Novel Strategies to Protect and Visualize Pancreatic ß Cells in Diabetes.

A common feature in the pathophysiology of different types of diabetes is the reduction of ß cell mass and/or impairment of ß cell function. Diagnosis and treatment of type 1 and type 2 diabetes is currently hampered by a lack of reliable techniques to restore ß cell survival, to improve insulin secretion, and to quantify ß cell mass in patients. Current new approaches may allow us to precisely and specifically visualize ß cells in vivo and provide viable therapeutic strategies to preserve, recover, and regenerate ß cells. In this review, we discuss recent protective approaches for ß cells and the advantages and limitations of current imaging probes in the field.

A nanobody-based nuclear imaging tracer targeting dipeptidyl peptidase 6 to determine the mass of human beta cell grafts in mice.

AIMS/HYPOTHESIS: Type 1 diabetes is characterised by a progressive decline in beta cell mass. This is also observed following implantation of pancreatic islet allografts, but there is no reliable information regarding the time course of beta cell loss. This is due to the limited availability of non-invasive pancreatic islet imaging techniques. We have previously described that dipeptidyl peptidase 6 (DPP6) is an alpha and beta cell-specific biomarker, and developed a camelid antibody (nanobody '4hD29') against it. We demonstrated the possibility to detect DPP6-expressing cells by single-photon emission computed tomography (SPECT)/ computed tomography (CT), but the correlation between the number of cells grafted and the SPECT signal was not assessed. Here, we investigate whether the 4hD29 nanobody allows us to detect different amounts of human pancreatic islets implanted into immune-deficient mice. In addition, we also describe the adaptation of the probe for use with positron emission tomography (PET). METHODS: DPP6 expression was assessed in human samples using tissue arrays and immunohistochemistry. The effect of the 4hD29 nanobody on cell death and glucose-stimulated insulin secretion was measured in EndoC-ßH1 cells and in human islets using Hoechst/propidium iodide staining and an anti-insulin ELISA, respectively. We performed in vivo SPECT imaging on severe combined immunodeficient (SCID) mice transplanted with different amounts of EndoC-ßH1 cells (2 × 106, 5 × 106 and 10 × 106 cells), human islets (1000 and 3000) or pancreatic exocrine tissue using 99mTc-labelled 4hD29 nanobody. This DPP6 nanobody was also conjugated to N-chlorosuccinimide (NCS)-1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), radiolabelled with either 67Ga (SPECT) or 68Ga (PET) and used in a proof-of-principle experiment to detect DPP6-expressing cells (Kelly neuroblastoma) grafted in SCID mice. RESULTS: The DPP6 protein is mainly expressed in pancreatic islets. Importantly, the anti-DPP6 nanobody 4hD29 allows non-invasive detection of high amounts of EndoC-ßH1 cells or human islets grafted in immunodeficient mice. This suggests that the probe must be further improved to detect lower numbers of islet cells. The 4hD29 nanobody neither affected beta cell viability nor altered insulin secretion in EndoC-ßH1 cells and human islets. The conversion of 4hD29 nanobody into a PET probe was successful and did not alter its specificity. CONCLUSIONS/INTERPRETATION: These findings suggest that the anti-DPP6 4hD29 nanobody may become a useful tool for the quantification of human islet grafts in mice and, pending future development, islet mass in individuals with diabetes.

Targeting tumor cells and neovascularization using RGD-functionalized magnetoliposomes.

PURPOSE: Magnetoliposomes (MLs) have shown great potential as magnetic resonance imaging contrast agents and as delivery vehicles for cancer therapy. Targeting the MLs towards the tumor cells or neovascularization could ensure delivery of drugs at the tumor site. In this study, we evaluated the potential of MLs targeting the αvß3 integrin overexpressed on tumor neovascularization and different tumor cell types, including glioma and ovarian cancer. METHODS: MLs functionalized with a Texas Red fluorophore (anionic MLs), and with the fluorophore and the cyclic Arginine-Glycine-Aspartate (cRGD; cRGD-MLs) targeting the αvß3 integrin, were produced in-house. Swiss nude mice were subcutaneously injected with 107 human ovarian cancer SKOV-3 cells. Tumors were allowed to grow for 3 weeks before injection of anionic or cRGD-MLs. Biodistribution of MLs was followed up with a 7T preclinical magnetic resonance imaging (MRI) scanner and fluorescence imaging (FLI) right after injection, 2h, 4h, 24h and 48h post injection. Ex vivo intratumoral ML uptake was confirmed using FLI, electron paramagnetic resonance spectroscopy (EPR) and histology at different time points post injection. RESULTS: In vivo, we visualized a higher uptake of cRGD-MLs in SKOV-3 xenografts compared to control, anionic MLs with both MRI and FLI. Highest ML uptake was seen after 4h using MRI, but only after 24h using FLI indicating the lower sensitivity of this technique. Furthermore, ex vivo EPR and FLI confirmed the highest tumoral ML uptake at 4 h. Last, a Perl's stain supported the presence of our iron-based particles in SKOV-3 xenografts. CONCLUSION: Uptake of cRGD-MLs can be visualized using both MRI and FLI, even though the latter was less sensitive due to lower depth penetration. Furthermore, our results indicate that cRGD-MLs can be used to target SKOV-3 xenograft in Swiss nude mice. Therefore, the further development of this particles into theranostics would be of interest.

Magnetoliposomes as Contrast Agents for Longitudinal in vivo Assessment of Transplanted Pancreatic Islets in a Diabetic Rat Model.

Magnetoliposomes (MLs) were synthesized and tested for longitudinal monitoring of transplanted pancreatic islets using magnetic resonance imaging (MRI) in rat models. The rat insulinoma cell line INS-1E and isolated pancreatic islets from outbred and inbred rats were used to optimize labeling conditions in vitro. Strong MRI contrast was generated by islets exposed to 50 µg Fe/ml for 24 hours without any increased cell death, loss of function or other signs of toxicity. In vivo experiments showed that pancreatic islets (50-1000 units) labeled with MLs were detectable for up to 6 weeks post-transplantation in the kidney subcapsular space. Islets were also monitored for two weeks following transplantation through the portal vein of the liver. Hereby, islets labeled with MLs and transplanted under the left kidney capsule were able to correct hyperglycemia and had stable MRI signals until nephrectomy. Interestingly, in vivo MRI of streptozotocin induced diabetic rats transplanted with allogeneic islets demonstrated loss of MRI contrast between 7-16 days, indicative of loss of islet structure. MLs used in this study were not only beneficial for monitoring the location of transplanted islets in vivo with high sensitivity but also reported on islet integrity and hereby indirectly on islet function and rejection.

Improved Labeling of Pancreatic Islets Using Cationic Magnetoliposomes.

Pancreatic islets (PIs) transplantation is an alternative approach for the treatment of severe forms of type 1 diabetes (T1D). To monitor the success of transplantation, it is desirable to follow the location of engrafted PIs non-invasively. In vivo magnetic resonance imaging (MRI) of transplanted PIs is a feasible cell tracking method; however, this requires labeling with a suitable contrast agent prior to transplantation. We have tested the feasibility of cationic magnetoliposomes (MLs), compared to commercial contrast agents (Endorem and Resovist), by labeling insulinoma cells and freshly isolated rat PIs. It was possible to incorporate Magnetic Ressonance (MR)-detectable amounts of MLs in a shorter time (4 h) when compared to Endorem and Resovist. MLs did not show negative effects on the PIs' viability and functional parameters in vitro. Labeled islets were transplanted in the renal sub-capsular region of healthy mice. Hypointense contrast in MR images due to the labeled PIs was detected in vivo upon transplantation, while MR detection of PIs labeled with Endorem and Resovist was only possible after the addition of transfection agents. These findings indicate that MLs are suitable to image PIs, without affecting their function, which is promising for future longitudinal pre-clinical and clinical studies involving the assessment of PI transplantation.