Heparin 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 systems to genetically
engineered viral vectors such as adeno-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 performance of the heparin-functionalized AAO
membranes, studies are performed of the capture of virus-like nanoparticles.

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.