Cytosolic delivery of nanolabels prevents their asymmetric inheritance and enables extended quantitative in vivo cell imaging

Long-term in vivo imaging of cells is crucial for the understanding of cellular fate in biological processes in cancer research, immunology, or in cell-based therapies such as beta cell transplantation, in type I diabetes or stem cell therapy. Traditionally, cell labeling with the desired contrast agent occurs ex vivo via spontaneous endocytosis, which is a variable and slow process that requires optimization for each particular label-cell type combination. Following endocytic uptake, the contrast agents mostly remain entrapped in the endolysosomal compartment, which leads to signal instability, cytotoxicity, and asymmetric inheritance of the labels upon cell division. Here, we demonstrate that these disadvantages can be circumvented by delivering contrast agents directly into the cytoplasm via vapor nanobubble photoporation. Compared to classic endocytic uptake, photoporation resulted in :50 and 3 times higher loading of fluorescent dextrans and quantum dots, respectively, with improved signal stability and reduced cytotoxicity: Most: interestingly, cytosolic delivery by iihotoporation prevented asymmetric inheritance of labels by daughter cells over subsequent cell' generations. Instead, unequal inheritance of endocytosed labels resulted in a dramatic increase in polydispersity of the amount of labels per cell with each cell division, hindering accurate quantification of cell numbers in vivo over time. The combined benefits of cell labeling by photoporation resulted in a marked improvement in long-term cell visibility in vivo where an insulin producing cell line (INS-1E cell line) labeled with fluorescent dextrans could be tracked for up to two months in Swiss nude mice compared to 2-Weeks for cells labeled by endocytosis

Longitudinal in vivo assessment of host-microbe interactions in a murine model of pulmonary aspergillosis

The fungus Aspergillus fumigatus is ubiquitous in nature and the most common cause of invasive pulmonary aspergillosis (IPA) in patients with a compromised immune system. The development of IPA in patients under immunosuppressive treatment or in patients with primary immunodeficiency demonstrates the importance of the host immune response in controlling aspergillosis. However, study of the host-microbe interaction has been hampered by the lack of tools for their non-invasive assessment. We developed a methodology to study the response of the host's immune system against IPA longitudinally in vivo by using fluorine-19 magnetic resonance imaging (F-19 MRI). We showed the advantage of a perfluorocarbon-based contrast agent for the in vivo labeling of macrophages and dendritic cells, permitting quantification of pulmonary inflammation in different murine IPA models. Our findings reveal the potential of F-19 MRI for the assessment of rapid kinetics of innate immune response against IPA and the permissive niche generated through immunosuppression

Longitudinal In Vivo Assessment of Host-Microbe Interactions in a Murine Model of Pulmonary Aspergillosis.

The fungus Aspergillus fumigatus is ubiquitous in nature and the most common cause of invasive pulmonary aspergillosis (IPA) in patients with a compromised immune system. The development of IPA in patients under immunosuppressive treatment or in patients with primary immunodeficiency demonstrates the importance of the host immune response in controlling aspergillosis. However, study of the host-microbe interaction has been hampered by the lack of tools for their non-invasive assessment. We developed a methodology to study the response of the host's immune system against IPA longitudinally in vivo by using fluorine-19 magnetic resonance imaging (19F MRI). We showed the advantage of a perfluorocarbon-based contrast agent for the in vivo labeling of macrophages and dendritic cells, permitting quantification of pulmonary inflammation in different murine IPA models. Our findings reveal the potential of 19F MRI for the assessment of rapid kinetics of innate immune response against IPA and the permissive niche generated through immunosuppression.We are grateful for the financial support by the following funding agencies: the European Commission Marie Curie (ITN) BetaTrain (289932), the Research Foundation Flanders (FWO, G.0A75.14, G.0B28.14, and G.069115N), the Agentschap voor Innovatie door Wetenschap en Technologie for the SBO NanoCoMIT (IWT SBO 140061), the European ERA-NET project “CryptoView” (third call of the FP7 programme Infect-ERA), and KU Leuven for PF 10/017 (IMIR)

Fluorine-19 Magnetic Resonance Imaging as a Potential Tool in Preclinical Research --- With Focus on Diabetes

Diabetes remains one of the increasing threats to human health with over 422 million people suffering from the disease all over the world. Despite the advancements in understanding the disease and improvements in treatment, diagnosis and early prevention with effective monitoring of pre- and post- therapy of the disease is still urgently needed. Hereby, the function and mass of pancreatic islets (PIs) and in particular their beta cells play an important role for monitoring the progression of the disease. Efforts have been made to stimulate research efforts to track them in a quantitative way. Hereby, magnetic resonance imaging (MRI) is considered as a safe (no ionizing radiation) prime aid with excellent soft tissue contrast and high-resolution with detailed anatomical information. In order to further enhance the sensitivity of MRI for cellular imaging, contrast agents are often utilized to label the targeted cells either by in situ labeling after systemic injection or ex vivo pre-labeling with subsequent transplantation. Recently, due to its unique feature of being background free, fluorine based contrast agents together with 19F MRI techniques have attracted attention, with first successful applications in immune cell tracking like dendritic cells and T cells in immunotherapy. This might also open new perspectives for diabetes research. The main aim of this PhD thesis was to contribute to the development and validation of PI and beta cell labeling with different fluorine-based contrast agents to follow their fate in vivo using 19F MRI. Based on the properties of the contrast agents, both in situ and ex vivo labeling strategy have been explored. In the context of in situ labeling, fluorine labeled D-mannoheptulose (19FMH) was tested for its high specificity towards GLUT-2 transporters, which are highly expressed by beta cells and hepatocytes. With the establishment of an in-house built, double-tuned radio-frequency coil, we could assess the distribution of 19FMH in vivo. Due to the quick clearance of the compound and relative low sensitivity of 19F MRI, limited in vivo signal were detected in the liver and potentially from the pancreas. Nevertheless, ex vivo 19F MR spectroscopy (MRS) confirmed the preferential uptake of 19FMH in tissue with high expression of the GLUT-2 transporter. These results suggest that synthesis of 18F-labeled FMH and detection by the highly sensitive PET could be a potential alternative solution for the future to overcome the sensitivity limitations of 19F MRI. Ex vivo labeling was applied to isolated PIs and INS-1E cells using liposomal particles containing a DiD fluorescent dye and perfluoro-5-crown-ether (PFCE) for both 19F MRI and fluorescence imaging (FLI) detection. In addition, lentiviral vector (LV) transduction of PIs and INS-1E cells was also performed for bioluminescent imaging (BLI). The PFCE labeling and LV transduction protocols were optimized for PIs and cells in vitro to be sufficient for in vivo imaging purposes without affecting the viability and functionality of PIs and INS-1E cells. Longitudinal multimodal in vivo imaging of transplanted PIs and cells at subcutaneous sites demonstrated the feasibility of longitudinal cell tracking for several months with PFCE labels. This presented imaging approach addresses a potential solution to overcome limitations in sensitivity, resolution and specificity of individual imaging techniques using a multimodal approach. The inherent sensitivity limitation of 19F MRI was also tackled by implementation of data analysis tools like the compressed sensing (CS) technique with a focus on low signal-to-noise ratio (SNR) images of PFCE labeled PIs and INS-1E cells. Both, offline simulation and CS acquisition with different reconstruction algorithms showed a three- to fourfold increase of SNR per time. These preliminary results not only demonstrated again that the CS technique together with appropriate reconstruction algorithms is useful to reduce the scan time for 19F MRI application with low SNR, but also provides evidence that by using the CS techniques, extra signal of interest could be detected, which might be missed otherwise by using conventional 19F MRI acquisition schemes within the same scan time. In this thesis, 19F MRI was also applied for a controlled release study from a pH-sensitive capsule. By combining 19F MRI and CT with fluoro-deoxyglucose (19FDG) and BaSO4 as contrast agents, respectively, information on early permeability of capsule material to water, the capsule’s integrity and its exact anatomical location of the site of release could be obtained both in vitro and in vivo in a hamster model. This novel imaging approach could serve as a valuable tool for understanding and evaluation of different models of controlled release mechanisms. This PhD work has added knowledge to the further understanding, improvement and validation of 19F MRI as a preclinical imaging tool for cell tracking and controlled release. The future of 19F MRI remains bright due to its versatile applications in different biomedical fields. This work will hopefully contribute to a solid basis not only for its future applications and development in preclinical research, but also for the translation of the technique to the clinic.nrpages: 134status: publishe

Comparison of different compressed sensing algorithms for low SNR F-19 MRI applications-Imaging of transplanted pancreatic islets and cells labeled with perfluorocarbons

Transplantation of pancreatic islets is a possible treatment option for patients suffering from Type I diabetes. In vivo imaging of transplanted islets is important for assessment of the transplantation site and islet distribution. Thanks to its high specificity, the absence of intrinsic background signal in tissue and its potential for quantification, 19 F MRI is a promising technique for monitoring the fate of transplanted islets in vivo. In order to overcome the inherent low sensitivity of 19 F MRI, leading to long acquisition times with low signal-to-noise ratio (SNR), compressed sensing (CS) techniques are a valuable option. We have validated and compared different CS algorithms for acceleration of 19 F MRI acquisition in a low SNR regime using pancreatic islets labeled with perfluorocarbons both in vitro and in vivo. Using offline simulation on both in vitro and in vivo low SNR fully sampled 19 F MRI datasets of labeled islets, we have shown that CS is effective in reducing the image acquisition time by a factor of three to four without seriously affecting SNR, regardless of the particular algorithms used in this study, with the exception of CoSaMP. Using CS, signals can be detected that might have been missed by conventional 19 F MRI. Among different algorithms (SPARSEMRI, OMMP, IRWL1, Two-level and CoSAMP), the two-level l1 method has shown the best performance if computational time is taken into account. We have demonstrated in this study that different existing CS algorithms can be used effectively for low SNR 19 F MRI. An up to fourfold gain in SNR/scan time could be used either to reduce the scan time, which is beneficial for clinical and translational applications, or to increase the number of averages, to potentially detect otherwise undetected signal when compared with conventional 19 F MRI acquisitions. Potential applications in the field of cell therapy have been demonstrated.status: publishe

Comparison of different compressed sensing algorithms for low SNR 19F MRI applications—Imaging of transplanted pancreatic islets and cells labeled with perfluorocarbons

Transplantation of pancreatic islets is a possible treatment option for patients suffering from Type I diabetes. In vivo imaging of transplanted islets is important for assessment of the transplantation site and islet distribution. Thanks to its high specificity, the absence of intrinsic background signal in tissue and its potential for quantification, 19F MRI is a promising technique for monitoring the fate of transplanted islets in vivo. In order to overcome the inherent low sensitivity of 19F MRI, leading to long acquisition times with low signal-to-noise ratio (SNR), compressed sensing (CS) techniques are a valuable option. We have validated and compared different CS algorithms for acceleration of 19F MRI acquisition in a low SNR regime using pancreatic islets labeled with perfluorocarbons both in vitro and in vivo. Using offline simulation on both in vitro and in vivo low SNR fully sampled 19F MRI datasets of labeled islets, we have shown that CS is effective in reducing the image acquisition time by a factor of three to four without seriously affecting SNR, regardless of the particular algorithms used in this study, with the exception of CoSaMP. Using CS, signals can be detected that might have been missed by conventional 19F MRI. Among different algorithms (SPARSEMRI, OMMP, IRWL1, Two-level and CoSAMP), the two-level l1 method has shown the best performance if computational time is taken into account. We have demonstrated in this study that different existing CS algorithms can be used effectively for low SNR 19F MRI. An up to fourfold gain in SNR/scan time could be used either to reduce the scan time, which is beneficial for clinical and translational applications, or to increase the number of averages, to potentially detect otherwise undetected signal when compared with conventional 19F MRI acquisitions. Potential applications in the field of cell therapy have been demonstrated.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Design and evaluation of theranostic perfluorocarbon particles for simultaneous antigen-loading and 19F-MRI tracking of dendritic cells

Perfluorocarbon (PFC) particles are currently on the rise as cell labeling agents for ¹⁹F-MRI tracking of dendritic cell (DC)-based vaccines. In this work, we design theranostic PFC particles for single-step loading of DCs with both antigenic protein and with a liquid PFC for ¹⁹F-MRI detection of the antigen-loaded cells. Upon addition to DCs in vitro, the antigen-loaded PFC particles are efficiently internalized, resulting in intracellular presence of up to 40 pmol ¹⁹F atoms per cell. At the same time, the DCs become loaded with antigenic proteins, that can be efficiently processed, without important effects on cell viability or altering the DC's phenotype and the cell's capacity to respond to danger signals. In addition, antigen-loaded PFC particle containing DCs are capable of inducing extensive proliferation of antigen-specific CD8⁺ T cells in vitro. Importantly, the antigen-coated PFC particles allow in vitro ¹⁹F-MRI-based detection of the antigen-containing DCs with detection limits as low as 10³ cells μl⁻¹. The dual-modality characteristics of the designed particles could assure that only those DCs that have taken up the antigen, and hence are responsible for an immune response, are traceable via ¹⁹F-MRI. Taken together, these novel dual-modality particles represent an interesting strategy in the development of a traceable DC vaccine

Tri-modal in vivo imaging of the rodent pancreatic islets transplanted in the subcutaneous site

status: publishe