Controlled Nano–Bio Interface of Functional Nanoprobes for in Vivo

Jan 23, 2019 - ‡Department of Radiology and §Department of Radiation Oncology, Molecular Imaging Program at Stanford (MIPS) and Bio-X Program, ...
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Controlled Nano-Bio Interface of Functional Nanoprobes for in Vivo Monitoring Enzyme Activity in Tumors Ziyan Sun, Kai Cheng, Yuyu Yao, Fengyu Wu, Jonathan Fung, Hao Chen, Xiaowei Ma, Yingfeng Tu, Lei Xing, Liming Xia, and Zhen Cheng ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.8b05825 • Publication Date (Web): 23 Jan 2019 Downloaded from http://pubs.acs.org on January 24, 2019

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is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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Controlled Nano-Bio Interface of Functional Nanoprobes for in Vivo Monitoring Enzyme Activity in Tumors Ziyan Sun,†,‡, Kai Cheng,‡,§, Yuyu Yao,‡ Fengyu Wu,‡, Jonathan Fung,‡ Hao Chen,‡ Xiaowei Ma,‡ Yingfeng Tu,‡ Lei Xing,§ Liming Xia,†* Zhen Cheng‡* †Department

of Radiology, Tongji Hospital, Tongji Medical College, Huazhong University of

Science and Technology, Wuhan, 430030, China ‡Department

of Radiology and §Department of Radiation Oncology, Molecular Imaging Program

at Stanford (MIPS) and Bio-X Program, Canary Center at Stanford for Cancer Early Detection, School of Medicine, Stanford University, Stanford, California 94305-5484, United States Department

of Nuclear Medicine, PET/CT Center, Affiliated Hospital of Qingdao University,

Qingdao, 266003, China

These

authors contributed equally.

*Corresponding Authors Zhen Cheng, Ph.D., Molecular Imaging Program at Stanford (MIPS), Canary Center at Stanford for Cancer Early Detection, Department of Radiology and Bio-X Program, School of Medicine, Stanford University, 1201 Welch Road, Lucas P095, Stanford, California 94305-5484, United States E-mail: [email protected]

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or Liming Xia, MD, Department of Radiology, Tongji Hospital,Tongji Medical College, Huazhong University of Science and Technology, 1095 Jiefang Avenue, Wuhan, 430030, China E-mail:[email protected]

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Abstract: Engineering inorganic nanoparticles with a biocompatible shell to improve their physicochemical properties is a vital step in taking advantage of their superior magnetic, optical and photothermal properties as multifunctional molecular imaging probes for disease diagnosis and treatment. The grafting/peeling-off strategy we developed for nanoparticle surface coating can fully control the targeting capability of functional nanoprobes by changing their colloidal behaviors such as diffusion and sedimentation rates at the desired sites. We demonstrated that a cleavable coating layer initially immobilized on the surface of magnetic resonance imaging probes not only makes the nanoparticles water-soluble but also can be selectively removed by specific enzymes, thereby resulting in a significant decrease of their water-solubility in an enzyme-rich environment. Upon removal of surface coating, the changes in hydrodynamic size and surface charges of nanoprobes as a result of interacting with biomolecules and proteins lead to dramatic changes in their in vivo colloidal behaviors (i.e., slow diffusion rates, tendency to aggregate and precipitate), which were quantitatively evaluated by examining changes in their hydrodynamic sizes, magnetic properties, and count rates during the size measurement. Because the retention time of nanoprobes within the tumor tissues depends on the uptake and excretion rate of the nanoprobes through the tumors, selective activation of nanoprobes by a specific enzyme resulted in much higher tumor accumulation and longer retention time within the tumors than that of the inactive nanoprobes, which passively passed through the tumors. The imaging contrast effect of tumors using activatable nanoprobes was significantly improved over using inactive probes. Therefore, the grafting/peeling-off strategy, as a general design approach for surface modification of nanoprobes, offers a promising and highly efficient way to render the nanoparticles suitable for targeted imaging of tumors.

KEYWORDS: colloidal behaviors, nano-bio interface, tumor monitoring, core/shell structure, iron oxide, matrix metalloproteinase-2

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As with newly developed biology and nanotechnology, the ability to manipulate nanoparticles at the biomolecular level to create functional platforms with superior magnetic, optical and photothermal properties has resulted in great progress in many facets of medicine including in vivo molecular imaging, diagnostics, and chemotherapy.1-6 As an essential component in these emerging fields, the nanoparticles have been extensively studied as delivery vehicles or imaging probes, and come in an almost infinite variety of sizes, shapes, and compositions with diversity of forms and functions.1-3, 7-10 Most efforts to improve their in vivo behaviors for targeted imaging and therapy have been focused on lengthening the particle circulation time, minimizing protein adsorption and mononuclear phagocyte system (MPS) uptake, and enhancing their targeting capability by optimizing the interfacial interactions between nanomaterials and biological systems.11-17 Engineering nanoparticles with a hydrophilic, neutral shell to improve their physicochemical properties is a vital step to keep their inherent properties as functional probes for disease diagnosis and treatment.18-26 Currently, the predominant strategy is to densely graft a hydrophilic polymer, most commonly poly (ethylene glycol) (PEG), to the surface of the nanoparticle

for

improving

particle

stability

and

water-solubility,

followed

by

immobilization/conjugation of targeting molecules to the PEG chain terminus for an increased uptake at the target.20, 27-36 Although this strategy is straightforward and proficient for many cases, there is a confinement in practice when the immobilized biomolecules on the particle surface adversely influence its pharmacokinetic and biodistribution after administration.11, 13, 37 Instead of following the conventional approaches by which the as-synthesized nanoparticles are uniformly dispersed in an aqueous solution after several coating steps for increased biocompatibility and stability, we consider their reverse processes: removing or dissociating the surface coatings in a controlled manner in vivo, by which we can manipulate their colloidal behaviors such as diffusion

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and sedimentation rates at the desired sites so as to control their targeting capability regardless of nanoparticle size, shape, density and composition. Although some recent studies have considered the effect of diffusion and sedimentation of nanoparticles for targeted delivery and imaging,38, 39 it is important to understand how the physical parameters of nanoparticles and biophysicochemical interactions at the nano-bio interface influence their colloidal properties in vitro and in vivo. The surface properties of nanoparticles, based on their physical and chemical characteristics such as composition, surface functionalization, size, shape and surface charge, could be quantitatively determined. However, characterizing the interactions between nanomaterial surfaces and biological systems at a nano-scale is still a big challenge because they are governed by a large number of long-range or short range colloidal forces and a series of dynamic biophysicochemical interactions, which could induce significant changes in particle colloidal behaviors such as diffusion rates, association/dissociation of particles, phase transformation and agglomeration in a physiological environment.1, 17, 26, 31, 40 In order to validate the mechanism for controlling particle’s colloidal behaviors, and to optimize the surface features for biocompatibility and safe use, we investigated the interactions at the nano-bio interface between the coating layer of the particles and biological system. To simplify the problem and eliminate some irrelevant influences from particle’s size and shape, we considered one of currently available nanoprobes, iron oxide nanoparticles (IONPs), to study the effect of sedimentation and diffusion of probes on tumor targeting and imaging in the living subjects by controlling interactions at the nano-bio interfaces. The IONPs we developed here have core/shell structures with highly monodisperse size and uniform shape.27 More importantly, their magnetic properties can be optimized without any change in shape, and their shells can be easily modified by oxidation, passivation, and functionalization with many types of linkers. Typically, a hydrophilic PEG coating layer

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immobilized on the external surface of the core/shell iron/iron oxide nanoparticles (Fe/IONPs) renders the particles water-soluble and biocompatible. In this case, a special peptide sequence incorporated between the PEG coating layer and the nanoparticles surface was designed to be preferentially cleaved in the presence of a target, in this case, a matrix metalloproteinase enzyme (MMP-2).40-42 The PEG coating layer can then be gradually peeled off from the nanoparticles, eventually resulting in a significant decrease of their water-solubility in a physiological condition. The changes in hydrodynamic size and surface charges of nanoprobes as a result of interacting with biomolecules and proteins lead to dramatic changes in their in vivo colloidal behaviors (i.e., slow diffuse rates, tendency to aggregate and precipitate). We quantitatively evaluated these changes by examining their hydrodynamic sizes, count rates, and magnetic properties. To further validate the enzyme-response of the nanoprobes, we functionalized the nanoprobe surfaces with either cleavable PEG linkers or noncleavable ones to construct active or inactive probes, respectively. The cleaved linkers terminated with fluorescent dyes from the probes were used as an indicator for monitoring the enzyme activities in vitro. The valid cleavage of the coating layers from the active and control nanoprobes with enzyme were judged by the changes in fluorescence intensity, particle sizes and count rates. Because of dramatic changes in their colloidal behaviors, the specific cellular uptakes of these active/control nanoprobes after incubation with MMP-2overexpressed cells could be differentiated with magnetic resonance imaging, relaxivity measurement, and elemental analysis. The grafting/peeling-off strategy we developed here can be used to control the targeting capability of functional nanoprobes by changing their colloidal behaviors such as diffusion and sedimentation rates at the desired sites. The PEGylated nanoprobes can passively accumulate into the tumors by extravasation of nanoprobes through increased permeability of the tumor vasculature

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and ineffective lymphatic drainage (EPR effect) (Figure 1). The retention time of nanoprobes within the tumor tissues depends on the ratio of the uptake and excretion rate of the nanoprobes through the tumors. Due to their enzyme-responsive colloidal behaviors, the active nanoprobes on activation by specific enzyme tend to aggregate and precipitate within the tumor tissues, thus resulting in much higher tumor accumulation and longer retention within the tumors than the inactive nanoprobes, which passively pass through the tumors (Figure 1). Therefore, the enzymeresponsive nanoprobes engineered by the grafting/peeling-off strategy can be used to selectively target specific tumors and monitor them over time in vivo. Finally, we evaluated the potential of these nanoprobes as targeted MR imaging contrast agents for noninvasively detecting and monitoring MMP-2-overexpressed carcinoma in living subjects.

Results Construction of Monodispersed Core/Shell Fe/IONPs As MR Imaging Probes. We prepared monodispersed core/shell Fe/IONPs as MR contrast agents by controlled oxidation of amorphous iron nanoparticles.27,

43, 44

Initially, an amorphous magnetite Fe3O4 layer around the Fe core

appeared when the as-synthesized Fe nanoparticles were exposed to air (Figure S4a). Due to nanoscale Kirkendall effect, the controlled oxidation of the as-synthesized iron nanoparticles in presence of the oxygen-transfer reagent resulted in core/shell and even hollow structures (Figure 2).45 We found that a dense shell of crystalline magnetite Fe3O4, once formed, can act as a passivation shell to protect the iron core from further oxidation. When the Fe nanoparticles were treated with a concentrated oxidizer at a high temperature (190C) for a short time (30 min), a robust and dense shell consisting of multiple domains of crystalline magnetite Fe3O4 was obtained for the Fe/IO core/shell structure (Fe/IONP). The oxidation process conditions, including the

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amount of oxidation reagent, heating temperature and time, play key roles in the formation of the crystalline iron oxide shell around iron core. Figure 2b showed the spherical Fe/IONPs were highly monodisperse and had an average diameter of 13.87  0.19 nm with a core of 7.98  0.15 nm as determined by TEM (Figure S4-S6 and Table S1). The hollow structures (HIONPs) were obtained at a harsher oxidation condition (Figure 2c). The geometrical parameters of both types of nanostructures, including their inner diameters, outer diameters, and core sizes, were determined by TEM analysis (Table S1). The hollow structures have a larger particle size than the core/shell ones because their Fe cores have been completely converted into the shells. The Dynamic Light Scattering (DLS) measurement also confirmed their uniform sizes and narrow size distributions of both core/shell and hollow nanostructures (Figure 2d and Table S1). HRTEM showed that the lattice spacing between two planes of the shells (b) is 2.961 Å, corresponding to the distance of two (220) planes of magnetite Fe3O4 (Figure 2e). To study the concentration-dependent chemical and physical properties of nanoprobes, we normalized the concentrations of nanoprobes based on either iron mass/molar concentrations or particle-number concentrations. The iron mass was determined by inductively coupled plasma mass spectrometer (ICP-MS), while the particle-number concentration was calculated based on the molar atomic weight of nanoprobe which was determined by geometrical parameters and relative densities of individual nanocrystals (Table S1). According to a weight of a single particle, the molar atomic weight of Fe/IONP is 4.22  0.22 × 106 g/mol. Similarly, the calculated molar atomic weight of HIONP is 4.97  0.25 × 106 g/mol. The iron oxide shell of nanoprobes was then capped with dopamine, offering terminal amine groups as reactive sites for subsequent PEGylation.46 The number of amine groups present on the nanoprobes were quantified using a spectrophotometric method.20 It is very important to quantify

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this amount for stoichiometric control of PEG loading. An average of 629  35 amines were measured per Fe/IONP, or 0.96 nm2 per amine via standardization versus 2-bromoethylamine hydrobromide at the same condition (Table S2 and Figure S7). There are more amine groups per particle found on the HIONP because they have slightly larger surface area than Fe/IONP. To make the particle water-soluble and biocompatible, we grafted the PEGylated peptides (MMP-2

substrate:

PEG2000-GGPLGVRGC-NH2

and

control

substrate:

PEG2000-

GGRGLPGVC-NH2) covalently on the external surface of amine-modified Fe/IONPs via the Cterminus cysteine using a heterobifunctional cross linker (Figure S7). The surface area coverage percentage of PEGylated peptides on the particle surface depended on the density of the surface amine groups, the concentration of PEGylated peptides and their accessibility to surface amines.20 To quantitatively determine loading levels, the amine-modified Fe/IONPs were further coated with fluorescent tag-(Rhodamine B) terminated PEG polymers instead of ones with methoxy terminus. To minimize artifacts originating from the fluorescence quenching effects with metal oxide surface, the PEG molecules were first detached from iron oxide surface through acid etching and then quantified spectrophotometrically in the supernatants (Figure S8). Three types of nanoparticles with different loading levels were obtained by changing the concentration of PEGylated peptides in the reaction. The maximum loading of PEGylated peptides on the surface was 512  27 per NP with a surface coverage of 81.4%. Accordingly, the nanoprobes with a low surface loading of PEG linkers were prepared for the stability comparison (Table S2). In vitro cytotoxicity of PEGylated nanoprobes was evaluated by a standard tetrazolium dye (MTT) based colorimetric assay of cell viability of HT-1080 cells. The results in Figure S9 showed low toxicity of both nanoprobes in the range of 1 - 100 µg Fe/mL at a standard physiological

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condition, and there was no significant difference in cell viability between control and active groups, indicating that both nanoprobes were biocompatible in the given concentration range.

Validation of Enzyme-Responsive MR Imaging Probes. To show that the activation of the probes in vitro can be followed spectrophotometrically, we prepared both the active and the control probes coated with the fluorescent-tag terminated and PEGylated peptides as described previously (Figure 3a). As shown in Figure 3b, MMP-Fe/IONPs had a broad absorption in the visible range (300 – 700 nm), contributed mainly by the iron/iron oxide cores. There was a shoulder peak centered at ca. 556 nm, which is characteristic absorption band of Rhodamine B. Compared to Rhodamine alone at the same concentration in an aqueous solution, RB-MMP-Fe/IONPs showed a very strong fluorescence intensity with an emission centered at ca. 580 nm. Although Rhodamine B has much high quantum yield in ethanol than in water, the changes in fluorescent intensities of RB-MMP-Fe/IONPs in water or ethanol were not obvious, probably due to the discrete physical mechanisms of fluorescent molecules on the NP’s surface (Figure 3c). The sensitivity and specificity of the MMP-2 responsive probes (MMP-Fe/IONPs) were first investigated in vitro. The cleavage efficiency of PEGylated peptide linkers from MMP-Fe/IONPs was judged by an increase in fluorescence intensity when the nanoparticles were treated with MMP-2. The cleaved linkers in the filtrates from the treated nanoprobes showed a significant increase of fluorescence emission over time (Figure 3d). Dose-dependent response of MMPFe/IONPs was further observed when the nanoprobes were incubated with different concentrations of MMP-2. The fluorescence intensity reached the maximum level after incubation of the nanoprobes with 10 µg/mL of MMP-2. As shown in Figure 3e, the kinetic studies showed that the activation of the nanoprobes displayed a proportional increase of fluorescence intensity in the first

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4 h of the incubation, reaching a maximum intensity after 5 h. More than 60 % of surface linkers have been cleaved from the active nanoprobes after 6 h incubation. These results indicated that the surface coating of the active nanoprobes can be efficiently peeled off by MMP-2 to release the dye-labeled residues. In contrast, Ctrl-Fe/IONPs showed almost no signal change in fluorescence intensity within the observation period, suggesting that Ctrl-Fe/IONPs remained inactive to MMP2.

Evaluation of the Colloidal Behaviors of Enzyme-Responsive Imaging Probes. We noticed that peeling the surface coating from the active nanoprobes during the kinetic study resulted in a significant decrease of their water-solubility and stability in the solution. To clarify the effect of coating-shell thickness and grafting density of the coating linkers on their colloidal behaviors, we prepared two types of nanoprobes: ones with three different coating densities (high, medium, and low loading) (Figure 4a-b and Table S2), which were designed to simulate different states of the treated nanoprobes; the other one with residue linkers (VRGC, the remaining sequence of the substrate undergoing cleavage), which was used to represent the completely cleaved nanoprobes (surface coverage = 0%). In Figure 4c-e, the average hydrodynamic size of the nanoprobes decreased as the surface coverage percentage increased. The high coating density of PEGylated peptide linkers on the surface can efficiently stabilize the nanoprobes in an aqueous medium; their size distribution was narrower than that of the other samples with the lower surface loading densities. The nanoprobes with 37% surface coverage, related to the late state of treated nanoprobes, showed significant larger mean particle size than ones with higher surface coating, indicating that there were many states of dispersions with large sizes in the sample (such as aggregates). Without the efficient coating, strong interactions between particles can influence the

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diffusion of nanoparticles and this can lead to non-specific aggregation that changes the mean size and size distribution. The similar trend was also found when examining their mean count rates (Figure 4e), which are sensitive to particle aggregation and sedimentation. To evaluate the in vitro colloidal behaviors of MMP-Fe/IONPs (i.e. diffusion rates, tendency to aggregate and precipitate), we investigated their hydrodynamic sizes, count rates, and states of dispersion (single, dimer, trimer, and so on) in an aqueous medium. The kinetic studies on these perimeters of MMP-Fe/IONPs after incubation with MMP-2 were shown in Figure 4f-h. The average hydrodynamic size of MMP-Fe/IONPs was initially 30.6 nm with a standard deviation of 0.9 nm, and TEM images showed they were individual, isolated nanoparticles (Figure 4h-0h). Their mean size increased to 58.1  25.3 nm after 2 h incubation with MMP-2. TEM images of sample aliquots from the reaction mixtures provided the qualitative analysis of the state of nanoparticle dispersions at the predetermined time intervals (Figure 4h). The initially monodisperse nanoparticles formed many different dispersions such as dimers, trimers, tetramers, and even aggregates. Moreover, the DLS count rates increased gradually from 111.9 to 168.0 kcps in the first 2 h, indicating that the volume percentages of large particles or aggregates gradually increased in the reaction mixture. After 2 h incubation, the nanoprobes underwent a proportional increase in size, and the average size quickly reached 254  35 nm in 5 h. A similar trend in the count rate of the nanoprobes was also observed in Figure 4g; their count rate continuously increased and reached the maximum within a short period, implying rapid aggregation and coagulation when the surface coating was peeled off. Many large and dense aggregates containing a large number of nanoparticles from several tens to several hundreds were found in TEM images of sample aliquots at the later time points (Figure 4h). The aggregate formation is a result of interparticle forces operating in the dispersion when the interparticle distance is small enough to

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interfere with each other. In contrast to the active nanoprobes, the control nanoprobes (CtrlFe/IONPs) showed a negligible increase in size or count rate for even up to 6 h, indicating that they did not coagulate and remain completely dispersed in the aqueous phase.

Magnetic Properties of Various Nanoprobes. To confirm the effectiveness of MMP-Fe/IONPs as an MRI contrast agent, we first studied T2-weighted MR images of the nanoprobes dispersed in an agar phantom at different concentrations of iron (mM of Fe). To compare the magnetic properties of the core/shell nanoprobes, we also prepared the hollow iron oxide nanoprobes with the same surface coating. Since the hollow iron oxide nanoparticles were directly derived from the core/shell iron oxide nanoparticles, the number of hollow iron oxide nanoparticles were the same as that of the core/shell ones at a certain molar concentration of iron measured by ICP-MS (in the Supporting Information (SI) section: Molar atomic weights of nanoprobes). As shown in Figure 5a, the core/shell MMP-Fe/IONP produced much improved negative contrast compared to the commercial contrast agent Ferumoxytol at a range of iron concentration from 0.031 to 2.0 mM. In addition, the contrast enhancement produced by the core/shell ones was slightly higher than that of the hollow ones, probably due to the ferromagnetic iron cores. Similar to T2 signal, T2* signal intensity of MMP-Fe/IONPs was reduced much faster than that of Ferumoxytol at the same iron concentration (Figure 5b). T2* decay underlying gradient echo imaging was used to efficiently detect inherently inhomogeneous samples with magnetic susceptibility effects caused by iron oxide nanoparticles. The transverse relaxivities (r2 or r2*) of various MRI contrast nanoprobes were obtained by graphing changes in relaxation rates (1/T2 or 1/T2*) at different concentrations (Figure 5c-d and Table S3). As expected, the core/shell MMP-Fe/IONP had an r2 of 277.2  8.2 mM-1S-1, more than four times than that of Ferumoxytol (68.7  2.5 mM-1S-1). Due to high mass

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magnetization and ferromagnetic iron cores, the core/shell nanoprobes showed higher relaxivities than the hollow ones. Moreover, the differences in relaxivity r2* became more obvious when we compared core/shell probes with the control ones (Table S3). Such a strong improvement in relaxivities will enable application of much lower doses of contrast agents for imaging.

Cellular Uptake and Activation of the Nanoprobes by MMP-2 in Vitro. As described previously, peeling the surface coating from the active nanoprobes resulted in dramatic changes in their colloidal behaviors such as slow diffusion velocity, tendency to aggregate and precipitate. It was noticed that the resultant large aggregates with slow diffusion rates and fast sedimentation rates under the influence of gravity would significantly affect the cellular uptake of nanoprobes when exposing cells to a suspension of these active nanoprobes. We quantitatively determined the specific cellular uptake of the active/control nanoprobes after incubation with MMP-2overexpressed cells (HT1080 cells) using the elemental analysis (ICP-MS), magnetic resonance imaging, and relaxivity measurement (Figure 6). The HT1080 fibrosarcoma tumor cells were chosen because of their reported high MMP-2 expression and the MMP-2 sensitivity of our developed nanoprobes.41, 47, 48 Based on the ICP results, we calculated the amount of nanoprobes taken up per cell. As seen in Figure 6a, the cellular uptake of MMP-Fe/IONPs was concentration dependent, showing a linear relation over a wide range of iron concentration. Although the cellular uptake of Ctrl-Fe/IONPs was also concentration dependent, it reached a plateau at approximately 0.8 pg Fe per cell. It was clear that the uptake values of MMP-Fe/IONPs were much higher than those of the control ones (Ctrl-Fe/IONPs) at the same dose. Dramatic increases in the cellular uptake of the nanoprobes were due to the in situ sedimentation of MMP-Fe/IONPs activated by excreted MMP-2 in the cell culture medium. As discussed in the previous section, the aggregated

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nanoprobes can sediment and have slower diffuse rates than the individual one, so the concentration of nanoprobes on the cell surface is higher than the initial bulk concentration.49 This will lead to an increase in the cellular uptake of nanoprobes during a standard incubation time. Approximately 30% ~ 40% of total MMP-Fe/IONPs in the medium were taken up by the cells, while only 10% of the control nanoprobes were associated or internalized within the cells (Figure 6b). The uptake efficiency (% of total) depends on the concentration of the nanoprobes and reached a maximum level with an increase of the nanoprobe concentration, and then gradually decreased. These observations are consequences of the nanoparticle aggregation and sedimentation, as well as the cellular uptake mechanism and kinetics.49 To further quantify the effect of diffusion and sedimentation of active nanoprobes on cellular uptake and evaluate the magnetic properties of nanoprobes internalized in the cells, we studied T2-weighted MR images of cell lysates containing nanoprobes in 0.5% agarose gel as conducted previously. The cell lysates containing MMPFe/IONPs showed significantly darker negative contrast than the control samples at the same doses (Figure 6c). Considering MR contrast enhancement together with the cellular uptake results, it is clear that the MMP-Fe/IONPs were efficiently activated and taken up by the cells. Since the MMPFe/IONPs were subject to slow diffusion and fast sedimentation when incubated with MMP-2 positive cells, they had more chances to be internalized by cells on the bottom of culture dishes compared to the control ones at the same doses. Figure 6d-e demonstrated the linear relationship between relaxation rates and iron concentrations in the cell lysates. The relaxivity of MMPFe/IONPs in the cells obtained from their linear regression analysis was 308.4 mM-1S-1, which was slightly higher than that of the control samples at the same condition (Figure 6e), and also higher than that of well-dispersed nanoprobes (Table S3). Such enhancement in T2 shortening effect was

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attributed to the aggregation of active nanoprobes which can be regarded as large magnetized particles whose magnetic moment increases with size in a certain range.50, 51

Noninvasive Monitoring of MMP2-Positive HT1080 Tumors in Vivo with MRI. To investigate tumor detection and monitoring in vivo behavior, mice bearing HT-1080 tumor xenograft model were intravenously administered with both active MMP-Fe/IONPs and inactive Ctrl-Fe/IONPs and their T2-weighted MR images were obtained at the predetermined time intervals. Representative T2-weighted MR transverse images of the mouse tumors prior to and after injection of either active or inactive nanoprobes were shown in Figure 7a-b. The hypointense signals within the tumors were clearly observed after 2 h postinjection of either active or inactive nanoprobes. It is clear that both active and inactive nanoprobes can passively target the tumor tissues with leaky vasculature by the EPR effect. More importantly, the contrast effect induced by active nanoprobes within the tumors was significantly higher than that of inactive ones throughout the entire period of measurement, indicating that there were more activated nanoprobes trapped within the tumors. Since the activated nanoprobes lost their water-solubility and tended to aggregate together, they diffused more slowly and had a longer retention time within the tumor tissues in comparison with those inactive nanoprobes, eventually leading to dramatic increases in the sensitivity and selectivity of MR imaging. We quantitatively analyzed the contrast effects by comparing the percentage reduction in the T2 signal produced by active nanoprobes in the regions of interest (ROIs) around the tumor areas relative to that observed for the control nanoprobes. The T2 signal reduction was defined as the percentage change in the mean tumor intensity between prescan and postscan images. After injection of either active or inactive nanoprobes, a continuous T2 signal reduction was observed in the first 4 h (Figure 7c). At 4 h postinjection, the T2 signal

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reduction of active nanoprobes reached the maximum value (57.3%), which is significantly higher than that of inactive nanoprobes (36.5%). After that, the T2 signal reduction gradually decreased but still remained at a relatively constant level over a 48-h period. Consistent with previous in vitro studies, the T2 signal reduction from inactive nanoprobes also indicated a mild EPR effect on the tumor site, but only to half the effectiveness of active nanoprobes. Compared to inactive nanoprobes, the active nanoprobes showed a significantly high contrast effect at each time point after injection. These results implied that the MMP-2 active nanoprobes as MR probes make it possible to actively reach and target the HT1080 tumor.47

In Vivo Biodistribution and Toxicity. We further investigated the in vivo biodistribution and clearance of nanoprobes in both active and control group right after MR imaging (at 48 h postinjection). The iron contents in the tumor and organs were determined by ICP-MS analysis. Because of the presence of endogenous iron in plasma and tissues, the control mice (n = 3) were used for “particle-free” correction. As seen in Figure 7d, the MMP-2 active nanoprobes were able to accumulate in the tumor sites, and their tumor uptake was significantly higher than that of control nanoprobes, which was consistent with the finding of the MR imaging. High accumulations of the nanoprobes in the liver and spleen were observed after 48 h postinjection in Figure S13, indicating that the nanoprobes tended to be trapped in the mononuclear phagocyte system (MPS) and hepatic excretion could be a major route of elimination of nanoprobes from mice. A pilot acute toxicity study was performed to evaluate the biocompatibility of the nanoprobes. The blood cell counts and serum biochemistry in the Table S4 were not significantly affected by the administration of the nanoprobes. Histologic and microscopic examination also revealed there was no common or uncommon toxic changes in the major parenchymal organs (Figure S14).

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Discussion A wide variety of strategies have been developed to improve the in vivo behaviors of nanoprobes for targeted imaging and therapy, including (1) the optimization of particle size, density and shape to extend the circulation time with favorable clearance characteristics, (2) the surface coating to minimize or eliminate unfavorable protein adsorption and MPS uptake, and (3) the immobilization of targeting ligands to enhance the targeting capability.25, 26, 31, 49 Many traditional approaches to coat and modify nanoparticles with PEG or biomolecules have established important trends on density and size of PEG linkers, surface charges and particle sizes.17, 18, 24, 32 It is an urgent need to take into consideration of the biophysicochemical interactions at the nano-bio interface because they substantially determine the performance feature, the uptake and in vivo fate of the nanoparticles.1, 26 Typical targeting strategies involve functionalizing and/or grafting the surface of nanoparticles with proteins or other biofunctional moieties after rendering the nanoparticles water-soluble. Although the conjugation of bio-moieties to nanoparticles in sequential steps is prevalent in an effort to determine their targeting ability, its reverse processes, removing or dissociating the coatings from the surface of nanoparticles, have intentionally been avoided in most cases in which the nanoparticle stability and colloidal properties could be seriously impaired by the dissociation of surface ligands. In this study, we focus on those reverse processes and developed a graft/peel-off strategy to characterize the nano-bio interface in a sophisticated way and to optimize the nanoparticle targeting ability by manipulating their colloidal behaviors such as diffusion and sedimentation rates at the desired sites. Finally, we have successfully demonstrated the graft/peel-off strategy can be used as a general approach for surface modification of nanoprobes for tumor imaging.

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The interactions at the nano-bio interface with the biological environment are critical in determining their biological outcomes. However, there is a lack of detailed information on the mechanisms that link the nanoparticle surface and the biological interactions for their targeting ability.1 Although there are many literatures on development of the tumor-environment-responsive nanocarriers (such as the “smart” probes, targeted molecular imaging probes, enzyme-triggered nanoprobes, etc.),1, 5, 16, 34, 35, 38, 40, 42 understanding nanoparticle/biological interactions for how the nanoparticle interfaces exchange with the surroundings still remains challenging. In order to characterize the nano-bio interface and guide the exploration of multifunctional nanoprobes, we focused on the colloidal forces and dynamic biophysicochemical interactions that most nanoparticles could encounter in the biological environment. With regard to it, we developed a simplified nanoplatform to study how these various forces and interactions affect the nanoparticle’s stability and targeting ability. Such a nanoplatform is composed of three important components: nanomaterial core, PEG layer, and more importantly, the cleavable linker (Figure 1). The PEG spacers are immobilized on the surface of the core via bifunctional, cleavable linkers. These PEG chains can form a hydrophilic dense layer on surfaces, which is expected to reduce nonspecific binding of environmental biomolecules and provide excellent solubility, stability and flexibility, regardless of the size, shape, and composition of the core. The key issue of grafting/peeling-off strategy we developed here is to control the solid-liquid interface, including changing the shell thickness of the coating, optimizing the grafting density, and modifying the surface hydrophobicity or hydrophilicity. The enzyme cleavable spacer played an important role in controlling the interactions at the nano-bio interface with the biological environment, not only allowing PEG chains to be grafted on the particle to change the surface properties but also enabling the selective recovery of previous surface characteristics at desired sites when PEG chains are

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cleaved from the particle surface in a controlled manner. Dramatic change in surface properties could significantly influence the sedimentation and diffusion velocities of the nanoparticles, eventually resulting in particle aggregation or sedimentation in a medium, which could be monitored by various special instruments (such as DLS and MRI). Since this approach mainly focuses on the solid-liquid interface, it is powerful and universally applicable and has many intrinsic advantages over other methods, including minimum nonspecific binding and facile modification process. Regardless of particle composition, this approach can render the nanoparticles with targeting ability by selectively controlling the ratio of sedimentation to diffusion velocities of nanoparticles at desired sites. Characterizing the nano-bio and solid-liquid interfaces in a quantitative and reliable way is one of the most important aspects of studying the tumor environment responsive nanocarriers and nanomedicines. By developing a facile and reliable strategy, we have the ability to control the surface chemistry, size, shape, and colloidal behaviors with molecular precision in order to understand how such physical and chemical parameters at the nano-bio and solid-liquid interfaces are associated with specific biological functions and mechanisms.1, 15, 21, 49 Since this approach is universally applicable and can be used for surface modification of many different particles, we chose the magnetic core/shell Fe/IONPs as imaging probes for this proof-of-concept study because of their easy synthesis, facile surface modification, tunable magnetic properties, and most importantly, high monodispersity. The core/shell structures provide an easy, promising way to optimize the magnetic moment by adjusting the size of iron cores and the thickness of iron oxide shells. The presence of the iron core in the core/shell structure induces a higher magnetization compared to iron oxide NPs at the same scale. The transverse relaxivity of the core/shell Fe/IONPs is more than four times that of commercial contrast agent, which can significantly reduce the dose

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and make it possible to achieve highly sensitive tumor cell detection and highly efficient cancer therapy. The passivating oxide shell is dense enough to protect the reactive iron core against further oxidation; this is responsible for chemically withstanding physiological conditions without property degradation in the detection or treatment time period. Moreover, the core/shell Fe/IONPs are highly monodisperse so that each individual NP has nearly identical physical and chemical properties for controlled biodistribution, bioelimination and contrast effects. In current study, we investigated the states of dispersion of MMP2-Fe/IONPs such as single, dimer, trimer, and aggregates in an aqueous medium using DLS and TEM to explain the disparity in the efficiency of the proteolysis and quantify the effects of diffusion and sedimentation on cellular uptake. In a given medium, typical interactions between magnetic nanoparticles involve van der Waals, electrostatic, magnetic, steric, and solvation forces. The total interaction potential energy between nanoparticles changes as a function of the surface-to-surface separation distance. It has been studied that the aggregate formation is a result of interparticle forces operating in the dispersion, especially when the interparticle distance is less than a certain value (cutoff distance, i.e. 1.2 times of the sum of their radii) (Figure 4b).39 There were individual isolated nanoparticles and loose aggregates of these particles initially observed in the aqueous dispersions when they were incubated with MMP2. The repulsive steric force caused by long hydrophilic PEG chains kept individual nanoparticles isolated in the medium, and the force decreased with a decrease in grafting density. The MMP2-induced proteolysis caused the peeling-off of PEG chains from the surface, resulting in a decrease of shell thickness and grafting density. When the interparticle distance between randomly selected particles in the dispersion is less than the cutoff distance, these two particles tend to aggregate and form a dimer. If one of particles is already a dimer, then the result is a trimer and so on. Moreover, rapid dehydration and aggregation will occur when two interacting

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particle surfaces become hydrophobic after the peeling-off of PEG chains. The diffusion and sedimentation velocities of the nanoparticles or aggregates strongly depend on their hydrodynamic sizes. The larger aggregates have a much slower diffusion velocity and faster sedimentation velocity than the smaller aggregates or single nanoparticles.49 Under the influence of gravity, the sedimentation of aggregates leads to the disparity in cellular uptake observed for different nanoparticles coated with either cleavable or noncleavable PEG layers. This strategy is definitely applicable for in vivo tumor monitoring because the activated nanoprobes with slow diffusion rates and fast sedimentation rates preferentially accumulated within MMP2-positive tumor with longer retention time compared to control ones. On the basis of the efficiency of the proteolysis on the solid-liquid interfaces and the detection sensitivity, we think those nanoprobes can selectively image the MMP2 activities within the tumors and monitor the outcomes for subsequent treatments.

Conclusions In conclusion, we have successfully demonstrated the graft/peel-off strategy can be used as a general approach for surface modification of nanoprobes for targeted tumor imaging. We took advantage of the reverse processes of surface functionalization, and controlled the solid-liquid interface to manipulate particle colloidal behaviors such as diffusion and sedimentation rates upon response to the high quantities of the active enzymes within tumors. Our study provides an important concept theory for the design of targeting nanoprobes for tumor imaging, improves our understanding of the interactions between nano-bio interface, and furthermore offers a promising way to apply a variety of nanoparticles for molecular imaging and biomedical applications.

Materials and Methods

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Materials. The heterobifunctional PEG2000 (amine-PEG-carboxymethyl, MW 2000, NH2-PEG2000-COOH) was purchased from Laysan Bio Inc. All N-Fmoc-protected amino acids were purchased from Advanced ChemTech (Louisville, KY, USA). Trifluoroacetic acid (TFA), Obenzotriazole-N,N,N',N'-tetramethyluronium

hexafluorophosphate

(HBTU),

hydroxybenzotriazole (HOBt), and 4-(2',4'-dimethoxyphenyl-Fmoc-aminomethyl)-phenoxy resin (Rink amide resin LS, 100-200 mesh, 1% DVB, 0.44 mmol/g) were purchased from Advanced Chemtech. Unless otherwise mentioned, all other chemicals were purchased from Sigma or Aldrich. Instruments. Mass spectra of synthetic polymers were recorded by a time-of-flight (TOF) mass spectrometer (AB SCIEX TOF/TOF 5800, Applied Biosystems) equipped with a matrix-assisted laser desorption ionization (MALDI) ion source. UV/vis absorption spectra were measured by a PerkinElmer Lambda 35 UV/vis spectrometer. The elemental analyses were performed using inductively coupled plasma mass spectrometer (ICP-MS, Thermo Scientific Xseries 2 Quadrupole). The high-resolution transmission electron microscope (HRTEM) and scanning transmission electron microscope (STEM) images were recorded with a FEI Tecnai G2 F20 XTWIN transmission electron microscope operating at 200 kV. Syntheses of MMP-2 Substrate (Ac-KGPLGVRGC-NH2), Mismatched Sequence (AcKGRGLPGVC-NH2), and Reside Sequence (VRGC-NH2). The MMP-2 substrate (AcKGPLGVRGC-NH2), mismatched sequence (Ac-KGRGLPGVC-NH2), and reside sequence (VRGC-NH2) were synthesized on a CS Bio CS336 instrument (CS Bio Company, Inc., Menlo Park, CA) using standard sold-phase Fmoc peptide chemistry. Further details can be found in SI. The purified peptides were characterized by MALDI-TOF. The Purity of the final product > 95%. Mass m/z [Ac-KGPLGVRGC-NH2 + H], calculated 927.51, found 927.47. Mass m/z [Ac-

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KGRGLPGVC-NH2 + H], mass m/z [Ac-KGRGLPGVC-NH2 + H], calculated 927.51, found 927.70. Mass m/z [VRGC-NH2 + H], calculated 433.23, found 433.25. The characterization data were shown in Figure S1c, S1d and S2.

Synthesis of PEGylated MMP-2. The multifunctional polyethylene glycols, including monomethoxyl polyethylene glycol (mPEG-2000-COOH) and heterofunctional polyethylene glycol (Fmoc-PEG-2000-COOH) were synthesized according to our previous publication with necessary modifications.20 Further details can be found in SI. The characterization data were shown in Figure S1 and S3. For mPEG-2000-COOH, purity > 98%; Mass m/z [mPEG2000-COOH + Na] when unit number n = 44, calculated 2050.33, found 2050.34. For Fmoc-PEG-2000-COOH, purity > 90%; Mass m/z [Fmoc-PEG2000-COOH + Na] (average unit number n ~ 45) when unit number n = 45, calculated 2301.43, found 2301.57. The procedure of the PEGylation of MMP-2 is as follows: after activated by EDC/NHS (3.1 mg/2 mg) at room temperature for 30 min, mPEG2000-COOH (32 mg, 16 µmol) was conjugated to the Lys group of MMP-2 substrate (AcKGPLGVRGC-NH2, 10 mg, 10.8 µmol) in anhydrous DMF (0.5 mL) containing 2% DIPEA at room temperature. The PEGylated peptide was further purified by (RP) HPLC on a C-18 column (see more details in the SI). Purity > 95%; mass m/z [mPEG2000-MMP2 (average unit number n ~ 44)], when unit number n = 44, calculated 2937.8, found 2938.1. The same procedure was performed for PEGylation of the control sequence. The characterization data were shown in Figure S1e and S1f. Conjugation of PEGylated MMP-2 with Rhodamine B. The conjugation of MMP-2 and Rhodamine B with PEG chain mainly involved three steps, including the protection of heterobifunctional PEG, conjugation of PEG on MMP-2 resin bead, cleavage and deprotection,

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and dye labeling. First, the heterobifunctional PEG-2000 (Fmoc-PEG-2000-COOH) was synthesized according to the procedure in the SI (Figure S3). Purity > 90%; Mass m/z [FmocPEG2000-COOH + Na] (average unit number n ~ 45) when unit number n = 45, calculated 2301.43, found 2301.57 (Figure S3). Second, the PEGylated MMP-2 substrate (PEG2000GGPLGVRGC-NH2) or PEGylated control sequence was synthesized on a CS Bio CS336 instrument using the same strategy as previously described for MMP-2 substrate. Fmoc-PEG2000COOH (230 mg, 0.1 mmol) after activated in a solution containing 0.5 mmol of HoBt and 0.5 M diisopropylcarbodiimide (DIC) in DMF was then coupled to the N-terminus of protected GGPLGVRGC-resin beads (0.05 mmol loading) in anhydrous DMF at room temperature. After overnight the resin beads were washed with DMF, DCM and methanol, and then dried under the vacuum. After deprotection of the Fmoc protecting group of the PEG linker, the PEGylated MMP2 substrate was cleaved and deprotected by 2 mL of a mixture of TFA/ethanedithiol/TIPS/water (92.5/2.5/2.5/2.5, v/v/v/v). After 3 h the crude peptide was filtered, and then precipitated with icecold anhydrous diethyl ether, and dried in vacuo. The crude product was purified by semipreparative reversed-phased high-performance liquid chromatography (RP-HPLC) on a C-18 column with a mobile phase (gradient 10~90%, 0.1% trifluoroacetic acid (TFA) in acetonitrile and 0.1% TFA de-ionized water) over 45 min at a flow rate of 4 mL/min. The fractions (retention time of fraction for product Rf = 17 ~19 min) were collected and lyophilized. The purified PEGylated peptide was characterized by MALDI-TOF. The Purity of the final product > 95%, NH2-PEG2000-MMP-2, average unit number n ~ 45, mass m/z [NH2-PEG2000-MMP-2] (average unit number n ~ 45) when unit number n = 45, calculated 2851.80, found 2851.65 (Figure 3c). Finally, the freshly synthesized NH2-PEG-2000-MMP-2 (50 mg, ~17 µmol) was dissolved in 1 mL of degassed PBS. Rhodamine B isothiocyanate (10.9 mg, 20.4 µmol) in 100 µL DMSO was added

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into the solution. Under the nitrogen atmosphere, the mixture was stirred at room temperature for 2 h. The crude product was purified by the same HPLC methods as previously described. The fractions (retention time of fraction for product Rf = 17 ~19 min) were collected and lyophilized. The purified PEGylated peptide with Rhodamine B (RB-PEG-2000-MMP-2) was directly used for surface modification of nanoparticles. Controlled Oxidation of as-Synthesized Fe/IO Nanoparticles. The 13 nm Fe/IONPs were synthesized according to the previous publication with a slight modification.27 The controlled oxidation process of Fe/IO nanoparticles was performed according to the previous publication with modifications (Figure S4-S6).27 Briefly, A 125 mL four-necked flask was purged with a nitrogen flow after 20 mL of 1-octadecene was added. A solution of trimethyl amine N-oxide (20 mg in 1 mL of ethanol) was then added in the flask. The mixture solution was heated at 130C for 1 h to remove any oxygen and moisture. A solution of as-synthesized Fe/IO nanoparticles (80 mg) in hexane was then quickly injected in the above mixture, and the resultant solution was kept at 130C for 20 min before heated up to 190C at a heating rate of 5C/min. The mixture was heated at 190C for 30 min before cooled to room temperature. The oxidation process conditions, including the amount of oxidation reagent, heating temperature and time, were optimized for the Fe/IO core/shell structures (Fe/IONPs). The oxidation process with higher heating temperature (210C), longer heating time (2h), and a larger amount of oxidation reagent (i.e. 30 mg of trimethyl amine N-oxide) compared to regular conditions can result in the hollow structures (HIONPs). Finally, forty milliliters of acetone were added into the mixture to precipitate the product once the solution was cooled to room temperature. The oxidized product was collected by centrifugation (8000 rpm for 8 min) and then redispersed in hexane. The washing step was repeated three times. The black product was stored in 10 mL of hexane with 0.02 mL of oleylamine.

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Conjugation of PEGylated MMP-2 or MMP2-residue on NH2-Fe/IONP or NH2-HIONP. The PEGylated MMP-2 peptide was conjugated on amine-modified iron oxide nanoparticles (NH2-Fe/IONP or NH2-HIONP) with an amine-to-sulfhydryl crosslinker [succinimidyl-4-(Nmaleimidomethyl) cyclohexane-1-carboxylate (SMCC)]. Briefly, 1 mg of amine-modified nanoparticles were suspended in 1 mL of DMSO containing 5 mM SMCC. The solution was stirred for 2 h at room temperature. The SMCC-modified iron oxide nanoparticle (SMCC-Fe/IONPs or SMCC-HIONPs) were purified and characterized as described above. Typically, the SMCCmodified nanoparticles were precipitated out by adding 10 mL of ethyl ether and collected by centrifugation (4000 rpm for 8 min). The nanoparticles were then washed with ethanol, followed by centrifugation. The SMCC-modified nanoparticles (SMCC-Fe/IONP or SMCC-HIONP) were redispersed in DMSO with nitrogen protection. The purified SMCC-Fe/IONPs or SMCC-HIONPs were suspended in 2 mL of DMF with 2 mg of PEGylated MMP-2 peptide under a nitrogen atmosphere. The resultant mixture was stirred for 24 h at room temperature with nitrogen protection. The product MMP2-Fe/IONPs or MMP2-HIONPs were collected by centrifuge and redissolved in the water. The water-soluble product (PEG-MMP2-Fe/IONPs or PEG-MMP2HIONPs) was further purified by a spin filter (MWCO = 30 kDa) and washed with water three times. Any small aggregates were removed by passing the particle solution through a syringe filter (0.22 µm). Similarly, the mismatched peptide-PEG was conjugated on amine-modified nanoparticles (NH2-Fe/IONPs or NH2-HIONPs) with SMCC linkers to provide the control samples (PEG-Ctrl-Fe/IONPs or PEG-Ctrl-HIONPs). The conjugation of MMP-2 residue on nanoparticles was performed using the same procedure. After conjugated with Fe/IONPs or HIONPs, the products were referred to as Residue-Fe/IONPs or Residue-HIONPs, respectively.

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In Vitro MMP-2 Activity Study (Fluorescent Methods). Active MMP-2 (full-length, recombinant, human MMP-2, 66 kDa, PF023) was purchased from EMD Chemicals. To determine the activation capacity of the nanoprobes, various amounts of MMP-2 were added into the active or the control probe solutions (5 nM in 100 µL PBS) in vials and the reaction mixtures was kept at 37C for 4 h. The filtrates of the reaction mixtures were collected using a spine filter (MWCO = 30 kDa). The fluorescence intensities of the filtrates were recorded on a TECAN Infinite M1000 microplate reader (TECAN Group Ltd.). Excitation wavelength was set at 550 nm, and the emission wavelength was set at 580 nm (Figure S10). The spectral measurement was done in the Fluoromax 4 (Horiba Scientific). A linear absorption versus concentration calibration curve was constructed by adding the known amounts of Rhodamine-B-PEG2000-MMP2 in the reaction solution under the same condition as described above. To measure the activation kinetics of the nanoprobes, the filtrates of the reaction mixtures were collected at the predetermined time intervals (0, 10, 30, 60, 90, 120, 180, 240, 300, and 360 min) using a spine filter (MWCO = 30 kDa). The fluorescence intensities of the filtrates were recorded on a TECAN Infinite M1000 microplate reader (TECAN Group Ltd.). Excitation wavelength was set at 550 nm, and the emission wavelength was set at 580 nm. The fluorescence spectra were measured in the 560- to 850-nm window. A linear absorption versus concentration calibration curve was constructed by adding the known amounts of Rhodamine-B-PEG2000-MMP2 in the reaction solution under the same condition as described above.

DLS Measurements for Stability Test of MMP2-Fe/IONPs and In Vitro MMP-2 Activity Study of MMP2-Fe/IONPs. The hydrodynamic diameters of various nanoparticles under investigation, including active nanoprobes, inactive nanoprobes, and residue-nanoprobes, were

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measured using a Zetasizer Nano ZS90 DLS system equipped with a 633 nm-red laser at a detection angle of 90. For each sample, three DLS measurements were conducted with a fixed 20 runs and each run lasts 10 s. A typical measurement sequence for MMP-2 activity of nanoprobes consisted of the following steps: the mixture of the nanoprobes (5 nM in PBS, 200 µl) and MMP-2 stock solution (1.5 pmol) was prepared directly in the cuvette, immediately homogenized by shaking for 2~3 seconds before placed in the DLS apparatus. After the temperature equilibration at 37C, the size changes and count rates were recorded. The mixture was incubated at 37. The interval between measurements was 30 min. Every nanoprobe-MMP2 system was monitors for at least 6 h, or until no further development was noticeable. After the measurements, the cuvettes were visually checked for macroscopic aggregates. The TEM-specimens of the aggregates and/or solution were prepared. To investigate the stability of the nanoprobes, the time development of the average hydrodynamic sizes and the count rates of various nanoprobes with different coatings, including active nanoprobes, inactive nanoprobes, and residue-nanoprobes, in the reaction buffer were monitored over 6 h at 37 (Figure S11).

DLS Measurements for Stability Test of MMP2-Fe/IONPs with Different Coating Densities. The hydrodynamic diameters and count rates of various nanoprobes, including MMP2-Fe/IONPs with different coating densities and residue modified Fe/IONPs (residue-Fe/IONPs) were under investigation at the same procedure as described previously. Typically, the solutions of the nanoprobes (5 nM in PBS, 200 µl) were prepared directly in the cuvettes and equilibrated at 37C before placed in the DLS apparatus. The size changes and count rates were recorded at 37 every hour over 6 h. After the measurements, the cuvettes were visually checked for macroscopic aggregates.

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Relaxivity Measurement of Phantom Samples. Relaxivity measurements of various nanoprobes, including MMP2-Fe/IONPs, Ctrl-Fe/IONPs, MMP2-HIONPs, were conducted in a dedicated small-animal MRI scanner equipped with custom-designed pulse sequences and radiofrequency coils. The MRI scanner consists of a superconducting magnet (Magnex Scientific) with magnetic bore size of 9 cm, 7.0 T field strength and a gradient (Resonance Research, Inc.) with 120 mm ID, 770 mT/m of maximum gradient amplitude, and 2,500 T/m/s of a maximum slew rate. A serial of concentrations (0, 0.0156, 0.0312, 0.0625, 0.125, 0.25, 0.5, and 1 mM Fe) of four types of nanoprobes (MMP2-Fe/IONPs, Ctrl-Fe/IONPs, MMP2-HIONPs and Ferumoxytol), were prepared by serial dilutions with 1% agarose gel and then were solidified in the 300 µL of vials. T2-wieighed MR images were obtained using fast-spin echo sequences under the following parameters: TE/TR = 40/4000 ms, 256 × 256 matrix, 1 NEX, 8 × 8 cm field of view with an inplane resolution of 313 × 313 µm2, and slice thickness of 1 mm. T2 maps were obtained using a spin-echo sequence with the following parameters: TE = 10, 20, 40, 60, 80, 100, 120, and 140 ms, TR = 4000 ms, FOV = 8 × 8 cm, 256 × 256 matrix, 1 NEX, and slice thickness of 1 mm. The T2 quantification was performed by curve-fitting the analytical equation: M(TE) = M0·exp(-TE/T2), where M(TE) is the signal intensity observed at a given echo time (TE). All data fittings were performed using a nonlinear least-squares algorithm implemented in the OriginPro 8.1 SR2 (OriginLab Co.) analysis software. T2*-weighted fast gradient recalled echo (FGRE) MR images were obtained using fast gradient recalled echo sequences under the following parameters: TE/TR = 9.1/300 ms, 256 × 256 matrix, 1 NEX, 8 × 8 cm field of view with an in-plane resolution of 313 × 313 µm2, and slice thickness of 1 mm. T2* maps were obtained using a fast gradient recalled echo (FGRE) sequence with the following parameters: TE = 1.8, 4.2, 6.6, 9.1, 11.3, 13.7, 16.1, and

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18.5 ms, TR = 300 ms, FOV = 8 × 8 cm, 256 × 256 matrix, 1 NEX, and slice thickness of 1 mm. The T2* quantification was performed by curve-fitting the analytical equation: M(TE) = M0·exp(TE/T2*) + C, where M(TE) is the signal intensity observed at a given echo time (TE), and C is a constant that reflects the background noise. All data fittings (Table S3) were performed using a nonlinear least-squares algorithm implemented in the OriginPro 8.1 SR2 (OriginLab Co.) analysis software.

Cellular Uptake and Activation of Nanoprobes by MMP-2 In Vitro. HT1080 cells were cultured in 6 cm culture dishes in an incubator until the cells reach 80~90% confluency (8 × 105 cells per dish). Both MMP2-Fe/IONP and Ctrl-Fe/IONP stock solutions were directly added into the growth media in the dishes instead of replacing them with fresh media. All of cell cultures were then incubated at 37C for 4 h in an incubator. After two times washing with cold PBS, the cells were harvested and suspended in 150 µL PBS and were sonicated for 2 h. The sonicated cell lysates containing nanoprobes were suspended in 0.5% agarose gel in 300 µL PCR tubes and the phantom study of MRI properties was conducted as the same procedure as described previously. After cell phantom study, the cell lysates in the agarose gel were dispersed in the concentrated nitric acid (70%) and heated at 90C for 30 min to completely dissolve them. After evaporating the excess nitric acid, the mixtures were diluted with 2% nitric acid solution in sample tubes. The iron concentrations were measured by Inductively Coupled Plasma Spectrometer (ICP). The cell uptakes of particles by HT1080 cells were calculated based on the total Fe amount of the incubation medium and the measured Fe amount within cells.

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MR Imaging of the Nanoprobe Activation In Vivo. All animal studies were conducted in accordance with the Guidelines for the Care and Use of Research Animals established by the Stanford University. Mice bearing HT1080 tumor were anesthetized with 2% isoflurane in oxygen and placed with prone position. MRI was performed using the same instrument, protocols, and conditions as in the phantom MRI study. MMP2-Fe/IONPs and Ctrl-Fe/IONPs were injected via tail vein into the HT-1080 tumor-bearing mice at a dose of 10 mg Fe/kg of mouse weight (n = 4). T2-weighted fast spin-echo MR images were acquired on a 7.0-T small animal MRI system under the following parameters: repetition time (TR) = 3000 ms, TE = 40 ms, echo train length = 8, FOV = 4×4 cm2, section thickness = 1 mm, flip angle = 90. MR images were acquired in both transverse and coronal direction preinjection and at 1, 2, 4, 24 and 48 h after injection. Transversal and coronal MR images were acquired and the signal intensities were measured in defined ROIs using OsiriX imaging software (OsiriX version 3.2; Apple Computer). The NIH standard was used for tumor imaging processing. The ROI analysis was performed according to the previous publications.52

ASSOCIATIED CONTECT Supporting Information: Detailed methodology, additional figures, and discussion are described in the Supporting Information. This material is available free of charge on the ACS Publications website at http://pubs.acs.org. AUTHOR INOFRMATION Corresponding Author

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*E-mail: [email protected] or [email protected] Author Contributions Z.

S. and K. C. contributed equally.

ACKNOWLEDGMENT This work was supported, in part, by the Office of Science (BER), U.S. Department of Energy (DE-SC0008397), NCI of Cancer Nanotechnology Excellence Grant CCNE-TR U54 CA119367, CA151459, NIH In vivo Cellular Molecular Imaging Center (ICMIC) grant P50 CA114747, and the National Natural Science Foundation of China (81471637, 81671656).

References: (1) Nel, A. E.; Madler, L.; Velegol, D.; Xia, T.; Hoek, E. M. V.; Somasundaran, P.; Klaessig, F.; Castranova, V.; Thompson, M. Understanding Biophysicochemical Interactions at the Nano-Bio Interface. Nat. Mater. 2009, 8, 543-557. (2) Boles, M. A.; Ling, D.; Hyeon, T.; Talapin, D. V. The Surface Science of Nanocrystals. Nat. Mater. 2016, 15, 141-153. (3) Howes, P. D.; Chandrawati, R.; Stevens, M. M. Bionanotechnology. Colloidal Nanoparticles as Advanced Biological Sensors. Science 2014, 346, 1247390-1247399. (4) Li, F.; Lu, J.; Kong, X.; Hyeon, T.; Ling, D. Dynamic Nanoparticle Assemblies for Biomedical Applications. Adv. Mater. 2017, 29, 1605897-1605926. (5) Pelaz, B.; Charron, G.; Pfeiffer, C.; Zhao, Y.; de la Fuente, J. M.; Liang, X. J.; Parak, W. J.; Del Pino, P. Interfacing Engineered Nanoparticles with Biological Systems: Anticipating Adverse Nano-Bio Interactions. Small 2013, 9, 1573-1584. (6) Kim, J.; Piao, Y.; Hyeon, T. Multifunctional Nanostructured Materials for Multimodal Imaging, and Simultaneous Imaging and Therapy. Chem. Soc. Rev. 2009, 38, 372-390. (7) Chauhan, V. P.; Stylianopoulos, T.; Martin, J. D.; Popović, Z.; Chen, O.; Kamoun, W. S.; Bawendi, M. G.; Fukumura, D.; Jain, R. K. Normalization of Tumour Blood Vessels Improves the Delivery of Nanomedicines in a Size-Dependent Manner. Nat. Nanotechnol. 2012, 7, 383-388. (8) Cheng, K.; Sano, M.; Jenkins, C. H.; Zhang, G.; Vernekohl, D.; Zhao, W.; Wei, C.; Zhang, Y.; Zhang, Z.; Liu, Y.; Cheng, Z.; Xing, L. Synergistically Enhancing the Therapeutic Effect of Radiation Therapy with Radiation Activatable and Reactive Oxygen Species-Releasing Nanostructures. ACS Nano 2018, 12, 4946-4958. (9) Cheng, K.; Chen, H.; Jenkins, C. H.; Zhang, G.; Zhao, W.; Zhang, Z.; Han, F.; Fung, J.; Yang, M.; Jiang, Y.; Xing, L.; Cheng, Z. Synthesis, Characterization, and Biomedical Applications of a

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Targeted Dual-Modal Near-Infrared-II Fluorescence and Photoacoustic Imaging Nanoprobe. ACS Nano 2017, 11, 12276-12291. (10) Cheng, K.; Yang, M.; Zhang, R.; Qin, C.; Su, X.; Cheng, Z. Hybrid Nanotrimers for Dual T1 and T2-Weighted Magnetic Resonance Imaging. ACS Nano 2014, 8, 9884-9896. (11) Mahmoudi, M.; Bertrand, N.; Zope, H.; Farokhzad, O. C. Emerging Understanding of the Protein Corona at the Nano-Bio Interfaces. Nano Today 2016, 11, 817-832. (12) Verderio, P.; Avvakumova, S.; Alessio, G.; Bellini, M.; Colombo, M.; Galbiati, E.; Mazzucchelli, S.; Avila, J. P.; Santini, B.; Prosperi, D. Delivering Colloidal Nanoparticles to Mammalian Cells: A Nano-Bio Interface Perspective. Adv. Healthc. Mater. 2014, 3, 957-976. (13) Ke, P. C.; Lin, S.; Parak, W. J.; Davis, T. P.; Caruso, F. A Decade of the Protein Corona. ACS Nano 2017, 11, 11773-11776. (14) Tonga, G. Y.; Saha, K.; Rotello, V. M. 25th Anniversary Article: Interfacing Nanoparticles and Biology: New Strategies for Biomedicine. Adv. Mater. 2014, 26, 359-370. (15) Tenzer, S.; Docter, D.; Kuharev, J.; Musyanovych, A.; Fetz, V.; Hecht, R.; Schlenk, F.; Fischer, D.; Kiouptsi, K.; Reinhardt, C.; Landfester, K.; Schild, H.; Maskos, M.; Knauer, S. K.; Stauber, R. H. Rapid Formation of Plasma Protein Corona Critically Affects Nanoparticle Pathophysiology. Nat. Nanotechnol. 2013, 8, 772-781. (16) Cai, P.; Leow, W. R.; Wang, X.; Wu, Y. L.; Chen, X. Programmable Nano-Bio Interfaces for Functional Biointegrated Devices. Adv. Mater. 2017, 29, 1605529-1605554. (17) Kreyling, W. G.; Abdelmonem, A. M.; Ali, Z.; Alves, F.; Geiser, M.; Haberl, N.; Hartmann, R.; Hirn, S.; de Aberasturi, D. J.; Kantner, K.; Khadem-Saba, G.; Montenegro, J.-M.; Rejman, J.; Rojo, T.; de Larramendi, I. R.; Ufartes, R.; Wenk, A.; Parak, W. J. In Vivo Integrity of PolymerCoated Gold Nanoparticles. Nat. Nanotechnol. 2015, 10, 619-623. (18) Jokerst, J. V.; Lobovkina, T.; Zare, R. N.; Gambhir, S. S. Nanoparticle Pegylation for Imaging and Therapy. Nanomedicine 2011, 6, 715-728. (19) Frey, N. A.; Peng, S.; Cheng, K.; Sun, S. Magnetic Nanoparticles: Synthesis, Functionalization, and Applications in Bioimaging and Magnetic Energy Storage. Chem. Soc. Rev. 2009, 38, 2532-2542. (20) Cheng, K.; Kothapalli, S. R.; Liu, H.; Koh, A. L.; Jokerst, J. V.; Jiang, H.; Yang, M.; Li, J.; Levi, J.; Wu, J. C.; Gambhir, S. S.; Cheng, Z. Construction and Validation of Nano Gold Tripods for Molecular Imaging of Living Subjects. J. Am. Chem. Soc. 2014, 136, 3560-3571. (21) Treuel, L.; Brandholt, S.; Maffre, P.; Wiegele, S.; Shang, L.; Nienhaus, G. U. Impact of Protein Modification on the Protein Corona on Nanoparticles and Nanoparticle–Cell Interactions. ACS Nano 2014, 8, 503-513. (22) Van Lehn, R. C.; Ricci, M.; Silva, P. H.; Andreozzi, P.; Reguera, J.; Voitchovsky, K.; Stellacci, F.; Alexander-Katz, A. Lipid Tail Protrusions Mediate the Insertion of Nanoparticles into Model Cell Membranes. Nat. Commun. 2014, 5, 4482-4492. (23) Kelly, P. M.; Åberg, C.; Polo, E.; O'Connell, A.; Cookman, J.; Fallon, J.; Krpetić, Ž.; Dawson, K. A. Mapping Protein Binding Sites on the Biomolecular Corona of Nanoparticles. Nat. Nanotechnol. 2015, 10, 472-479. (24) Dai, Q.; Wilhelm, S.; Ding, D.; Syed, A. M.; Sindhwani, S.; Zhang, Y.; Chen, Y. Y.; MacMillan, P.; Chan, W. C. W. Quantifying the Ligand-Coated Nanoparticle Delivery to Cancer Cells in Solid Tumors. ACS Nano 2018, 12, 8423-8435. (25) Shin, T.-H.; Cheon, J. Synergism of Nanomaterials with Physical Stimuli for Biology and Medicine. Acc. Chem. Res. 2017, 50, 567-572.

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(26) Polo, E.; Collado, M.; Pelaz, B.; del Pino, P. Advances toward More Efficient Targeted Delivery of Nanoparticles in Vivo: Understanding Interactions between Nanoparticles and Cells. ACS Nano 2017, 11, 2397-2402. (27) Cheng, K.; Peng, S.; Xu, C.; Sun, S. Porous Hollow Fe3O4 Nanoparticles for Targeted Delivery and Controlled Release of Cisplatin. J. Am. Chem. Soc. 2009, 131, 10637-10644. (28) Kircher, M. F.; de la Zerda, A.; Jokerst, J. V.; Zavaleta, C. L.; Kempen, P. J.; Mittra, E.; Pitter, K.; Huang, R.; Campos, C.; Habte, F.; Sinclair, R.; Brennan, C. W.; Mellinghoff, I. K.; Holland, E. C.; Gambhir, S. S. A Brain Tumor Molecular Imaging Strategy Using a New Triple-Modality MRI-Photoacoustic-Raman Nanoparticle. Nat. Med. 2012, 18, 829-834. (29) Pelaz, B.; del Pino, P.; Maffre, P.; Hartmann, R.; Gallego, M.; Rivera-Fernández, S.; de la Fuente, J. M.; Nienhaus, G. U.; Parak, W. J. Surface Functionalization of Nanoparticles with Polyethylene Glycol: Effects on Protein Adsorption and Cellular Uptake. ACS Nano 2015, 9, 69967008. (30) Wilhelm, S.; Tavares, A. J.; Dai, Q.; Ohta, S.; Audet, J.; Dvorak, H. F.; Chan, W. C. W. Analysis of Nanoparticle Delivery to Tumours. Nat. Rev. Mater. 2016, 1, 16014-16025. (31) Carril, M.; Padro, D.; del Pino, P.; Carrillo-Carrion, C.; Gallego, M.; Parak, W. J. In Situ Detection of the Protein Corona in Complex Environments. Nat. Commun. 2017, 8, 1542-1546. (32) del Pino, P.; Yang, F.; Pelaz, B.; Zhang, Q.; Kantner, K.; Hartmann, R.; Martinez de Baroja, N.; Gallego, M.; Möller, M.; Manshian, B. B.; Soenen, S. J.; Riedel, R.; Hampp, N.; Parak, W. J. Basic Physicochemical Properties of Polyethylene Glycol Coated Gold Nanoparticles That Determine Their Interaction with Cells. Angew. Chem., Int. Ed. 2016, 55, 5483-5487. (33) Xie, C.; Zhen, X.; Lyu, Y.; Pu, K. Nanoparticle Regrowth Enhances Photoacoustic Signals of Semiconducting Macromolecular Probe for in Vivo Imaging. Adv. Mater. 2017, 29, 17036931703699. (34) Miao, Q.; Xie, C.; Zhen, X.; Lyu, Y.; Duan, H.; Liu, X.; Jokerst, J. V.; Pu, K. Molecular Afterglow Imaging with Bright, Biodegradable Polymer Nanoparticles. Nat. Biotechnol. 2017, 35, 1102-1110. (35) Lyu, Y.; Zhen, X.; Miao, Y.; Pu, K. Reaction-Based Semiconducting Polymer Nanoprobes for Photoacoustic Imaging of Protein Sulfenic Acids. ACS Nano 2017, 11, 358-367. (36) Yin, C.; Zhen, X.; Fan, Q.; Huang, W.; Pu, K. Degradable Semiconducting Oligomer Amphiphile for Ratiometric Photoacoustic Imaging of Hypochlorite. ACS Nano 2017, 11, 41744182. (37) Salvati, A.; Pitek, A. S.; Monopoli, M. P.; Prapainop, K.; Bombelli, F. B.; Hristov, D. R.; Kelly, P. M.; Aberg, C.; Mahon, E.; Dawson, K. A. Transferrin-Functionalized Nanoparticles Lose Their Targeting Capabilities When a Biomolecule Corona Adsorbs on the Surface. Nat. Nanotechnol. 2013, 8, 137-143. (38) Matsumura, S.; Aoki, I.; Saga, T.; Shiba, K. A Tumor-Environment-Responsive Nanocarrier That Evolves Its Surface Properties Upon Sensing Matrix Metalloproteinase-2 and Initiates Agglomeration to Enhance T(2) Relaxivity for Magnetic Resonance Imaging. Mol. Pharm. 2011, 8, 1970-1974. (39) Kumar, S.; Ravikumar, C.; Bandyopadhyaya, R. State of Dispersion of Magnetic Nanoparticles in an Aqueous Medium: Experiments and Monte Carlo Simulation. Langmuir : the ACS journal of surfaces and colloids 2010, 26, 18320-18330. (40) Harris, T. J.; von Maltzahn, G.; Derfus, A. M.; Ruoslahti, E.; Bhatia, S. N. Proteolytic Actuation of Nanoparticle Self-Assembly. Angew. Chem., Int. Ed. 2006, 45, 3161-3165.

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(41) Levi, J.; Kothapalli, S. R.; Bohndiek, S.; Yoon, J. K.; Dragulescu-Andrasi, A.; Nielsen, C.; Tisma, A.; Bodapati, S.; Gowrishankar, G.; Yan, X.; Chan, C.; Starcevic, D.; Gambhir, S. S. Molecular Photoacoustic Imaging of Follicular Thyroid Carcinoma. Clin. Cancer Res. 2013, 19, 1494-1502. (42) Harris, T. J.; von Maltzahn, G.; Lord, M. E.; Park, J.-H.; Agrawal, A.; Min, D.-H.; Sailor, M. J.; Bhatia, S. N. Protease-Triggered Unveiling of Bioactive Nanoparticles. Small 2008, 4, 13071312. (43) Peng, S.; Wang, C.; Xie, J.; Sun, S. Synthesis and Stabilization of Monodisperse Fe Nanoparticles. J. Am. Chem. Soc. 2006, 128, 10676-10677. (44) Cheong, S.; Ferguson, P.; Feindel, K. W.; Hermans, I. F.; Callaghan, P. T.; Meyer, C.; Slocombe, A.; Su, C. H.; Cheng, F. Y.; Yeh, C. S.; Ingham, B.; Toney, M. F.; Tilley, R. D. Simple Synthesis and Functionalization of Iron Nanoparticles for Magnetic Resonance Imaging. Angew. Chem., Int. Ed. 2011, 50, 4206-4209. (45) Peng, S.; Sun, S. Synthesis and Characterization of Monodisperse Hollow Fe3O4 Nanoparticles. Angew. Chem., Int. Ed. 2007, 46, 4155-4158. (46) Xie, J.; Wang, J.; Niu, G.; Huang, J.; Chen, K.; Li, X.; Chen, X. Human Serum Albumin Coated Iron Oxide Nanoparticles for Efficient Cell Labeling. Chem. Comm. 2010, 46, 433-435. (47) Bremer, C.; Tung, C.-H.; Weissleder, R. In Vivo Molecular Target Assessment of Matrix Metalloproteinase Inhibition. Nat. Med. 2001, 7, 743-748. (48) Levi, J.; Kothapalli, S. R.; Ma, T.-J.; Hartman, K.; Khuri-Yakub, B. T.; Gambhir, S. S. Design, Synthesis, and Imaging of an Activatable Photoacoustic Probe. J. Am. Chem. Soc. 2010, 132, 11264-11269. (49) Cho, E. C.; Zhang, Q.; Xia, Y. The Effect of Sedimentation and Diffusion on Cellular Uptake of Gold Nanoparticles. Nat. Nanotechnol. 2011, 6, 385-391. (50) Colombo, M.; Ronchi, S.; Monti, D.; Corsi, F.; Trabucchi, E.; Prosperi, D. Femtomolar Detection of Autoantibodies by Magnetic Relaxation Nanosensors. Anal. Biochem. 2009, 392, 96102. (51) Perez, J. M.; Josephson, L.; O'Loughlin, T.; Högemann, D.; Weissleder, R. Magnetic Relaxation Switches Capable of Sensing Molecular Interactions. Nat. Biotechnol. 2002, 20, 816820. (52) Yang, M.; Cheng, K.; Qi, S. B.; Liu, H. G.; Jiang, Y. X.; Jiang, H.; Li, J. B.; Chen, K.; Zhang, H. M.; Cheng, Z. Affibody Modified and Radiolabeled Gold-Iron Oxide Hetero-Nanostructures for Tumor PET, Optical and MR Imaging. Biomaterials 2013, 34, 2796-2806.

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Figures:

Figure 1. Schematic illustration of the controlled colloidal behaviors by which nanoprobes can selectively target specific tumors and monitor them over time in vivo. Functional nanoprobes are shown as representative core/shell nanospheres with either active coating (active probe) or inactive coating (inactive probe). The nanoprobes can passively accumulate into the tumors by extravasation of nanoprobes through increased permeability of the tumor vasculature and ineffective lymphatic drainage (EPR effect). The surface coating on the active probes can be cleaved by the enzymes over-expressed within specific tumors, thereby resulting in a significant decrease of their water-solubility in a physiological condition. Upon removing PEG coating, the changes in hydrodynamic size and surface charges of nanoprobes as a result of interacting with biomolecules and proteins can lead to dramatic changes in their in vivo colloidal behaviors (i.e., slow diffuse rates, tendency to aggregate and sediment). The retention time of nanoprobes within the tumor tissues depends on the ratio of the uptake and excretion rate of the nanoprobes through the tumors. The active nanoprobes on activation by specific enzyme can result in much higher tumor accumulation and longer retention within the tumors than the inactive nanoprobes, which passively pass through the tumors. The red-colored symbols at the top of linkers represent various types of tags such as fluorescent dyes or radiotracers. The released linkers with tags can be used as an indicator for monitoring the enzyme activities of diseases at a molecular level.

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Figure 2. Monodispersed core/shell Fe/IONPs. (a) Schematic illustration of controlled oxidation of as-synthesized Fe/IONPs. The core/shell Fe/IONPs were obtained by oxidation of assynthesized Fe/IO nanoparticles in a controlled manner. The complete oxidation of as-synthesized Fe/IO nanoparticles resulted in the hollow IONPs. (b) TEM images of oxidized core/shell Fe/IONPs. (c) TEM images of hollow IONPs (HIONPs). (d) Size distributions of various nanoprobes (as-synthesized Fe/IONPs, Fe/IONPs, and HIONPs) in hexane measured using Dynamic Light Scattering (DLS) method. (e) TEM images of the oxidized core-shell Fe/IONPs showing the uniform Fe3O4 coating on the Fe core. The lattice spacing between two planes of the shells (b) is 2.961 Å, corresponding to the distance of two (220) planes of magnetite Fe3O4.

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Figure 3. Validation of enzyme-responsive imaging probes. (a) Schematic illustration of construction and in vitro evaluation of enzyme-responsive imaging probes. The amine-modified core/shell Fe/IONPs were conjugated with either cleavable dye-PEG linkers or noncleavable dyePEG linkers to form active MMP2-Fe/IONPs or inactive Ctrl-Fe/IONPs, respectively. The PEGylated peptide linkers can make the as-synthesized nanoprobes water-soluble. The surface PEG coating on the active probes can be cleaved by the enzymes over-expressed within specific tumors. The cleaved linkers with dyes were measured as fluorescence intensity over time to monitor the enzyme activity. (b) UV-vis spectra of MMP2-Fe/IONPs, Rhodamine B (RB), and RD-MMP2-Fe/IONPs in water. (c) Fluorescence spectra of Rhodamine B (RB) and RD-MMP2Fe/IONPs at the same concentration of dye in water or in ethanol. (d) Activation of the MMP-

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Fe/IONPs by MMP-2 in vitro. Complete cleavage of linkers from the MMP-Fe/IONPs as judged by an increase in fluorescence intensity was achieved using 2 µg/mL MMP-2 at 37C for 5 h. (e) Time course study on MMP-cleavage capacity of MMP-Fe/IONPs and Ctrl-Fe/IONPs (mean  SD, n = 3).

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Figure 4. Evaluation of the colloidal behaviors of enzyme-responsive imaging probes. (a) Schematic illustration of in vitro evaluation of enzyme-responsive imaging nanoprobes. (b), Schematic representation of two particles with diameter of d and layer thickness (L) separated by a surface-to-surface distance (s) and a center-to-center distance (D). The particle dispersity of various nanoprobes: (c) Hydrodynamic sizes of various nanoprobes, including MMP2-Fe/IONPs with different coating densities (see more information in Table S2). (d) Hydrodynamic sizes of various nanoprobes depend on the surface coating densities. (e) Count rates of various nanoprobes depend on the surface coating densities. Time-dependent changes in average sizes (f) and count rates (g) of MMP2-Fe/IONPs and Ctrl-Fe/IONPs following incubation with MMP-2. (f) Plots of time-dependent changes in average sizes of MMP2-Fe/IONPs and Ctrl-Fe/IONPs (5 nM) following incubation with MMP-2 (1.5 pmol) in PBS buffer. (g) Plots of time-dependent changes in DLS count rates of MMP2-Fe/IONPs and Ctrl-Fe/IONPs (5 nM) following incubation with MMP-2 (1.5 pmol) in PBS buffer. (h) TEM images of time-dependent changes in particle dispersity of MMP2-Fe/IONPs following incubation with MMP-2 in buffer at 0, 1, 2, 3, 4, 5, and 6 h. (h-2h), Representative TEM image of MMP2-Fe/IONPs incubated with MMP-2 at 2 h. Scale bars = 100 nm.

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Figure 5. Magnetic properties of various nanoprobes, including MMP2-Fe/IONPs, MMP2HIONPs, and Ferumoxytol. (a) Phantom images acquired from T2-weighted MRI scans (TE/TR = 40/4000 ms for spin-echo measurement) for MMP2-Fe/IONPs, HIONPs, and Ferumoxytol at different iron concentrations (0, 0.031, 0.0625, 0.125, 0.25, 0.5, 1.0, and 2.0 mM [Fe]). (b) Phantom images acquired from T2*-weighted MRI scans (TE/TR = 9.1/300 ms for gradient-echo measurement) for MMP2-Fe/IONPs, HIONPs, and Ferumoxytol at different iron concentrations. (c) R2 relexivity curves of MMP2-Fe/IONPs, HIONPs, and Ferumoxytol. Relaxivity rates r2 were obtained from slopes of linear fits of the curves of 1/T2 vs. Fe concentrations (mean  SD, n = 3). See more information about fitting data in Table S3. (d) The R2* relexivity curves of MMP2Fe/IONPs, HIONPs, and Ferumoxytol. Relaxivity rates r2* were obtained from slopes of linear fits of the curves of 1/T2 vs. Fe concentrations (mean  SD, n = 3).

Figure 6. Cellular uptake and activation of the nanoprobes by MMP-2 in vitro. (a) Cellular uptakes of MMP2-Fe/IONPs and Ctrl-Fe/IONPs by HT1080 cells after 4h incubation. HT1080 cells were treated with a serial of concentrations of MMP2-Fe/IONP and Ctrl-Fe/IONP for 4 h. (b) Uptake

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efficiency (% of total) of MMP2-Fe/IONP and Ctrl-Fe/IONP by HT1080 cells at different incubation concentrations. (c) Phantom images acquired from T2-weighted MRI scans (TE/TR = 40/4000 ms) for MMP2-Fe/IONPs and Ctrl-Fe/IONP at a serial of iron concentrations in the cell lysates. (d) and (e) R2 relexivity curves of MMP2-Fe/IONPs and Ctrl-Fe/IONP. Relaxivity rates r2 were obtained from slopes of linear fits of the curves of 1/T2 vs. Fe concentrations within cells (mean  SD, n = 3).

Figure 7. Noninvasive monitoring of MMP2-positive HT1080 tumors in vivo with MRI. (a) T2weighted MR axial images of HT1080 tumor bearing mice (n = 4) before and after intravenous injection of MMP2-Fe/IONPs (10 mg/kg mouse). The top row shows gray scale images, and the

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bottom row shows the pseudo-colored image (NIH look-up tables (LUTs)). (b) T2-weighted MR axial images of HT1080 tumor bearing mice (n = 4) before and after intravenous injection of CtrlFe/IONPs (10 mg/kg mouse). The top row shows gray scale images, and the bottom row shows the pseudo-colored image (NIH look-up tables (LUTs)). (c) MRI quantification analysis of tumors uptake of MMP2-Fe/IONPs (red line and circles) and Ctrl-Fe/IONPs (green line and cubes) across time (0, 1, 2, 4, 24, and 48 h postinjection) (mean  SD, n = 4 per treatment group). * p < 0.01. (d) Distribution of both MMP2-Fe/IONPs and Ctrl-Fe/IONPs in the tumor was quantified by ICP-MS at 48 h postinjection. Because of the presence of endogenous iron in plasma and tissues, the control mice (n = 3) were used for “particle-free” correction.

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