Exendin-4-Conjugated Manganese Magnetism-Engineered Iron Oxide Nanoparticles as a Potential Magnetic Resonance Imaging Contrast Agent for Tracking Transplanted β-Cells

To specifically detect and trace transplanted islet β-cells by magnetic resonance imaging (MRI), we conjugated manganese magnetism-engineered iron oxide nanoparticles (MnMEIO NPs) with exendin-4 (Ex4) which specifically binds glucagon-like peptide-1 receptors on the surface of β-cells. The size distribution of MnMEIO and MnMEIO-Ex4 NPs were 67.8 ± 1.3 and 70.2 ± 2.3 nm and zeta potential 33.3 ± 0.5 and 0.6 ± 0.1 mV, respectively. MnMEIO and MnMEIO-Ex4 NPs with iron content ≤ 40 μg/mL did not affect MIN6 β-cell viability and insulin secretion. Positive iron staining was found in MIN6 β-cells loaded with MnMEIO-Ex4 NPs but not in those with MnMEIO NPs. A transmission electron microscope confirmed MnMEIO-Ex4 NPs were distributed in the cytoplasm of MIN6. In vitro MR images revealed a loss of signal intensity in MIN6 β-cells labeled with MnMEIO-Ex4 NPs but not with MnMEIO NPs. After transplantation of islets labeled with MnMEIO-Ex4, the graft under kidney capsule could be visualized on MRI as persistent hypointense areas up to 17 weeks. Moreover, histology of the islet graft showed positive staining for insulin, glucagon and iron. Our results indicate MnMEIO-Ex4 NPs are safe and effective for the detection and long-term monitoring of transplanted β-cells by MRI.


Characterization of MnMEIO and MnMEIO-Ex4 NPs
The hydrodynamic size and zeta potential were determined by dynamic light-scattering (DLS) with Zetasizer (Nano ZS90, Malvern Instruments, Malvern, UK). The iron concentration of ferrofluid was photometrically analyzed by o-phenanthroline and the absorbance was read at 510 nm [48]. Energy-dispersive spectra (EDS) of MnMEIO and MnMEIO-Ex4 NPs were measured by an energy-dispersive spectrometer (S-3000N, Hitachi, Tokyo, Japan).

Culture of MIN6 Cells
MIN6 cells, obtained from Prof. Susumu Seino at Kobe University, Kobe, Japan, were cultured in DMEM medium with supplementation of 10% fetal bovine serum (FBS). The cells were incubated at 37 °C in the presence of 5% carbon dioxide. The medium was changed every 3 days and cells were passaged weekly.

In Vitro Cytotoxicity Assay of MnMEIO and MnMEIO-Ex4 NPs
Cell death was determined by Propidium iodide (PI) which only enters cells with damaged membranes. For flowmetric analysis, MIN6 cells were stained with PI (50 μg/mL) and then analyzed by flow cytometry using a FACSCalibur (Becton Dickinson, Oxford, UK) equipped with a single argon ion laser emitting an excitation light at 488 nm wavelength. Data of 10,000 cells were obtained at a low flow rate and then analyzed by a software (CellQuest) [28,32].

Characterization of MnMEIO and MnMEIO-Ex4 NPs
The hydrodynamic size and zeta potential were determined by dynamic light-scattering (DLS) with Zetasizer (Nano ZS90, Malvern Instruments, Malvern, UK). The iron concentration of ferrofluid was photometrically analyzed by o-phenanthroline and the absorbance was read at 510 nm [48]. Energy-dispersive spectra (EDS) of MnMEIO and MnMEIO-Ex4 NPs were measured by an energy-dispersive spectrometer (S-3000N, Hitachi, Tokyo, Japan).

Culture of MIN6 Cells
MIN6 cells, obtained from Prof. Susumu Seino at Kobe University, Kobe, Japan, were cultured in DMEM medium with supplementation of 10% fetal bovine serum (FBS). The cells were incubated at 37 • C in the presence of 5% carbon dioxide. The medium was changed every 3 days and cells were passaged weekly.

In Vitro Cytotoxicity Assay of MnMEIO and MnMEIO-Ex4 NPs
Cell death was determined by Propidium iodide (PI) which only enters cells with damaged membranes. For flowmetric analysis, MIN6 cells were stained with PI (50 µg/mL) and then analyzed by flow cytometry using a FACSCalibur (Becton Dickinson, Oxford, UK) equipped with a single argon ion laser emitting an excitation light at 488 nm wavelength. Data of 10,000 cells were obtained at a low flow rate and then analyzed by a software (CellQuest) [28,32].

Insulin Secretion of MIN6 Cells
MIN6 cells were incubated with MnMEIO or MnMEIO-Ex4 NPs (40 µg/mL) for 4 h. Then, the culture supernatant was measured with an insulin RIA kit.

Cellular Uptake of MnMEIO and MnMEIO-Ex4 NPs
MIN6 cells, 3T3 fibroblasts and RAW macrophages were incubated overnight with MnMEIO and MnMEIO-Ex4 NPs. After a washing process to remove the excess of NPs, the cells were stained with Prussian blue (5% potassium ferrocyanide and 5% HCl) and the labeling efficiency was examined using a microscope. Cells containing intracellular blue particles were considered labeled [28].

Transmission Electron Microscope (TEM) Measurements
The core size and size distribution of MnMEIO and MnMEIO-Ex4 NPs were measured by a TEM (JEOL JEM-2000 EX II, Tokyo, Japan) at a voltage of 100 kV. Aqueous solutions of NPs were drop-casted onto a 200-mesh copper grid and the grid was air-dried at room temperature before TEM measurements [46].
MIN6 cells treated with NPs were prepared as following for TEM analysis: 1 × 10 6 cells grown on glass coverslips were incubated with MnMEIO or MnMEIO-Ex4 NPs (10 µg/mL) for 24 h. After being washed with PBS to remove unbounded NPs, cells were trypsinized, washed and fixed in 2.5% glutaraldehyde for 2 h. Fixed cells were stained with 1% osmium tetroxide at room temperature for 1 h. Then, they were dehydrated by alcohol and embedded in epoxy resin for sectioning. Ultra-thin sections were stained with uranyl acetate and lead citrate and then examined with an electron microscope (Hitachi H-7500) [28].
Elemental analysis was performed by electron energy loss spectroscopy (EELS) in a TEM (JEOL 2010F, Tokyo, Japan) equipped with a Schottkytype emission gun and a Gatan imaging filter (GIF Trim, Pleasanton, CA, USA) [32].

In Vitro MR Scanning
MR imaging were obtained with a 7.0 T MRI (Bruker, Ettlingen, Germany). 1 × 10 6 MIN6 cells were incubated overnight with MnMEIO or MnMEIO-Ex4 NPs at 37 • C. Samples were scanned by using a fast gradient recalled echo pulse sequence (repetition time (TR)/echo time (TE) = 881 msec/9.37 msec). The contrast enhancement was calculated by the following equation: contrast enhancement (%) = (SIpost − SIpre)/SIpre × 100, where SIpost is the signal intensity measured from within the phantom of cells incubated with the contrast agents, MnMEIO and MnMEIO-Ex4 NPs, and SIpre is the signal intensity for cells alone [28,30].

Animals
Male inbred C57BL/6 mice were purchased from the National Laboratory Animal Center, Taipei, Taiwan. Mice aged 8-12 weeks were used as islet donors and recipients. The animal experiment was approved by the Institutional Animal Care and Use Committee of Chang Gung Memorial Hospital, Taoyuan, Taiwan (No. 2015062902).

Islet Isolation and Labeling
With the mouse under anesthesia with sodium amobarbital, each pancreas was injected with 1.5 mg/mL of collagenase, and then removed and incubated in a water bath at 37 • C. Islets were purified by a density gradient Histopaque ® -1077, and then handpicked under a dissecting microscope [28,[31][32][33].
Isolated islets were incubated overnight in the RPMI-1640 medium containing 30 or 40 µg/mL MnMEIO and MnMEIO-Ex4 NPs at 37 • C in a 5% CO 2 atmosphere. After that, islets were washed with culture medium and then used for islet transplantation.

Islet Transplantation
We syngeneically transplanted islets labeled with MnMEIO-Ex4 NPs beneath the kidney capsule of three nondiabetic C57BL/6 mice, each with 300, 600 or 700 islets. Islets in PE-50 tubing were centrifuged. With the mouse under anesthesia, a lumbar incision was made to expose the left kidney. Capsule was cut in the lower pole, and the tip of PE-50 tubing was advanced beneath the capsule to the upper pole, the final injection site of islets. The capsule was left unsutured [28,[31][32][33].

In Vivo MR Scanning
After transplantation, serial MR imaging were carried out on a 7.0 T MRI scanner in three recipients using a surface coil with the following parameters for the gradient-recalled echo sequence: slice thickness = 0.5 mm, TR = 3700 msec, TE = 37 msec. MR signal intensity of the graft at the left kidney and the mirror area at the right kidney, a within-subject control, was calculated [28][29][30][31][32][33].

Islet Graft Removal and Histological Studies
The six-hundred-and seven-hundred-islet grafts were removed at 49 and 120 days after transplant, respectively. With the mouse under anesthesia, an abdominal incision was made to expose the left kidney. Then, the graft was excised with the adherent capsule.
The removed graft was fixed in a formalin solution and then processed for paraffin embedding and sectioning. Sections were stained for endocrine αand β-cells with glucagon and insulin antibodies and for iron with Prussian blue [29][30][31][32][33].

Statistical Analysis
In vivo MR signal intensity was expressed as the mean and standard deviation (SD). Statistical analyses were conducted with the software PASW Statistics 21 (IBM SPSS Statistics for Windows; Armonk, NY, USA: IBM Corp.). The normal distribution of the variable was checked with the Kolmogorov-Smirnov test. A pair comparison of mean values of the graft at the left kidney and the mirror area at the right kidney, the unpaired student's t-test was carried out if both samples passed the normality test. The Mann-Whitney U test (Wilcoxon test) was used if any one sample of the comparison pair failed with the normality test. A p-value less than 0.05 was considered significant.

Materials
Hydrodynamic Size (nm) Zeta Potential (mV)  Three curves in black, green and red indicate three measurements.

Effects of MnMEIO and MnMEIO-Ex4 NPs on MIN6 Cell Viability and Insulin Secretion
We utilized PI staining to analyze the toxicity of MnMEIO and MnMEIO-Ex4 NPs to MIN6 cells. Figure

Cellular Uptake of MnMEIO and MnMEIO-Ex4 NPs
We first examined the uptake of MnMEIO NPs by non-β cells, 3T3 fibroblasts ( Figure  4A,B) and RAW macrophages ( Figure 4C,D). After being incubated overnight with NPs, cells were stained with Prussian blue. There was no intracellular iron staining in 3T3 fibroblasts ( Figure 2B) but it was positive in RAW macrophages ( Figure 4D). We then examined the uptake of MnMEIO and MnMEIO-Ex4 by MIN6 β-cells ( Figure 4E-I). There

Effects of MnMEIO and MnMEIO-Ex4 NPs on MIN6 Cell Viability and Insulin Secretion
We utilized PI staining to analyze the toxicity of MnMEIO and MnMEIO-Ex4 NPs to MIN6 cells. Figure 3A shows MIN6 cell viability maintained in culture with 20 and 40 µg/mL of MnMEIO and MnMEIO-Ex4 NPs, however, some cytotoxicity appeared in MIN6 cells with 80 µg/mL of MnMEIO NPs. Based on this finding; we used MnMEIO and MnMEIO-Ex4 NPs ≤ 40 µg/mL for the rest of our in vitro and in vivo experiments. Three curves in black, green and red indicate three measurements.

Effects of MnMEIO and MnMEIO-Ex4 NPs on MIN6 Cell Viability and Insulin Secretion
We utilized PI staining to analyze the toxicity of MnMEIO and MnMEIO-Ex4 NPs to MIN6 cells. Figure

Cellular Uptake of MnMEIO and MnMEIO-Ex4 NPs
We first examined the uptake of MnMEIO NPs by non-β cells, 3T3 fibroblasts ( Figure  4A,B) and RAW macrophages ( Figure 4C,D). After being incubated overnight with NPs, cells were stained with Prussian blue. There was no intracellular iron staining in 3T3 fibroblasts ( Figure 2B) but it was positive in RAW macrophages ( Figure 4D). We then examined the uptake of MnMEIO and MnMEIO-Ex4 by MIN6 β-cells ( Figure 4E-I). There

Insulin secretion of MIN6 cells was measured 4 h after incubating MIN6 cells with
MnMEIO and MnMEIO-Ex4 NPs (40 µg/mL). The insulin levels were not significantly different among MIN6 cells with or without MnMEIO and MnMEIO-Ex4 NPs ( Figure 3B).

Cellular Uptake of MnMEIO and MnMEIO-Ex4 NPs
We first examined the uptake of MnMEIO NPs by non-β cells, 3T3 fibroblasts ( Figure 4A,B) and RAW macrophages ( Figure 4C,D). After being incubated overnight with NPs, cells were stained with Prussian blue. There was no intracellular iron staining in 3T3 fibroblasts ( Figure 2B) but it was positive in RAW macrophages ( Figure 4D). We then examined the uptake of MnMEIO and MnMEIO-Ex4 by MIN6 β-cells ( Figure 4E-I). There was no iron stain found in MIN6 cells with MnMEIO loading (Figure 4F,G). In contrast, the blue spots were observed in MnMEIO-Ex4 NPs-labeled MIN6 cells ( Figure 4H,I), especially those with 40 µg/mL ( Figure 4I). Our TEM results further confirmed electron dense particles in the cytoplasm of MnMEIO-Ex4 NPs-loaded MIN6 cells ( Figure 5A). Moreover, elemental analysis by EELS demonstrated the presence of manganese and iron in MIN6 cells labeled with MnMEIO-Ex4 NPs ( Figure 5B). Nanomaterials 2021, 11, 3145 7 of 19 was no iron stain found in MIN6 cells with MnMEIO loading (Figure 4F,G). In contrast, the blue spots were observed in MnMEIO-Ex4 NPs-labeled MIN6 cells ( Figure 4H,I), especially those with 40 μg/mL ( Figure 4I). Our TEM results further confirmed electron dense particles in the cytoplasm of MnMEIO-Ex4 NPs-loaded MIN6 cells ( Figure 5A). Moreover, elemental analysis by EELS demonstrated the presence of manganese and iron in MIN6 cells labeled with MnMEIO-Ex4 NPs ( Figure 5B).

In Vitro MR Images of MnMEIO and MnMEIO-Ex4 NPs and NPs-Labeled MIN6 Cells
We performed in vitro MRI on agar, MnMEIO and MnMEIO-Ex4 NPs ( Figure 6A) as well as MIN6 cells incubated with or without MnMEIO and MnMEIO-Ex4 NPs ( Figure  6B). As expected, there was a background image of the agar (a negative control) and a dark image of MnMEIO and MnMEIO-Ex4 NPs (a positive control) ( Figure 6A). In contrast to a background image in MIN6 cells and MnMEIO NPs-labeled MIN6 cells, MnMEIO-Ex4 NPs-labeled MIN6 cells appeared as dark spots, especially those with 40 μg/mL ( Figure 6B).

In Vitro MR Images of MnMEIO and MnMEIO-Ex4 NPs and NPs-Labeled MIN6 Cells
We performed in vitro MRI on agar, MnMEIO and MnMEIO-Ex4 NPs ( Figure 6A) as well as MIN6 cells incubated with or without MnMEIO and MnMEIO-Ex4 NPs ( Figure 6B). As expected, there was a background image of the agar (a negative control) and a dark image of MnMEIO and MnMEIO-Ex4 NPs (a positive control) ( Figure 6A). In contrast to a background image in MIN6 cells and MnMEIO NPs-labeled MIN6 cells, MnMEIO-Ex4 NPs-labeled MIN6 cells appeared as dark spots, especially those with 40 µg/mL ( Figure 6B).

In Vitro MR Images of MnMEIO and MnMEIO-Ex4 NPs and NPs-Labeled MIN6 Cells
We performed in vitro MRI on agar, MnMEIO and MnMEIO-Ex4 NPs ( Figure 6A) as well as MIN6 cells incubated with or without MnMEIO and MnMEIO-Ex4 NPs ( Figure  6B). As expected, there was a background image of the agar (a negative control) and a dark image of MnMEIO and MnMEIO-Ex4 NPs (a positive control) ( Figure 6A). In contrast to a background image in MIN6 cells and MnMEIO NPs-labeled MIN6 cells, MnMEIO-Ex4 NPs-labeled MIN6 cells appeared as dark spots, especially those with 40 μg/mL ( Figure 6B).

In Vivo MR Images of MnMEIO-Ex4 NPs-Labeled Islets after Transplantation
Since MIN6 cells did not uptake MnMEIO NPs in our in vitro study, islets labeled with MnMEIO-Ex4 NPs were used for syngeneic transplantation in three nondiabetic C57BL/6 mice. In our pilot study, we transplanted 300 islets labeled with MnMEIO-Ex4 NPs under the left kidney capsule of one mouse and performed MRI on the second day ( Figure 7). On MR scans, the graft of MnMEIO-Ex4 NPs-labeled islets (indicated by arrows) was visualized as a distinct hypointense area located at the implantation site. Then, we performed long-term studies by transplanting 600 and 700 islets labeled with MnMEIO-Ex4 NPs into each of two mice and followed-up MRI between 1 and 7 weeks in the former ( Figure 8) and between 1 and 17 weeks in the latter (Figure 9). In both mice, the abovementioned hypointense areas were persistent on MR scans. Our quantification analysis further revealed that, in two mice, the MR signal intensity of the MnMEIO-Ex4 NPs-labeled islet grafts made a persistent 30-70% ( Figure 8C) and 70-90% reduction ( Figure 9C) of MR signal as compared to the same area in the contralateral kidney at all time points (p = 0.000). This indicates the potential for long-term monitoring of islet isografts with the use of MnMEIO-Ex4 NPs.

In Vivo MR Images of MnMEIO-Ex4 NPs-Labeled Islets after Transplantation
Since MIN6 cells did not uptake MnMEIO NPs in our in vitro study, islets labeled with MnMEIO-Ex4 NPs were used for syngeneic transplantation in three nondiabetic C57BL/6 mice. In our pilot study, we transplanted 300 islets labeled with MnMEIO-Ex4 NPs under the left kidney capsule of one mouse and performed MRI on the second day (Figure 7). On MR scans, the graft of MnMEIO-Ex4 NPs-labeled islets (indicated by arrows) was visualized as a distinct hypointense area located at the implantation site. Then, we performed long-term studies by transplanting 600 and 700 islets labeled with MnMEIO-Ex4 NPs into each of two mice and followed-up MRI between 1 and 7 weeks in the former (Figure 8) and between 1 and 17 weeks in the latter (Figure 9). In both mice, the above-mentioned hypointense areas were persistent on MR scans. Our quantification analysis further revealed that, in two mice, the MR signal intensity of the MnMEIO-Ex4 NPs-labeled islet grafts made a persistent 30-70% ( Figure 8C) and 70-90% reduction ( Figure 9C) of MR signal as compared to the same area in the contralateral kidney at all time points (p = 0.000). This indicates the potential for long-term monitoring of islet isografts with the use of MnMEIO-Ex4 NPs.

Histological Studies of the MnMEIO-Ex4 NPs-Labeled Islet Graft
MnMEIO-Ex4 NPs-labeled islet grafts were removed from two recipients at 7 and 17 weeks after transplantation, respectively. To investigate the graft microscopically, we used a glucagon and an insulin antibody to stain islet α-and β-cells, respectively, and Prussian blue to stain iron. There were abundant insulin-and glucagon-positive cells in islet grafts ( Figure 10A,B,D,E). Moreover, these grafts were also stained positive for iron ( Figure 10C,F).

Histological Studies of the MnMEIO-Ex4 NPs-Labeled Islet Graft
MnMEIO-Ex4 NPs-labeled islet grafts were removed from two recipients at 7 and 17 weeks after transplantation, respectively. To investigate the graft microscopically, we used a glucagon and an insulin antibody to stain islet αand β-cells, respectively, and Prussian blue to stain iron. There were abundant insulin-and glucagon-positive cells in islet grafts ( Figure 10A,B,D,E). Moreover, these grafts were also stained positive for iron ( Figure 10C,F).

Discussion
The β-cell-specific imaging is important for understanding the fate of β-cells after islet transplantation. In this study, we conjugated MnMEIO NPs with Ex4 constructing a potential β-cell-specific MRI probe, MnMEIO-Ex4 NPs, and demonstrated its targeting properties to MIN6 β-cell line in vitro and transplanted islet β-cells in vivo. An ideal cell tracking agent should not associate with incidental adverse effects. Previously, Lee et al. reported MnMEIO NPs were biologically nontoxic to HeLa and HepG2 cell lines [41]. In the present study, we further demonstrated the concentration of MnMEIO and MnMEIO-Ex4 NPs ≤ 40 μg/mL did not affect MIN6 cell viability and insulin secretion, which is essential for in vivo islet transplantation.
The size of MnMEIO and MnMEIO-Ex4 NPs is around 70 nm which is between two previously commercial SPIO NPs, ferumoxides (120-180 nm) and ferucarbotran (30 nm) [26]. SPIO NPs are efficiently internalized by different cell types through uptake routes including pinocytosis, phagocytosis and receptor-mediated endocytosis [26]. In non-β cells, RAW macrophages instead of 3T3 fibroblasts could uptake MnMEIO NPs, implying MnMEIO NPs enter cells via phagocytosis but not by pinocytosis. In MIN6 β-cells, MnMEIO-Ex4 but not MnMEIO NPs were taken up, indicating MIN6 cells uptake MnMEIO NPs through receptor-mediated endocytosis. These densely packed MnMEIO-Ex4 NPs in MIN6 cells were confirmed by the TEM and elemental analysis by EELS. They were responsible for producing a local magnetic field and resulted in higher contrast in MR images as dark spots, which were reported corresponding to the locations of single loaded cells [28]. This visualization of MnMEIO-Ex4 NPs-labeled MIN6 cells is fundamental for detecting islet grafts by in vivo MRI.
In our in vivo study, we syngeneic transplanted islets labeled with MnMEIO-Ex4 NPs under the left kidney capsule of three nondiabetic C57BL/6 mice. On MR scans, all

Discussion
The β-cell-specific imaging is important for understanding the fate of β-cells after islet transplantation. In this study, we conjugated MnMEIO NPs with Ex4 constructing a potential β-cell-specific MRI probe, MnMEIO-Ex4 NPs, and demonstrated its targeting properties to MIN6 β-cell line in vitro and transplanted islet β-cells in vivo. An ideal cell tracking agent should not associate with incidental adverse effects. Previously, Lee et al. reported MnMEIO NPs were biologically nontoxic to HeLa and HepG2 cell lines [41]. In the present study, we further demonstrated the concentration of MnMEIO and MnMEIO-Ex4 NPs ≤ 40 µg/mL did not affect MIN6 cell viability and insulin secretion, which is essential for in vivo islet transplantation.
The size of MnMEIO and MnMEIO-Ex4 NPs is around 70 nm which is between two previously commercial SPIO NPs, ferumoxides (120-180 nm) and ferucarbotran (30 nm) [26]. SPIO NPs are efficiently internalized by different cell types through uptake routes including pinocytosis, phagocytosis and receptor-mediated endocytosis [26]. In non-β cells, RAW macrophages instead of 3T3 fibroblasts could uptake MnMEIO NPs, implying MnMEIO NPs enter cells via phagocytosis but not by pinocytosis. In MIN6 β-cells, MnMEIO-Ex4 but not MnMEIO NPs were taken up, indicating MIN6 cells uptake MnMEIO NPs through receptor-mediated endocytosis. These densely packed MnMEIO-Ex4 NPs in MIN6 cells were confirmed by the TEM and elemental analysis by EELS. They were responsible for producing a local magnetic field and resulted in higher contrast in MR images as dark spots, which were reported corresponding to the locations of single loaded cells [28]. This visualization of MnMEIO-Ex4 NPs-labeled MIN6 cells is fundamental for detecting islet grafts by in vivo MRI.
In our in vivo study, we syngeneic transplanted islets labeled with MnMEIO-Ex4 NPs under the left kidney capsule of three nondiabetic C57BL/6 mice. On MR scans, all MnMEIO-Ex4 NPs-labeled islet grafts were visualized as a distinct hypointense area located at the implantation site. Even though, we found that the hypointense area was bigger on the second day than those at 1 week and later after transplantation, that may be due to islet loss caused by death from anoxia and the lack of engraftment soon after implantation [49]. The persistent hypointense areas on the MR images during 7-and 17-week follow-ups are consistent with our 18-week observation in CSPIO-labeled islet isografts [28,29]. However, MnMEIO-Ex4-labeled islet grafts had smaller hypointense areas on MR images. This could be explained firstly by the strong binding of cationic chitosan with anionic cell surface which enhanced internalization of CSPIO NPs via endocytosis [50]. As shown in Figure 11, there were more CSPIO NPs adhered to islet surface than MnMEIO and MnMEIO-Ex4 NPs did. Secondly, the uptake of MnMEIO-Ex4 NPs is β-cell specific but CSPIO NPs can be taken up by any cells in islets. Therefore, there were fewer MnMEIO-Ex4 NPs-containing cells than CSPIO NPs-containing cells in the islet graft. Our quantification analysis in two mice further confirmed a persistent reduction of the MR signal intensity of the MnMEIO-Ex4 NPs-labeled islet grafts as compared to the same area in the contralateral kidney during 7-and 17-week follow-ups, respectively. This indicates the potential for long-term monitoring of islet isografts with the use of MnMEIO-Ex4 NPs. Although the Ex4-NP probes were used for in vivo MR imaging implanted insulinoma [41] and native pancreatic β-cells [42,43], to the best of our knowledge, we are the first to apply this strategy by using MnMEIO-Ex4 NPs to image islet grafts for a long period of time.
Nanomaterials 2021, 11, 3145 16 MnMEIO-Ex4 NPs-labeled islet grafts were visualized as a distinct hypointense are cated at the implantation site. Even though, we found that the hypointense area was ger on the second day than those at 1 week and later after transplantation, that ma due to islet loss caused by death from anoxia and the lack of engraftment soon afte plantation [49]. The persistent hypointense areas on the MR images during 7-and 17follow-ups are consistent with our 18-week observation in CSPIO-labeled islet isog [28,29]. However, MnMEIO-Ex4-labeled islet grafts had smaller hypointense areas on images. This could be explained firstly by the strong binding of cationic chitosan anionic cell surface which enhanced internalization of CSPIO NPs via endocytosis [50 shown in Figure 11, there were more CSPIO NPs adhered to islet surface than MnM and MnMEIO-Ex4 NPs did. Secondly, the uptake of MnMEIO-Ex4 NPs is β-cell sp but CSPIO NPs can be taken up by any cells in islets. Therefore, there were f MnMEIO-Ex4 NPs-containing cells than CSPIO NPs-containing cells in the islet graft quantification analysis in two mice further confirmed a persistent reduction of the signal intensity of the MnMEIO-Ex4 NPs-labeled islet grafts as compared to the same in the contralateral kidney during 7-and 17-week follow-ups, respectively. This indi the potential for long-term monitoring of islet isografts with the use of MnMEIO-Ex4 Although the Ex4-NP probes were used for in vivo MR imaging implanted insulin [41] and native pancreatic β-cells [42,43], to the best of our knowledge, we are the fi apply this strategy by using MnMEIO-Ex4 NPs to image islet grafts for a long peri time. Figure 11. Binding of MnMEIO, MnMEIO-Ex4 and CSPIO NPs to islets. Islets were incubated overnight with MnMEIO NPs 40 μg/mL, MnMEIO-Ex4 NPs 40 μg/mL and CSPIO 10 μg/mL, respectively, and then stained with Prussian blue.
The histological studies of MnMEIO-Ex4 NPs-labeled islet grafts revealed abun insulin-and glucagon-positive cells in islet grafts, indicating the existence of functio islets at 7 and 17 weeks after transplantation, respectively. In addition, positive iron s ing implies that MnMEIO-Ex4 NPs in transplanted islets were responsible for positiv images. Taken together, MnMEIO-Ex4 NPs are potential for the in vivo molecular ta ing of transplanted β-cells.

Conclusions
To detect and trace transplanted β-cells by MRI, we conjugated MnMEIO NPs exendin-4 which can specifically bind the GLP-1 receptors on β-cells. MnMEIO-Ex4 did not affect MIN6 cell viability and insulin secretion and could be specifically take by MIN6 cells. Both MnMEIO-Ex4-labeled MIN6 cells and islet grafts showed positiv images in vitro and after transplantation, respectively. Moreover, histology of the graft showed positive staining for insulin, glucagon and iron. Our results indicate MnMEIO-Ex4 NPs are safe and effective for the detection and long-term monitori transplanted β-cells by MRI.  The histological studies of MnMEIO-Ex4 NPs-labeled islet grafts revealed abundant insulin-and glucagon-positive cells in islet grafts, indicating the existence of functioning islets at 7 and 17 weeks after transplantation, respectively. In addition, positive iron staining implies that MnMEIO-Ex4 NPs in transplanted islets were responsible for positive MR images. Taken together, MnMEIO-Ex4 NPs are potential for the in vivo molecular targeting of transplanted β-cells.

Conclusions
To detect and trace transplanted β-cells by MRI, we conjugated MnMEIO NPs with exendin-4 which can specifically bind the GLP-1 receptors on β-cells. MnMEIO-Ex4 NPs did not affect MIN6 cell viability and insulin secretion and could be specifically taken up by MIN6 cells. Both MnMEIO-Ex4-labeled MIN6 cells and islet grafts showed positive MR images in vitro and after transplantation, respectively. Moreover, histology of the islet graft showed positive staining for insulin, glucagon and iron. Our results indicate that MnMEIO-Ex4 NPs are safe and effective for the detection and long-term monitoring of transplanted β-cells by MRI.