“…5 a, right). Interestingly, we observed that the fluorescence signal of Cy5 PFCs taken up by monocytes and EpiSCs slightly decreased over time most likely by a reduction of the fluorescence signal upon internalization and quenching in the late endosomal system 29 . Figure 5 ( a ) Cellular uptake of PFCs with increasing amounts of PEGylation by EpiSCs and human primary monocytes.…”
Section: Resultsmentioning
confidence: 84%
“…PFCs containing the fluorescent dye Cy5 ( Cy5 PFCs) or rhodamine ( Rho PFCs) were prepared as described previously 28 , 29 . The PFC preparations contained 10 mM phosphate buffer (7 mM Na 2 HPO 4 , 3 mM NaH 2 PO 4, pH 7.4 isotonized with 2.5% [w/w] glycerol), 35 mM phospholipid (Lipoid S75; Lipoid GmbH, Ludwigshafen, Germany) and 0.01 mol% Cy5- or rhodamine-labelled lipids.…”
After myocardial infarction (MI), epicardial cells reactivate their embryonic program, proliferate and migrate into the damaged tissue to differentiate into fibroblasts, endothelial cells and, if adequately stimulated, to cardiomyocytes. Targeting epicardium-derived stromal cells (EpiSC) by specific ligands might enable the direct imaging of EpiSCs after MI to better understand their biology, but also may permit the cell-specific delivery of small molecules to improve the post-MI healing process. Therefore, the aim of this study was to identify specific peptides by phage display screening to enable EpiSC specific cargo delivery by active targeting. To this end, we utilized a sequential panning of a phage library on cultured rat EpiSCs and then subtracted phage that nonspecifically bound blood immune cells. EpiSC specific phage were analyzed by deep sequencing and bioinformatics analysis to identify a total of 78 300 ± 31 900 different, EpiSC-specific, peptide insertion sequences. Flow cytometry of the five most highly abundant peptides (EP1, -2, –3, -7 or EP9) showed strong binding to EpiSCs but not to blood immune cells. The best binding properties were found for EP9 which was further studied by surface plasmon resonance (SPR). SPR revealed rapid and stable association of EpiSCs with EP9. As a negative control, THP-1 monocytes did not associate with EP9. Coupling of EP9 to perfluorocarbon nanoemulsions (PFCs) resulted in the efficient delivery of 19F cargo to EpiSCs and enabled their visualization by 19F MRI. Moreover, active targeting of EpiSCs by EP9-labelled PFCs was able to outcompete the strong phagocytic uptake of PFCs by circulating monocytes. In summary, we have identified a 7-mer peptide, (EP9) that binds to EpiSCs with high affinity and specificity. This peptide can be used to deliver small molecule cargos such as contrast agents to permit future in vivo tracking of EpiSCs by molecular imaging and to transfer small pharmaceutical molecules to modulate the biological activity of EpiSCs.
“…5 a, right). Interestingly, we observed that the fluorescence signal of Cy5 PFCs taken up by monocytes and EpiSCs slightly decreased over time most likely by a reduction of the fluorescence signal upon internalization and quenching in the late endosomal system 29 . Figure 5 ( a ) Cellular uptake of PFCs with increasing amounts of PEGylation by EpiSCs and human primary monocytes.…”
Section: Resultsmentioning
confidence: 84%
“…PFCs containing the fluorescent dye Cy5 ( Cy5 PFCs) or rhodamine ( Rho PFCs) were prepared as described previously 28 , 29 . The PFC preparations contained 10 mM phosphate buffer (7 mM Na 2 HPO 4 , 3 mM NaH 2 PO 4, pH 7.4 isotonized with 2.5% [w/w] glycerol), 35 mM phospholipid (Lipoid S75; Lipoid GmbH, Ludwigshafen, Germany) and 0.01 mol% Cy5- or rhodamine-labelled lipids.…”
After myocardial infarction (MI), epicardial cells reactivate their embryonic program, proliferate and migrate into the damaged tissue to differentiate into fibroblasts, endothelial cells and, if adequately stimulated, to cardiomyocytes. Targeting epicardium-derived stromal cells (EpiSC) by specific ligands might enable the direct imaging of EpiSCs after MI to better understand their biology, but also may permit the cell-specific delivery of small molecules to improve the post-MI healing process. Therefore, the aim of this study was to identify specific peptides by phage display screening to enable EpiSC specific cargo delivery by active targeting. To this end, we utilized a sequential panning of a phage library on cultured rat EpiSCs and then subtracted phage that nonspecifically bound blood immune cells. EpiSC specific phage were analyzed by deep sequencing and bioinformatics analysis to identify a total of 78 300 ± 31 900 different, EpiSC-specific, peptide insertion sequences. Flow cytometry of the five most highly abundant peptides (EP1, -2, –3, -7 or EP9) showed strong binding to EpiSCs but not to blood immune cells. The best binding properties were found for EP9 which was further studied by surface plasmon resonance (SPR). SPR revealed rapid and stable association of EpiSCs with EP9. As a negative control, THP-1 monocytes did not associate with EP9. Coupling of EP9 to perfluorocarbon nanoemulsions (PFCs) resulted in the efficient delivery of 19F cargo to EpiSCs and enabled their visualization by 19F MRI. Moreover, active targeting of EpiSCs by EP9-labelled PFCs was able to outcompete the strong phagocytic uptake of PFCs by circulating monocytes. In summary, we have identified a 7-mer peptide, (EP9) that binds to EpiSCs with high affinity and specificity. This peptide can be used to deliver small molecule cargos such as contrast agents to permit future in vivo tracking of EpiSCs by molecular imaging and to transfer small pharmaceutical molecules to modulate the biological activity of EpiSCs.
“…We observed a rapid decline in the in vivo fluorescence signal within the first days after administration. Previous animal model studies to have used other imaging probes report the dissociation of MR and fluorescence signals, a similar effect possibly caused by lysosomal degradation of fluorescent dyes 51,52 .…”
As a natural polysaccharide polymer, glycogen possesses suitable properties for use as a nanoparticle carrier in cancer theranostics. not only it is inherently biocompatible, it can also be easily chemically modified with various moieties. Synthetic glycogen conjugates can passively accumulate in tumours due to enhanced permeability of tumour vessels and limited lymphatic drainage (the EPR effect). For this study, we developed and examined a glycogen-based carrier containing a gadolinium chelate and near-infrared fluorescent dye. Our aim was to monitor biodistribution and accumulation in tumourbearing rats using magnetic resonance and fluorescence imaging. Our data clearly show that these conjugates possess suitable imaging and tumour-targeting properties, and are safe under both in vitro and in vivo conditions. Additional modification of glycogen polymers with poly(2-alkyl-2-oxazolines) led to a reduction in the elimination rate and lower uptake in internal organs (lower whole-body background: 45% and 27% lower MRI signals of oxazoline-based conjugates in the liver and kidneys, respectively compared to the unmodified version). Our results highlight the potential of multimodal glycogen-based nanopolymers as a carrier for drug delivery systems in tumour diagnosis and treatment. The majority of anticancer drugs are highly toxic and adversely affect healthy cells due to a lack of specific targeting 1,2. Therefore, a safe drug delivery system capable of accumulating and providing controlled drug release at the tumour site is highly desirable. Nanoparticles represent a versatile carrier for drug delivery due to their ability to passively accumulate in solid tumours. This occurs via the enhanced permeability and retention (EPR) effect, a relatively universal phenomenon whereby nano-sized structures (up to approx. 200 nm in size) become entrapped in tumour tissue due to either leaky endothelia or aberrant tumour vessel architecture. Restricted or even completely absent lymphatic drainage in tumours limits the release and passive accumulation of compounds 3. Nanotherapeutic carrier can accumulate in tumours via the EPR effect without any targeting ligand 4-6. Their size above the renal threshold (molecular weight > ca 45 kDa/hydrodynamic diameter > 8 nm) leads to prolonged circulation in the bloodstream increasing the probability of accumulation at the target site 3. Importantly, polymers can control the release of drugs, being composed of a structure responsive to external stimuli, such as pH changes in acidic cancer-cell environments 7 , the presence of enzymes, and redox potential 8,9. Thus to minimise the drug release in the bloodstream and the cytotoxic effect on healthy cells 10-12. One limitation of nanoparticles is that they tend to opsonise, clearing quickly through the reticuloendothelial system in the liver and spleen 13. However, this can be mitigated by surface modification: for instance, grafting nanoparticles with poly(ethylene oxide) (PEO) (known also as poly(ethylene glycol)-PEG) is a well-established pr...
“…This can be compensated by data resampling strategies that improve image quality and suppress background [3]. Quantification of the fluorine MR signal will also be essential for most of the conceivable applications [4][5][6][7][8][9][10][11]. Therefore, the necessary technologies such as B 1 mapping to correct for non-uniform radiofrequency fields [12] or development of new radio frequency coil technologies [13,14] .…”
mentioning
confidence: 99%
“…In this special issue, a dissociation of the fluorescence and the fluorine label was shown signal over time, both in vitro and in vivo. This has a critical impact on the interpretation of long-term experiments validated by standard fluorescent techniques [6]. Fluorine-rich probes can also act as labels within microcapsules impregnated within cells for in vivo applications; e.g., microcapsules containing and protecting stem cells from immune rejection following transplantation [8].…”
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