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

Abstract

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.

Scheme 1. (a) The construction procedure and detection principle of fluorescent turn-on nanosensor MnIO@pep-FITC. (b) The detailed structure of MnIO@pep-FITC. The illustration is not drawn to scale.

Fig. 1. TEM micrographs of (a) MnIO, (b) MnIO@PEG, (c) MnIO@pep-FITC, respectively. (d) Magnetic hysteresis loop of MnIO@pep-FITC.

Fig. 2. (a) Fluorescence spectra of 100 μg mL⁻¹ MnIO@pep-FITC in the presence of 60 ng mL⁻¹ trypsin at various incubation times. Inset is F_R of various incubation times. (b) Fluorescence spectra of MnIO@pep-FITC in the presence of various concentrations of trypsin following incubation for 90 min. (c) F_R of MnIO@pep-FITC as a function of trypsin concentration. (d) The selectivity of the MnIO@pep-FITC. The inset of (d) is the corresponding fluorescence spectra. The error bars mean standard deviations (n = 3).

Fig. 3. Fluorescence spectra of (a) HCT116 cells and (b) NCM460 cells cultured with 100 μg mL⁻¹ MnIO@pep-FITC for various times (0–4 h), and the lysates of normal cultured HCT116 cells and NCM460 cells were used as backgrounds, respectively. (c) Corresponding ICP-MS results of HCT116 cells and NCM460 cells, and control groups are the results of normal cultured HCT116 cells and NCM460 cells. (d) The fluorescence spectra of 100 μg mL⁻¹ MnIO@pep-FITC incubated with the lysates of various amounts of HCT116 cells. Inset is the corresponding calibration curve of F_Rversus cell numbers in 1 mL reaction solution. The error bars mean standard deviations (n = 3).

Fig. 4. Fluorescence micrographs of HCT116 cells co-cultured with 100 μg mL⁻¹ MnIO@pep-FITC in the MF for 4 h, (a) bright field mode, (b) FITC mode and (c) merging image. The scale bar is 100 μm.

Fig. 5. (a) In vivo MR images of HCT116 tumor-bearing nude mice injected intravenously with MnIO@pep-FITC (0 h means pre-injection). (b and c) In vivo fluorescence images of HCT116 tumor-bearing nude mice with intravenous injection of MnIO@pep-FITC (0 h means pre-injection, (b) large tumor (126 mm³ in volume) and (c) ultrasmall tumor (ca. 4.9 mm³ in volume)). (d) Relative MR signal values of tumor after intravenous injection of MnIO@pep-FITC at different time intervals. (e) Quantification of fluorescence intensities at the tumor sites of (b) and (c) after intravenous injection. The error bars mean standard deviations (n = 3).