Comparison of different transfection methods for mrna delivery in articular joint cells.

Purpose: Tissues in the joint express a large amount of extracellular matrix (ECM) which interferes with transfecting agents. Currently employed methods do not result in a satisfactory transfection of ECM-rich tissue. Biodegradable and bioresponsive polymer-based nanoparticles (Nanogel, 20Med Therapeutics) can be used as an oligonucleotide delivery technology and be optimized for transfection of specific cells. This makes it a promising candidate for in vivo delivery of osteoarthritis-modulating mRNA drugs. In this study, different transfection methods were evaluated in vitro for different cell types that are present in the joint. We hypothesize that use of the Nanogel allows for a better ECM-penetration due its electrochemical charge. Methods: Lipofectamine MessengerMAX (Invitrogen; 1.5μl/well of 24 well plate) and NEON electroporation (Thermo Fisher; 990 Volt, 40 ms) were compared to different concentrations of the Nanogel (20Med Therapeutics) agent. Experiments were performed in monolayer culture in TC24 well plate with an area of 1,92 cm2 as well as 3D pellets (200,000 cells/pellet) and tissue explants (15±4 mg cartilage tissue). Primary human bone marrow stromal cells (hBMSC), human synovial derived stem cells (hSDSC), bovine chondrocytes (bCH), rat tendon derived stem/progenitor cells (rTDSPC) and C28/I2 chondrocyte cell line at the concentration 15,000/cm2 were tested with different concentrations of Nanogel (15 μg/ml to 120 μg/ml) loaded with chemically modified ARCA capped mRNA encoding for EGFP or tdTomato (240 μg/well to 1920 μg/well mRNA). These mRNAs were provided by Ethris GmbH. Successful transfection resulted in the translation of the respective fluorescent protein, which was evaluated by confocal microscopy (Zeiss, LSM800) and flow cytometry (BD, FACSAria III). Transfected cells were co-stained with Calcein (5 μM) and DAPI (0.625 μg/ml) to discriminate between live and dead cells. Metabolic activity at 24, 48, 72 and 96 hours after transfection was evaluated with alamarBlue. 3D pellets of bCH and hSDSC were cultured in DMEM-HG, ITS 1%, NEA 1% P/S 1%, 50 μg/ml ascorbic acid, 10-7M dexamethasone and with or without 10 ng/ml TGFβ for 7 or 14 days before transfection; after 24 hours, pellets and tissue explant were fixed, cut with cryostat and stained with Safranin-O/Fast green to check matrix deposition. IHC staining against EGFP and tdTomato was performed in order to assess the penetration depth of transfecting agents. Results: All tested methods resulted in successful mRNA transfection and protein production in a concentration-dependent manner. Nanogel exposure for 4 hours with a subsequent medium change did not affect cell viability as compared to lipofectamine and electroporation; however, the transfection efficiency varied based on the cell type. BMSCs and C28/I2 showed high protein expression for all the methods; on the contrary, bCH and hSDSC transfected with 30 μg/ml Nanogel and 480 ng mRNA showed a limited intensity in protein expression. For that reason, higher concentrations of Nanogel and mRNA were tested, showing a comparable efficiency as in the BMSCs and C28/I2 cells. It was observed that longer exposure to Nanogel (≥24 hours) resulted in a time- and concentration-dependent decrease in metabolic activity irrespective of the transfected mRNA. Interesting, when we tested the Nanogel on 3D pellet only those that expressed higher ECM showed a positivity for the transfection. ECM deposition was correlated with penetration depth of the transfection reagents to evaluate the feasibility of cell transfection in matrix-rich tissues. Optimization of Nanogel:mRNA-ratio allowed us to achieve optimal transfection efficacy and minimal cytotoxicity based on chemical modification. Conclusions: Nanogel transfection proved successful for various cell types present in the joint and may be a promising method for tissues that exhibit a complex dense matrix. It will be further developed for application in the joint in vivo. Four hours exposure with Lipofectamine reduced the cell viability, which was not observed when using the Nanogel. In addition, although the NEON electroporation does affect cell viability less strongly than lipofectamine, electroporation can only be used in vitro for cells in suspension which makes it unsuitable for in vivo use. Our observation and optimization on the Nanogel will pave the way to further applications. Local delivery of therapeutic mRNAs for the modulation of osteoarthritis will be further studied in vitro and in vivo.