Supramolecular Organization Predicts Protein Nanoparticle Delivery to Neutrophils for Acute Lung Inflammation Diagnosis and Treatment

Abstract

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.

SciScore for 10.1101/2020.04.15.037564: (What is this?)

Please note, not all rigor criteria are appropriate for all manuscripts.

Table 1: Rigor

Institutional Review Board Statement	IACUC: Animal and human study protocols: All animal studies were carried out in strict accordance with Guide for the Care and Use of Laboratory Animals as adopted by National Institute of Health and approved by University of Pennsylvania Institutional Animal Care and Use Committee (IACUC). IRB: Gift of Life obtained the relevant permissions for research use of the discarded lungs, and in conjunction with the University of Pennsylvania’s Institutional Review Board ensured that all relevant ethical standards were met.
Randomization	not detected.
Blinding	not detected.
Power Analysis	not detected.
Sex as a biological variable	Nanoparticle Tracing in Inflammatory Disease Mouse Models: Nanoparticle or protein biodistributions were tested by injecting radiolabeled nanoparticles or protein (suspended to 100 µL in PBS or 0.9% saline at a dose of 2.5 mg/kg with tracer quantities of radiolabeled material) in C57BL/6 male mice from Jackson Laboratories.

Table 2: Resources

Antibodies
Sentences	Resources
Populations of intravascular vs. extravascular leukocytes were assessed by subsequent stain of fixed cell suspensions with PerCP-conjugated anti-CD45 antibody and/or APC-conjugated clone 1A8 anti-Ly6G antibody.	anti-CD45 suggested: None
To accomplish staining of fixed cells, 100 µL aliquots of the cell suspensions described above were pelleted at 400xg for 5 minutes, then resuspended in labeled antibody diluted in FACS buffer (1:150 dilution for APC-conjugated anti-Ly6G antibody and 1:500 dilution for PerCP-conjugated anti-CD45 antibody).	anti-Ly6G suggested: None
Staining buffer used was a 1:1000 dilution of stock antibody solution (APC anti-mouse CD45; Alexa Fluor 488 anti-mouse Ly6G, Biolegend) into FACS buffer.	anti-mouse CD45 suggested: None anti-mouse Ly6G suggested: None
Cells were re-suspended in 200 µL FACS buffer for staining with APC-conjugated anti-human CD45, applied by 20-minute incubation with a 1:500 dilution of the antibody stock.	anti-human CD45 suggested: None
Sections were stained with PerCP-conjugated anti-rat secondary antibody and neutrophil-associated fluorescence was observed with epifluorescence microscopy.	anti-rat suggested: None
Experimental Models: Organisms/Strains
Sentences	Resources
Nanoparticle Tracing in Inflammatory Disease Mouse Models: Nanoparticle or protein biodistributions were tested by injecting radiolabeled nanoparticles or protein (suspended to 100 µL in PBS or 0.9% saline at a dose of 2.5 mg/kg with tracer quantities of radiolabeled material) in C57BL/6 male mice from Jackson Laboratories.	C57BL/6 suggested: None
Male C57BL/6J mice, 6-8 weeks old, were purchased from Jackson Laboratories.	C57BL/6J suggested: None
Software and Algorithms
Sentences	Resources
Single stain controls allowed automatic generation of compensation matrices in FCS Express software.	FCS Express suggested: (FCS Express, RRID:SCR_016431)
SPECT and CT data, in NIFTI format, were opened with ImageJ software (FIJI package)	ImageJ suggested: (ImageJ, RRID:SCR_003070) FIJI suggested: (Fiji, RRID:SCR_002285)
Three-dimensional reconstructions of the lung CT data, and co-registrations of SPECT data with lung CT data, were generated as above with ImageJ’s 3D plugin applied to CT data cropped and partitioned for lung contrast.	ImageJ’s suggested: None
Images were recorded in SlideBook software and opened in ImageJ (FIJI distribution) for composition in movies with co-registration of the four fluorescent channels.	SlideBook suggested: (SlideBook , RRID:SCR_014300)

Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.

Results from TrialIdentifier: No clinical trial numbers were referenced.

Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

Results from JetFighter: Please consider improving the rainbow (“jet”) colormap(s) used on pages 40, 34, 43 and 36. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.

Results from rtransparent:

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Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
No protocol registration statement was detected.

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Supramolecular Organization Predicts Protein Nanoparticle Delivery to Neutrophils for Acute Lung Inflammation Diagnosis and Treatment

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