Macromolecular therapeutics and nano-sized drug delivery systems often require localisation to specific intracellular compartments. In particular, efficient endosomal escape, retrograde trafficking, or late endocytic/lysosomal activation are often prerequisites for pharmacological activity. The aim of this study was to define a fluorescence microscopy technique able to confirm the localisation of water-soluble polymeric carriers to late endocytic intracellular compartments. Three polymeric carriers of different molecular weight and character were studied: dextrin (Mw~50,000 g/mol), a N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer (Mw approximately 35,000 g/mol) and polyethylene glycol (PEG) (Mw 5000 g/mol). They were labelled with Oregon Green (OG) (0.3-3 wt.%; <3% free OG in respect of total). A panel of relevant target cells were used: THP-1, ARPE-19, and MCF-7 cells, and primary bovine chondrocytes (currently being used to evaluate novel polymer therapeutics) as well as NRK and Vero cells as reference controls. Specific intracellular compartments were marked using either endocytosed physiological standards, Marine Blue (MB) or Texas-red (TxR)-Wheat germ agglutinin (WGA), TxR-Bovine Serum Albumin (BSA), TxR-dextran, ricin holotoxin, C6-7-nitro-2,1,3-benzoxadiazol-4-yl (NBD)-labelled ceramide and TxR-shiga toxin B chain, or post-fixation immuno-staining for early endosomal antigen 1 (EEA1), lysosomal-associated membrane proteins (LAMP-1, Lgp-120 or CD63) or the Golgi marker GM130. Co-localisation with polymer-OG conjugates confirmed transfer to discreet, late endocytic (including lysosomal) compartments in all cells types. The technique described here is a particularly powerful tool as it circumvents fixation artefacts ensuring the retention of water-soluble polymers within the vesicles they occupy.

Objective. The major proteases responsible for aggrecan turnover in articular cartilage are the aggrecanases (ADAMTS-4 and ADAMTS-5). Although several studies have demonstrated C-terminal truncation of these aggrecanases, the mechanism and importance of this processing are poorly understood. The objective of this study was to further investigate ADAMTS-4 and ADAMTS-5 C-terminal truncation in a porcine model in vitro culture system.Methods. Chondrocyte-agarose cultures with well-established extracellular matrices were treated with or without interleukin-1 (IL-1), for a variety of different culture time periods. Cultures were analyzed for release of sulfated glycosaminoglycan, aggrecanasegenerated interglobular domain (IGD)-aggrecan cleavage, and the presence of ADAMTS-4 and ADAMTS-5 isoforms. Inhibition of aggrecanase activity with monoclonal antibodies, tissue inhibitor of metalloproteinases 3 (TIMP-3), and cycloheximide pretreatment were used to identify ADAMTS isoforms involved in IGDaggrecan catabolism.Results. Multiple isoforms, including possible zymogens, of ADAMTS-4 and ADAMTS-5 were sequestered within the extracellular matrix formed by 3-week chondrocyte-agarose cultures. IL-1 exposure induced production of a low molecular weight (37 kd) isoform of ADAMTS-4. This isoform was capable of degrading exogenous aggrecan at the IGD-aggrecanase site, was inhibited by TIMP-3, was blocked after preincubation with an antibody to a sequence in the catalytic domain of ADAMTS-4, and required de novo synthesis in the presence of IL-1 for its generation.Conclusion. In porcine chondrocyte-agarose cultures, a 37-kd ADAMTS-4 isoform appears to be the major matrix protease responsible for the IGDaggrecanase activity detected in response to exposure to IL-1. This conclusion contradicts that of recent studies of transgenic knockout mice and highlights the need to determine the roles of the different aggrecanase(s) in human disease.Degradation of cartilage is one of the major pathologic features of arthropathies such as osteoarthritis and rheumatoid arthritis and involves proteolysis of the major structural elements of cartilage, aggrecan, and type II collagen. Aggrecanolysis has been attributed to ADAMTS-4 and ADAMTS-5 (1). Aggrecan comprises 3 globular domains (G1, G2, and G3) intersected by 2 rod-like segments, the interglobular domain (IGD), and the 2 glycosaminoglycan (GAG) attachment regions, CS1 and CS2, respectively, whose charge density provides the tissue with its water-imbibing properties (2). Aggrecan degradation occurs as an early event in the pathogenesis of osteoarthritis. Cleavage occurs within the IGD at Glu 373 -Ala 374 , resulting in release of the GAG-rich regions to the synovial fluid (3). Both ADAMTS-4 and ADAMTS-5 (and, to a lesser extent, ADAMTS-1, ADAMTS-8, and ADAMTS-9) have demonstrated proteolytic cleavage at this IGD-aggrecan site (4-7).ADAMTS-4 was first isolated from interleukin-1 (IL-1)-stimulated bovine explant culture medium as a

The ability of a polymer therapeutic to access the appropriate subcellular location is crucial to its efficacy and is defined to a large part by the many and complex cellular biological and biochemical barriers such that a construct must traverse. It is shown here that model dextrin conjugates are able to pass through a cartilaginous extracellular matrix into chondrocytes, with little perturbation of the matrix structure, indicating that targeting of potential therapeutics through a cartilaginous extracellular matrix should be proven possible. Rapid chondrocytic targeting of drugs which require intra cellularisation for their activity and uniform extracellular concentrations of drugs with an extracellular target, is thus enabled though polymer conjugation.