(387g) Investigating Bio-Nano Interactions of Pegylated Cationic Polyamidoamine (PAMAM) Dendrimers within Articular Cartilage

Osteoarthritis (OA) is a joint disease characterized by loss of articular cartilage resulting in pain and decreased mobility. Therapeutics can be delivered directly to the joint, however, it is challenging for drugs conjugated to larger nanocarriers to penetrate cartilage deep enough to reach cells, and molecules that successfully penetrate are cleared rapidly and do not reside long enough to have a therapeutic effect. We have identified polyamidoamine (PAMAM) dendrimers as ideal nanocarriers to overcome delivery challenges to cartilage- they are positively charged which facilitates transport through the negatively charged matrix, small enough (4.5nm – 6.7nm) to penetrate cartilage, and contain end groups that are easily functionalized. Analysis of dendrimer delivery efficacy must consider how synovial fluid-derived proteins and biomolecules interact with dendrimers. Herein, we propose that after injection to the intra-articular space, synovial fluid-derived proteins can adsorb to the surface of nanocarriers forming a nanocarrier-protein complex that affects targeting and transport properties. Our goal is to understand how dendrimer-protein interactions affect biological outcomes of dendrimer uptake by cartilage and determine if there is a change in physiochemical properties of dendrimers in synovial fluid.

To study the biological outcome of dendrimers, we used an ex vivo bovine cartilage model where we assessed dendrimer uptake and desorption on cartilage, as well as the kinetics of uptake. We determined uptake of dendrimers into cartilage decreases as the concentration of synovial fluid increases, however varying PEG density and/or PEG chain length mitigates this (Fig. 1A). Recent work has determined the number of available amines on the surface accessible to the physiological environment (ie: cartilage or synovial fluid) is a key property that governs the degree of dendrimer interactions with cartilage, where a higher number of amines means more interactions are possible with cartilage surface. Using a salt screening assay, we observed that for unPEGylated dendrimers as the protein concentration increases, there is a decrease in critical salt concentration, defined as the lowest salt concentration to screen electrostatic interactions between dendrimer and cartilage (Fig. 1B). This value correlates to the number of amines available on the dendrimer surface. Taken together this suggests protein adsorption to dendrimer is blocking amines on the surface. Interestingly, for the PEGylated dendrimers, despite protein being present, there is little to no change in critical salt concentration likely due to PEG repelling proteins. In a kinetic uptake study, we demonstrate that protein adsorption reduces the first-order rate constant of dendrimer uptake (Fig. 1C). We also used an electrophoresis-based technique combined with mass spectrometry (LC-MS) to identify the proteins interacting with dendrimers. In a preliminary analysis, proteins were categorized by biological function, and percentages were calculated based on spectral counts (Fig. 1D). For unPEGylated dendrimers, relative to synovial fluid only, there is an increased percentage of complement, coagulation, and acute inflammation proteins. For PEGylated dendrimers, complement and apolipoproteins increase with increasing percent PEG, while coagulation and acute inflammation proteins decrease. Work is ongoing to study how protein-dendrimer interactions affect the depth of dendrimer penetration in cartilage; after initial uptake of dendrimer to cartilage, we anticipate observing competitive loss of bound proteins due to higher charge density of the matrix.

Overall, we have determined dendrimer uptake into cartilage is a balance between PEG, protein, and cartilage. Our data suggest that proteins are a physical barrier to uptake on cartilage, and interact with the dendrimer surface. However, for PEGylated dendrimers, when there is sufficient shielding PEG prevents proteins from altering accessible amines which enables the dendrimer to interact with the cartilage surface. This work is further evidence for us to consider how the biological environment alters the intrinsic surface properties of nanocarriers; understanding these mechanisms can improve the design of nanocarriers and optimize their targeting and transport capabilities.