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Sepsis is characterized by a dysregulated immune response and is very difficult to treat. In the cecal ligation and puncture (CLP) mouse model, we show that nanomedicines can effectively alleviate systemic and local septic events by targeting neutrophils. Specifically, by decorating the surface of clinical-stage dexamethasone liposomes with cyclic arginine-glycine-aspartic acid (cRGD) peptides, we promote their engagement with neutrophils in the systemic circulation, leading to their prominent accumulation at primary and secondary sepsis sites. cRGD-targeted dexamethasone liposomes potently reduce immature circulating neutrophils and neutrophil extracellular traps in intestinal sepsis induction sites and the liver. Additionally, they mitigate inflammatory cytokines systemically and locally while preserving systemic IL-10 levels, contributing to lower IFN-γ/IL-10 ratios as compared to control liposomes and free dexamethasone. Our strategy addresses sepsis at the cellular level, illustrating the use of neutrophils both as a therapeutic target and as a chariot for drug delivery.

Microbubbles (MB) are routinely used ultrasound (US) contrast agents that have recently attracted increasing attention as stimuli-responsive drug delivery systems. To better understand MB-based drug delivery, we studied the role of drug hydrophobicity and molecular weight on MB loading, shelf-life stability, US properties, and drug release. Eight model drugs, varying in hydrophobicity and molecular weight, were loaded into the shell of poly(butyl cyanoacrylate) (PBCA) MB. In the case of drugs with progesterone as a common structural backbone (i.e., for corticosteroids), loading capacity and drug release correlated well with hydrophobicity and molecular weight. Conversely, when employing drugs with no structural similarity (i.e., four different fluorescent dyes), loading capacity and release did not correlate with hydrophobicity and molecular weight. All model drug-loaded MB formulations could be equally efficiently destroyed upon exposure to US. Together, these findings provide valuable insights on how the physicochemical properties of (model) drug molecules affect their loading and retention in and US-induced release from polymeric MB, thereby facilitating the development of drug-loaded MB formulations for US-triggered drug delivery.

Nanomedicines are sub-micrometer-sized carrier materials designed to improve the biodistribution of i.v. administered (chemo-) therapeutic agents. In recent years, ever more efforts in the nanomedicine field have employed optical imaging (OI) techniques to monitor biodistribution and target site accumulation. Thus far, however, the longitudinal assessment of nanomedicine biodistribution using OI has been impossible, due to limited light penetration (in the case of 2D fluorescence reflectance imaging; FRI) and to the inability to accurately allocate fluorescent signals to nonsuperficial organs (in the case of 3D fluorescence molecular tomography; FMT). Using a combination of high-resolution microcomputed tomography (μCT) and FMT, we have here set out to establish a hybrid imaging protocol for noninvasively visualizing and quantifying the accumulation of near-infrared fluorophore-labeled nanomedicines in tissues other than superficial tumors. To this end, HPMA-based polymeric drug carriers were labeled with Dy750, their biodistribution and tumor accumulation were analyzed using FMT, and the resulting data sets were fused with anatomical μCT data sets in which several different physiologically relevant organs were presegmented. The robustness of 3D organ segmentation was validated, and the results obtained using 3D CT-FMT were compared to those obtained upon standard 3D FMT and 2D FRI. Our findings convincingly demonstrate that combining anatomical μCT with molecular FMT facilitates the noninvasive assessment of nanomedicine biodistribution.

Abstract: Microbubbles (MB) are routinely used as contrast agents for functional and molecular ultrasound (US) imaging. For molecular US imaging, MB are functionalized with antibodies or peptides, in order to visualize receptor expression by angiogenic or inflamed endothelium. In general, initial in vitro binding studies with targeted MB are performed using phase contrast microscopy. Difficulties in the identification of MB in standard phase contrast microscopy, however, generally result in high variability, high observer dependency, and low reproducibility. To overcome these shortcomings, we here describe a simple post-loading strategy for labeling polymer-based MB with fluorophores, and we show that the use of rhodamine-loaded MB in combination with fluorescence microscopy substantially reduces the variability and the observer dependency of in vitro binding studies. In addition, we demonstrate that rhodamine-loaded MB can also be used for in vivo and ex vivo experimental setups, e.g., for analyzing MB binding to inflamed carotids using two-photon laser scanning microscopy, and for validating the binding of VEGFR2-targeted MB to tumor endothelium. These findings demonstrate that fluorescently labeled MB substantially facilitate translational molecular US studies, and they suggest that a similar synthetic strategy can be exploited for preparing drug-loaded MB, to enable image-guided, targeted, and triggered drug delivery to tumors and to sites of inflammation.