(350e) Functionalization of Anodized Aluminum Oxide Membranes for Affinity-Based Nanoparticle Capture

Nanoparticles are of growing interest as therapeutic devices, ranging from synthetic nanoparticle drug delivery or theranostic systems to genetically engineered viral vectors such as anedo-associated viruses (AAVs). While these nanoparticle-based therapeutics have a high degree if efficacy for treating diseases, their separation from synthesis media is often a bottleneck in developing and scaling up processes for their manufacture. Centrifugation is frequently used at lab scale to separate nanoparticles, but this technology is not able to provide selective separations based on surface functionality and is difficult to scale. Adsorptive separations are more tunable based on charge and surface functional groups, but can be difficult to effectively design for the capture of 20-100 nm particles.

Here, anodized aluminum oxide (AAO) membranes are developed as a platform for affinity-based nanoparticle capture. These membranes provide a high density of straight, uniform pores through a chemically stable aluminum oxide framework that can be tuned by synthesis conditions. In the current study, 200 nm pores are used as a baseline. As a model affinity ligand, heparin is conjugated to the surface of the membranes. In the first step, membranes are functionalized with aminopropyl groups using aminopropyltriethoxysilane (APTES) to react with hydroxyl groups created on the AAO surface through a hydroxylation procedure. The density of amines in the resulting membranes is quantified using an acid orange II binding / release assay at pH values compatible with the chemistry of AAO. Heparin is subsequently attached via its carboxylic acid groups by using EDC-NHS coupling to form imide bonds. The density of attached heparin is measured by using binding of methylene blue (a cationic probe) followed by its release by charge screening.

To test the functional performance of the heparin-functionalized AAO membranes, studies are performed of the capture of a heparin-specific protein and of a virus-like nanoparticle. For proof of concept of heparin functionality, fluorescently labeled fibroblast growth factor 2 (FGF-2) is used due to its selective interactions with heparin. The ability of the membranes to bind and subsequently release FGF-2 is used to show that heparin not only provides a negative charge to the particles but also retains its biofunctionality. The ability to capture nanoparticles is tested using silica nanoparticles (SNPs) with a size similar to that of AAV vectors (18 ± 4 nm), functionalized with amines to provide charged sites for interaction with heparin. The virus-like particles are fluorescently labeled to allow their capture and release to be readily quantified. Pressure-driven flux measurements with dead end filtration show the effects of functionalization on the flux of media with and without virus-like SNPs, and to demonstrate the capture effectiveness and capacity of the membranes towards virus-like SNPs.