Production, characterization and biodistribution of therapeutic high-density lipoprotein-like nanoparticles reconstituted with or without histidine-tagged recombinant ApoA1

High-density lipoproteins (HDLs) are known for their cardiovascular protection due to apolipoprotein A-1 (ApoA1), their primary protein. ApoA1 promotes cholesterol reverse transport and exhibits antioxidant and anti-inflammatory properties. Although increasing HDL levels has not consistently reduced cardiovascular mortality in clinical trials, reconstituted HDL (rHDL) nanoparticles containing ApoA1 show potential in treating acute inflammation, such as in ischemic stroke, sepsis, and even COVID-19. ApoA1 is commonly produced in bacteria due to its simplicity and potential therapeutic optimisation. Addition of a histidine tag to recombinant ApoA1 may improve purification, stability and therapeutic efficacy, although its functional impact remains a subject of debate. In this study, ApoA1 with a poly-histidine tag (His-rApoA1) was produced in a clear coli system for simplified purification, followed by an evaluation of the tag's effects on rHDL nanoparticle properties. rHDL and His-rHDL nanoparticles were prepared using the sodium cholate dialysis method, combining recombinant rApoA1 or His-rApoA1 with phosphatidylcholine at a 1:75 M ratio. Nuclear magnetic resonance confirmed that both forms of rApoA1 structurally resembled plasma ApoA1, whether lipid-free or in nanoparticle form. Dynamic light scattering and electron microscopy revealed nanoparticle sizes around 7 nm with native HDL-like morphology. Testing on endothelial cells (EA.hy926) showed rapid uptake of rHDL and His-rHDL while preserving cell viability. Additionally, both nanoparticles reduced interleukin-6 and ICAM-1 expression in cells, demonstrating their anti-inflammatory and protective effects, unaffected by the poly-histidine tag. Intravenous injection in mice shows homogeneous distribution of His-rHDL in the liver, lungs, and spleen, with no cytotoxicity, indicating potential use for treating inflammatory diseases.