Peptide modified manganese-doped iron oxide nanoparticles as a sensitive fluorescence nanosensor for non-invasive detection of trypsin activity in vitro and in vivo

Herein, a fluorescence turn-on nanosensor (MnIO@pep-FITC) has been proposed for detecting trypsin activity in vitro and in vivo through covalently immobilizing an FITC modified peptide substrate of trypsin (pep-FITC) on manganese-doped iron oxide nanoparticle (MnIO NP) surfaces via a polyethylene glycol (PEG) crosslinker. The conjugation of pep-FITC with MnIO NPs results in the quenching of FITC fluorescence. After trypsin cleavage, the FITC moiety is released from the MnIO NP surface, leading to a remarkable recovery of FITC fluorescence signal. Under the optimum experimental conditions, the recovery ratio of FITC fluorescence intensity is linearly dependent on the trypsin concentration in the range of 2 to 100 ng mL−1 in buffer and intracellular trypsin in the lysate of 5 × 102 to 1 × 104 HCT116 cells per mL, respectively. The detection limit of trypsin is 0.6 ng mL−1 in buffer or 359 cells per mL HCT116 cell lysate. The MnIO@pep-FITC is successfully employed to noninvasively monitor trypsin activity in the ultrasmall (ca. 4.9 mm3 in volume) BALB/c nude mouse-bearing HCT116 tumor by in vivo fluorescence imaging with external magnetic field assistance, demonstrating that it has excellent practicability.


Introduction
As paradigms of enzyme catalysis, serine (Ser) proteases play critical roles in a variety of biological and physiological processes such as cell differentiation, cell apoptosis, blood coagulation, atherosclerosis, inammation and cancer. [1][2][3][4][5][6] The Ser proteases normally contain a nucleophilic Ser residue at their active site, which has nucleophilic activity against the peptide bond resulting in the digestion of some proteins implicated in vital activities. Trypsin (EC 3.4.21.4), a kind of Ser protease produced by the pancreas, is the most popular digestive enzyme, which is not only involved in the digestion of dietary proteins but also induces proteolytic cascades by activating other proteases, such as matrix metalloproteinases (MMPs) through the selective hydrolysis of polypeptide chains of arginine (Arg) or lysine (Lys) in the C-terminal. [7][8][9] It is found that trypsin expression is signicantly increased in several human cancer cells of the stomach, colon, lung and breast. 10 Recently, trypsin has been regarded as a potential prognosticator for cancer patients because dysfunction of trypsin has been linked to the malignancy of several tumors including colorectal cancer (CRC), gastric cancer and pancreatic cancer. 9,[11][12][13][14][15][16] For instance, 5 year survival rate of patients with trypsin-positive CRC is lower than that of patients with trypsinnegative CRC. 11 Therefore, timely detection of trypsin activity is critical for diagnosis and treatment of CRC.
At present, a large number of methods/assays have been developed for quantication of trypsin levels in vitro including chromatography, 17 radioimmunoassay 18 and biosensors with different detection principles. [19][20][21][22][23][24][25][26][27][28][29][30][31][32][33][34][35][36][37][38] Among of these techniques, peptide substrate-based biosensors have been considered as a promising way to determine trypsin activity in different matrixes since peptide substrates exhibit several unique merits, such as easy synthesis, low cost, high stability in harsh environments and suitable to conjugate/modify with other label molecules. [33][34][35][36][37][38] For example, a variety of peptide-based uorescence turn-on/off strategies have been developed for sensing trypsin activity with high sensitivity and selectivity. [32][33][34] Unfortunately, most of asdeveloped sensing platforms are only capable of trypsin activity detection in vitro, which does not necessarily reect the quantitative information on trypsin expression in a solid tumor. It is still a challenge to develop peptide-based biosensors for measurement of tumor-related trypsin activity in vivo since it is difficult to efficiently transport peptide substrate to tumor site.
Recently, magnetic nanoparticles (MNPs) have been successfully employed to fabricate Förster resonance energy transfer (FRET) sensing platforms for detecting MMP-9 activity both in vitro and in vivo because there is inherent spectral overlap interference of uorescent dyes and MNPs. 39 The MNPbased FRET sensing platform enables to quantitatively map MMP-9 activity across the entire tumor because MNPs can efficiently accumulate in tumors through the enhanced permeability and retention (EPR) effect. In particular, the accumulation amount of MNPs in the tumor site can be further enhanced with the aid of an external magnetic eld (MF). [40][41][42] The phenomenon may increase the detection sensitivity of MNP-based FRET sensing platforms.
Herein, a peptide-functionalized manganese-doped iron oxide nanoparticle-based uorescence turn-on sensing platform (MnIO@pep-FITC) has been constructed for noninvasive detection of trypsin activity in vivo through conjugation of FITC modied peptide substrate on the MnIO NP surface. Under the specic digestion of trypsin, the FITC was liberated from MnIO@pep-FITC, and quenched uorescence could be recovered in response to trypsin activity. With the aid of an external MF, we demonstrate that MnIO@pep-FITC can be used to visualize ultrasmall (ca. 4.9 mm 3 in volume) trypsin-positive subcutaneous tumor (HCT116) in BALB/c nude mouse through intravenous administration, showing great promising application in the diagnosis of tumor.

Fabrication of MnIO@pep-FITC
The OA capped MnFe 2 O 4 nanoparticles (MnIO NPs) were rstly synthesized by the literature reported strategy with slight modications (see ESI † for details). 43 For transferring the hydrophilic OA capped MnIO NPs to aqueous medium, 20 mg OA capped MnIO NPs were mixed with 20 mg DIB-PEG-COOH in 5 mL CHCl 3 and stirred at room temperature for 4 h. Then, the DIB-PEG-COOH modied MnIO NPs (MnIO@PEG) were collected by centrifugation (6000 rpm, 10 min), washed with the 10 mL mixture of hexane and CHCl 3 (6000 rpm, 10 min, 3 times), and redispersed in 1 mL H 2 O.

Sensing performance of MnIO@pep-FITC in buffer
For trypsin detection, 100 mg mL À1 MnIO@pep-FITC were incubated with various concentrations of trypsin in 500 mL PBS (10 mmol L À1 , pH 7.4) at 37 C for 90 min. Subsequently, the uorescence spectra of mixtures were measured on a QE65 Pro ber optic spectrometer with 490 nm as the excitation wavelength. To investigate its selectivity, MnIO@pep-FITC were incubated with 100 ng mL À1 a series of enzymes including matrix metalloproteinases (MMP-2, MMP-7 and MMP-9), caspase-3 and caspase-9 under optimized conditions.

Intracellular trypsin activity detection
The HCT116 and NCM460 cells (1.5 Â 10 4 cells per well) were seeded in the 48-well microtiter plate and cultured in 300 mL fresh culture medium for 24 h. Aer washed with PBS (3 times), the cells were incubated with 100 mg mL À1 MnIO@pep-FITC in 300 mL fresh culture medium at 37 C for various time (0, 1, 2, 3 and 4 h), respectively. Then, the uorescence imaging was performed with a reconstructive Nikon Ti-S uorescent microscope at the FITC channel. For quantitative measurements, the MnIO@pep-FITC stained cells were washed with PBS (300 mL, 3 times), detached from the 48-well plate, collected by centrifugation (1000 rpm, 5 min), redispersed in 100 mL lysis buffer, diluted by 400 mL PBS, and subjected to uorescence measurement. In addition, to investigate the intracellular trypsin detection sensitivity, the unstained HCT116 cells were also treated as previously described, and then the cell lysates containing different cell numbers were incubated with 100 mg mL À1 MnIO@pep-FITC in 500 mL PBS at 37 C for 90 min. The uorescence spectra of mixture were then measured at the excitation wavelength of 490 nm. For the in vitro MF-assisted uorescence imaging, a magnet was placed under the center of the cell culture dish while 100 mg mL À1 MnIO@pep-FITC were added into the dish. Aer co-cultured for 4 h, the cells were washed with 1 mL PBS (3 times), and subjected to image by a reconstructive Nikon Ti-S uorescent microscope at the FITC channel.

In vivo toxicity evaluation
All animal procedures were performed in accordance with the Guidelines for Care and Use of Laboratory Animals of Jilin University and experiments were approved by the Animal Ethics Committee of Jilin University. For histology analysis, 200 mL 0.9 wt% NaCl solution with or without 10 mg kg À1 MnIO@pep-FITC were injected intravenously into the healthy mice as experimental group and control group, respectively. The healthy mice were anatomized at 30 days post-injection, and the major organs including heart, liver, spleen, lung and kidneys were collected for hematoxylin and eosin (H&E) staining. Furthermore, blood biochemistry assay was also employed to evaluate the biosafety of MnIO@pep-FITC.

In vivo uorescence and magnetic resonance (MR) imaging
Female BALB/c nude mice (6 to 8 weeks old) were selected for establishing a HCT116 tumor model and injected subcutaneously with HCT116 cells (3 Â 10 6 cells in 100 mL PBS) on the right anks of mice. A 0.5 T magnet was placed around the tumor site for generate external MF. For in vivo MR imaging, the HCT116 tumor-bearing mice (tumor size: 113 mm 3 ) were injected intravenously with NaCl solution (0.9 wt%, 200 mL) containing 10 mg kg À1 MnIO@pep-FITC. Aer placed in the MF for 15 min, T 2 -weighted MR images of mice were collected by using a GE Signa 3 T MR unit with the following imaging parameters: repetition time (TR), 240 ms; echo time (TE), 15.9 ms; eld of view, 120 mm Â 72 mm; slice thickness, 2.0 mm. For in vivo uorescence imaging, the tumor-bearing mice (tumor size: 4.9 mm and 126 mm 3 ) were injected intravenously with NaCl solution (0.9 wt%, 200 mL) containing 10 mg kg À1 MnIO@pep-FITC. Subsequently, the mice were placed in the MF for 15 min and then the in vivo uorescence imaging was collected by the Davinch Invivo HR imaging system at predetermined time intervals (excitation wavelength: 490 nm; emission wavelength: 520 nm, exposure time: 5 s). For quantitative analysis, the regions of interest (ROI) were drawn over tumors and measured by the ImageJ soware. The tumor volumes were calculated according to the following formula: The size of as-synthesized OA capped MnIO NPs is 30.5 AE 2.9 nm in diameters (as shown in Fig. 1a). The X-ray diffraction (XRD) measurement indicates that the diffraction peaks of MnIO NPs is consistent with the manganese ferrite standard card (MnFe 2 O 4 , JCPDS 73-1964, as shown in Fig. S1 †). The IR bands at 1443 cm À1 and 1653 cm À1 are attributed to C]C symmetric stretching vibration band of aromatic ring of the DIB group (as shown in Fig. S2 †), suggesting that DIB-PEG-COOH is modied on the MnIO NPs surface. The IR band at 2088 cm À1 of FITC (vibration band of isothiocyanate) is observed in the FTIR spectrum of MnIO@pep-FITC (as shown in Fig. S2 †), indicating that pep-FITC is successfully conjugated to MnIO-PEG. The size and morphology of MnIO NP exhibits negligible changes aer modication with DIB-PEG-COOH and pep-FITC, respectively (as shown in Fig. 1b and c). The hydrodynamic sizes of MnIO-PEG and MnIO@pep-FITC are 53.77 AE 6.82 and 64.95 AE 4.87 (as shown in Table S1 †), respectively, indicating that they have good monodispersity. Because of the out-layer of carboxyl group, the MnIO-PEG has relatively low zeta potential (À12.8 AE 1.01 mV, as shown in Table S1 †). The zeta potential of MnIO@pep-FITC (À7.09 AE 0.56 mV) is higher than that of MnIO-PEG (as shown in Table S1 †), also conrming the

Sensing performance of MnIO@pep-FITC in buffer
The uorescence intensity of FITC is gradually increased as the reaction time prolongs and reaches saturation at approximately 90 min, when the concentrations of trypsin and MnIO@pep-FITC are kept as constants (as shown in Fig. 2a). Taking together with the cytotoxicity of MnIO@pep-FITC, the trypsin is detected under following conditions, 90 min reaction time with 100 mg mL À1 MnIO@pep-FITC. The uorescence intensity of FITC is increased proportionally with the concentration of trypsin increasing (as shown in Fig. 2b). The uorescence recovery ratio (F R ) has a good linear t to the trypsin concentration within the range from 2 ng mL À1 to 100 ng mL À1 (as shown in the Fig. 2c). Here, F R ¼ DF/F 0 , DF ¼ F À F 0 , F 0 and F are the uorescence intensities of FITC of MnIO@pep-FITC before and aer trypsin digestion. The limit of detection is calculated to be 0.6 ng mL À1 or 25.2 pmol L À1 (3 times the standard deviation of F R of blank solution), which is lower than those of literature reported. [46][47][48] The performance of the method is better than or comparable to those of other methods (as shown in Table S2 †). In order to evaluate its selectivity, MnIO@pep-FITC was exposed to ve proteases including MMP-2, MMP-7, MMP-9, caspase-3 and caspase-9, which possibly coexist with trypsin in real samples (such as cells and tumor tissues). The F R values of those interferences are much lower than that of trypsin (as shown in the Fig. 2d), suggesting that MnIO@pep-FITC has good selectivity.

Detection of intracellular trypsin activity
Prior to the detection of intracellular trypsin activity, the NCM460 cells (normal colonic epithelial cell) and HCT116 cells (CRC cell) were selected to evaluate the cytotoxicity of MnIO@pep-FITC by traditional MTT assay. The cell viabilities of NCM460 cells and HCT116 cells are still above 90% aer incubated with 100 mg mL À1 MnIO@pep-FITC for 24 h (as shown in Fig. S5 †). The result indicates that MnIO@pep-FITC has low cytotoxicity.
For demonstrating its sensing capability, MnIO@pep-FITC was employed for evaluating intracellular trypsin activities of NCM460 cells and HCT116 cells. As expected, the FITC uorescence signals of cells were increased with increasing incubation time, when the cells were incubated with a certain concentration of MnIO@pep-FITC (as shown in Fig. 3a, b, S6 and S7 †). Under the same experimental condition, the result of ICP-MS measurement demonstrates that there is no signicant difference of internalization amounts between HCT116 cells and NCM460 cells (as shown in Fig. 3c), while the uorescence intensity of HCT116 cells is much higher than that of NCM460  cells. The results demonstrate that HCT116 cells express higher level of trypsin than that of NCM460 cells. In addition, the uorescence intensity of mixtures is gradually increased with increasing cell numbers when MnIO@pep-FITC were incubated with HCT116 cell lysates. And the F R value shows a linear correlation with the cell lysate within the range of 5 Â 10 2 to 1 Â 10 4 cells per mL with a detection limit of 359 cells per mL. The result also demonstrates that MnIO@pep-FITC has high sensitivity for detection of intracellular trypsin activity.
Furthermore, there is an apparent local accumulation of MnIO@pep-FITC guided by the MF (i.e., only the cells localized close to the magnet show strong uorescence signal (as shown in Fig. 4)). All of the experimental results suggest that MnIO@pep-FITC could be used to sensitively visualize tumor through in situ monitoring of trypsin activity in cancer cells with the aid of external MF.

Detection of trypsin activity in vivo
Before detection of trypsin activity in vivo, the toxicity of MnIO@pep-FITC was evaluated by blood test and histology analysis. There is little difference in blood examinations between control group and MnIO@pep-FITC treated group (as shown in Table S3 †). No hemolytic phenomenon is observed by hemolysis test (as shown in Fig. S8 †). The histology analysis suggests that there is neither noticeable tissue damage nor inammation of MnIO@pep-FITC on major organs (as shown in Fig. S9 †). These results demonstrate that MnIO@pep-FITC has good biocompatibility.
For demonstrating its sensing capability in vivo, MnIO@pep-FITC were injected into in the HCT116 tumor-bearing BALB/c nude mice through the tail veins with external MF assistance, respectively. The in vivo T 2 -weighted MR and uorescence imaging were performed aer intravenous injection of MnIO@pep-FITC. Generally, the tumor site displays a clearly signal enhancement on both of the MR images and FITC uorescence images at 1 h post-injection of MnIO@pep-FITC (as shown in Fig. 5). The MR signal enhancement is gradually decreased aer reaching maximum MR signal enhancement at 2 h post-injection (as shown in Fig. 5a and d). The uorescence intensity of FITC continuously increases until it reaches the maximum value at 4 h post-injection before decaying (as shown in Fig. 5b and e). The delay of maximum uorescence intensity of FITC to maximum MR signal can be understood by the cleavage kinetics of trypsin (as shown in Fig. 2a) because the  uorescence intensity of FITC is mainly governed by the trypsin activity. The results suggest that MnIO@pep-FITC can be employed to in situ monitor trypsin activity of tumor by in vivo uorescence imaging. Encouraging by its strong trypsin detection capacity, MnIO@pep-FITC was injected into nude micebearing ultrasmall HCT116 tumor xenogras (4.9 mm 3 in volume) through the tail veins. Strong uorescence signal enhancement of tumor site was successfully observed at 4 h post-injection (as shown in Fig. 5c and e). The result further demonstrates that MnIO@pep-FITC could be used as an efficient tool to diagnose tumor through sensitive measurement of trypsin activity.
The in vivo MR images of mice organs were recorded for evaluating the in vivo clearance pathway of the MnIO@pep-FITC. During the whole experiment process, the MR signals of liver exhibit signicant variation, while MR signals of kidneys show negligible change (as shown in the Fig. S10 and S11 †). Only a small amount of MnIO@pep-FITC accumulated in the kidney and were excreted within 24 hours. The MR signal of liver decreased by 42% at 5 h post-injection. And aer 24 hours, the degree of MR signal reduction returned to 31%. The result demonstrates that MnIO@pep-FITC are metabolized by the liver.

Conclusions
In summary, a uorescence turn-on nanosensor, MnIO@pep-FITC has been developed for highly sensitive and selective determination of trypsin activity both in vitro and in vivo. The results indicate that the MnIO@pep-FITC has relatively low detection limits for both of pure trypsin and intracellular trypsin integrated with a reasonable dynamic range. In particular, the MnIO@pep-FITC enables to noninvasively visualize BALB/c nude mouse-bearing ultrasmall (ca. 4.9 mm 3 in volume) trypsin-positive subcutaneous tumor by in vivo uorescence imaging with the aid of an external MF. Combination with its excellent biocompatibility and long-term stability, the MnIO@pep-FITC shows great promise for screening trypsinpositive tumors at early stage.

Conflicts of interest
There are no conicts to declare.