Osteoarthritis is a prevalent and debilitating disease that involves pathological contributions from numerous joint tissues and cells. The joint is a challenging arena for drug delivery, since the joint has poor bioavailability for systemically administered drugs and experiences rapid clearance of therapeutics after intra-articular injection. Moreover, each tissue within the joint presents unique barriers to drug localization. In this review, the various applications of nanotechnology to overcome these drug delivery limitations are investigated. Nanomaterials have reliably shown improvements to retention profiles of drugs within the joint space relative to injected free drugs. Additionally, nanomaterials have been modified through active and passive targeting strategies to facilitate interactions with and localization within specific joint tissues such as cartilage and synovium. Last, the limitations of drawing cross-study comparisons, the implications of synovial fluid, and the potential importance of multi-modal therapeutic strategies are discussed. As emerging, cell-specific disease modifying osteoarthritis drugs continue to be developed, the need for targeted nanomaterial delivery will likely become critical for effective clinical translation of therapeutics for osteoarthritis.

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Zipper-interacting protein kinase (ZIPK) has been implicated in Ca2؉ -independent smooth muscle contraction, although its specific role is unknown. The addition of ZIPK to demembranated rat caudal arterial strips induced an increase in force, which correlated with increases in LC 20 and MYPT1 phosphorylation. However, because of the number of kinases capable of phosphorylating LC 20 and MYPT1, it has proven difficult to identify the mechanism underlying ZIPK action. Therefore, we set out to identify bona fide ZIPK substrates using a chemical genetics method that takes advantage of ATP analogs with bulky substituents at the N 6 position and an engineered ZIPK capable of utilizing such substrates.32 P-Labeled 6-phenyl-ATP and ZIPK-L93G mutant protein were added to permeabilized rat caudal arterial strips, and substrate proteins were detected by autoradiography following SDS-PAGE. Mass spectrometry identified LC 20 as a direct target of ZIPK in situ for the first time. Tissues were also exposed to 6-phenyl-ATP and ZIPK-L93G in the absence of endogenous ATP, and putative ZIPK substrates were identified by Western blotting. LC 20 was thereby confirmed as a direct target of ZIPK; however, no phosphorylation of MYPT1 was detected. We conclude that ZIPK is involved in the regulation of smooth muscle contraction through direct phosphorylation of LC 20 .Smooth muscle plays an important role in the regulation of diverse physiological processes, including vascular tone, gastrointestinal motility, penile erection, bronchial diameter, and parturitional/postparturitional myometrial contraction. All smooth muscle tissues rely on the Ca 2ϩ /calmodulin-dependent activation of myosin light chain kinase (MLCK) 6 and subsequent phosphorylation of the 20-kDa myosin regulatory light chains (LC 20 ) at Ser-19 to initiate actomyosin cross-bridge cycling and force development (1). On the other hand, relaxation is induced by dephosphorylation of LC 20 by myosin light chain phosphatase (MLCP), a type 1 protein serine/threonine phosphatase (2, 3). Contraction of multiple smooth muscle tissues has frequently been observed in the absence of an increase in cytosolic free Ca 2ϩ concentration in response to a variety of stimuli (4). This phenomenon, commonly referred to as Ca 2ϩ sensitization, involves alteration of the MLCK:MLCP activity ratio in favor of the kinase, which can be achieved by the following mechanisms: (i) activation of MLCK by a mechanism not involving Ca 2ϩ /calmodulin, e.g. phosphorylation of MLCK by proline-directed kinases (5, 6); (ii) an increase in Ca 2ϩ -independent LC 20 kinase activity (7); and (iii) inhibition of MLCP either directly by phosphorylation of inhibitory residues (Thr-697 and/or Thr-855) in the myosin phosphatase targeting subunit 1 (MYPT1) of MLCP (8 -10) or indirectly by phosphorylation of the protein kinase C-potentiated inhibitory protein for heterotrimeric MLCP of 17 kDa (CPI-17) at Thr-38 (11, 12). The observation that intact and permeabilized smooth muscle tissues exhibit Ca 2ϩ -independent contracti...

A major hurdle limiting the ability to treat and cure osteoarthritis, a common and debilitating disease, is rapid joint clearance and limited cartilage targeting of intra-articular therapies. Nano-scale drug carriers have the potential to improve therapeutic targeting and retention in the joint after direct injection, however, there still lacks a fundamental understanding of how the physiochemical properties of nanoparticles (NPs) influence localization to the degenerating cartilage and how joint conditions such as disease state and synovial fluid impact NP biodistribution. The goal of this study was to assess how physicochemical properties of NPs influence their interactions with joint tissues and, ultimately, cartilage localization. Ex vivo models of joint tissues were used to study how poly(lactide-co-glycolide) (PLGA) and polystyrene (PS) NP size, charge, and surface chemistry influence cartilage retention under normal and disease-mimicking conditions. Of the particles investigated, PLGA NPs surface-modified with a quaternary ammonium cation had the greatest retention within cartilage explants, however, retention was diminished 2 to 2.9-fold in arthritic tissue and in the presence of synovial fluid. Interactions with synovial fluid induced changes to NP surface properties and colloidal stability in vitro. The impact of NP charge on "off-target" synoviocyte uptake was also dependent on synovial fluid interactions. The results suggest that the design of nanocarriers for targeted drug delivery within the joint cannot be based on a single parameter such as zeta potential or size, and that the fate of injected delivery systems will likely be influenced by the disease state of the joint and the presence of synovial fluid.

Understanding intra-articular biodistribution is imperative as candidate osteoarthritis (OA) drugs become increasingly site-specific. Cartilage has been identified as opportunistic for therapeutic intervention, but poses numerous barriers to drug delivery. To facilitate drug delivery to cartilage, nanoscale vehicles have been designed with different features that target the tissue's matrix. However, it is unclear if these targeting strategies are influenced by OA and the associated structural changes that occur in cartilage. The goal of this work was to study the effectiveness of different cartilage-targeting nanomaterials with respect to cartilage localization and retention, and to determine how these outcomes change in OA. To address these questions, a nanoparticle (NP) system was developed, and the formulation was tuned to possess three distinct cartilage-targeting strategies: (1) passive targeting cationic NPs for electrostatic attraction to cartilage, (2) active targeting NPs with binding peptides for collagen type II, and (3) untargeted neutrally-charged NPs. Ex vivo analyses with bovine cartilage explants demonstrated that targeting strategies significantly improved NP associations with both healthy and OA-like cartilage. In vivo studies with collagenase-induced OA in rats revealed that disease state influenced joint biodistribution for all three NP formulations. Importantly, the extent of cartilage accumulation for each NP system was affected by disease differently; with active NPs, but not passive NPs, cartilage accumulation was increased in OA relative to healthy knees. Together, this work suggests that NPs can be strategically designed for site-specific OA drug delivery, but the biodistribution of the NPs are influenced by the disease conditions into which they are delivered.