Osteoarthritis (OA) is a progressive disease that causes significant pain and suffering and for which there are limited medical treatment options. Although effective disease-modifying OA drugs (DMOADs) are critically needed, none has successfully emerged in the clinic [1
]. The reasons for this failure are multifold, including the fact that primary OA is a complex, multifactorial disease with incompletely understood pathogenesis [2
Much interest has recently focused on the role of inflammation and inflammatory cytokines in the pathogenesis of OA [3
]. It is posited that an imbalance between inflammatory (catabolic) and anabolic factors leads to cartilage degeneration, a hallmark of OA. Thus, blockade of catabolic cytokines such as tumor necrosis factor alpha (TNF-α) and interleukin 1 beta (IL-1β) has gained attention as potential therapy. Indeed, intra-articular (IA) administration of IL-1 receptor antagonist (IL-1ra) exhibits disease-modifying effects in a rodent model of OA [3
]. However, in clinical studies, the administration of commercially available IL-1ra (Anakinra, Kineret®
) had no therapeutic effect in established knee OA [4
] and so far, only offers short-term improvement in pain and function if administered within the first month following knee injury (NCT00332254) [5
]. Thus, novel approaches that promote and/or maintain joint homeostasis to mitigate OA progression are highly desirable.
Wingless and the name Int-1 (WNT) comprises an evolutionarily conserved family of glycoproteins that signal through different pathways. Beta (β)-catenin-dependent (or canonical) WNT signaling leads to the nuclear translocation of β-catenin and transcriptional activation of target genes that regulate many crucial aspects of cell fate and function during embryogenesis [6
] Non-canonical, β-catenin-independent WNT signaling pathways and function on the other hand are less well understood [7
]. The WNT signaling pathway has been implicated in the pathogenesis of OA [8
]. WNT3a-dependent activation of the canonical β-catenin-dependent WNT signaling pathway stimulates catabolic activities, resulting in an OA-like phenotype [9
]. In contrast, WNT16-deficiency leads to more severe OA in a rodent model, with increased chondrocyte apoptosis and decreased expression of lubricin [11
], an essential joint lubricant [12
]. In vivo injection of recombinant WNT16 in a Xenopus assay buffers the activation of canonical WNT3a [11
]. Thus, we posit that overexpression of WNT16 antagonizes canonical WNT signaling to halt cartilage loss and mitigate the progression of OA.
The delivery of ribonucleic acids (RNA), small interfering RNA (siRNA), messenger RNA (mRNA), and microRNA (miRNA) into cells has been attempted using a variety of platforms, including lipid-based nanocarriers [13
]. However, once taken up inside the cells, these particles are often trapped inside endosomes, with slow release of the RNA structures. Herein, we employ a cytolytic peptide, melittin, that has been modified to significantly attenuate its pore-forming capacity while maintaining its ability to insert into membrane bilayers [14
], as well as improving its interactions with oligonucleotides [17
]. Our previous work has shown that the modified peptide, called p5RHH, forms a self-assembled nanostructure that facilitates endosomal escape and rapidly delivers siRNA to the cytoplasm, to down-modulate specific gene expression in vitro and in vivo [17
]. To our knowledge, the delivery of mRNA using p5RHH for WNT16 overexpression has not been reported. Herein, we present a proof-of-concept study showing that WNT16 mRNA can be efficiently delivered to articular cartilage and that its overexpression modulates cartilage homeostasis ex vivo.
The challenge of delivering nanotherapeutics to cartilage in effective doses in vivo is well known [22
]. Critical barriers include inefficient delivery to the chondrocytes residing in the avascular cartilage tissue and the dense ECM that excludes large particles from entering the deeper layers in order to deliver the therapeutic cargo. We have employed the amphipathic cationic peptide p5RHH that is a modified version of the natural peptide melittin, which rapidly forms a biocompatible and stable nanocomplex upon mixing of the peptide and nucleotide components [17
]. The mechanism by which the modified melittin-derived peptide forms self-assembled nanostructures has been previously described [17
]. In brief, modifications to p5RHH, with the addition of histidine and arginine moieties, enhance electrostatic interactions, permitting formation of noncovalent hydrogen bonds between oligonucleotides and the peptide [24
]. The complex protects the RNA from degradation and once taken up inside the cell, the peptide can facilitate endosomal escape and coordinated release of nucleic acid structures into the cytoplasm [18
]. Herein we show for the first time that p5RHH can also complex with mRNA structures (up to ~1100 nt) to form stable NPs of ~65 nm, small enough to penetrate cartilage for delivery and translation of the mRNA. An HA coating further enhances cellular uptake without retarding the ability of the NPs to deeply penetrate human cartilage. The versatility of the platform to incorporate short and long nucleotide structures significantly broadens the range of clinical applications for this technology.
Approaches to OA treatment have recently shifted toward anabolic pathways that promote cartilage repair and homeostasis. Fibroblast growth factors (FGFs) are important regulators of cartilage development and homeostasis [25
]. IA injection of FGF-18 in a rat meniscal tear model induces new cartilage formation [26
]. Sprifermin (AS902330), a recombinant form of human FGF-18 injected IA in patients with advanced or end-stage OA shows early promise; however, durability of response at two years was uncertain [27
]. Likewise, excessive (β-catenin-dependent) canonical WNT activation leads to cartilage breakdown and increases risk of OA [8
]. A small molecule inhibitor of the WNT pathway (SM04690) shows protective and regenerative effects in an OA animal model [30
] and has the potential to be disease modifying in knee OA; however, long-term effects are still unknown (ongoing trials NCT03727022). In the present study, we show that overexpression of WNT16 suppresses canonical β-catenin/WNT3a signaling. We envision that the p5RHH platform, by its ability to accommodate a wide range of oligonucleotide structures (siRNA, mRNA, and others) without the need for backbone or end-piece alterations, will enable the delivery of a “cocktail” of factors (anti-inflammatory and anabolic) that should control cartilage loss and maintain homeostasis, mitigating OA progression.
In conclusion, we have shown that melittin-derived p5RHH peptide self-assembles with mRNA to form stable nanostructures that deeply penetrate cartilage for efficient expression of WNT16 ex vivo. These results hold promise that this approach will overcome the shortcoming of slow RNA release encountered by lipid-based NPs [13
]. In future studies, we will test the effectiveness of WNT16 overexpression in maintaining cartilage homeostasis in vivo.
4. Materials and Methods
4.1. Preparation of HA-Coated p5RHH-mRNA NPs
Ten milligrams of sodium hyaluronic acid (part# HA1M-1, Lifecore Biomedical, Chaska, MN, USA) was dissolved in 1 mL HBSS with Ca++/Mg++ by sonification for 60 min and ultracentrifuged at 90,000g for 40 m. The supernatant was aliquoted and stored at −80 °C until use. p5RHH peptide (VLTTGLPALISWIRRRHRRHC, provided by Genscript, Piscataway, NJ, USA) was dissolved at 10 mM in DNase-, RNase-, and protease-free sterile purified water (Cellgro at Corning, Tewksbury, MA, USA) and stored in 10 μL aliquots at −80 °C until use.
The p5RHH-Cy3-labeled siRNA NPs were prepared as previously described [20
]. The p5RHH-mRNA NPs were prepared as follows: 1 μg of Cy5-eGFP mRNA (TriLink Biotechnologies, San Diego, CA, USA), or 1, 2, or 4 μg of WNT16 mRNA (TriLink Biotechnologies) in HBSS with Ca++/Mg++ were added to 10 μmol of p5RHH peptide (in a total volume of 100 μL), mixed well, and incubated at 37 °C for 40 min. After incubation, 5 μL of HA was added to the self-assembled NPs and placed on ice for 5 min. This mixture was diluted into a total volume of 500 µL with culture medium for in vitro transfection. NP size was measured by TEM and zeta potential by DLS. To calculate the actual spherical volume of the NPs from their “flattened” shape acquired during the TEM drying process, we used the formula for the volume of a right cylinder V = πr2
h, where V = volume, r = radius, and h (height). Height was assumed to be 1/5th of their flattened diameter. The radius for a sphere of the same volume as the right cylinder was calculated from the formula V = 4/3πr3
4.2. Human Cartilage Explant Culture
Human cartilage explants were obtained from patients, through a protocol (ID # 201104119 approved 01/10/2019) approved by the Washington University in St. Louis Institutional Review Board (IRB), at the time of total knee arthroplasty. All study participants provided written informed consent. The de-identified cartilage tissues were washed several times with HBSS containing antibiotics, then incubated in Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F12) (1:1) medium containing 10% fetal bovine serum (FBS), penicillin/streptomycin (100 U/0.1 mg/mL), amphotericin B (0.25 µg/mL), and ciproflaxin (10 μg/mL) in a 6-well plate at 37 °C and 5% CO2 for 2–3 days. The explants were then transferred to a 96-well plate and subsequently exposed to the aforementioned p5RHH-mRNA NPs for 48 h. The excess NPs were washed off after 48 h incubation. The cartilage explants were harvested and then embedded in Tissue-Tek optimal cutting temperature (O.C.T.) compound (Sakura Finetek, Torrance, CA, USA) and sectioned for analysis.
4.3. In Vitro NP Uptake by Bone-Marrow-Derived Macrophages (BMMϕ)
All animal experiments were performed in compliance with guidelines and protocols approved by the Division of Comparative Medicine at Washington University in St. Louis. The animal protocol (Animal Welfare Assurance # A-3381-01, approved 02/28/2019) is subjected to annual review and approval by The Animal Studies Committee of Washington University. Bone marrow from C57BL/6 WT mice (Cat# 000664, Jackson Laboratory, Bar Harbor, ME, USA) was cultured in complete RPMI-1640 medium with 10% fetal bovine serum containing recombinant murine granulocyte-macrophage colony stimulating factor (GM-CSF) (10 μg/mL, Cat# PMC2015, Thermo Fisher Scientific, Waltham, MA, USA) for 7 days at 37 °C. Cultured cells were plated in 12-well plates at 0.5 × 106 cells/well overnight. The cells were starved for 30 min prior to stimulation with 10 µg/mL lipopolysaccharide (LPS) (Cat# L2762, Sigma-Aldrich, St. Louis, MO, USA) for 15 min. The cells were subsequently cultured in Opti-Minimum Essential Medium (MEM) (Thermo Fischer Scientific) containing HA-coated Cy3-labeled NPs or uncoated Cy3-labeled NPs at 37 °C for the indicated times. The cells were collected with ethylenediaminetetraacetic acid (EDTA) solution (1:10 dilution with phosphate buffered saline (PBS), spun down, resuspended in Flow Cytometry Staining (FACS) buffer, and analyzed by flow cytometry. For confocal analysis, cells were cultured and fixed with 4% paraformaldehyde in PBS, permeabilized with 0.1% Triton X-100/PBS, and blocked with 8% BSA. F-actin (Cat# T7471, 1:200 dilution, Invitrogen at Thermo Fischer Scientific) was added and the cells mounted with VECTASHIELD containing 4’, 6-diamidino-2-phenylindole (DAPI) (Cat# D3571 Molecular Probes at Thermo Fischer Scientific). The images were captured by a ZEISS LSM 880 confocal laser scanning microscope.
4.4. TUNEL Assay
The apoptotic assay was performed to identify DNA fragmentation associated with terminal deoxynucleotidyl transferase-mediated (dUTP) nick end labeling (TUNEL). Detection of apoptotic cells was performed on non-fixed frozen cartilage sections using an in situ cell death detection kit (Cat#: 11–684-795–910, Roche at Millipore Sigma, St. Louis, MO, USA). In brief, the cartilage sections were rinsed with PBS, then permeabilized with 0.5% TWEEN-20/PBS for 15 min, and blocked with 8% BSA solution. Freshly prepared TUNEL reaction mixture, according to the manufacturer’s protocol, was applied to the sections for 1 h at 37 °C, rinsed 3 to 5 times with PBS, and probed with COL2 antibody (1:200 dilution, generously provided by L. J. Sandell and M. F. Rai, Washington University, St. Louis), followed by tetramethylrhodamine (TRITC)-conjugated anti-rat secondary antibody (1:100 dilution, Cat# 712-295-153, Jackson Immuno Research, West Grove, PA, USA), and counterstained with DAPI (1:1000 dilution, Vector Laboratories, Burlingame, CA, USA). The sections were mounted with VECTASHIELD mounting medium with DAPI (Cat#: H-1200, Vector Laboratories). The TUNEL+ cells were enumerated across non-overlapped fields. Data represent 6–8 sections per cartilage and 4–6 patients per treatment.
4.5. Confocal Microscopy
After incubation with eGFP mRNA NPs for 48 h, the cartilage explants were harvested and sectioned. Frozen sections (9 μm) were rinsed, fixed, and covered with VECTASHIELD mounting medium with DAPI (1:1000, Vector Laboratories) at room temperature. The images were acquired with a ZEISS LSM 880 confocal laser scanning microscope—15 to 20 cells per section and 3–4 sections were analyzed with the software ZEN. The data was presented as the mean fluorescent intensity per cell.
Formalin-fixed, O.C.T-embedded 9 μm sections of human cartilage explants were probed with WNT16 (1:100 dilution, Cat# LS-A9629, LifeSpan Biosciences, Seattle, WA, USA), WNT3A (1:100 dilution, Cat# OABF00803, Aviva Systems Biology, San Diego, CA, USA), Lubricin (1:200 dilution, Cat# 55463, MP Biomedicals, Irvine, CA, USA), or β-catenin (1:100, Cat# ab16051, Abcam, Cambridge, MA, USA) at room temperature for 1 h. After washing, the sections were incubated with the corresponding horse radish peroxidase (HRP)-conjugated secondary antibodies for 1 h. Data presented were derived from 6–8 cartilage sections. The pattern was confirmed on 4–6 independent human cartilage explants.
Comparisons between two groups were performed by Student’s t-test, and between multiple groups (≥3) by one-way ANOVA followed by Bonferroni’s correction for multiple comparisons. Differences between experimental groups at a p value of <0.05 were considered significant.