Preparation and characterisation of PTP-Fe3O4-IRDye800CW nanoparticles
To ensure active targeting, DSPE-PEG-PTP and DSPE-PEG-CON were first synthesised by conjugating PTP to DSPE-PEG-NHS with an active carbonyl ester group via stable amide bonds, which was demonstrated using Fourier-transform infrared spectroscopy (FTIR) spectra (Supplementary Fig. 1a, 1b). And then, PTP-Fe3O4-IRDye800CW nanoparticles were generated by coating the surface of oleic acid-modified Fe3O4 with phospholipids of DSPE-PEG-PTP, DSPE-IRDye800CW, and DSPC. This process ensured that the resulting system could disperse well in aqueous solutions through the thin film hydration method, in which the mass ratio of oleic acid-modified Fe3O4 and phospholipids was 1:2. Meanwhile, the molar ratio of DSPE-PEG-PTP and DSPE-IRDye800CW was 30:10:60. The thin film containing all these materials was hydrated with water and sonicated to obtain stable and dispersed nanoparticles. Transmission electron microscopy (TEM) observation demonstrated that the uniform size of PTP-Fe3O4-IRDye800CW nanoparticles was ~ 20 nm with square morphology (Fig. 2a), and dynamic light scattering revealed an average particle size of 27.2 nm (Supplementary Fig. 1c). The slightly larger size is likely due to the surface coating layer, which disrupts nanoparticle aggregation. The control group of Con-Fe3O4-IRDye800CW nanoparticles was prepared using the same procedure. The Con-Fe3O4-IRDye800CW was similar in size (~ 25.6 nm) and morphology (Fig. 2b, Supplementary Fig. 1d); thus, were well suited for subsequent comparison. The FTIR spectra of PTP-Fe3O4-IRDye800CW and Con-Fe3O4-IRDye800CW nanoparticles were further used to investigate the composition of the surface coating of Fe3O4. The spectra displayed characteristic peaks of DSPE-PEG-PTP or DSPE-PEG-CON and DSPE-IRDye800CW, which indicated successful modifications of the targeting group and fluorescent dye onto the nanoparticles (Fig. 2c, 2d).
The absorbance spectra of aqueous PTP-Fe3O4-IRDye800CW and Con-Fe3O4-IRDye800CW nanoparticles were further investigated. These spectra exhibited a wide and elevated absorption between and 550–850 nm and a maximum absorption at ~ 780 nm, which was slightly red-shifted compared to that of the free IRDye800CW. The highest peak was at 774 nm (Fig. 2e). This shift might be due to the interaction between IRDye800 CW and the cored Fe3O4. The IRDye800CW fluorescence emission spectra further indicated that both PTP-Fe3O4-IRDye800CW and Con-Fe3O4-IRDye800CW nanoparticles had similar fluorescence to free IRDye800CW upon light irradiation (the excitation and emission wavelengths were 740 nm and 795 nm, respectively) (Fig. 2f), confirming their ability to be used as fluorescent imaging probes for biological applications. Dispersed stability is an important way to mimic the in vivo environment. PTP-Fe3O4-IRDye800CW nanoparticles were well dispersed in various biological media without any precipitation, such as DMEM, FBS, RPMI-1640 cell medium, PBS, and deionised (DI) water (Supplementary Fig. 1e). These findings revealed that thes nanoparticles can be further utilized for the following imaging studies.
In vitro FMI/MPI/MRI properties of PTP-Fe3O4-IRDye800CW nanoparticles
The IRDye800CW on the PTP-Fe3O4-IRDye800CW nanoparticles can be utilized for FMI, and Fe3O4 can be utilized for MPI and T2-weighted MRI dual-modality imaging. To assess the FMI, MPI, and MRI T2 relaxivity properties of PTP-Fe3O4-IRDye800CW nanoparticles, we initially conducted FMI, MPI, and MRI measurements in the phantom. PTP-Fe3O4-IRDye800CW nanoparticles were prepared at different Fe concentrations, as identified by ICP-OES, to analyse the relevance of FMI, MPI, and MRI observations. The IRDye800CW fluorophore was conjugated to Fe3O4 at a ratio of 50:1. The fluorescence intensity was linearly correlated with the increased concentration of PTP-Fe3O4-IRDye800CW samples at Fe concentrations ranging from 0.3125–2.5000 µg/mL, as shown in Fig. 2g (Y = 2.990 × 108 X + 3.947 × 107, R2 = 0.998, P < 0.0001). However, with the increase in sample concentration, the fluorescence intensity tended to reach a plateau at 20.00 µg/mL due to the quenching effect. The MPI linear regression results and corresponding images are demonstrated in Fig. 2h, which shows that MPI signals increased with an increase in sample concentration (Y = Y = 400.9 × X + 18.38, R2 = 0.9997, P < 0.0001). Moreover, Fig. 2i shows the T2-weighted MRI of nanoparticles with reference to the sample concentration. The T2-weighted MR images of all the samples became dimming with incremental concentrations, indicating the samples as T2-negative contrast agents. The r1 of PTP-Fe3O4-IRDye800CW nanoparticles was calculated to be 44.4192 mM− 1s− 1 at 7.0 T.
Characterization of Plectin-1 expression in murine and human tumour PDAC xenografts
To validate plectin-1 as a specific molecular target for PDAC, the expression and location of human and murine pancreatic cell lines and tumour tissues were analysed. In BxPC3 human pancreatic tumour xenografts obtained from nude mice, high plectin-1 expression levels were detected by means of immunohistochemistry (Fig. 3a). Similarly, high expression of plectin-1 observed in pan02 murine pancreatic tumour xenografts from C57 mice (Fig. 3a). We found that plectin-1 was expressed at a high level in PDAC but not in other major organs, such as, the heart, liver, spleen, lung, and kidney (Fig. 3b). Flow cytometry data further quantified the cell surface and intracellular expression of plectin-1 in the pancreatic cancer cell lines. In Pan02 cells, 21.11% of the plectin-1 expression was observed on the cell surface, whereas 90.05% was observed in the cells. Similarly, 29.27% of the plectin-1 expression was observed on the BxPC3 cell surface, while 99.80% of the plectin-1 expression was observed intracellularly (Fig. 3c).
In vitro targeting specificity and cytotoxicity of PTP-Fe3O4-IRDye800CW nanoparticles
Cells were incubated with different concentrations of nanoparticles (2.5–80 µg/mL) for 24 h and their viability was examined. The data showed that no significant cytotoxicity was observed (Fig. 3d). Further, the binding specificity of PTP-Fe3O4-IRDye800CW nanoparticles for both murine (Pan02-Luc) and human (BxPC3-Luc) pancreatic cancer cells was assessed. Two pancreatic cancer cell lines were incubated with PTP-Fe3O4-fluorescein isothiocyanate (FITC) and Con-Fe3O4-FITC as the control, and laser scanning confocal microscopy was performed to examine their targeting ability. The pancreatic cancer cell uptake was obviously higher for PTP-Fe3O4-FITC nanoparticles than for Con-Fe3O4-FITC nanoparticles, which confirmed the specific pancreatic cancer cell targeting ability of the PTP peptide (Fig. 3e). The cytotoxicity assay was analyzed using the CCK-8 assay for both murine (Pan02-Luc) and human (BxPC3-Luc) pancreatic cancer cells. In general, the data showed that the PTP-Fe3O4-IRDye800CW nanoparticles possessed potential targeted imaging properties with good biocompatibility and biosafety.
In vivo MPI, FMI and MRI multimodality imaging for subcutaneous PDAC mouse model
MPI-FMI-MRI triple-modality imaging was initially performed on an in vivo subcutaneous mouse PDAC model. After intratumoural injection of PTP-Fe3O4-IRDye800CW or Con-Fe3O4-IRDye800CW nanoparticles, the FMI data demonstrated that PTP-Fe3O4-IRDye800CW had the highest fluorescence intensity at 4 h post-injection and almost equal fluorescence intensity with Con-Fe3O4-IRDye800CW nanoparticles, and the fluorescence intensity decreased at 8 h post-injection. The FMI data demonstrated that PTP-Fe3O4-IRDye800CW also had a longer retention effect compared to the Con-Fe3O4-IRDye800CW nanoparticles (Fig. 4a). Quantitative analysis of the fluorescence intensity data was identical with the in vivo observations (Fig. 4b). The difference between the two sets of NFI (%) appeared 2 days post-injection (PTP-Fe3O4-IRDye800CW group: 43.68% ± 5.72% vs. Con-Fe3O4-IRDye800CW group: 31.79 ± 4.65%, P < 0.05), and we detected the NFI (%) until 7 days in the PTP-Fe3O4-IRDye800CW group (PTP-Fe3O4-IRDye800CW group: 30.41 ± 7.77% vs. Con-Fe3O4-IRDye800CW group: 13.13 ± 0.44%, P < 0.05). These results demonstrated that PTP-Fe3O4-IRDye800CW imaging nanoparticles showed better targeting and retention effects than Con-Fe3O4-IRDye800CW nanoparticles in the tumours.
Moreover, subcutaneous pancreatic tumour-bearing mice were simultaneously and dynamically monitored using MPI. MPI signal changes were observed for both PTP-Fe3O4-IRDye800CW and Con-Fe3O4-IRDye800CW nanoparticles at 4 h post-injection (Fig. 4c). Similarly, the MPI signals of the Con-Fe3O4-IRDye800CW nanoparticles were metabolised fast, and the PTP-Fe3O4-IRDye800CW group showed better targeted imaging at 2 d post-injection (PTP-Fe3O4-IRDye800CW group: 87.90 ± 5.55% vs. Con-Fe3O4-IRDye800CW group: 64.91 ± 6.97%, P < 0.01), whereas the PTP-Fe3O4-IRDye800CW group demonstrated homogeneous MPI signal distribution inside tumours and longer retention until 7 days (PTP-Fe3O4-IRDye800CW group: 72.66 ± 3.27% vs. Con-Fe3O4-IRDye800CW group: 49.95 ± 7.98%, P < 0.05). A quantitative analysis of MPI signals was further performed. The PTP-Fe3O4-IRDye800CW group retained 90.40% MPI signals, while the Con-Fe3O4-IRDye800CW group retained 64.91%. Moreover, the MPI signals decreased to half of that of the Con-Fe3O4-IRDye800CW group, but more than 72.66% of the MPI signal was observed in the PTP-Fe3O4-IRDye800CW group after a 7d metabolism (Fig. 4d).
As the representative clinical imaging modality, MPI was also performed. The results in Fig. 4e distinctly show that T2-weighted signals for the PTP-Fe3O4-IRDye800CW targeting probe in the coronal position gradually diffused through the whole PDAC xenografts, but the MRI signal was restricted to the injection site for the Con-Fe3O4-IRDye800CW group. Notably, the increased darker T2-weighted signals and prolonged retention caused by the PTP-Fe3O4-IRDye800CW targeting probe were retained for 7 d post-injection. In contrast, almost all the Con-Fe3O4-IRDye800CW probe stayed at the injection site for a short period of time and then was metabolised quickly. Compared with FMI, which provides only 2D information about the probe’s metabolism, MRI offers more valuable and accurate anatomical details, which can locate the probe position within the tumour site.
To verify the in vivo triple-modality imaging observations, the tumours and major organs were obtained for further ex vivo multimodality imaging. As shown in Fig. 5a, ex vivo FMI images of the tumours and major organs from the mice injected with nanoparticles were obtained 2 days post-injection. Stronger fluorescence signals were observed in the resected tumours of the PTP-Fe3O4-IRDye800CW group than in the Con-Fe3O4-IRDye800CW group. No signals were detected in the liver, kidney, or spleen because the probes were intratumourally injected. To further confirm that the MPI signal biodistribution, we performed ex vivo MPI scanning at 2 days post-injection. Tumours in the PTP-Fe3O4-IRDye800CW group had stronger MPI signals than those in the Con-Fe3O4-IRDye800CW group (Fig. 5b). Finally, histological and Prussian blue staining were performed, and the data demonstrated that more Fe-positive staining was evenly distributed inside the tumour in the PTP-Fe3O4-IRDye800CW group than in the Con-Fe3O4-IRDye800CW group (Fig. 5c). Furthermore, the liver and spleen tissues of the Con-Fe3O4-IRDye800CW group showed a small amount of Fe-positive staining in sporadic areas, but staining was barely detected in the PTP-Fe3O4-IRDye800CW group.
For the biosafety evaluation, we performed H&E staining of the major organs as well as liver and renal function. No abnormalities were found in major organs, such as the heart, liver, spleen, lung, and kidney (Supplementary Fig. 2a). In addition, ALT, AST, and ALP were tested for liver function. For renal function, serum creatinine (Scr) and blood urea nitrogen (BUN) levels were measured. The data showed that there were no differences between the two groups and the healthy normal mice group (Supplementary Fig. 2b).
In vivo MPI, FMI and MRI multimodality imaging for orthotopic PDAC mouse model
Based on our previous findings, we further performed targeted multimodality imaging of orthotopic PDAC xenografts, which can mimic the real tumour growth environment. The orthotopic mouse model was established using Pan02-Luc cells, and BLI was carried out to confirm the successful establishment of the orthotopic PDAC model (Supplementary Fig. 3a and 3b). The in vivo biodistribution and targeting specificity of the nanoparticles were investigated. The Pan02-Luc orthotopic PDAC tumour-bearing C57 mice were intratumourally injected with the imaging nanoparticles, and MPI-FMI-MRI triple-modality imaging was then carried out at different time points. As shown in Fig. 6a, FMI of both the PTP-Fe3O4-IRDye800CW and Con-Fe3O4-IRDye800CW groups showed the highest fluorescence signal at tumour sites at 4 h post-injection, which then reduced thereafter. The fluorescence signals in the PTP-Fe3O4-IRDye800CW group decreased more slowly compared to those in the Con-Fe3O4-IRDye800CW group. The difference in fluorescence intensity between two groups at the tumour site peaked at 2 d post-injection (PTP-Fe3O4-IRDye800CW group: 64.6 ± 7.03% vs. Con-Fe3O4-IRDye800CW group: 41.99 ± 0.12%, P < 0.01). Moreover, statistical difference of the fluorescence intensity between two groups could last for 7 d (PTP-Fe3O4-IRDye800CW group: 45.73 ± 6.42% vs. Con-Fe3O4-IRDye800CW group: 32.93 ± 0.76%, P < 0.05) (Fig. 6b). However, the problems associated with FMI of deep tumours are susceptible with light scattering and limited imaging depth etc [32]. Therefore, MPI is needed to overcome this problem.
MPI was further performed in vivo. As shown in Fig. 6c and 6d, the two groups displayed better MPI signals at 2 d post-injection (PTP-Fe3O4-IRDye800CW group: 85.72 ± 1.53% vs. Con-Fe3O4-IRDye800CW group: 74.41 ± 1.91%, P < 0.01), while the signals lasted up to 7 d post-injection in the PTP-Fe3O4-IRDye800CW group rather than Con-Fe3O4-IRDye800CW group (68.78 ± 7.75% vs. 49.66 ± 7.39%, P < 0.05). Moreover, 3D MPI/CT images showed a more intense and homogeneous distribution of the PTP-Fe3O4-IRDye800CW nanoparticles (Supplementary Video 1) compared to the Con-Fe3O4-IRDye800CW nanoparticles (Supplementary Video 2) on the orthotopic PDAC tumours with details of the spatial positions of the whole body.
T2-weighted MRI was performed at the same time points. As shown in Fig. 6e, the PTP-Fe3O4-IRDye800CW nanoparticles were distributed throughout the tumour instead of being confined to the injection site as observed in the Con-Fe3O4-IRDye800CW group. Additionally, the PTP-Fe3O4-IRDye800CW nanoparticles were present for over 7 days, showing longer retention than the Con-Fe3O4-IRDye800CW nanoparticles. The MRI signals were consistent with the results of MPI and FMI. However, because of its intrinsic negative signals and susceptibility artifacts, MRI lacks the sensitivity to distinguish small or early-stage tumours and hypointense areas [33]. Consequently, it is necessary to achieve more comprehensive imaging information through multimodal imaging.
To further verify the in vivo multimodality imaging observations, we collected the pancreas with the tumours and major organs (spleen, liver, lung, heart, and kidney) of the mice at 2 d post-injection and examined their signal intensity. For ex vivo FMI, the fluorescence signals of tumours in the PTP-Fe3O4-IRDye800CW group were obviously stronger than those in the Con-Fe3O4-IRDye800CW group (Fig. 7a). In addition, the ex vivo MPI signals were further validated the tumours of PTP-Fe3O4-IRDye800CW group showed higher MPI signals than those of the Con-Fe3O4-IRDye800CW group, suggesting that the PTP-Fe3O4-IRDye800CW group showed longer signal retention than the Con-Fe3O4-IRDye800CW group (Fig. 7b). H&E and Prussian blue staining were further conducted, and the observations indicated that more Fe-positive staining was within the tumour in the PTP-Fe3O4-IRDye800CW group than the Con-Fe3O4-IRDye800CW group (Fig. 7c).