Abstract Lipid nanoparticles (LNPs) are the most clinically advanced nonviral RNA-delivery vehicles, though challenges remain in fully understanding how LNPs interact with biological systems.In vivo, proteins form an associated corona on LNPs that redefines their physicochemical properties and influences delivery outcomes. Despite its importance, the LNP protein corona is challenging to study owing to the technical difficulty of selectively recovering soft nanoparticles from biological samples. Herein, we developed a quantitative, label-free mass spectrometry-based proteomics approach to characterize the protein corona on LNPs. Critically, this protein corona isolation workflow avoids artifacts introduced by the presence of endogenous nanoparticles in human biofluids. We applied continuous density gradient ultracentrifugation for protein-LNP complex isolation, with mass spectrometry for protein identification normalized to protein composition in the biofluid alone. With this approach, we quantify proteins consistently enriched in the LNP corona including vitronectin, C-reactive protein, and alpha-2-macroglobulin. We explore the impact of these corona proteins on cell uptake and mRNA expression in HepG2 human liver cells, and find that, surprisingly, increased levels of cell uptake do not correlate with increased mRNA expression in part likely due to protein corona-induced lysosomal trafficking of LNPs. Our results underscore the need to consider the protein corona in the design of LNP-based therapeutics. Abstract Figure 
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                            Correlative Imaging of 3D Cell Culture on Opaque Bioscaffolds for Tissue Engineering Applications
                        
                    
    
            Abstract Three-dimensional (3D) tissue engineering (TE) is a prospective treatment that can be used to restore or replace damaged musculoskeletal tissues such as articular cartilage. However, current challenges in TE include identifying materials that are biocompatible and have properties that closely match the mechanical properties and cellular environment of the target tissue, while allowing for 3D tomography of porous scaffolds as well as their cell growth and proliferation characterization. This is particularly challenging for opaque scaffolds. Here we use graphene foam (GF) as a 3D porous biocompatible substrate which is scalable, reproduceable, and a suitable environment for ATDC5 cell growth and chondrogenic differentiation. ATDC5 cells are cultured, maintained, and stained with a combination of fluorophores and gold nanoparticle to enable correlative microscopic characterization techniques, which elucidate the effect of GF properties on cell behavior in a three-dimensional environment. Most importantly, our staining protocols allows for direct imaging of cell growth and proliferation on opaque GF scaffolds using X-ray MicroCT, including imaging growth of cells within the hollow GF branches which is not possible with standard fluorescence and electron microscopy techniques. Abstract Figure 
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                            - Award ID(s):
- 1950305
- PAR ID:
- 10588210
- Publisher / Repository:
- bioRxiv
- Date Published:
- Volume:
- 6
- Issue:
- 9
- Page Range / eLocation ID:
- 3717-3725
- Format(s):
- Medium: X
- Institution:
- bioRxiv
- Sponsoring Org:
- National Science Foundation
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