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Nucleolin (NCL) plays an important role in tumor vascular development. An increased endothelial expression level of NCL has been related to cancer aggressiveness and prognosis and has been detected clinically in advanced tumors. Here, with a peptide targeted to NCL (F3 peptide), we created an NCL-targeted microbubble (MB) and compared the performance of F3-conjugated MBs with non-targeted (NT) MBs both in vitro and in vivo. In an in vitro study, F3-conjugated MBs bound 433 times more than NT MBs to an NCL-expressing cell line, while pretreating cells with 0.5 mM free F3 peptide reduced the binding of F3-conjugated MBs by 84%, n = 4, p

Advancements in liposomal drug delivery have produced long circulating and very stable drug formulations. These formulations minimize systemic exposure; however, unfortunately, therapeutic efficacy has remained limited due to the slow diffusion of liposomal particles within the tumor and limited release or uptake of the encapsulated drug. Here, the carboxyl-terminated CRPPR peptide, with affinity for the receptor neuropilin-1 (NRP), which is expressed on both endothelial and cancer cells, was conjugated to liposomes to enhance the tumor accumulation. Using a pH sensitive probe, liposomes were optimized for specific NRP binding and subsequent cellular internalization using in vitro cellular assays. Liposomes conjugated with the carboxyl-terminated CRPPR peptide (termed C-LPP liposomes) bound to the NRP-positive primary prostatic carcinoma cell line (PPC-1) but did not bind to the NRP-negative PC-3 cell line, and binding was observed with liposomal peptide concentrations as low as 0.16mol%. Binding of the C-LPP liposomes was receptor-limited, with saturation observed at high liposome concentrations. The identical peptide sequence bearing an amide terminus did not bind specifically, accumulating only with a high (2.5mol%) peptide concentration and adhering equally to NRP positive and negative cell lines. The binding of C-LPP liposomes conjugated with 0.63mol% of the peptide was 83-fold greater than liposomes conjugated with the amide version of the peptide. Cellular internalization was also enhanced with C-LPP liposomes, with 80% internalized following 3h incubation. Additionally, fluorescence in the blood pool (~40% of the injected dose) was similar for liposomes conjugated with 0.63mol% of carboxyl-terminated peptide and non-targeted liposomes at 24h after injection, indicating stable circulation. Prior to doxorubicin treatment, in vivo tumor accumulation and vascular targeting were increased for peptide-conjugated liposomes compared to non-targeted liposomes based on confocal imaging of a fluorescent cargo, and the availability of the vascular receptor was confirmed with ultrasound molecular imaging. Finally, over a 4-week course of therapy, tumor knockdown resulting from doxorubicin-loaded, C-LPP liposomes was similar to non-targeted liposomes in syngeneic tumor-bearing FVB mice and C-LPP liposomes reduced doxorubicin accumulation in the skin and heart and eliminated skin toxicity. Taken together, our results demonstrate that a carboxyl-terminated RXXR peptide sequence, conjugated to liposomes at a concentration of 0.63mol%, retains long circulation but enhances binding and internalization, and reduces toxicity.

Infectious diseases caused by invasive, environmentally persistent fungal pathogens have increasingly endangered global biodiversity, yet disease management remains a major conservation challenge. A prominent example is white‐nose syndrome (WNS), a disease caused by the invasive fungal pathogen Pseudogymnoascus destructans (Pd) that has devastated populations of multiple North American bat species, but for which few effective management tools exist. Here, we propose that strategies to delay environmental transmission of Pd during early winter could limit WNS disease effects across winter, benefitting bats. We used a small captive experiment and a multi‐year field trial on wild, free‐ranging bats to assess an environmental control strategy to manage Pd within its environmental reservoir in Pennsylvania, USA, where the pathogen has become endemic. The strategy centers on the application of Polyethylene Glycol 8000 (PEG) to roost substrates in summer, prior to bat hibernation, as a means to disrupt environmental transmission to bats in early winter. In the captive experiment, environmental transmission of Pd to immunologically naïve little brown myotis (Myotis lucifugus) occurred from roost substrates inoculated with Pd, but the application of PEG to these substrates effectively blocked this transmission. In the field trial, Pd load and infection extent both declined substantially in free‐ranging M. lucifugus after treatment relative to controls, with declines exceeding effects of inter‐site and inter‐annual variation. Pathogen prevalence and load also declined substantially after PEG treatment in big brown bats (Eptesicus fuscus). No negative effects of PEG treatment were observed in body condition or colony counts of bats or in the microbial community. Together, these results are consistent with effective environmental control of Pd and reduced WNS disease effects in bats within contaminated hibernacula. The results also highlight the potential of carefully designed environmental control strategies for managing environmentally persistent pathogens. [ABSTRACT FROM AUTHOR]