ABS 033: Construction of 22-NBD-Cholesterol-Loaded Lipoprotein Amphiphile Nanoparticles

Uchenna Abba ¹ , Claire J. Stewart ², Sara Suchanti ², Francesca Starvaggi ³, Naima G. Sharaf ²

¹ Department of Chemical Engineering, Stanford University
² Department of Biology, Stanford University
³ Department of Chemistry, Stanford University

The Van Wickle Journal (2026) Volume 2, ABS033

Introduction: Nanoparticles are widely explored for drug delivery applications, but their use is often limited by instability, toxicity, and difficulty with scaling and functionalization. There is a strong need for delivery systems that are both stable, versatile, and biologically compatible.

In this work, we developed a nanoparticle delivery platform that harnesses the self-assembly of bacterial lipoproteins into higher-order structures. Using the hydrophobic interactions that drive lipoprotein self-assembly, we engineered amphiphile nanoparticles capable of incorporating 22-NBD-cholesterol, a fluorescent cholesterol analog, as a model hydrophobic cargo. Our primary discovery is that bacterial lipoproteins can be formulated into stable, nanoscale particles that non-covalently form a linkage to cholesterol-like molecules while maintaining structural integrity.

Fluorescence was used as a central quantification to evaluate cargo incorporation and nanoparticle performance. By measuring fluorescence intensity at defined excitation and emission wavelengths, we confirmed successful incorporation of 22-NBD-cholesterol into complete nanoparticles. Fluorescence increased with cholesterol concentration, enabling direct quantification of cargo loading. Control formulations lacking either protein or lipid showed minimal signal, demonstrating that cholesterol incorporation depends on the fully assembled nanoparticle structure. Additional size and stability measurements revealed that moderate cholesterol loading enhances nanoparticle stability, while excessive cholesterol leads to destabilization.

Overall, this study establishes bacterial lipoprotein amphiphile nanoparticles as a stable, tunable, and fluorescently trackable delivery system for hydrophobic molecules. By combining biological self-assembly with fluorescence-based quantification, this platform provides a foundation for future studies in further characterization, cellular uptake, and therapeutic delivery. The approach offers a promising path toward safer and more effective nanoparticle-based drug delivery technologies.

Methods: Lipoprotein amphiphile constructs were expressed in Escherichia coli and purified through cell lysis, detergent-based solubilization, centrifugation, and size-exclusion chromatography. Purified protein was characterized using SDS-PAGE, nano dynamic light scattering (DLS), and NanoDrop spectrophotometry to assess protein integrity, size distribution, and concentration. Nanoparticles were assembled using a thin-film hydration method containing protein, polar lipids, and 22-NBD-cholesterol. Fluorescence microscopy was used to evaluate incorporation of the fluorescent cholesterol analog within the nanoparticle structure. Control formulations lacking individual nanoparticle components were prepared to investigate their contributions to nanoparticle assembly and structural stability. Additionally, thermal stability assays were performed to assess the stabilizing effects of cholesterol on the nanoparticle.

Results: We have constructed a stable lipoprotein amphiphile nanoparticle. 22-NBD-Cholesterol has been proven to be incorporated into the nanoparticle and has been quantified through fluorescence intensity testing. We have proven that 22-NBD-Cholesterol provides a form of thermal stability for the nanoparticles.

Discussion: Results demonstrated successful association of cholesterol with the lipoprotein nanoparticles, supporting the feasibility of the engineered biomaterial platform. Future studies will further characterize nanoparticle stability and functionality. Freeze–thaw assays will evaluate stability under cold-chain storage conditions. Lyophilization studies will assess long-term storage stability and structural integrity following dehydration and reconstitution. Further thermal unfolding and heating assays will determine transition temperatures associated with protein and lipid conformational changes. A bacterial cell line model will also be developed to investigate endosomal escape capabilities. Parallel formulations containing non-lipidated proteins will be evaluated to determine the broader robustness and versatility of the assembly approach.