Peptide ligands for targeting and retention of nanoparticles and protein therapeutics in articular cartilage

Osteoarthritis (OA) is one of the most common causes of pain and disability. Currently, non-surgical treatment is limited to analgesic and anti-inflammatory drugs, which are not capable of either modifying the disease process or regenerate damaged parts in articular cartilage. From a clinical point of view, small focal lesions in articular cartilage are frequent incidental findings mainly in the hip and knee but also in other joints during open and arthroscopic joint preserving surgical procedures. There is therefore an obvious demand for (bio)-pharmaceutical treatment options to complement surgical procedures in order to regenerate damaged parts in articular cartilage and generally attenuate disease progression. However, pharmaceutical approaches are generally associated with low bioavailabilities of the therapeutic molecules in the cartilage matrix, the primary site of the disease process in OA. The low bioavailability is due to the fact that the joint assumes a remote location being connected to the systemic circulation via the synovial fluid, which is an ultrafiltrate of blood secreted into the joint space. Articular cartilage, i.e. the lining of the joint surfaces providing nearly frictionless motion, is avascular and therefore not connected to the systemic circulation at all and thus depends on nutrients from the synovial fluid being transported to the cells, i.e. chondrocytes, by convective transport, which is inherently impaired due to its dense extracellular matrix. In addition, small molecules which may end up in the joint space via the synovial fluid systemically or after local administration by intra-articular injection are rapidly cleared out of the joint through lymphatic uptake. In order for (bio)-pharmaceutical treatment options to be efficient and effective, the therapeutic needs to reach the site of the disease process, here articular cartilage, and stay there long enough to exert its desired function. At first, we sought to engineer a nanoparticle-based drug delivery system which is capable of targeting the cartilage matrix and is subsequently immobilized there to release its cargo over time. Towards this end, we used a technique called peptide-on-phage display to carry out rounds of affinity selection against bovine cartilage in order to discover potential 6-mer peptides as bioaffinity ligands to a cartilage extracellular matrix component. Phage display relies on the genotype-phenotype linkage of inserts in the gene coding for minor coat protein of the phage virion, which is then displayed as a fusion protein on its surface. The sequence displayed by recovered binding phages can therefore be determined by sequencing of the phage DNA. After five rounds of affinity selection, i.e. recovering the binding phages, amplifying and re-exposing them to bovine articular cartilage, the peptide sequence WYRGRL could be discovered, which appeared in 94 out of 96 sequenced phage clones. WYRGRL was shown to bind specifically to articular car