This study shows that supramolecular arrangement of proteins in nanoparticle structure predicts nanoparticle accumulation in neutrophils in acute lung inflammation (ALI). We observed homing to inflamed lungs for a variety of
n
anoparticles with
a
gglutinated
p
rotein (NAPs), defined by arrangement of protein in or on the nanoparticles via; a) hydrophobic interactions; b) crosslinking; c) electrostatic interactions. Nanoparticles with symmetric protein arrangement (
e.g.
, viral capsids) had no selectivity for inflamed lungs. Flow cytometry and immunohistochemistry showed NAPs have tropism for pulmonary neutrophils. Protein-conjugated liposomes were engineered to recapitulate NAP tropism for pulmonary neutrophils. NAP uptake in neutrophils was shown to depend on complement opsonization. We; a) demonstrate diagnostic imaging of ALI with NAPs; b) show NAP tropism for inflamed human donor lungs; c) show NAPs can remediate pulmonary edema in ALI. This work demonstrates structure-dependent tropism for neutrophils drives NAPs to inflamed lungs and shows NAPs can detect and treat ALI.
Complement opsonization is among the biggest challenges facing nanomedicine. Nearly instantly after injection into blood, nanoparticles are opsonized by the complement protein C3, leading to clearance by phagocytes, fouling of targeting moieties, and release of anaphylatoxins. While surface polymers such as poly(ethylene glycol) (PEG) partially decrease complement opsonization, most nanoparticles still suffer from extensive complement opsonization, especially when linked to targeting moieties. To ameliorate the deleterious effects of complement, two of mammals’ natural regulators of complement activation (RCAs), Factors H and I, are here conjugated to the surface of nanoparticles. In vitro, Factor H or I conjugation to PEG‐coated nanoparticles decrease their C3 opsonization, and markedly reduce nanoparticle uptake by phagocytes. In an in vivo mouse model of sepsis‐induced lung injury, Factor I conjugation abrogates nanoparticle uptake by intravascular phagocytes in the lungs, allowing the blood concentration of the nanoparticle to remain elevated much longer. For nanoparticles targeted to the lung's endothelium by conjugation to anti‐ICAM antibodies, Factor I conjugation shifts the cell‐type distribution away from phagocytes and toward endothelial cells. Finally, Factor I conjugation abrogates the severe anaphylactoid responses common to many nanoparticles, preventing systemic capillary leak and preserving blood flow to visceral organs and the brain. Thus, conjugation of RCAs, like Factor I, to nanoparticles is likely to help in nanomedicine's long battle against complement, improving several key parameters critical for clinical success.
Acute lung inflammation has severe morbidity, as seen in COVID-19 patients. Lung inflammation is accompanied or led by massive accumulation of neutrophils in pulmonary capillaries ("margination"). We sought to identify nanostructural properties that predispose nanoparticles to accumulate in pulmonary marginated neutrophils, and therefore to target severely inflamed lungs. We designed a library of nanoparticles and conducted an in vivo screen of biodistributions in naive mice and mice treated with lipopolysaccharides. We found that supramolecular organization of protein in nanoparticles predicts uptake in inflamed lungs. Specifically, nanoparticles with agglutinated protein (NAPs) efficiently home to pulmonary neutrophils, while protein nanoparticles with symmetric structure (e.g. viral capsids) are ignored by pulmonary neutrophils. We validated this finding by engineering protein-conjugated liposomes that recapitulate NAP targeting to neutrophils in inflamed lungs. We show that NAPs can diagnose acute lung injury in SPECT imaging and that NAP-like liposomes can mitigate neutrophil extravasation and pulmonary edema arising in lung inflammation. Finally, we demonstrate that ischemic ex vivo human lungs selectively take up NAPs, illustrating translational potential. This work demonstrates that structure-dependent interactions with neutrophils can dramatically alter the biodistribution of nanoparticles, and NAPs have significant potential in detecting and treating respiratory conditions arising from injury or infections.
Background: Extreme thrombocytosis (EXT, platelet count > 1000 × 10 3 /μL) is an uncommon but potentially clinically significant finding. Primary EXT in the setting of myeloproliferative disorders is linked to thrombotic and/or bleeding complications more frequently than secondary EXT, which typically occurs in reaction to infection, inflammation, or iron deficiency. However, comorbidities have been reported in adults with secondary EXT. Clinical implications of EXT in children are not well defined, as prior studies targeted small and/or specialized pediatric populations. Objectives: Our objectives were to determine etiologies and sequelae of EXT in a hospitalized general pediatric patient population. Patients and Methods: We retrospectively analyzed EXT cases from a single-center pediatric cohort of ~80 000 patients over 8 years. Results: Virtually all cases (99.8%) were secondary in nature, and most were multifactorial. Many cases of EXT occurred in children under 2 years old (47%) and/or during critical illness (55%). No thrombotic or bleeding events directly resulted from EXT, confirming a paucity of clinical complications associated with EXT in pediatric patients. There were indications that neonatal hematopoiesis and individual genetic variation influenced some cases, in addition to certain diagnoses (eg, sickle cell anemia) and clinical contexts (eg, asplenia). Conclusion: Our findings confirm that thrombotic events related to EXT are rare in pediatric patients, which can inform the use of empiric anti-platelet therapy.
Neutrophils are critical mediators of host defense in pathogen-induced and sterile inflammation. Excessive neutrophil activation has been associated with increased host pathology through collateral organ damage. The beneficial aspects of neutrophil activation, particularly in sterile inflammation, are less well defined. We observed accumulation of nuclear debris in the lungs of neutropenic mice exposed to acid-induced injury compared to wild-type. Size analysis of DNA-debris showed that neutropenic mice were unable to degrade extracellular DNA fragments. In addition, we found that neutrophils are able to differentially express DNA-degrading and repair-associated genes and proteins. Once neutrophils are at sites of lung inflammation they are able to phagocytose and degrade extracellular DNA. This neutrophil-dependent DNA degradation occurs in a MyD88-dependent pathway. The increased DNA-debris in neutropenic mice was associated with dysregulated alveolar repair and the phenotype is rescued by intra-tracheal administration of DNAseI. Thus, we show a novel mechanism as part of the inflammatory response, in which neutrophils engulf and degrade extracellular DNA fragments and allow for optimal organ repair.
Extreme thrombocytosis (ET, platelet count >1000 x 10^3/ul) is an uncommon clinical finding 1. Primary ET is associated with myeloproliferative disorders, such as essential thrombocythemia 2. Secondary ET is more common and occurs in reaction to infection, inflammation, or iron deficiency. Bleeding and thrombotic complications more frequently arise in primary ET cases 1, but have been reported with secondary ET in adults 3. Etiologies and complications associated with ET in children are less well-defined, as prior pediatric studies have been relatively small or restricted to specialized patient populations 4,5. We aimed to characterize ET in a large, single-center pediatric cohort.
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