Systemic delivery of murine SOD2 mRNA to experimental abdominal aortic aneurysm mitigates expansion and rupture.

Background: Oxidative stress is implicated in the pathogenesis and progression of abdominal aortic aneurysm (AAA). Antioxidant delivery as a therapeutic for AAA is of substantial interest although clinical translation of antioxidant therapy has met with significant challenges due to limitations in achieving sufficient antioxidant levels at the site of AAA. We posit that nanoparticle-based approaches hold promise to overcome challenges associated with systemic administration of antioxidants. Methods: We employed a peptide-based nanoplatform to overexpress a key modulator of oxidative stress, superoxide dismutase 2 (SOD2). The efficacy of systemic delivery of SOD2 mRNA as a nanotherapeutic agent was studied in two different murine AAA models. Unbiased mass spectrometry-enabled proteomics and high-dimensional bioinformatics were used to examine pathways modulated by SOD2 overexpression. Results: The murine SOD2 mRNA sequence was mixed with p5RHH, an amphipathic peptide capable of delivering nucleic acids in vivo to form self-assembled nanoparticles of ∼55 nm in diameter. We further demonstrated that the nanoparticle was stable and functional up to four weeks following self-assembly when coated with hyaluronic acid. Delivery of SOD2 mRNA mitigated the expansion of small AAA and largely prevented rupture. Mitigation of AAA was accompanied by enhanced SOD2 protein expression in aortic wall tissue. Concomitant suppression of nitric oxide, inducible nitric oxide synthase expression, and cell death was observed. Proteomic profiling of AAA tissues suggests that SOD2 overexpression augments levels of microRNAs that regulate vascular inflammation and cell apoptosis, inhibits platelet activation/aggregation, and downregulates mitogen-activated protein kinase signaling. Gene set enrichment analysis shows that SOD2 mRNA delivery is associated with activation of oxidative phosphorylation, lipid metabolism, respiratory electron transportation, and tricarboxylic acid cycle pathways. Conclusions: These results confirm that SOD2 is key modulator of oxidative stress in AAA. This nanotherapeutic mRNA delivery approach may find translational application in the medical management of small AAA and the prevention of AAA rupture.

Elucidating allergic reaction mechanisms in response to SARS-CoV-2 mRNA vaccination in adults.

BACKGROUND: During the COVID-19 pandemic, novel nanoparticle-based mRNA vaccines were developed. A small number of individuals developed allergic reactions to these vaccines although the mechanisms remain undefined. METHODS: To understand COVID-19 vaccine-mediated allergic reactions, we enrolled 19 participants who developed allergic events within 2 h of vaccination and 13 controls, nonreactors. Using standard hemolysis assays, we demonstrated that sera from allergic participants induced stronger complement activation compared to nonallergic subjects following ex vivo vaccine exposure. RESULTS: Vaccine-mediated complement activation correlated with anti-polyethelyne glycol (PEG) IgG (but not IgM) levels while anti-PEG IgE was undetectable in all subjects. Depletion of total IgG suppressed complement activation in select individuals. To investigate the effects of vaccine excipients on basophil function, we employed a validated indirect basophil activation test that stratified the allergic populations into high and low responders. Complement C3a and C5a receptor blockade in this system suppressed basophil response, providing strong evidence for complement involvement in vaccine-mediated basophil activation. Single-cell multiome analysis revealed differential expression of genes encoding the cytokine response and Toll-like receptor (TLR) pathways within the monocyte compartment. Differential chromatin accessibility for IL-13 and IL-1B genes was found in allergic and nonallergic participants, suggesting that in vivo, epigenetic modulation of mononuclear phagocyte immunophenotypes determines their subsequent functional responsiveness, contributing to the overall physiologic manifestation of vaccine reactions. CONCLUSION: These findings provide insights into the mechanisms underlying allergic reactions to COVID-19 mRNA vaccines, which may be used for future vaccine strategies in individuals with prior history of allergies or reactions and reduce vaccine hesitancy.

Targeting cathepsin C ameliorates murine acetaminophen-induced liver injury.

Acetaminophen (APAP) overdosing is a major cause of acute liver failure worldwide and an established model for drug-induced acute liver injury (ALI). While studying gene expression during murine APAP-induced ALI by 3'mRNA sequencing (massive analysis of cDNA ends, MACE), we observed splenic mRNA accumulation encoding for the neutrophil serine proteases cathepsin G, neutrophil elastase, and proteinase-3 - all are hierarchically activated by cathepsin C (CtsC). This, along with increased serum levels of these proteases in diseased mice, concurs with the established phenomenon of myeloid cell mobilization during APAP intoxication. Objective: In order to functionally characterize CtsC in murine APAP-induced ALI, effects of its genetic or pharmacological inhibition were investigated. Methods and Results: We report on substantially reduced APAP toxicity in CtsC deficient mice. Alleviation of disease was likewise observed by treating mice with the CtsC inhibitor AZD7986, both in short-term prophylactic and therapeutic protocols. This latter observation indicates a mode of action beyond inhibition of granule-associated serine proteases. Protection in CtsC knockout or AZD7986-treated wildtype mice was unrelated to APAP metabolization but, as revealed by MACE, realtime PCR, or ELISA, associated with impaired expression of inflammatory genes with proven pathogenic roles in ALI. Genes consistently downregulated in protocols tested herein included cxcl2, mmp9, and angpt2. Moreover, ptpn22, a positive regulator of the toll-like receptor/interferon-axis, was reduced by targeting CtsC. Conclusions: This work suggests CtsC as promising therapeutic target for the treatment of ALI, among others paradigmatic APAP-induced ALI. Being also currently evaluated in phase III clinical trials for bronchiectasis, successful application of AZD7986 in experimental APAP intoxication emphasizes the translational potential of this latter therapeutic approach.

The complement regulator CD55 modulates TLR9 signaling and supports survival in marginal zone B cells.

Marginal zone (MZ) B cells bridge innate and adaptive immunity by sensing bloodborne antigens and producing rapid antibody and cytokine responses. CD55 is a membrane-bound complement regulator that interferes with complement activation, an important component of innate immunity. CD55 also regulates adaptive immunity-CD55 downregulation is critical for germinal center reactions. MZ B cells also express low CD55, but its role in MZ B cell function is unknown. Using germline knockout mice, we found that similar numbers of MZ B cells are initially established in 3-week-old CD55-deficient mice compared to wild-type (WT) mice. However, MZ B cells fail to accumulate as mice age and undergo increased apoptosis. Following ex vivo stimulation of MZ B cells through Toll-like receptor 9, we observed a proinflammatory phenotype with increased IL-6 expression. These findings demonstrate a critical role for CD55 in supporting MZ B cell survival while also regulating cellular function.

Rapamycin Perfluorocarbon Nanoparticle Mitigates Cisplatin-Induced Acute Kidney Injury.

For nearly five decades, cisplatin has played an important role as a standard chemotherapeutic agent and been prescribed to 10-20% of all cancer patients. Although nephrotoxicity associated with platinum-based agents is well recognized, treatment of cisplatin-induced acute kidney injury is mainly supportive and no specific mechanism-based prophylactic approach is available to date. Here, we postulated that systemically delivered rapamycin perfluorocarbon nanoparticles (PFC NP) could reach the injured kidneys at sufficient and sustained concentrations to mitigate cisplatin-induced acute kidney injury and preserve renal function. Using fluorescence microscopic imaging and fluorine magnetic resonance imaging/spectroscopy, we illustrated that rapamycin-loaded PFC NP permeated and were retained in injured kidneys. Histologic evaluation and blood urea nitrogen (BUN) confirmed that renal structure and function were preserved 48 h after cisplatin injury. Similarly, weight loss was slowed down. Using western blotting and immunofluorescence staining, mechanistic studies revealed that rapamycin PFC NP significantly enhanced autophagy in the kidney, reduced the expression of intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1), as well as decreased the expression of the apoptotic protein Bax, all of which contributed to the suppression of apoptosis that was confirmed with TUNEL staining. In summary, the delivery of an approved agent such as rapamycin in a PFC NP format enhances local delivery and offers a novel mechanism-based prophylactic therapy for cisplatin-induced acute kidney injury.

Hydrogel Encapsulation of Genome-Engineered Stem Cells for Long-Term Self-Regulating Anti-Cytokine Therapy.

Biologic therapies have revolutionized treatment options for rheumatoid arthritis (RA) but their continuous administration at high doses may lead to adverse events. Thus, the development of improved drug delivery systems that can sense and respond commensurately to disease flares represents an unmet medical need. Toward this end, we generated induced pluripotent stem cells (iPSCs) that express interleukin-1 receptor antagonist (IL-1Ra, an inhibitor of IL-1) in a feedback-controlled manner driven by the macrophage chemoattractant protein-1 (Ccl2) promoter. Cells were seeded in agarose hydrogel constructs made from 3D printed molds that can be injected subcutaneously via a blunt needle, thus simplifying implantation of the constructs, and the translational potential. We demonstrated that the subcutaneously injected agarose hydrogels containing genome-edited Ccl2-IL1Ra iPSCs showed significant therapeutic efficacy in the K/BxN model of inflammatory arthritis, with nearly complete abolishment of disease severity in the front paws. These implants also exhibited improved implant longevity as compared to the previous studies using 3D woven scaffolds, which require surgical implantation. This minimally invasive cell-based drug delivery strategy may be adapted for the treatment of other autoimmune or chronic diseases, potentially accelerating translation to the clinic.

Side-chain modification of collagen-targeting peptide prevents dye aggregation for improved molecular imaging of arthritic joints.

Near-infrared (NIR) dye-peptide conjugates are widely used for tissue-targeted molecular fluorescence imaging of pathophysiologic conditions. However, the significant contribution of both dye and peptide to the net mass of these bioconjugates implies that small changes in either component could alter their photophysical and biological properties. Here, we synthesized and conjugated a type I collagen targeted peptide, RRANAALKAGELYKCILY, to either a hydrophobic (LS1000) or hydrophilic (LS1006) NIR fluorescent dye. Spectroscopic analysis revealed rapid self-assembly of both LS1000 and LS1006 in aqueous media to form stable dimeric/H aggregates, regardless of the free dye's solubility in water. We discovered that replacing the cysteine residue in LS1000 and LS1006 with acetamidomethyl cysteine to afford LS1001 and LS1107, respectively, disrupted the peptide's self-assembly and activated the previously quenched dye's fluorescence in aqueous conditions. These results highlight the dominant role of the octadecapeptide, but not the dye molecules, in controlling the photophysical properties of these conjugates by likely sequestering or extruding the hydrophobic or hydrophilic dyes, respectively. Application of the compounds for imaging collagen-rich tissue in an animal model of inflammatory arthritis showed enhanced uptake of all four conjugates, which retained high collagen-binding affinity, in inflamed joints. Moreover, LS1001 and LS1107 improved the arthritic joint-to-background contrast, suggesting that reduced aggregation enhanced the clearance of these compounds from non-target tissues. Our results highlight a peptide-driven strategy to alter the aggregation states of molecular probes in aqueous solutions, irrespective of the water-solubilizing properties of the dye molecules. The interplay between the monomeric and aggregated forms of the conjugates using simple thiol-modifiers lends the peptide-driven approach to diverse applications, including the effective imaging of inflammatory arthritis joints.

Peptide-siRNA nanoparticles targeting NF-κB p50 mitigate experimental abdominal aortic aneurysm progression and rupture.

Abdominal aortic aneurysm (AAA) is a progressive vascular condition associated with high risk of mortality if left untreated. AAA is an inflammatory process with excessive local production of extracellular matrix degrading enzymes, leading to dilatation and rupture of the abdominal aorta. We posit that targeting NF-κB, a signaling pathway that controls inflammation, will halt AAA progression and prevent rupture. In an elastase-induced AAA model we observed that NF-κB activation increased progressively post-elastase perfusion. Unexpectedly, we found that AAA progression was marked by predominant nuclear accumulation of the NF-κB p50 subunit at the exclusion of p65. Using the amphipathic peptide p5RHH to form nanocomplexes with siRNA, we sought to mitigate AAA progression by knocking down the expression of different NF-κB subunits. We found that the administration of NF-κB p65 siRNA was only beneficial when given early (day 3 post-elastase perfusion) while p50 siRNA was still effective in mitigating elastase-induced AAA even when delivery was delayed until day 5. Additionally, systemic delivery of p50 siRNA, but not p65 siRNA decreased the risk of aortic rupture and sudden death in the transforming growth factor-beta blockade model of AAA. In both murine models, knockdown of NF-κB was accompanied by a significant decrease in leukocyte infiltrates, inflammatory cytokine release, inducible nitric oxide synthase expression, and cell apoptosis. These results suggest that the NF-κB p50 and p65 subunits contribute differentially at different stages of disease and the timing of in vivo siRNA delivery was of critical importance. The results also provide a rationale for selective targeting of p50 for more specific therapeutic intervention in the medical treatment of small AAA.

Proteomic Identification of Potential Target Proteins of Cathepsin W for Its Development as a Drug Target for Influenza.

Influenza A virus (IAV) coopts numerous host factors for efficient replication. The cysteine protease cathepsin W (CTSW) has been identified as one host factor required for IAV entry, specifically for the escape of IAVs from late endosomes. However, the substrate specificity of CTSW and the proviral mechanism are thus far unknown. Here, we show that intracellular but not secreted CTSW promotes viral entry. We reveal 79 potential direct and 31 potential indirect cellular target proteins of CTSW using the high-throughput proteomic approach terminal amine isotopic labeling of substrates (TAILS) and determine the cleavage motif shared by the substrates of CTSW. Subsequent integration with data from RNA interference (RNAi) screens for IAV host factors uncovers first insights into the proviral function of CTSW. Notably, CTSW-deficient mice display a 25% increase in survival and a delay in mortality compared to wild-type mice upon IAV infection. Altogether, these findings support the development of drugs targeting CTSW as novel host-directed antiviral therapies. IMPORTANCE Influenza viruses are respiratory pathogens and pose a constant threat to human health. Although antiviral drugs are available for influenza, the emergence and spread of drug-resistant viruses is cause for concern. Therefore, the development of new antivirals with lower chances of their target viruses acquiring resistance is urgently needed to reduce the high morbidity and mortality caused by influenza. Promising alternatives to drugs targeting viral proteins are those directed against host factors required for viral replication. The cysteine protease cathepsin W (CTSW) is an important host factor for IAV replication, and its proteolytic activity is required for fusion of viral and endosomal membranes. In this work, we identify a number of hitherto unknown CTSW substrates, providing new insights into virus-host interactions, and reveal that CTSW might also play a proviral role in an in vivo model. These results support the development of CTSW as a drug target for next-generation antivirals against influenza.

Caspase-1-driven neutrophil pyroptosis and its role in host susceptibility to Pseudomonas aeruginosa.

Multiple regulated neutrophil cell death programs contribute to host defense against infections. However, despite expressing all necessary inflammasome components, neutrophils are thought to be generally defective in Caspase-1-dependent pyroptosis. By screening different bacterial species, we found that several Pseudomonas aeruginosa (P. aeruginosa) strains trigger Caspase-1-dependent pyroptosis in human and murine neutrophils. Notably, deletion of Exotoxins U or S in P. aeruginosa enhanced neutrophil death to Caspase-1-dependent pyroptosis, suggesting that these exotoxins interfere with this pathway. Mechanistically, P. aeruginosa Flagellin activates the NLRC4 inflammasome, which supports Caspase-1-driven interleukin (IL)-1ß secretion and Gasdermin D (GSDMD)-dependent neutrophil pyroptosis. Furthermore, P. aeruginosa-induced GSDMD activation triggers Calcium-dependent and Peptidyl Arginine Deaminase-4-driven histone citrullination and translocation of neutrophil DNA into the cell cytosol without inducing extracellular Neutrophil Extracellular Traps. Finally, we show that neutrophil Caspase-1 contributes to IL-1ß production and susceptibility to pyroptosis-inducing P. aeruginosa strains in vivo. Overall, we demonstrate that neutrophils are not universally resistant for Caspase-1-dependent pyroptosis.

Safety Profile of Rapamycin Perfluorocarbon Nanoparticles for Preventing Cisplatin-Induced Kidney Injury.

Cancer treatment-induced toxicities may restrict maximal effective dosing for treatment and cancer survivors' quality of life. It is critical to develop novel strategies that mitigate treatment-induced toxicity without affecting the efficacy of anti-cancer therapies. Rapamycin is a macrolide with anti-cancer properties, but its clinical application has been hindered, partly by unfavorable bioavailability, pharmacokinetics, and side effects. As a result, significant efforts have been undertaken to develop a variety of nano-delivery systems for the effective and safe administration of rapamycin. While the efficacy of nanostructures carrying rapamycin has been studied intensively, the pharmacokinetics, biodistribution, and safety remain to be investigated. In this study, we demonstrate the potential for rapamycin perfluorocarbon (PFC) nanoparticles to mitigate cisplatin-induced acute kidney injury with a single preventative dose. Evaluations of pharmacokinetics and biodistribution suggest that the PFC nanoparticle delivery system improves rapamycin pharmacokinetics. The safety of rapamycin PFC nanoparticles was shown both in vitro and in vivo. After a single dose, no disturbance was observed in blood tests or cardiac functional evaluations. Repeated dosing of rapamycin PFC nanoparticles did not affect overall spleen T cell proliferation and responses to stimulation, although it significantly decreased the number of Foxp3+CD4+ T cells and NK1.1+ cells were observed.

Non-invasive monitoring of arthritis treatment response via targeting of tyrosine-phosphorylated annexin A2 in chondrocytes.

BACKGROUND: The development and optimization of therapies for rheumatoid arthritis (RA) is currently hindered by a lack of methods for early non-invasive monitoring of treatment response. Annexin A2, an inflammation-associated protein whose presence and phosphorylation levels are upregulated in RA, represents a potential molecular target for tracking RA treatment response. METHODS: LS301, a near-infrared dye-peptide conjugate that selectively targets tyrosine 23-phosphorylated annexin A2 (pANXA2), was evaluated for its utility in monitoring disease progression, remission, and early response to drug treatment in mouse models of RA by fluorescence imaging. The intraarticular distribution and localization of LS301 relative to pANXA2 was determined by histological and immunohistochemical methods. RESULTS: In mouse models of spontaneous and serum transfer-induced inflammatory arthritis, intravenously administered LS301 showed selective accumulation in regions of joint pathology including paws, ankles, and knees with positive correlation between fluorescent signal and disease severity by clinical scoring. Whole-body near-infrared imaging with LS301 allowed tracking of spontaneous disease remission and the therapeutic response after dexamethasone treatment. Histological analysis showed preferential accumulation of LS301 within the chondrocytes and articular cartilage in arthritic mice, and colocalization was observed between LS301 and pANXA2 in the joint tissue. CONCLUSIONS: We demonstrate that fluorescence imaging with LS301 can be used to monitor the progression, remission, and early response to drug treatment in mouse models of RA. Given the ease of detecting LS301 with portable optical imaging devices, the agent may become a useful early treatment response reporter for arthritis diagnosis and drug evaluation.

A genome-engineered bioartificial implant for autoregulated anticytokine drug delivery.

Biologic drug therapies are increasingly used for inflammatory diseases such as rheumatoid arthritis but may cause significant adverse effects when delivered continuously at high doses. We used CRISPR-Cas9 genome editing of iPSCs to create a synthetic gene circuit that senses changing levels of endogenous inflammatory cytokines to trigger a proportional therapeutic response. Cells were engineered into cartilaginous constructs that showed rapid activation and recovery in response to inflammation in vitro or in vivo. In the murine K/BxN model of inflammatory arthritis, bioengineered implants significantly mitigated disease severity as measured by joint pain, structural damage, and systemic and local inflammation. Therapeutic implants completely prevented increased pain sensitivity and bone erosions, a feat not achievable by current clinically available disease-modifying drugs. Combination tissue engineering and synthetic biology promises a range of potential applications for treating chronic diseases via custom-designed cells that express therapeutic transgenes in response to dynamically changing biological signals.

Amelioration of Posttraumatic Osteoarthritis in Mice Using Intraarticular Silencing of Periostin via Nanoparticle-Based Small Interfering RNA.

OBJECTIVE: Recent evidence delineates an emerging role of periostin in osteoarthritis (OA), since its expression after knee injury is detrimental to the articular cartilage. We undertook this study to examine whether intraarticular (IA) knockdown of periostin would ameliorate posttraumatic OA in a murine model. METHODS: Posttraumatic OA was induced in 10-week-old male C57BL/6J mice (n = 24) by destabilization of the medial meniscus (DMM), and mice were analyzed 8 weeks after surgery. Periostin expression was inhibited by small interfering RNA (siRNA) delivered IA using a novel peptide-nucleotide polyplex. Following histologic assessment of the mouse knee cartilage, the extent of cartilage degeneration was determined using Osteoarthritis Research Society International (OARSI) cartilage damage score, and severity of synovitis was also assessed. Bone changes were measured using micro-computed tomography. The effect and mechanism of periostin silencing were investigated in human chondrocytes that had been stimulated with interleukin-1ß (IL-1ß) with or without the IκB kinase 2 inhibitor SC-514. RESULTS: Periostin expression in mice with posttraumatic OA was significantly abolished using IA delivery of a peptide-siRNA nanoplatform. OARSI cartilage damage scores were significantly lower in mice receiving periostin siRNA (mean ± SEM 10.94 ± 0.66) compared to untreated mice (22.38 ± 1.30) and mice treated with scrambled siRNA (22.69 ± 0.87) (each P = 0.002). No differences in the severity of synovitis were observed. Subchondral bone sclerosis, bone volume/total volume, volumetric bone mineral density, and heterotopic ossification were significantly lower in mice that had received periostin siRNA treatment. Immunostaining of cartilage revealed that periostin knockdown reduced the intensity of DMM-induced matrix metalloproteinase 13 (MMP-13) expression and also diminished the phosphorylation of p65 and immunoreactivity of the aggrecan neoepitope DIPEN. Periostin knockdown also suppressed IL-1ß-induced MMP-13 and ADAMTS-4 expression in chondrocytes. Mechanistically, periostin-induced MMP-13 expression was abrogated by SC-514, demonstrating a link between periostin and NF-κB. CONCLUSION: IA delivery of the periostin-siRNA nanocomplex represents a promising clinical approach to mitigate the severity of joint degeneration in OA. Our findings may thus provide an unequivocal scientific rationale for longitudinal studies of this approach. Utilizing a cartilage-specific gene-knockout strategy will further illuminate the functional role of periostin in OA.

Size-Dependent Effective Diffusivity in Healthy Human and Porcine Joint Synovium.

Intra-articular drug delivery can be effective in targeting a diseased joint but is hampered by rapid clearance times from the diarthrodial joint. The synovium is a multi-layered tissue that surrounds the diarthrodial joint and governs molecular transport into and out of the joint. No models of drug clearance through synovium exist to quantify diffusivity across solutes, tissue type and disease pathology. We previously have developed a finite element model of synovium as a porous, permeable, fluid-filled tissue and used an inverse method to determine urea's effective diffusivity (Deff) in de-vitalized synovium explants.22 Here we apply this method to determine Deff from unsteady diffusive transport of model solutes and confirm the role of molecular weight in solute transport. As molecular weight increased, Deff decreased in both human and porcine tissues, with similar behavior across the two species. Unsteady transport was well-described by a single exponential transient decay in concentration, yielding solute half-lives (t1/2) that compared favorably with the Deff determined from the finite element model fit. Determined values for Deff parallel prior observations of size-dependent in vivo drug clearance and provide an intrinsic parameter with greater ability to resolve size-dependence in vitro. Thus, this work forms the basis for understanding the influence of size on drug transport in synovium and can guide future studies to elucidate the role of charge and tissue pathology on the transport of therapeutics in healthy and pathological human synovium.

Adipose tissue is a critical regulator of osteoarthritis.

Osteoarthritis (OA), the leading cause of pain and disability worldwide, disproportionally affects individuals with obesity. The mechanisms by which obesity leads to the onset and progression of OA are unclear due to the complex interactions among the metabolic, biomechanical, and inflammatory factors that accompany increased adiposity. We used a murine preclinical model of lipodystrophy (LD) to examine the direct contribution of adipose tissue to OA. Knee joints of LD mice were protected from spontaneous or posttraumatic OA, on either a chow or high-fat diet, despite similar body weight and the presence of systemic inflammation. These findings indicate that adipose tissue itself plays a critical role in the pathophysiology of OA. Susceptibility to posttraumatic OA was reintroduced into LD mice using implantation of a small adipose tissue depot derived from wild-type animals or mouse embryonic fibroblasts that undergo spontaneous adipogenesis, implicating paracrine signaling from fat, rather than body weight, as a mediator of joint degeneration.

Induction of WNT16 via Peptide-mRNA Nanoparticle-Based Delivery Maintains Cartilage Homeostasis.

Osteoarthritis (OA) is a progressive joint disease that causes significant disability and pain and for which there are limited treatment options. We posit that delivery of anabolic factors that protect and maintain cartilage homeostasis will halt or retard OA progression. We employ a peptide-based nanoplatform to deliver Wingless and the name Int-1 (WNT) 16 messenger RNA (mRNA) to human cartilage explants. The peptide forms a self-assembled nanocomplex of approximately 65 nm in size when incubated with WNT16 mRNA. The complex is further stabilized with hyaluronic acid (HA) for enhanced cellular uptake. Delivery of peptide-WNT16 mRNA nanocomplex to human cartilage explants antagonizes canonical ß-catenin/WNT3a signaling, leading to increased lubricin production and decreased chondrocyte apoptosis. This is a proof-of-concept study showing that mRNA can be efficiently delivered to articular cartilage, an avascular tissue that is poorly accessible even when drugs are intra-articularly (IA) administered. The ability to accommodate a wide range of oligonucleotides suggests that this platform may find use in a broad range of clinical applications.

Combined Experimental Approach and Finite Element Modeling of Small Molecule Transport Through Joint Synovium to Measure Effective Diffusivity.

Trans-synovial solute transport plays a critical role in the clearance of intra-articularly (IA) delivered drugs. In this study, we present a computational finite element model (FEM) of solute transport through the synovium validated by experiments on synovial explants. Unsteady diffusion of urea, a small uncharged molecule, was measured through devitalized porcine and human synovium using custom-built diffusion chambers. A multiphasic computational model was constructed and optimized with the experimental data to extract effective diffusivity for urea within the synovium. A monotonic decrease in urea concentration was observed in the donor bath over time, with an effective diffusivity found to be an order of magnitude lower in synovium versus that measured in free solution. Parametric studies incorporating an intimal cell layer with varying thickness and varying effective diffusivities were performed, revealing a dependence of drug clearance kinetics on both parameters. The findings of this study indicate that the synovial matrix impedes urea solute transport out of the joint with little retention of the solute in the matrix.

Nanotherapy Targeting NF-κB Attenuates Acute Pain After Joint Injury.

Inflammation after joint injury leads to joint responses that result in eventual osteoarthritis development. Blockade of inflammation, by suppressing NF-κB expression, has been shown to reduce joint injury-induced chondrocyte apoptosis and reactive synovitis in vivo. Herein, we demonstrate that the suppression of NF-κB p65 expression also significantly mitigates the acute pain sensitivity induced by mechanical injury to the joint. These results suggest that early intervention with anti-NF-κB nanotherapy mitigates both structural and pain-related outcomes, which in turn may impact the progression of post-traumatic osteoarthritis.