Evaluation of nanostructured vectors for the treatment of osteoarticular pathologies
Abstract
Purpose: One of the major problems in treatment of osteoarticular diseases is to reach cells inside the matrix to provide drug. Indeed, cartilage is an avascular tissue with a few cells feed by diffusion through a dense protein network (collagens, glycosaminoglycans). In this work we have designed polymeric nanoparticles (NPs) of poly (D, L-lactic/glycolic acid)(PLGA) synthesized by a double emulsion method, which are biocompatible, biodegradable and can encapsulate water-soluble agents. Our NPs are labelled with BSA coupled to a fluorescent dye (Cyanine-3) to follow them by epifluorescent microscopy. As articular cells expressed CD44, one receptor of hyaluronic acid (HA)(a main component of synovial fluid), the nanoparticles are recovered with HA in order to enhance targeting of cells. Here, we have studied the internalization kinetics of “empty” nanoparticles, and we have evaluated their neutrality on chondrocytes matrix synthesis, mesenchymal stem cell (MSC) differentiation and inflammatory response. Innocuity has been also evaluated in healthy animals, after direct intraarticular injection of labelled nanoparticles (inflammatory response, Extracellular matrix integrity).
Methods: Articular cells (chondrocytes, synoviocytes) and MSC are isolated from human donors, and cultured as primary culture. First, cells are exposed with 100 μg/mL of NPs from 2 to 12 hours. At the end of the kinetic immunofluorescence pictures with DAPI (nuclear staining) are realized to assess of the internalization of NPs, expression of inflammatory markers (IL1ß, TNFα and Cox2) are monitored by RT-qPCR analysis, and confirmed by PGE2 and nitrites measurement in supernatant.
In other hand we evaluate, with pellets culture system, the effect of NPs exposition on extracellular matrix synthesis by chondrocytes, with RT-qPCR analysis of specific markers (Col2, Aggrecan and COMP), and by histological study of pellets (Alcian blue staining of proteoglycans, Sirius Red staining of collagen). Finally by growing MSC into 3 different differentiation media, we investigate if NPs pre-treatment can interfere with differentiation ability of MSC onto chondrogenic, adipogenic or osteogenic pathway. RT-qPCR assays for differentiation markers according to culture conditions and specific staining of lipid vesicles or calcium deposits, allow us to confirm the differentiation of cells.
Intraarticular injections were realized in healthy rat’s Knees. Structure of joint (synovium, cartilage, subchondral bone) was assessed by histological studies, performed at 7 and 10 days after injection (single and repeated).
Results: For the different cell types, NPs are found into cytoplasm after 6 hours of exposition.