Background: Cyclotides are useful scaffolds to stabilize bioactive peptides. Results: Four melanocortin analogues of kalata B1 were synthesized. One is a selective MC4R agonist. Conclusion:The analogues retain the native kalata B1 scaffold and introduce a designed pharmacological activity, validating cyclotides as protein engineering scaffolds. Significance: A novel type of melanocortin agonist has been developed, with potential as a drug lead for treating obesity.
Immunosuppressive cells in the tumor microenvironment allow cancer cells to escape immune recognition and support cancer progression and dissemination. To improve therapeutic efficacy, we designed a liposomal oxaliplatin formulation (PCL8-U75) that elicits cytotoxic effects toward both cancer and immunosuppressive cells via protease-mediated, intratumoral liposome activation. The PCL8-U75 liposomes displayed superior therapeutic efficacy across all syngeneic cancer models in comparison to free-drug and liposomal controls. The PCL8-U75 depleted myeloid-derived suppressor cells and tumor-associated macrophages in the tumor microenvironment. The combination of improved cancer cell cytotoxicity and depletion of immunosuppressive populations of immune cells is attractive for combination with immune-activating therapy. Combining the PCL8-U75 liposomes with a TLR7 agonist induced immunological rejection of established tumors. This combination therapy increased intratumoral numbers of cancer antigen–specific cytotoxic T cells and Foxp3− T helper cells. These results are encouraging toward advancing liposomal drug delivery systems with anticancer and immune-modulating properties into clinical cancer therapy.
Liposomes are nanoparticles used in drug delivery that distribute over several days in humans and larger animals. Radiolabeling with long-lived positron emission tomography (PET) radionuclides, such as manganese-52 (Mn, T½=5.6days), allow the imaging of this biodistribution. We report optimized protocols for radiolabeling liposomes with Mn, through both remote-loading and surface labeling. For comparison, liposomes were also remote-loaded and surface labeled with copper-64 (Cu, T½=12.7h) through conventional means. The chelator DOTA was used in all cases. The in vivo stability of radiometal chelates is widely debated but studies that mimic a realistic in vivo setting are lacking. Therefore, we employed these four radiolabeled liposome types as platforms to demonstrate a new concept for such in vivo evaluation, here of the chelates Mn-DOTA andCu-DOTA. This was done by comparing "shielded" remote-loaded with "exposed" surface labeled variants in a CT26 tumor-bearing mouse model. Remote loading (90min at 55°C) and surface labeling (55°C for 2h) of Mn gave excellent radiolabeling efficiencies of 97-100% and 98-100% respectively, and the liposome biodistribution was imaged by PET for up to 8days. Liposomes with surface-conjugatedMn-DOTA exhibited a significantly shorter plasma half-life (T=14.4h) when compared to the remote-loaded counterpart (T=21.3h), whereas surface-conjugated Cu-DOTA cleared only slightly faster and non-significantly, when compared to remote-loaded (17.2±2.9h versus 20.3±1.2h). From our data, we conclude the successful remote-loading of liposomes withMn, and furthermore that Mn-DOTA may be unstable in vivo whereasCu-DOTA appears suitable for quantitative imaging.
SummaryGlucagon is secreted from pancreatic α cells, and hypersecretion (hyperglucagonemia) contributes to diabetic hyperglycemia. Molecular heterogeneity in hyperglucagonemia is poorly investigated. By screening human plasma using high-resolution-proteomics, we identified several glucagon variants, among which proglucagon 1-61 (PG 1-61) appears to be the most abundant form. PG 1-61 is secreted in subjects with obesity, both before and after gastric bypass surgery, with protein and fat as the main drivers for secretion before surgery, but glucose after. Studies in hepatocytes and in β cells demonstrated that PG 1-61 dose-dependently increases levels of cAMP, through the glucagon receptor, and increases insulin secretion and protein levels of enzymes regulating glycogenolysis and gluconeogenesis. In rats, PG 1-61 increases blood glucose and plasma insulin and decreases plasma levels of amino acids in vivo. We conclude that glucagon variants, such as PG 1-61, may contribute to glucose regulation by stimulating hepatic glucose production and insulin secretion.
Liposomal drug delivery systems are designed to avoid the body's natural clearance machinery and are typically achieved by incorporation lipid‐anchored poly(ethylene glycol) (PEG) polymers. When preparing such liposomes it is intrinsically assumed that all available PEG‐lipid is incorporated evenly into every liposome of a preparation, however this assumption remains unchallenged. Thus, a quantitative understanding of how PEG‐lipid incorporation conditions affect the PEG density on individual liposomes can provide novel insights on how to optimize an important parameter of liposomal circulation kinetics and therapeutic efficacy. Here a sensitive fluorescence‐based single liposome assay is employed that allows, for the first time, the quantification of the PEG‐lipid density on individual liposomes. The assay is used to demonstrate that incubation time and temperature, liposome membrane lipid saturation state and PEG anchoring moiety all affect the effective PEG surface density. Furthermore, the unique ability of the assay to investigate single liposomes within the ensemble, allows to quantify significant variations in PEG surface density between individual liposomes. Thus, the assay provides crucial insights on how to improve both the overall PEG surface density and reduce the liposome‐to‐liposome variation, facilitating the design and development of more uniform, controllable, and efficacious PEG‐lipid containing liposomal drug delivery systems.
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