Resolving sepsis-induced immunoparalysis via trained immunity by targeting interleukin-4 to myeloid cells.

Immunoparalysis is a compensatory and persistent anti-inflammatory response to trauma, sepsis or another serious insult, which increases the risk of opportunistic infections, morbidity and mortality. Here, we show that in cultured primary human monocytes, interleukin-4 (IL4) inhibits acute inflammation, while simultaneously inducing a long-lasting innate immune memory named trained immunity. To take advantage of this paradoxical IL4 feature in vivo, we developed a fusion protein of apolipoprotein A1 (apoA1) and IL4, which integrates into a lipid nanoparticle. In mice and non-human primates, an intravenously injected apoA1-IL4-embedding nanoparticle targets myeloid-cell-rich haematopoietic organs, in particular, the spleen and bone marrow. We subsequently demonstrate that IL4 nanotherapy resolved immunoparalysis in mice with lipopolysaccharide-induced hyperinflammation, as well as in ex vivo human sepsis models and in experimental endotoxemia. Our findings support the translational development of nanoparticle formulations of apoA1-IL4 for the treatment of patients with sepsis at risk of immunoparalysis-induced complications.

Employing nanobodies for immune landscape profiling by PET imaging in mice.

Noninvasive immunoimaging holds great potential for studying and stratifying disease as well as therapeutic efficacy. Radiolabeled single-domain antibody fragments (i.e., nanobodies) are appealing probes for immune landscape profiling, as they display high stability, rapid targeting, and excellent specificity, while allowing extremely sensitive nuclear readouts. Here, we present a protocol for radiolabeling an anti-CD11b nanobody and studying its uptake in mice by a combination of positron emission tomography imaging, ex vivo gamma counting, and autoradiography. Our protocol is applicable to nanobodies against other antigens. For complete details on the use and execution of this protocol, please see Priem et al. (2020), Senders et al. (2019), or Rashidian et al. (2017).

Trained Immunity-Promoting Nanobiologic Therapy Suppresses Tumor Growth and Potentiates Checkpoint Inhibition.

Trained immunity, a functional state of myeloid cells, has been proposed as a compelling immune-oncological target. Its efficient induction requires direct engagement of myeloid progenitors in the bone marrow. For this purpose, we developed a bone marrow-avid nanobiologic platform designed specifically to induce trained immunity. We established the potent anti-tumor capabilities of our lead candidate MTP10-HDL in a B16F10 mouse melanoma model. These anti-tumor effects result from trained immunity-induced myelopoiesis caused by epigenetic rewiring of multipotent progenitors in the bone marrow, which overcomes the immunosuppressive tumor microenvironment. Furthermore, MTP10-HDL nanotherapy potentiates checkpoint inhibition in this melanoma model refractory to anti-PD-1 and anti-CTLA-4 therapy. Finally, we determined MTP10-HDL's favorable biodistribution and safety profile in non-human primates. In conclusion, we show that rationally designed nanobiologics can promote trained immunity and elicit a durable anti-tumor response either as a monotherapy or in combination with checkpoint inhibitor drugs.

Fluorescent nanoparticles for the accurate detection of drug delivery.

INTRODUCTION: The application of intravenously administered nanoparticle (NP) therapies is increasingly being explored for a variety of diseases. The key to their success lies in a thorough understanding of nanoparticle pharmacological behavior, specificity and elimination kinetics. Fluorescent imaging techniques provide exciting opportunities to gain insight into such NP behavior in complex biological systems, at macroscopic as well as microscopic levels. AREAS COVERED: In this review, we will summarize NP labeling methods in relation to their established and emerging fluorescent imaging modalities for in vitro, in vivo, and ex vivo studies of NP behavior. We will highlight novel applications and discuss recent developments in techniques such as fluorescence molecular tomography (FMT), Förster resonance energy transfer (FRET), and Raman imaging. Finally, we will provide a perspective on the challenges and future directions of NP-based fluorescent labeling and imaging in nanotherapeutics. EXPERT OPINION: Commonly used in preclinical applications, fluorescent imaging of NPs can be achieved with minimal invasiveness and low toxicity in a multiplex fashion. Increasingly applied in the study of NP biodistribution, dissociation, and elimination behavior, fluorescent imaging allows fluid longitudinal tracking and visualization of NP interactions at the level of the whole animal, target organs/tissues, and individual cells.

HDL-mimetic PLGA nanoparticle to target atherosclerosis plaque macrophages.

High-density lipoprotein (HDL) is a natural nanoparticle that exhibits an intrinsic affinity for atherosclerotic plaque macrophages. Its natural targeting capability as well as the option to incorporate lipophilic payloads, e.g., imaging or therapeutic components, in both the hydrophobic core and the phospholipid corona make the HDL platform an attractive nanocarrier. To realize controlled release properties, we developed a hybrid polymer/HDL nanoparticle composed of a lipid/apolipoprotein coating that encapsulates a poly(lactic-co-glycolic acid) (PLGA) core. This novel HDL-like nanoparticle (PLGA-HDL) displayed natural HDL characteristics, including preferential uptake by macrophages and a good cholesterol efflux capacity, combined with a typical PLGA nanoparticle slow release profile. In vivo studies carried out with an ApoE knockout mouse model of atherosclerosis showed clear accumulation of PLGA-HDL nanoparticles in atherosclerotic plaques, which colocalized with plaque macrophages. This biomimetic platform integrates the targeting capacity of HDL biomimetic nanoparticles with the characteristic versatility of PLGA-based nanocarriers.

Targeted labeling of an early-stage tumor spheroid in a chorioallantoic membrane model with upconversion nanoparticles.

In vivo detection of cancer at an early-stage, i.e. smaller than 2 mm, is a challenge in biomedicine. In this work target labeling of an early-stage tumor spheroid (∼500 µm) is realized for the first time in a chick embryo chorioallantoic membrane (CAM) model with monoclonal antibody functionalized upconversion nanoparticles (UCNPs-mAb).

A statin-loaded reconstituted high-density lipoprotein nanoparticle inhibits atherosclerotic plaque inflammation.

Inflammation is a key feature of atherosclerosis and a target for therapy. Statins have potent anti-inflammatory properties but these cannot be fully exploited with oral statin therapy due to low systemic bioavailability. Here we present an injectable reconstituted high-density lipoprotein (rHDL) nanoparticle carrier vehicle that delivers statins to atherosclerotic plaques. We demonstrate the anti-inflammatory effect of statin-rHDL in vitro and show that this effect is mediated through the inhibition of the mevalonate pathway. We also apply statin-rHDL nanoparticles in vivo in an apolipoprotein E-knockout mouse model of atherosclerosis and show that they accumulate in atherosclerotic lesions in which they directly affect plaque macrophages. Finally, we demonstrate that a 3-month low-dose statin-rHDL treatment regimen inhibits plaque inflammation progression, while a 1-week high-dose regimen markedly decreases inflammation in advanced atherosclerotic plaques. Statin-rHDL represents a novel potent atherosclerosis nanotherapy that directly affects plaque inflammation.

Synthesis of polymer-lipid nanoparticles for image-guided delivery of dual modality therapy.

For advanced treatment of diseases such as cancer, multicomponent, multifunctional nanoparticles hold great promise. In the current study we report the synthesis of a complex nanoparticle (NP) system with dual drug loading as well as diagnostic properties. To that aim we present a methodology where chemically modified poly(lactic-co-glycolic) acid (PLGA) polymer is formulated into a polymer-lipid NP that contains a cytotoxic drug doxorubicin (DOX) in the polymeric core and an anti-angiogenic drug sorafenib (SRF) in the lipidic corona. The NP core also contains gold nanocrystals (AuNCs) for imaging purposes and cyclodextrin molecules to maximize the DOX encapsulation in the NP core. In addition, a near-infrared (NIR) Cy7 dye was incorporated in the coating. To fabricate the NP we used a microfluidics-based technique that offers unique NP synthesis conditions, which allowed for encapsulation and fine-tuning of optimal ratios of all the NP components. NP phantoms could be visualized with computed tomography (CT) and near-infrared (NIR) fluorescence imaging. We observed timed release of the encapsulated drugs, with fast release of the corona drug SRF and delayed release of a core drug DOX. In tumor bearing mice intravenously administered NPs were found to accumulate at the tumor site by fluorescence imaging.