(676g) Engineered Green Fluorescent Proteins: Cartilage-Targeted Delivery Nanocarriers That Provide Insights on the Effects of Charge on Transport into Dense Charged Tissues

Rees, H. A., Harvard and MIT
Kim, S. E., MIT
Olsen, B. D., Massachusetts Institute of Technology
Liu, D. R., Harvard and MIT
Post-traumatic Osteoarthritis (PTOA) is a degenerative disease that affects cartilage and associated joint tissues. PTOA is triggered by a traumatic joint injury and there are currently no disease modifying osteoarthritis drugs to slow down or halt disease progression. A major challenge for developing such a therapeutic is associated with drug delivery into cartilage. Systemic delivery is ineffective because cartilage does not have a blood supply. If drugs are injected directly into the joint (intra-articular therapy), they are rapidly cleared by the synovium leading to poor cartilage penetration. Cationic delivery carriers such as Avidin have significantly improved the uptake, penetration and retention of low molecular weight drugs through electrostatic interactions with negatively charged aggrecan proteoglycans in cartilage.

We have characterized the precise effect of carrier charge on delivery efficacy into cartilage in isolation of other factors such as size and shape that can influence transport. While higher cationic charge leads to enhanced transport due to higher Donnan partitioning at the cartilage-synovial fluid interface, subsequent electrostatic binding interactions between the nanocarrier and cartilage matrix can slow transport. We studied the use of supercharged green fluorescent proteins (S-GFPs) engineered to have net charge ranging from +9 to +36 at physiological pH while having nearly identical sizes and shapes. As controls, we used three engineered GFP variants having net neutral charge but different surface charge distributions. All the S-GFPs had higher quantitative cartilage uptake compared to the controls. Amongst the S-GFPs, the lower charged variants (net charge +9 and +15) had significantly higher net uptake and penetration into cartilage compared to higher charged variants (net charge +25 and +36). These trends were observed both in bovine cartilage and in human cartilage obtained from two donors with no or early osteoarthritic cartilage. Two of the three neutral variants did not show preferential uptake into cartilage while the third variant had an uptake ratio (defined as the ratio of concentration inside the tissue to the final bath concentration) that was an order of magnitude higher than the other two. This demonstrated that, in addition to net cationic charge, surface charge distribution also plays a very important role in carrier transport in cartilage. Cellular uptake of S-GFPs into cartilage cells increased monotonically with higher cationic charge, which was the opposite of the trends observed in tissue-level uptake and penetration experiments. Based on these results, +9 GFP was found to be the optimal delivery carrier for drugs with ECM or cell surface receptor targets. For drugs with intracellular targets, a variant with intermediate charge, such as the +15 GFP, would provide a better balance between tissue penetration and cellular uptake.