Fe3O4-pepB-Gd, and Fe3O4-pepA(D)-Gd) solution was added into the serum free media. We set the concentration of NP-substrates in the media at 0.2 mM (iron concentration) because of the excellent SNR as shown in the previous section. After incubation for 0, 2, 4, 8 h, the media were collected and performed T1 measurements and T1-weighted imaging (Figure 6). Fe3O4-pepA-Gd exhibited apparent T1 decrease as well as 2.5 times increase in phantom brightness, indicating the significant T1 relaxivity recovery because of Gd chelates escaping from the local magnetic field of Fe3O4. Although slight T1 decrease was detected for Fe3O4-pepB-Gd, it

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was hard to distinguish among their phantoms, reflecting the significance of signal amplification. There were almost no signal changes for Fe3O4-pepA(D)-Gd during incubation that ascribed the T1 relaxivity recovery to peptide cleavage. To further confirm the cleavage of peptide by MMP, enzyme inhibition experiments were performed by pretreating HT-1080 cells with 10 µM Batimastat, a potent, broad spectrum inhibitor of MMP by mimicking the structure of substrates.57 The retaining T1 relaxation time suggested the sensing specificity of our NP-substrate. We also collected the serum free media (without NP-substrate conjugates) after 4 h culture and detected the relative gelatinolytic activity by gelatin zymograms and found the media equal to 76.8 nM MMP-2 (experimental details and quantitative analysis, see Figure S6). There was a substantial lapse of MMP-2 quantity in the media between gelatin zymograms and Fe3O4-pepA-Gd assay. This was attributed to various MMPs secreted by HT-1080 cells, and the different enzymatic specificity from gelatin to Peptide A. The cellular sensing experiments proved that Fe3O4-pepA-Gd conjugates could sensitively detect the MMP activity and expression level. In vivo sensing. Apart from the excellent in vitro MMP-2 analysis efficiency and favorable sensing performance at cellular level, Fe3O4-pepA-Gd also served as an excellent candidate for in vivo MMP sensing. Visualization of MMP expressing quantity at tumor tissues would be helpful to distinguish malignancy and evaluate metastasis probability. We tested the in vivo sensing capacity in mice bearing HT-1080 xenografts by intratumoral injection of 50 µL of Fe3O4-pepA-Gd and ([Fe] 0.2 mM) or noncleavable Fe3O4-pepA(D)-Gd. Then we acquired T1-weighted MR images before and after the injection on a 7 T MRI scanner. With Fe3O4-pepA-Gd, the tumor area (dashed lines) showed obvious signal enhancement in both the coronal and axial planes at 2 and 4 h p.i., compared with pre-injection (Figure 7a). Tumor area signal enhancement were quantified to be 36.2% and 77.6% at 2 and 4 h p.i. in coronal planes, 38.4% and 51.9% at 2 and 4 h p.i. in axial planes, respectively. Gradual T1 relaxivity recovery suggested that the overexpressed MMP in HT-1080 xenografts cleaved the linking peptide and released

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attached Gd chelates. By contrary, signal enhancement did not occur in the tumor area for Fe3O4pepA(D)-Gd, even decreased in coronal and axial planes (Figure 7b). The D-type amino acids in the sequence inhibited MMP hydrolysis as well as T1 relaxivity recovery. The signal decease was attributed to the T2 effect of Fe3O4 nanocrystals. The in vivo sensing experiments proved that Fe3O4-pepA-Gd conjugates can sensitively probe the MMP expression level in situ tumor tissues, and may guide the cancer diagnosis and metastasis monitoring.

CONCLUSION In summary, we reported a novel strategy based on T1 relaxivity recovery for kinetic MMP-2 sensing in vitro and at cellular level. After the linking peptides were cleaved by MMP-2, Gd chelates were released from the surface of superparamagnetic NP and regained their T1-relaxation shortening effect. Benefiting from the effective signal amplification, detection limit and sensitivity were significantly improved. The relationship between T1 relaxation contribution and the concentration of released Gd chelates made it possible for real-time monitoring of enzymatic kinetic processes. We also proved the MMP sensing capacity of Fe3O4-pepA-Gd conjugates in cell cultures and small living subjects. This sensing system based on T1 relaxivity recovery may be broadly applicable to detect MMP with enhanced sensitivity and specificity, and may become a truly portable, easy-to-use, and low-cost solution for point-of-care IVD of associated diseases in future.

ASSOCIATED CONTENT Supporting Information Detailed experimental procedures, TEM of as-synthesized Fe3O4-SiO2 and Fe3O4-pepA-Gd, assembly process of Fe3O4-pepA-Gd conjugates, kinetic parameters of the enzymatic processes, SNR analysis of

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the T1-weighted MRI phantoms ([Fe] = 0.1, 0.2, 0.4, 0.8 mM), MTT assays of HT-1080 cells with nanoconjugates. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author *Fax: (+) 86-592-2189959, Tel: (+) 86-592-2180278. *E-mail: [email protected]. Author Contributions #

Author X.Z., H.L., and L.W. contributed equally to this work.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT This work was supported by the National Key Basic Research Program of China (2013CB933901, 2014CB744502, and 2014CB932004), National Natural Science Foundation of China (21521004, 21602186, 21327001, 81370042, and 81430041), and Fok Ying Tung Education Foundation (142012).

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Figure 1. Schematic illustration of MMP-2 detection using Fe3O4-pepA-Gd conjugates based on T1 relaxivity recovery. Superparamagnetic Fe3O4 nanocrystals induce the local magnetic fields under an external field, and act as a quencher to influence the T1 spin alignment of attached Gd chelates. MMP-2 cleaves the linking peptide and releases multiple Gd chelates from superparamagnetic Fe3O4. Gd chelates regain their T1 relaxivity as escaping from local magnetic fields induced by superparamagnetic Fe3O4.

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Figure 2. (a) T1 relaxivity of multi-Gd-pepA and single-Gd-pepB complexes to investigate their T1 relaxivity contribution after released into bulk solution. The almost 1-fold increase in r1 value of multiGd-pepA over multi-Gd-pepB was attributed to its longer rotational correlation time (tR). (b) T1 relaxivity measurement to confirm T1 relaxivity recovery of Gd chelates after Fe3O4-pepA-Gd and Fe3O4-pepB-Gd conjugates were cleaved by MMP-2 (see results and discussion for details). This experiment indicated that analysis sensitivity would be improved with the effective signal amplification.

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Figure 3. (a) The processes of enzymatic reactions were monitored by measuring T1 of solutions containing Fe3O4-pepA-Gd conjugates and MMP-2 over time. The initial concentrations of NP-substrate (as iron concentrations) were 0.1, 0.2, 0.4 and 0.8 mM with MMP-2 concentrations of 12 (black), 6 (red), 3 (blue) and 1.5 nM (magenta), respectively. All experiments were carried out in triplicate. (b) Concentration curves of released multi-Gd-pepA complex in enzyme-time course. Concentrations of released multi-Gd complex were derived from its contribution in T1 relaxivity. Cyan dashed curves were the best fit with the integrated MM equation.

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Figure 4. Initial hydrolysis rates of Fe3O4-pepA-Gd conjugates by MMP-2 as a function of initial substrate concentrations. The data were fitted with Eq. (3) (dashed lines).

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Figure 5. The brightness gradients of T1-weighted MRI phantoms reflected the hydrolysis process of Fe3O4-pepA-Gd conjugates by MMP-2. The initial concentrations of NP-substrate (as iron concentrations) were (a) 0.1, (b) 0.2, (c) 0.4 and (d) 0.8 mM. Phantoms gradually brightened with time, indicating the activatable T1 relaxivity recovery.

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Figure 6. (a) T1 measurements and (b) T1-weighted phantom imaging investigated cellular sensing capabilities of Fe3O4-pepA-Gd, Fe3O4-pepB-Gd and Fe3O4-pepA(D)-Gd conjugates. Compared with Fe3O4-pepB-Gd and Fe3O4-pepA(D)-Gd, Fe3O4-pepA-Gd exhibited a great potential to detect MMP-2 at cellular level with 2.5 times better SNR. This ascribed the T1 relaxivity recovery to the linking peptide cleavage. The inhibition experiment was performed by pretreating HT-1080 cells with Batimastat for 10 min before Fe3O4-pepA-Gd incubation. Each experiment was carried out in triplicate.

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Figure 7. In vivo MMP sensing study of (a) Fe3O4-pepA-Gd and (b) Fe3O4-pepA(D)-Gd. T1-weighted MR images at both coronal and axial planes were acquired at 2 and 4 h after intratumoral injection of 50 µL of Fe3O4-pepA-Gd and non-cleavable Fe3O4-pepA(D)-Gd ([Fe] 0.2 mM) into HT-1080 xenografts. Dashed lines indicated the tumor areas, where signal changes were quantified by MR SNR analysis after administration (n = 3, data represents mean ± s.d.).

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TOC

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Activatable T1 Relaxivity Recovery Nanoconjugates for Kinetic and

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