A Modular Albumin-Oligonucleotide Biomolecular Assembly for Delivery of Antisense Therapeutics

Antisense nucleic acid drugs are susceptible to nuclease degradation, rapid renal clearance, and short circulatory half-life. In this work, we introduce a modular-based recombinant human albumin-oligonucleotide (rHA-cODN) biomolecular assembly that allows incorporation of a chemically stabilized therapeutic gapmer antisense oligonucleotide (ASO) and FcRn-driven endothelial cellular recycling. A phosphodiester ODN linker (cODN) was conjugated to recombinant human albumin (rHA) using maleimide chemistry, after which a complementary gapmer ASO, targeting ADAMTS5 involved in osteoarthritis pathogenesis, was annealed. The rHA-cODN/ASO biomolecular assembly production, fluorescence labeling, and purity were confirmed using polyacrylamide gel electrophoresis. ASO release was triggered by DNase-mediated degradation of the linker strand, reaching 40% in serum after 72 h, with complete release observed following 30 min of incubation with DNase. Cellular internalization and trafficking of the biomolecular assembly using confocal microscopy in C28/I2 cells showed higher uptake and endosomal localization by increasing incubation time from 4 to 24 h. FcRn-mediated cellular recycling of the assembly was demonstrated in FcRn-expressing human microvascular endothelial cells. ADAMTS5 in vitro silencing efficiency reached 40%, which was comparable to free gapmer after 72 h incubation with human osteoarthritis patients’ chondrocytes. This work introduces a versatile biomolecular modular-based “Plug-and-Play” platform potentially applicable for albumin-mediated half-life extension for a range of different types of ODN therapeutics

The Added Value of the “Co” in Co-Culture Systems in Research on Osteoarthritis Pathology and Treatment Development

Osteoarthritis (OA) is a highly prevalent disease and a major health burden. Its development and progression are influenced by factors such as age, obesity or joint overuse. As a whole organ disease OA affects not only cartilage, bone and synovium but also ligaments, fatty or nervous tissue surrounding the joint. These joint tissues interact with each other and understanding this interaction is important in developing novel treatments. To incorporate and study these interactions in OA research, several co-culture models have evolved. They combine two or more cell types or tissues and investigate the influence of amongst others inflammatory or degenerative stimuli seen in OA. This review focuses on co-cultures and the differential processes occurring in a given tissue or cell as a consequence of being combined with another joint cell type or tissue, and/or the extent to which a co-culture mimics the in vivo processes. Most co-culture models depart from synovial lining and cartilage culture, but also fat pad and bone have been included. Not all of the models appear to reflect the postulated in vivo OA pathophysiology, although some of the discrepancies may indicate current assumptions on this process are not entirely valid. Systematic analysis of the mutual influence the separate compartments in a given model exert on each other and validation against in vivo or ex vivo observation is still largely lacking and would increase their added value as in vitro OA models

Nerves are more abundant than blood vessels in the degenerate human intervertebral disc

Chronic low back pain (LBP) is the most common cause of disability worldwide. New ideas surrounding LBP are emerging that are based on interactions between mechanical, biological and chemical influences on the human IVD. The degenerate IVD is proposed to be innervated by sensory nerve fibres and vascularised by blood vessels, and it is speculated to contribute to pain sensation. However, the incidence of nerve and blood vessel ingrowth, as well as whether these features are always associated, is unknown. We investigated the presence of nerves and blood vessels in the nucleus pulposus (NP) of the IVD in a large population of human discs

Celecoxib alleviates nociceptor sensitization mediated by interleukin-1beta-primed annulus fibrosus cells.

PURPOSE This study aims to analyze the effect of pro-inflammatory cytokine-stimulated human annulus fibrosus cells (hAFCs) on the sensitization of dorsal root ganglion (DRG) cells. We further hypothesized that celecoxib (cxb) could inhibit hAFCs-induced DRG sensitization. METHODS hAFCs from spinal trauma patients were stimulated with TNF-α or IL-1β. Cxb was added on day 2. On day 4, the expression of pro-inflammatory and neurotrophic genes was evaluated using RT-qPCR. Levels of prostaglandin E2 (PGE-2), IL-8, and IL-6 were measured in the conditioned medium (CM) using ELISA. hAFCs CM was then applied to stimulate the DRG cell line (ND7/23) for 6 days. Then, calcium imaging (Fluo4) was performed to evaluate DRG cell sensitization. Both spontaneous and bradykinin-stimulated (0.5 μM) calcium responses were analyzed. The effects on primary bovine DRG cell culture were performed in parallel to the DRG cell line model. RESULTS IL-1ß stimulation significantly enhanced the release of PGE-2 in hAFCs CM, while this increase was completely suppressed by 10 µM cxb. hAFCs revealed elevated IL-6 and IL-8 release following TNF-α and IL-1β treatment, though cxb did not alter this. The effect of hAFCs CM on DRG cell sensitization was influenced by adding cxb to hAFCs; both the DRG cell line and primary bovine DRG nociceptors showed a lower sensitivity to bradykinin stimulation. CONCLUSION Cxb can inhibit PGE-2 production in hAFCs in an IL-1β-induced pro-inflammatory in vitro environment. The cxb applied to the hAFCs also reduces the sensitization of DRG nociceptors that are stimulated by the hAFCs CM

Cell uptake and intracellular trafficking of bioreducible poly(amidoamine) nanoparticles for efficient mRNA translation in chondrocytes

Disulfide-containing poly(amidoamine) (PAA) is a cationic and bioreducible polymer, with potential use as a nanocarrier for mRNA delivery in the treatment of several diseases including osteoarthritis (OA). Successful transfection of joint cells with PAA-based nanoparticles (NPs) was shown previously, but cell uptake, endosomal escape and nanoparticle biodegradation were not studied in detail. In this study, C28/I2 human chondrocytes were transfected with NPs co-formulated with a PEG-polymer coating and loaded with EGFP mRNA for confocal imaging of intracellular trafficking and evaluation of transfection efficiency. Compared with uncoated NPs, PEG-coated NPs showed smaller particle size, neutral surface charge, higher colloidal stability and superior transfection efficiency. Furthermore, endosomal entrapment of these PEG-coated NPs decreased over time and mRNA release could be visualized both in vitro and in live cells. Importantly, cell treatment with modulators of the intracellular reducing environment showed that glutathione (GSH) concentrations affect translation of the mRNA payload. Finally, we applied a D-optimal experimental design to test different polymer-to-RNA loading ratios and dosages, thus obtaining an optimal formulation with up to ≈80% of GFP-positive cells and without toxic effects. Together, the biocompatibility and high transfection efficiency of this system may be a promising tool for intra-articular delivery of therapeutical mRNA in OA treatment

Drug delivery in intervertebral disc degeneration and osteoarthritis : Selecting the optimal platform for the delivery of disease-modifying agents

Acknowledgement We would like to acknowledge Prof. Gerjo Van Osch and prof Molly Stevens for their careful and critical revision of the manuscript. We wish to thank all principal investigators of the TargetCaRe consortium for their enormous support during the years: Prof G. van Osch. Prof. Mauro Alini, Prof. Bruce Caterson, Dr. Alan Chan, Prof. Cosimo De Bari, Prof. Ron Heeren, Prof. Kennet Howard, Prof. Marcelle Machluf, Prof. Molly M. Stevens and Prof. Avner Yayon. This work was supported by European Union's Horizon 2020 Research And Innovation Programme under Marie Sklodowska Curie Grant agreement no. 642414.Peer reviewedproofPublisher PD

Cell uptake and intracellular trafficking of bioreducible poly(amidoamine) nanoparticles for efficient mRNA translation in chondrocytes

Disulfide-containing poly(amidoamine) (PAA) is a cationic and bioreducible polymer, with potential use as a nanocarrier for mRNA delivery in the treatment of several diseases including osteoarthritis (OA). Successful transfection of joint cells with PAA-based nanoparticles (NPs) was shown previously, but cell uptake, endosomal escape and nanoparticle biodegradation were not studied in detail. In this study, C28/I2 human chondrocytes were transfected with NPs co-formulated with a PEG-polymer coating and loaded with EGFP mRNA for confocal imaging of intracellular trafficking and evaluation of transfection efficiency. Compared with uncoated NPs, PEG-coated NPs showed smaller particle size, neutral surface charge, higher colloidal stability and superior transfection efficiency. Furthermore, endosomal entrapment of these PEG-coated NPs decreased over time and mRNA release could be visualized both in vitro and in live cells. Importantly, cell treatment with modulators of the intracellular reducing environment showed that glutathione (GSH) concentrations affect translation of the mRNA payload. Finally, we applied a D-optimal experimental design to test different polymer-to-RNA loading ratios and dosages, thus obtaining an optimal formulation with up to ≈80% of GFP-positive cells and without toxic effects. Together, the biocompatibility and high transfection efficiency of this system may be a promising tool for intra-articular delivery of therapeutical mRNA in OA treatment

Hyperosmolar expansion medium improves nucleus pulposus cell phenotype

Background:Repopulating the degenerated intervertebral disc (IVD) with tissue-spe-cific nucleus pulposus cells (NPCs) has already been shown to promote regenerationin various species. Yet the applicability of NPCs as cell-based therapy has been ham-pered by the low cell numbers that can be extracted from donor IVDs and theirpotentially limited regenerative capacity due to their degenerated phenotype. Tooptimize the expansion conditions, we investigated the effects of increasing culturemedium osmolarity during expansion on the phenotype of dog NPCs and their abilityto produce a healthy extracellular matrix (ECM) in a 3D culture model.Methods:Dog NPCs were expanded in expansion medium with a standard osmolar-ity of 300 mOsm/L or adjusted to 400 or 500 mOsm/L in both normoxic and hypoxicconditions. Following expansion, NPCs were cultured in a 3D culture model in chon-drogenic culture medium with a standard osmolarity. Read-out parameters includedcell proliferaton rate, morphology, phenotype and healthy ECM production.Results:Increasing the expansion medium osmolarity from 300 to 500 mOsm/Lresulted in NPCs with a more rounded morphology and a lower cell proliferation rateaccompanied by the expression of several healthy NPC and progenitor markers atgene (KRT18, ACAN, COL2, CD73, CD90) and protein (ACAN, PAX1, CD24, TEK,CD73) level. The NPCs expanded at 500 mOsm/L were able to retain most of theirphenotypic markers and produce healthy ECM during 3D culture independent of theoxygen level used during expansion. Conclusions:Altogether, our findings show that increasing medium osmolarity duringexpansion results in an NPC population with improved phenotype, which couldenhance the potential of cell-based therapies for IVD regeneration