TitleTargeting tie-2 receptor with rebastinib (DCC-2036) for angiogenesis inhibition in early-stage arthritis: enhanced efficacy through liposomal sustained release

TitleTargeting tie-2 receptor with rebastinib (DCC-2036) for angiogenesis inhibition in early-stage arthritis: enhanced efficacy through liposomal sustained release | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article TitleTargeting tie-2 receptor with rebastinib (DCC-2036) for angiogenesis inhibition in early-stage arthritis: enhanced efficacy through liposomal sustained release Minuk Jeong, Heung-Myong Woo, Jang-Hyuk Yun, Junhyung Kim This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7380099/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 02 Dec, 2025 Read the published version in Inflammopharmacology → Version 1 posted 4 You are reading this latest preprint version Abstract The prevalence of arthritis continues to increase, which has driven research on new therapeutic approaches. However, existing treatments often have limitations. Angiogenesis and pathological changes in the synovium are the key contributors to the early development of arthritis. Rebastinib, a tie-2 receptor inhibitor, blocks the activation of tie2-expressing macrophages, which are involved in angiogenesis. Although previous studies have highlighted the importance of angiogenesis in early arthritis, few have focused on targeting the tie-2 receptor to slow disease progression. In this study, we evaluated the effects of rebastinib encapsulated in pH-dependent liposomes in a rabbit model of surgically induced arthritis. Additionally, we investigated the efficacy of a pH-dependent liposomal formulation, developed using microfluidic technology for sustained drug release. The results demonstrated that rebastinib-loaded pH-dependent liposomes were stable and provided controlled release and rebastinib effectively inhibited the progression of early stage arthritis in this model. Statistical analyses were performed using SPSS software (IBM Corp., Armonk, NY, USA), and significance was assessed using one-way ANOVA. In conclusion, rebastinib encapsulated in pH-dependent liposomes holds promise as a potential therapeutic strategy for the treatment of early arthritis, offering both stability and efficacy in disease suppression. Early-stage arthritis inflammation liposomes drug delivery tie-2 receptor inhibitor angiogenesis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Osteoarthritis (OA), also known as osteoarthrosis or degenerative joint disease, is a multifaceted disorder that affects the entire joint structure, encompassing ,cartilage, synovium, subchondral bone, and associated tissues (Anderson et al. 2020 ). Despite the hallmark features of deterioration and functional impairment of articular cartilage, the pathogenesis of OA is underpinned by a complex interplay of biochemical, cellular, and mechanical factors. However, the primary mechanisms triggering OA onset remain unclear (Nelson et al. 2014 ). Current therapeutic approaches focus predominantly on alleviating symptoms, with no existing interventions capable of halting or reversing the underlying degenerative processes (Su et al. 2020 ) Recent investigations have underscored the role of angiogenesis as a key driver of OA progression, mediated by dynamic interactions between endothelial cells, pericytes, and the extracellular matrix (Ribatti et al. 2011 ; Su et al. 2021 ). The Angiopoietin (Ang)/Tie-2 signaling pathway has been identified as a critical regulator of angiogenesis, governing vascular remodeling, and endothelial barrier stability (Maisonpierre et al. 1997 ). Rebastinib (DCC-2036), a highly selective and potent Tie-2 receptor tyrosine kinase inhibitor, exhibits a unique mechanism of action by stabilizing Tie-2 in its inactive conformation through an allosteric "switch control pocket." Extensive pharmacokinetic and safety evaluations have reported its efficacy with a half-life of approximately 10 h, supporting its potential utility in therapeutic applications (Cortes et al. 2016 ; Feng et al. 2023 ; Harney et al. 2017 ) Liposomes, nanoscale vesicular drug carriers composed of phospholipid bilayers, have significant promise in OA management (Goldberg and Klein, 2012 ; Sivan et al. 2010 ). Beyond their ability to serve as boundary lubricants and reduce cartilage friction and wear through the hydration of their phospholipid head groups, liposomes also offer the advantage of controlled and sustained drug release, enabling the localized delivery of therapeutic agents. These attributes have made liposomes promising platforms for the development of disease-modifying OA treatments (Ji et al. 2019 ). Microfluidic technology has revolutionized liposome fabrication by enabling precise control of particle size, uniformity, and encapsulation efficiency. This method produces highly reproducible nanoformulations with tailored physicochemical properties, making it an ideal platform for the encapsulation of therapeutic agents, including liposome-based drug delivery systems (Chiesa et al. 2020 ; Wang et al. 2018 ). Building on these advancements, this study posits that the encapsulation of rebastinib into liposomes using microfluidics offers a novel therapeutic approach for early stage OA. By targeting the Tie-2 signaling pathway, this strategy aims to mitigate angiogenesis-driven joint degeneration. To evaluate this hypothesis, we encapsulated rebastinib in liposomes (Lipo@Reba) and compared the efficacy of free and encapsulated Rebastinib in a surgically induced anterior cruciate ligament transection (ACLT) rabbit model of OA (Fig. 1 ). Therapeutic outcomes were assessed using imaging modalities, including radiography and arthroscopy, coupled with detailed histological analyses, to elucidate the effects of the drug on joint pathology. Materials and methods Unless otherwise specified, all reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA). Lipid reagents were purchased from Avanti Polar Lipids (Alabaster, AL). Dialysis membrane tubing (MWCO 3.5 kDa) was purchased from Thermo Fisher Scientific (Waltham, MA, USA). Rebastinib (DCC-2036) was purchased from MedChemExpress (Monmouth Junction, NJ, USA). Production of liposomes using a microfluidics system Liposomes were synthesized using an automated dolomite microfluidic system (Dolomite, Royston, UK) equipped with a 5-input chip, allowing precise mixing of the lipid and aqueous phases. The lipid solution (10 mM) consisted of dioleoylphosphatidylethanolamine (DOPE), cholesteryl hemisuccinate (CHEMS), and PEG-2000 at a DOPE: CHEMS: PEG-2000 molar ratio of 63.3:31.6:5, with PEG-2000 accounting for 5% of the total molar composition. To ensure homogeneous distribution, rebastinib and lipids were dissolved in a chloroform:ethanol (1:4, v/v) solvent mixture. The aqueous phase was prepared as a 1× phosphate-buffered saline (PBS) solution adjusted to pH 7.4. The lipid and aqueous phases were mixed in a microfluidic device at a flow rate ratio of 25:75 (lipid:PBS) to promote the formation of stable liposomes. Following liposome formation, the dispersion was purified using dialysis tubing (MWCO, 3.5 kDa) in a PBS reservoir. Dialysis was performed at a 25-fold dilution rate for 12 h, with PBS refreshed every 4 h to remove residual solvents and unencapsulated materials. Mild agitation during the process prevented aggregation and maintained a uniform dispersion. Dynamic light scattering Liposome size was determined using dynamic light scattering (Malvern Zetasizer Nano-ZSP, Malvern Instruments Ltd., UK). Measurements were conducted at 25°C, with flow rate ratios of 1:2, 1:3, and 1:4, under each condition (n = 3). Transmission electron microscopy (TEM) Liposome morphology was visualized using a transmission electron microscope (TEM, JEM-2100F, Japan) operated at 200 kV. Samples were prepared by negative staining with 1% phosphotungstic acid and air-dried before imaging. High performance liquid chromatography (HPLC) Rebastinib quantification was conducted using reverse-phase HPLC (Agilent 1260 series, Santa Clara, CA, USA) equipped with a C-18 column (4.6 × 150 mm, 3.5 µm). Analyses were performed under isocratic conditions at a flow rate of 1.0 mL/min, detection at 254 nm, and a runtime of 30 min. A calibration curve was established in the range of 0.01–0.5 mg/mL. Encapsulation efficiency of rebastinib Lipid solutions were prepared to achieve rebastinib concentrations of 5 and 10 mg/mL, and liposomes were fabricated using a previously described method using a microfluidic device. Unencapsulated drugs were removed by diluting the final samples and liposomes were completely disrupted using a sonicator. The disrupted liposome solution was diluted with acetonitrile and samples were analyzed for rebastinib concentration using reverse-phase HPLC. Encapsulation efficiency was calculated according to the following equation: Encapsulation efficiency = \(\:\frac{\varvec{E}\varvec{n}\varvec{t}\varvec{r}\varvec{a}\varvec{p}\varvec{p}\varvec{e}\varvec{d}\:\varvec{d}\varvec{r}\varvec{u}\varvec{g}\left(\varvec{w}\varvec{t}\right)}{\varvec{T}\varvec{o}\varvec{t}\varvec{a}\varvec{l}\:\varvec{d}\varvec{r}\varvec{u}\varvec{g}\left(\varvec{w}\varvec{t}\right)}\:\) x 100% In vitro drug release Drug release kinetics were assessed by incubating rebastinib-loaded liposomes in 700 mL of PBS buffer sink (pH 7.4) at 37°C after removing unencapsulated drug. Liposomes were placed in a dialysis cassette and shaken at 100 rpm. Samples (0.6 mL) were collected at time intervals of 4, 8, 24, 48, and 96 h. The concentration of rebastinib was quantified using HPLC. Animals New Zealand White rabbits, aged 16 weeks and weighing 2.5–2.9 kg, were used in this study. The rabbits were purchased from an experimental animal supplier (Narabiotech, Seoul, Republic of Korea) and housed in a regularly ventilated temperature-controlled environment under a 12-hour light/12-hour dark cycle. The rabbits were acclimated for a period after arrival. All experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of Kangwon National University. Safety test of rebastinib The drug concentration used for compartmental injections was based on a previous study that used a selective small-molecule Tie2 inhibitor for subretinal injections (Liu, Lin et al. 2008). Twelve New Zealand White rabbits were randomly divided into two groups and injected with 0.4 mL of PBS or 0.4 mL of 1.33 mg rebastinib (in 0.4 mL) into the stifle joint. Injections were repeated two weeks later. The rabbits were allowed to move freely after the injections, and all behavior and conditions were monitored. The rabbits were sacrificed six weeks later under anesthesia, followed by drug administration. After euthanasia, body weight was recorded, and approximately 2 mL of blood was collected from the ear vein for complete blood cell count and blood chemistry analysis. Cartilage samples were collected from the medial tibial plateau and synovial samples were obtained from the suprapatellar pouch. The therapeutic effect in a rabbit ACLT model Unilateral ACLT was performed on twenty-four New Zealand White rabbits. Before and after surgery, animals received subcutaneous injections of enrofloxacin (5 mg/kg) and tramadol (4 mg/kg). Deep anesthesia was induced via intramuscular (IM) injection of ketamine (35 mg/kg) and xylazine (5 mg/kg) and maintained with isoflurane (1–3.5%) administered via endotracheal intubation. ACLT was performed by a skilled veterinarian using the method described in a previous study (Levillain, Boulocher et al. 2015). Briefly, a longitudinal skin incision was made on the right knee joint to expose the capsule. The joint capsule was then opened through a longitudinal incision between the medial collateral and patellar ligaments. Complete rupture of the anterior cruciate ligament was confirmed by using the anterior drawer sign (manual horizontal dislocation) before closing the articular capsule. The operated leg was not immobilized and the rabbits were allowed to move freely in their individual cages after surgery. Three days after surgery, the rabbits were randomly divided into four groups (n = 6). The three groups received intra-articular injections of the following formulations (0.4 mL) once every two weeks: PBS, rebastinib solution (3.33 mg/mL), and rebastinib in liposomes (Lipo@Reba, 6.27 mM). Finally, the patients were divided into the following groups: ACLT group (surgery only, no intra-articular treatment), PBS group (PBS intra-articular injection), Reba group (rebastinib intra-articular injection), and Lipo@Reba group (Lipo@Reba intra-articular injection). Six weeks after surgery, radiographic imaging and arthroscopy of the joints were performed. The rabbits were sacrificed after six weeks of treatment with anesthesia, followed by KCl. After sacrifice, the right stifle joints and organs were collected from each rabbit for toxicity assessment. Arthroscopic examination Intra-articular examination using arthroscopy was performed following the methods described in previous studies (Peters, Wilke et al. 2018). The rabbits were positioned in dorsal recumbency and the limb to be examined was placed ventrally toward the examiner. A 5 cm radius around the stifle joint was shaved to prepare the site. The limb was fully extended and a 20-gauge 1-inch needle was inserted into the joint space, 2 mm proximal to the tibial plateau and immediately medial to the patellar ligament. The joint capsule was then distended with 3 mL lactated Ringer’s solution. A stab incision was made at the same site using a No. 11 surgical blade positioned one-third of the distance from the tibial crest to the patellar base and immediately medial to the patellar ligament. A 1.9 mm arthroscope sheath with a blunt obturator (Arthrex Inc., Florida, USA) was inserted into the joint, directing the obturator proximally and axially until it rested between the patella and femur. While maintaining the obturator in position, a 20-gauge, 1-inch needle was inserted for fluid egress, positioned in the suprapatellar pouch medially or laterally, 3–4 mm proximal to the superior patellar border. The joint was visualized using a 1.9 x 58 mm, 30° forward-oblique arthroscope (Arthrex Inc., Florida, USA). Joint distension was maintained by pressurizing 2 L of lactated Ringer’s solution to 30–50 mm Hg, which was controlled using arthroscopic equipment. The trochlear groove was centered on the monitor, and the stifle joint was gradually flexed to an angle of 30–45° with the scope directed distally for a comprehensive examination. The medial and lateral sides of the joint capsule in the suprapatellar pouch were imaged and scored by two blinded observers using the OARSI scoring system by two blinded observers (Cook, Kuroki et al. 2010). In addition, the femorotibial joint surfaces were imaged to indirectly observe the cartilage and meniscus. Radiographic assessment Six weeks post-surgery, rabbit knee joints were scanned using an X-ray imager (DRGEM Co., Korea). The articular surface and joint space widths were analyzed across the different groups. Six weeks post-surgery, the knee joints were harvested and assessed using high-resolution micro-CT (Quantum GX2 MicroCT, PerkinElmer, Inc., USA) with an isometric resolution of 76 µm. The scanning parameters included a voltage of 90 kV and a current of 88 µA. All samples were analyzed using Analyze 14.0 software. Based on the division of the knee joint into four major regions (i.e., medial femoral condyle, lateral femoral condyle, medial tibial plateau, and lateral tibial plateau), the medial tibial plateau was broadly defined as the osteochondral unit that includes the subchondral bone, spanning from the medial end of the tibial articular surface to its midline. The bone volume fraction (BV/TV) and trabecular thickness (Tb.Th) were obtained through software analysis. Histological examination and OARSI score Synovial specimens were fixed in 10% formaldehyde for 24 h, subjected to conventional dehydration, and embedded in paraffin. Serial 5-µm sections were prepared. Cartilage specimens were similarly fixed in 10% formaldehyde, decalcified, and embedded in paraffin, followed by the preparation of serial 5-µm sections. Synovial and cartilage sections were stained with hematoxylin-eosin (H&E). Additionally, the cartilage sections were subjected to Safranin O-fast green staining. The OA damage observed in the Safranin O-fast green-stained cartilage sections and H&E-stained synovial sections was evaluated according to the widely accepted OARSI scoring system [46,47] (Pritzker, Gay et al. 2006, Laverty, Girard et al. 2010). For cartilage assessment, the OARSI score considers four stages (extent of involvement) and six grades (depth of lesion), assigning scores from 0 (normal) to 24 (severe). For synovial assessment, the degree of synoviocyte proliferation and hypertrophy observed in H&E-stained sections was evaluated using the OARSI scoring system. All assessments were performed by two blinded observers. Statistical analysis All data are expressed as mean ± SD. The statistical software package SPSS v29 (IBM Corp., Armonk, NY, USA) was used for all analyses. An independent-samples t-test was used to compare the two group-design experiments. One-way analysis of variance was used to compare multiple group experiments. OARSI scores were compared using a chi-square test. P < 0.05 was considered statistically significant. Results Microfluidic fabrication and characterization of liposome Liposomes were synthesized using a microfluidic system (Dolomite, Royston, UK) equipped with a five-input microfluidic chip. Transmission electron microscopy (TEM) revealed uniform spherical particles with smooth surfaces (Fig. 2 a). When the lipid solution contained rebastinib concentrations of 5 mg/mL and 10 mg/mL, the encapsulation efficiencies were comparable, yielding 88.64 ± 4.23% and 87.95 ± 1.51%, respectively (Fig. 2 b). The size of the liposomes decreased as the flow rate ratio of the lipid phase to the aqueous phase increased, with mean sizes of 192.26 ± 2.65 nm (1:2), 159.6 ± 1.41 nm (1:3), and 147.46 ± 2.87 nm (1:4) (Fig. 2 c). The rebastinib release profile demonstrated an initial rapid phase, releasing approximately 80% of the drug within the first 24 h, followed by sustained release over the subsequent 96-hour period, as quantified by HPLC analysis (Fig. 2 d). Safety test of rebastinib Six weeks post-treatment, hematological parameters and biochemical markers showed no statistically significant differences between the rebastinib group and PBS group (p > 0.05) (Table 1 ). Animals from both groups exhibited normal physiological behavior, including good physical activity, stable appetite, and absence of adverse effects such as alopecia, skin infections, or diarrhea. Body weight remained consistent between groups throughout the study period (p > 0.05) (Fig. 3 b). Histological evaluation of the synovial tissue using H&E staining revealed that both groups had synovial layers consisting of 1–2 cells, with no evidence of synovial or vascular hyperplasia. Cartilage sections stained with H&E and safranin O-Fast Green were intact and hyaline cartilage structure, clearly defined tidal lines, and normal chondrocyte morphology with no significant structural abnormalities in either group were apparent (Fig. 3 a). Table 1 Blood cell count and biochemical test results of the experimental and control groups (mean ± SD) Groups Num White blood cell (WBC) (K/µL) Red blood cell (RBC) (M/µL) Platelet (PLT) (K/µL) Aspartate aminotransferase (AST) (U/L) Blood urea nitrogen (BUN) (mg/dL) PBS group 6 9.96 ± 3.41 6.30 ± 0.80 342 ± 116 24.67 ± 14.22 17.61 ± 3.12 Rebastinib group 6 9.27 ± 2.54 6.25 ± 0.42 392 ± 90 17.33 ± 3.67 15.64 ± 1.30 No statistical signification was detected among all parameters Radiographic assessment The "fat pad sign," indicative of synovial effusion compressing the fat pad within the stifle joint, was evident in radiographic assessments of the ACLT and PBS groups (Fuller et al. 2014). The Reba group exhibited reduced effusion, whereas the Lipo@Reba group exhibited minimal effusion (Fig. 4 a). Micro-CT imaging performed with coronal plane alignment revealed substantial osteophyte formation in both ACLT and PBS groups. In contrast, the presence of osteophytes was negligible in both Reba and Lipo@Reba groups (Fig. 4 b). Quantitative analysis of the medial tibial plateau bone volume fraction (BV/TV) indicated no significant difference between the ACLT and PBS groups (p > 0.05), with both exhibiting similar trends. However, the Reba and Lipo@Reba groups displayed significantly lower BV/TV values than the ACLT and PBS groups, with the Lipo@Reba group exhibiting the lowest values (p < 0.05)(Fig. 4 c). Trabecular thickness (Tb.Th) followed a similar pattern, although statistical significance was observed only between the Lipo@Reba group and the ACLT and PBS groups (p < 0.05)(Fig. 4 d). Arthroscopic examination All experimental rabbits were arthroscopically evaluated prior to euthanasia. Examination of the femorotibial joint surfaces revealed no significant macroscopic differences across groups; however, fissure lines were observed on the medial tibial plateau in both the ACLT (no treatment) and PBS (intra-articular PBS injection) groups, along with partial fibrillation of the meniscus. In contrast, the Reba group (rebastinib intra-articular injection) and Lipo@Reba group (liposome-encapsulated rebastinib injection) demonstrated relatively smooth cartilage surfaces with reduced evidence of damage (Fig. 5 a). Examination of synovial tissue revealed pronounced differences between groups (Fig. 5 b). The ACLT and PBS groups exhibited severe pathological changes including thickened synovial tissue, fibrillation, and discoloration. In contrast, the synovium in the Reba and Lipo@Reba groups displayed substantially improved morphology, retaining an opalescent white appearance with minimal discoloration. OARSI gross scoring indicated significant differences between the groups (Fig. 5 c); the Reba group showed a notable reduction in pathological changes compared to the ACLT and PBS groups (p < 0.05), while the Lipo@Reba group exhibited the lowest scores, significantly differing from all other groups (p < 0.05). Histological examination and OARSI scores Histological analysis of the synovial tissue in the Reba group revealed a synovial lining comprising two to three layers of synovial cells accompanied by abundant loose connective tissue, minimal fibrous tissue, and slight vascular hyperplasia within the subsynovial layer. The Lipo@Reba group exhibited similar histological features, but with a thinner synovial lining (1–2 layers) and more prominent loose subsynovial connective tissue. Conversely, the ACLT and PBS groups displayed a marked increases in synovial cell proliferation, resulting in the formation of multiple synovial layers. Cartilage analysis using H&E and safranin O-fast green staining revealed distinct differences between groups. The Reba group showed mild superficial fibrillation, a well-preserved proteoglycan layer, a prominent tidal line, and intact cartilage structure. The Lipo@Reba group demonstrated an even smoother cartilage surface, superior chondrocyte organization, and a dense and vividly stained proteoglycan layer. In contrast, the ACLT and PBS groups showed reduced proteoglycan staining, irregular cartilage surfaces, and Safranin O staining. Discussion In the early stages of OA, subchondral bone undergoes significant pathological remodeling, driven in part by increased angiogenesis. This process facilitates vascular invasion at the osteochondral junction, contributing to cartilage degradation through inflammatory cell infiltration and matrix remodeling. Imaging techniques such as micro-computed tomography (µCT) and radiography commonly reveal increased subchondral bone density and sclerosis, indicative of early disease progression. Histologically, vascular channels penetrating the calcified cartilage layer often co-occur with sensory nerve fibers, which are implicated in OA-associated pain. Over time, these changes compromise the structural integrity of the osteochondral unit, exacerbating disease symptoms and progression (Goldring and Goldring, 2016 ; Saito et al. 2012 ; Stewart and Kawcak, 2018 ). The synovium is similarly affected by pathological changes during early OA that are largely driven by increased angiogenesis. Elevated levels of pro-inflammatory cytokines and angiogenic mediators, such as VEGF, promote neovascularization and immune cell recruitment, and increase vascular permeability. Histological findings in early OA often include synovial lining hyperplasia, mononuclear cell infiltration, and microvessel proliferation within the sublining layer. These changes contribute to chronic inflammation and synovial thickening, thereby perpetuating OA progression by facilitating sustained cytokine infiltration and immune cell activity (Mathiessen and Conaghan, 2017 ; Tak, 2001 ). Despite advances in our understanding of osteochondral and synovial pathologies in OA, the precise mechanisms and initiating factors of early OA remain unclear. Previous studies have demonstrated that inhibition of VEGF and PDGF signaling reduces angiogenesis in the subchondral bone and synovium, thereby mitigating OA progression (Goldring and Goldring, 2016 ; Li et al. 2019 ; Su et al. 2020 ). Given the close interplay between VEGF/PDGF-mediated angiogenesis and the Ang-Tie-2 pathway, targeting Tie-2 is a promising therapeutic strategy (Enholm et al. 1997 ; Fagiani and Christofori, 2013 ; Peng et al. 2020 ). Moreover, anti-angiogenic therapies targeting VEGF alone can induce hypoxic conditions and inadvertently promote pro-angiogenic factors and resistance mechanisms in diseased tissues (Bergers and Hanahan, 2008 ; Rigamonti et al. 2014 ). These findings underscore the rationale for targeting the Ang-Tie-2 pathway using rebastinib. In this study, we demonstrated that rebastinib effectively mitigated early osteoarthritic changes. Although radiographic evaluations have limited reliability for objectively assessing arthritis, differences in the fat pad sign provide indirect indicators of joint pathology (Fife et al. 1991 ). Micro-CT analysis revealed significantly fewer osteophytes in the rebastinib-treated group, indicating reduced pathological remodeling in the subchondral bone. Quantitative metrics, such as BV/TV and Tb.Th, further support the efficacy of rebastinib in alleviating OA-associated bone changes. Additionally, arthroscopic evaluation highlighted smoother cartilage surfaces and reduced fibrillation in the rebastinib-treated groups. Improvements in synovial morphology, supported by the OARSI gross scores and histological analyses, further confirmed the therapeutic benefits of rebastinib in targeting angiogenesis-driven joint degeneration. The encapsulation of rebastinib in liposomes (Lipo@Reba) further enhanced its therapeutic efficacy. The Lipo@Reba group exhibited significantly improved metrics across multiple parameters, including BV/TV, Tb.Th, and synovial morphology. Histological examination revealed that liposome-encapsulated rebastinib provided superior preservation of cartilage and synovial integrity, outperforming free rebastinib. These findings highlight the potential of liposomal delivery systems to maximize drug efficacy and extend therapeutic benefits. This study had several limitations. First, the relatively small sample size may have limited the generalizability of our findings. Second, a comparative evaluation of rebastinib with other antiangiogenic agents was not conducted. Third, the effects of varying rebastinib concentrations were not explored. Fourth, additional optimization of the liposome preparation conditions could provide more comprehensive insights. Lastly, in vitro analyses of the cellular effects of rebastinib in joint tissues were not performed. Future research should address these limitations by employing diverse experimental conditions and evaluating the effects of rebastinib in different models. Conclusions In conclusion, rebastinib, with its selective Tie-2 inhibition and favorable pharmacokinetics, demonstrated significant potential for the modification of early osteoarthritic progression. The integration of liposomal delivery further amplified its therapeutic effect, positioning liposome-encapsulated Tie-2 inhibitors as promising options for OA treatment. With further validation, this approach could offer a transformative alternative to conventional OA therapies. Declarations Conflict of interests: The authors declare no conflicts of interest. Ethic approval This research was approved by the Institutional Animal Care and Use Committee of Kangwon National University (Approval No. KW-240202-1). Informed Consent Statement : Not applicable Funding: This research was funded by the Basic Science Research Program through the National Research Foundation of Korea (NRF), funded by the Ministry of Education (RS-2023-00210585), and by a 2023 Research Grant from Kangwon National University(202305040001). Author Contributions: Conceptualization, M.J. and J.K.; methodology, M.J. and J.H.Y.; software, M.J.; validation, M.J.; formal analysis, M.J.; investigation, M.J.; resources: M.J., data curation, M.J..; writing—original draft preparation, M.J.; writing—review and editing, J.K.; visualization, M.J. and H.W.; supervision, J.K.; project administration: J.K. All authors have read and agreed to the published version of the manuscript Data availability: All the data are discussed in the manuscript. References Anderson KL, Zulch H, O'Neill DG, Meeson RL, Collins LM (2020) Risk factors for canine osteoarthritis and its predisposing arthropathies: a systematic review. 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Theranostics 10(1):426. http://doi.org/10.7150/thno.34126 Ribatti D, Nico B, Crivellato E (2011) The role of pericytes in angiogenesis. Int J Dev Biol 55(3):261–268. http://doi.org/10.1387/ijdb.103167dr Rigamonti N, Kadioglu E, Keklikoglou I, Rmili CW, Leow CC, De Palma M (2014) Role of angiopoietin-2 in adaptive tumor resistance to VEGF signaling blockade. Cell Rep 8(3):696–706. http://doi.org/10.1016/j.celrep.2014.06.059 Saito M, Sasho T, Yamaguchi S, Ikegawa N, Akagi R, Muramatsu Y, Mukoyama S, Ochiai N, Nakamura J, Nakagawa K (2012) Angiogenic activity of subchondral bone during the progression of osteoarthritis in a rabbit anterior cruciate ligament transection model. Osteoarthr Cartil 20(12):1574–1582. https://doi.org/10.1016/j.joca.2012.08.023 Sivan S, Schroeder A, Verberne G, Merkher Y, Diminsky D, Priev A, Maroudas A, Halperin G, Nitzan D, Etsion I (2010) Liposomes act as effective biolubricants for friction reduction in human synovial joints. 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Genes 9(6):283. https://doi.org/10.3390/genes9060283 Cite Share Download PDF Status: Published Journal Publication published 02 Dec, 2025 Read the published version in Inflammopharmacology → Version 1 posted Editorial decision: Major revisions 08 Sep, 2025 Reviewers invited by journal 22 Aug, 2025 Editor assigned by journal 20 Aug, 2025 First submitted to journal 15 Aug, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7380099","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":504127078,"identity":"8dfd3a4b-eee4-4884-92e3-81c3b199406b","order_by":0,"name":"Minuk Jeong","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA00lEQVRIiWNgGAWjYBAC9gYgwQNnGVgQ1sJzAKYFzDKQIEWLRAKYJEKL9OGDD95UHM6Tj3x+dcOPAgkG/vbuBPxa+NKSDeecOVxseDun7GYP0GESZ85uwKvFnofHTJq37XDixtk5aTd4gFoMJHLxa+Hh4f/+G6xl5pm0m3+I08LDxgzSMl+C/dhtIm1hM5accyY9cQNPDtttGQMJHoJ+4eFhfvjhTYV14vz2489uvvljI8ff3otfCxwYHOAxAJtBnHIQkG9gf0C86lEwCkbBKBhRAACKH0ZBf+0T0gAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0009-0007-7703-2093","institution":"Kangwon National University","correspondingAuthor":true,"prefix":"","firstName":"Minuk","middleName":"","lastName":"Jeong","suffix":""},{"id":504127079,"identity":"685ad031-9bad-4af2-8743-2cae30971ffb","order_by":1,"name":"Heung-Myong Woo","email":"","orcid":"","institution":"Kangwon National University","correspondingAuthor":false,"prefix":"","firstName":"Heung-Myong","middleName":"","lastName":"Woo","suffix":""},{"id":504127080,"identity":"b6260ed4-d79b-4055-b26c-4206985206e1","order_by":2,"name":"Jang-Hyuk Yun","email":"","orcid":"","institution":"Kangwon National University","correspondingAuthor":false,"prefix":"","firstName":"Jang-Hyuk","middleName":"","lastName":"Yun","suffix":""},{"id":504127081,"identity":"459b0464-cb21-4101-95db-1c06cf246a3c","order_by":3,"name":"Junhyung Kim","email":"","orcid":"","institution":"Kangwon National University","correspondingAuthor":false,"prefix":"","firstName":"Junhyung","middleName":"","lastName":"Kim","suffix":""}],"badges":[],"createdAt":"2025-08-15 09:22:19","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7380099/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7380099/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s10787-025-02081-6","type":"published","date":"2025-12-02T15:57:31+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":90312801,"identity":"1dc397dd-2884-428e-9594-7102db65af4a","added_by":"auto","created_at":"2025-09-01 10:04:32","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":4121241,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Schematic of chemical structures of rebastinib. (b) Schematic illustaration showing the microfluidic fabrication of rebastinib-loaded liposome (Lipo@Reba) and rebastinib-loaded liposome. (c) Schematic of Treatment of Lipo@Reba in rabbit osteoarthritis via intraarticular injection and its inhibition mechanism in joint angiogenesis\u003c/p\u003e","description":"","filename":"image1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7380099/v1/2488494b755e89fe7d864b81.jpeg"},{"id":90312435,"identity":"fef1f2ca-68ea-4d5b-83f8-2e614f49f1fd","added_by":"auto","created_at":"2025-09-01 09:56:32","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1455745,"visible":true,"origin":"","legend":"\u003cp\u003eCharacterization of liposome. (a) TEM image of liposomes: dispersed liposome (left panel), mono-liposome (right panel). (b) The encapsulation efficiency of rebastinib in liposomes (Rebastinib concentration in lipid solution : 5mg/ml, 10mg/ml). (c) Z-average and polydispersity index(PDI) of liposomes measured by DLS. (d) In vitro release of rebastinib from liposomes\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-7380099/v1/9bfe1bf985c9b832519867bd.png"},{"id":90312440,"identity":"d45d9ff2-f8f0-4197-8f89-dce55c7f8859","added_by":"auto","created_at":"2025-09-01 09:56:32","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":8210336,"visible":true,"origin":"","legend":"\u003cp\u003eSafety test of rebastinib. (a) H\u0026amp;E and safranin O-fast green staining of synovium and articular cartilage. The synovium and articular cartilage were normal in the PBS group. The Rebastinib-treated group did not show cartilage degenerative changes. Scale bar, 100μm (in left panels) and 200μm (in middle and right panels). (b) Body weight did not show and statistical significant difference between the rebastinib group and PBS group. (c) Cartilage OARSI score showed no statistically significant difference between the rebastinib group and PBS group. n = 6 per group\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-7380099/v1/90824f3759da181850eec58e.png"},{"id":90312436,"identity":"acaaf637-6f59-4afa-b48e-bd574f349691","added_by":"auto","created_at":"2025-09-01 09:56:32","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2916074,"visible":true,"origin":"","legend":"\u003cp\u003eRebastinib and rebastinib in liposome reduce osteophyte burden and subchondral bone changes in rabbit ACLT models (n = 6 per group). (a) Representative radiographic images of stifle joints at week 6 post-surgery. (b) Representative micro-CT images, showing the osteophytes in red arrows. (c) The quantitative analysis of bone volume fraction (BV/TV) in four groups 6 weeks post-surgery; n = 6 per group. *p \u0026lt; 0.05, ACLT group, PBS group, Reba group compared to the Lipo@Reba group. #p \u0026lt; 0.05, ACLT group, PBS group compared to the Reba group. (d) The quantitative analysis of trabecular bone thickness (Tb. Th) in four groups 6 weeks post-surgery; n = 6 per group. *p \u0026lt; 0.05, ACLT group, PBS group compared to the Lipo@Reba group\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-7380099/v1/4315ee90f86c76d672bde5f6.png"},{"id":90312803,"identity":"8844cdc3-b6a1-4bed-b8c8-dddf2f19c552","added_by":"auto","created_at":"2025-09-01 10:04:32","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":3952600,"visible":true,"origin":"","legend":"\u003cp\u003eRepresentative arthroscopic images of the femorotibial joint surfaces (a) and synovium (b) of the right knee joint from each group. Fissure lines (black arrow) were observed on the medial tibial plateau (MTP) in the ACLT and PBS groups. Additionally, fibrillation of the meniscus (arrowhead) was evident in some cases. (c) OARSI gross scores assessed via arthroscopy demonstrated significantly fewer arthritic lesions in the Reba and Lipo@Reba groups compared to the ACLT and PBS groups. Data are presented as mean ± SD (n = 6 per group). *p \u0026lt; 0.05, Reba group and Lipo@Reba group compared to the ACLT group. #p \u0026lt; 0.05, Reba group and Lipo@Reba group compared to the PBS group. †p \u0026lt; 0.05, Lipo@Reba group compared to the Reba group. Abbreviations: MTP, medial tibial plateau; MFC, medial femoral condyle\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-7380099/v1/5a2db5db3aa9f33c9043e4ac.png"},{"id":90312443,"identity":"e2975e1f-664e-4d8d-9b74-050acf0eba02","added_by":"auto","created_at":"2025-09-01 09:56:32","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":9005739,"visible":true,"origin":"","legend":"\u003cp\u003eHistological evaluation of efficacy of rebastinib. (a) H\u0026amp;E and Safranin O-fast green staining of the synovium and articular cartilage. The ACLT and PBS groups exhibited severe cartilage degenerative changes and synovium hyperplasia, while the Reba group and Lipo@Reba group showed fewer cartilage degenerative changes and less synovium hyperplasia. Scale bar, 100 μm (in top panels), 200 μm (in middle and bottom panels). (b) The synovial proliferation score shows that synovium hyperplasia was severe in the ACLT and PBS groups, while it was significantly reduced in the Reba group and even more so in the Lipo@Reba group. This indicates that rebastinib effectively inhibits inflammatory synovial proliferation, and liposomes enhance this inhibitory effect further; n = 6 per group. *p \u0026lt; 0.05, Reba group and Lipo@Reba group compared to the ACLT group. #p \u0026lt; 0.05, Reba group and Lipo@Reba group compared to the PBS group. †p \u0026lt; 0.05, Lipo@Reba group compared to the Reba group. (c) In the Cartilage OARSI score, the extent of cartilage damage was severe in the ACLT and PBS groups but significantly less in the Reba and Lipo@Reba groups, with the least damage observed in the Lipo@Reba group; n = 6 per group. *p \u0026lt; 0.05, Reba group and Lipo@Reba group compared to the ACLT group. #p \u0026lt; 0.05, Reba group and Lipo@Reba group compared to the PBS group. †p \u0026lt; 0.05, Lipo@Reba group compared to the Reba group\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-7380099/v1/65fcc39e6d5d62b6dbd5824f.png"},{"id":97724013,"identity":"757fc2fa-85ed-4a77-8877-8ae26cc4bc84","added_by":"auto","created_at":"2025-12-08 16:10:56","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":28625308,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7380099/v1/dc98d03a-30f5-4f62-8486-ba6e4fe2a068.pdf"}],"financialInterests":"","formattedTitle":"TitleTargeting tie-2 receptor with rebastinib (DCC-2036) for angiogenesis inhibition in early-stage arthritis: enhanced efficacy through liposomal sustained release","fulltext":[{"header":"Introduction","content":"\u003cp\u003eOsteoarthritis (OA), also known as osteoarthrosis or degenerative joint disease, is a multifaceted disorder that affects the entire joint structure, encompassing ,cartilage, synovium, subchondral bone, and associated tissues (Anderson et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Despite the hallmark features of deterioration and functional impairment of articular cartilage, the pathogenesis of OA is underpinned by a complex interplay of biochemical, cellular, and mechanical factors. However, the primary mechanisms triggering OA onset remain unclear (Nelson et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Current therapeutic approaches focus predominantly on alleviating symptoms, with no existing interventions capable of halting or reversing the underlying degenerative processes (Su et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2020\u003c/span\u003e)\u003c/p\u003e\u003cp\u003eRecent investigations have underscored the role of angiogenesis as a key driver of OA progression, mediated by dynamic interactions between endothelial cells, pericytes, and the extracellular matrix (Ribatti et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Su et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The Angiopoietin (Ang)/Tie-2 signaling pathway has been identified as a critical regulator of angiogenesis, governing vascular remodeling, and endothelial barrier stability (Maisonpierre et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e1997\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eRebastinib (DCC-2036), a highly selective and potent Tie-2 receptor tyrosine kinase inhibitor, exhibits a unique mechanism of action by stabilizing Tie-2 in its inactive conformation through an allosteric \"switch control pocket.\" Extensive pharmacokinetic and safety evaluations have reported its efficacy with a half-life of approximately 10 h, supporting its potential utility in therapeutic applications (Cortes et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Feng et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Harney et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2017\u003c/span\u003e)\u003c/p\u003e\u003cp\u003eLiposomes, nanoscale vesicular drug carriers composed of phospholipid bilayers, have significant promise in OA management (Goldberg and Klein, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Sivan et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Beyond their ability to serve as boundary lubricants and reduce cartilage friction and wear through the hydration of their phospholipid head groups, liposomes also offer the advantage of controlled and sustained drug release, enabling the localized delivery of therapeutic agents. These attributes have made liposomes promising platforms for the development of disease-modifying OA treatments (Ji et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eMicrofluidic technology has revolutionized liposome fabrication by enabling precise control of particle size, uniformity, and encapsulation efficiency. This method produces highly reproducible nanoformulations with tailored physicochemical properties, making it an ideal platform for the encapsulation of therapeutic agents, including liposome-based drug delivery systems (Chiesa et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Wang et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eBuilding on these advancements, this study posits that the encapsulation of rebastinib into liposomes using microfluidics offers a novel therapeutic approach for early stage OA. By targeting the Tie-2 signaling pathway, this strategy aims to mitigate angiogenesis-driven joint degeneration. To evaluate this hypothesis, we encapsulated rebastinib in liposomes (Lipo@Reba) and compared the efficacy of free and encapsulated Rebastinib in a surgically induced anterior cruciate ligament transection (ACLT) rabbit model of OA (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Therapeutic outcomes were assessed using imaging modalities, including radiography and arthroscopy, coupled with detailed histological analyses, to elucidate the effects of the drug on joint pathology.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003eUnless otherwise specified, all reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA). Lipid reagents were purchased from Avanti Polar Lipids (Alabaster, AL). Dialysis membrane tubing (MWCO 3.5 kDa) was purchased from Thermo Fisher Scientific (Waltham, MA, USA). Rebastinib (DCC-2036) was purchased from MedChemExpress (Monmouth Junction, NJ, USA).\u003c/p\u003e\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eProduction of liposomes using a microfluidics system\u003c/h2\u003e\u003cp\u003eLiposomes were synthesized using an automated dolomite microfluidic system (Dolomite, Royston, UK) equipped with a 5-input chip, allowing precise mixing of the lipid and aqueous phases. The lipid solution (10 mM) consisted of dioleoylphosphatidylethanolamine (DOPE), cholesteryl hemisuccinate (CHEMS), and PEG-2000 at a DOPE: CHEMS: PEG-2000 molar ratio of 63.3:31.6:5, with PEG-2000 accounting for 5% of the total molar composition. To ensure homogeneous distribution, rebastinib and lipids were dissolved in a chloroform:ethanol (1:4, v/v) solvent mixture. The aqueous phase was prepared as a 1\u0026times; phosphate-buffered saline (PBS) solution adjusted to pH 7.4. The lipid and aqueous phases were mixed in a microfluidic device at a flow rate ratio of 25:75 (lipid:PBS) to promote the formation of stable liposomes.\u003c/p\u003e\u003cp\u003eFollowing liposome formation, the dispersion was purified using dialysis tubing (MWCO, 3.5 kDa) in a PBS reservoir. Dialysis was performed at a 25-fold dilution rate for 12 h, with PBS refreshed every 4 h to remove residual solvents and unencapsulated materials. Mild agitation during the process prevented aggregation and maintained a uniform dispersion.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eDynamic light scattering\u003c/h3\u003e\n\u003cp\u003eLiposome size was determined using dynamic light scattering (Malvern Zetasizer Nano-ZSP, Malvern Instruments Ltd., UK). Measurements were conducted at 25\u0026deg;C, with flow rate ratios of 1:2, 1:3, and 1:4, under each condition (n\u0026thinsp;=\u0026thinsp;3).\u003c/p\u003e\n\u003ch3\u003eTransmission electron microscopy (TEM)\u003c/h3\u003e\n\u003cp\u003eLiposome morphology was visualized using a transmission electron microscope (TEM, JEM-2100F, Japan) operated at 200 kV. Samples were prepared by negative staining with 1% phosphotungstic acid and air-dried before imaging.\u003c/p\u003e\n\u003ch3\u003eHigh performance liquid chromatography (HPLC)\u003c/h3\u003e\n\u003cp\u003eRebastinib quantification was conducted using reverse-phase HPLC (Agilent 1260 series, Santa Clara, CA, USA) equipped with a C-18 column (4.6 \u0026times; 150 mm, 3.5 \u0026micro;m). Analyses were performed under isocratic conditions at a flow rate of 1.0 mL/min, detection at 254 nm, and a runtime of 30 min. A calibration curve was established in the range of 0.01\u0026ndash;0.5 mg/mL.\u003c/p\u003e\n\u003ch3\u003eEncapsulation efficiency of rebastinib\u003c/h3\u003e\n\u003cp\u003eLipid solutions were prepared to achieve rebastinib concentrations of 5 and 10 mg/mL, and liposomes were fabricated using a previously described method using a microfluidic device. Unencapsulated drugs were removed by diluting the final samples and liposomes were completely disrupted using a sonicator. The disrupted liposome solution was diluted with acetonitrile and samples were analyzed for rebastinib concentration using reverse-phase HPLC.\u003c/p\u003e\u003cp\u003eEncapsulation efficiency was calculated according to the following equation:\u003c/p\u003e\u003cp\u003eEncapsulation efficiency = \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\frac{\\varvec{E}\\varvec{n}\\varvec{t}\\varvec{r}\\varvec{a}\\varvec{p}\\varvec{p}\\varvec{e}\\varvec{d}\\:\\varvec{d}\\varvec{r}\\varvec{u}\\varvec{g}\\left(\\varvec{w}\\varvec{t}\\right)}{\\varvec{T}\\varvec{o}\\varvec{t}\\varvec{a}\\varvec{l}\\:\\varvec{d}\\varvec{r}\\varvec{u}\\varvec{g}\\left(\\varvec{w}\\varvec{t}\\right)}\\:\\)\u003c/span\u003e\u003c/span\u003ex 100%\u003c/p\u003e\u003cp\u003e\u003cb\u003eIn vitro\u003c/b\u003e \u003cb\u003edrug release\u003c/b\u003e\u003c/p\u003e\u003cp\u003eDrug release kinetics were assessed by incubating rebastinib-loaded liposomes in 700 mL of PBS buffer sink (pH 7.4) at 37\u0026deg;C after removing unencapsulated drug. Liposomes were placed in a dialysis cassette and shaken at 100 rpm. Samples (0.6 mL) were collected at time intervals of 4, 8, 24, 48, and 96 h. The concentration of rebastinib was quantified using HPLC.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eAnimals\u003c/h2\u003e\u003cp\u003eNew Zealand White rabbits, aged 16 weeks and weighing 2.5\u0026ndash;2.9 kg, were used in this study. The rabbits were purchased from an experimental animal supplier (Narabiotech, Seoul, Republic of Korea) and housed in a regularly ventilated temperature-controlled environment under a 12-hour light/12-hour dark cycle. The rabbits were acclimated for a period after arrival. All experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of Kangwon National University.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eSafety test of rebastinib\u003c/h3\u003e\n\u003cp\u003eThe drug concentration used for compartmental injections was based on a previous study that used a selective small-molecule Tie2 inhibitor for subretinal injections (Liu, Lin et al. 2008). Twelve New Zealand White rabbits were randomly divided into two groups and injected with 0.4 mL of PBS or 0.4 mL of 1.33 mg rebastinib (in 0.4 mL) into the stifle joint. Injections were repeated two weeks later. The rabbits were allowed to move freely after the injections, and all behavior and conditions were monitored. The rabbits were sacrificed six weeks later under anesthesia, followed by drug administration. After euthanasia, body weight was recorded, and approximately 2 mL of blood was collected from the ear vein for complete blood cell count and blood chemistry analysis. Cartilage samples were collected from the medial tibial plateau and synovial samples were obtained from the suprapatellar pouch.\u003c/p\u003e\n\u003ch3\u003eThe therapeutic effect in a rabbit ACLT model\u003c/h3\u003e\n\u003cp\u003eUnilateral ACLT was performed on twenty-four New Zealand White rabbits. Before and after surgery, animals received subcutaneous injections of enrofloxacin (5 mg/kg) and tramadol (4 mg/kg). Deep anesthesia was induced via intramuscular (IM) injection of ketamine (35 mg/kg) and xylazine (5 mg/kg) and maintained with isoflurane (1\u0026ndash;3.5%) administered via endotracheal intubation. ACLT was performed by a skilled veterinarian using the method described in a previous study (Levillain, Boulocher et al. 2015). Briefly, a longitudinal skin incision was made on the right knee joint to expose the capsule. The joint capsule was then opened through a longitudinal incision between the medial collateral and patellar ligaments. Complete rupture of the anterior cruciate ligament was confirmed by using the anterior drawer sign (manual horizontal dislocation) before closing the articular capsule. The operated leg was not immobilized and the rabbits were allowed to move freely in their individual cages after surgery. Three days after surgery, the rabbits were randomly divided into four groups (n\u0026thinsp;=\u0026thinsp;6). The three groups received intra-articular injections of the following formulations (0.4 mL) once every two weeks: PBS, rebastinib solution (3.33 mg/mL), and rebastinib in liposomes (Lipo@Reba, 6.27 mM). Finally, the patients were divided into the following groups: ACLT group (surgery only, no intra-articular treatment), PBS group (PBS intra-articular injection), Reba group (rebastinib intra-articular injection), and Lipo@Reba group (Lipo@Reba intra-articular injection). Six weeks after surgery, radiographic imaging and arthroscopy of the joints were performed. The rabbits were sacrificed after six weeks of treatment with anesthesia, followed by KCl. After sacrifice, the right stifle joints and organs were collected from each rabbit for toxicity assessment.\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eArthroscopic examination\u003c/h2\u003e\u003cp\u003eIntra-articular examination using arthroscopy was performed following the methods described in previous studies (Peters, Wilke et al. 2018). The rabbits were positioned in dorsal recumbency and the limb to be examined was placed ventrally toward the examiner. A 5 cm radius around the stifle joint was shaved to prepare the site. The limb was fully extended and a 20-gauge 1-inch needle was inserted into the joint space, 2 mm proximal to the tibial plateau and immediately medial to the patellar ligament. The joint capsule was then distended with 3 mL lactated Ringer\u0026rsquo;s solution. A stab incision was made at the same site using a No. 11 surgical blade positioned one-third of the distance from the tibial crest to the patellar base and immediately medial to the patellar ligament. A 1.9 mm arthroscope sheath with a blunt obturator (Arthrex Inc., Florida, USA) was inserted into the joint, directing the obturator proximally and axially until it rested between the patella and femur. While maintaining the obturator in position, a 20-gauge, 1-inch needle was inserted for fluid egress, positioned in the suprapatellar pouch medially or laterally, 3\u0026ndash;4 mm proximal to the superior patellar border. The joint was visualized using a 1.9 x 58 mm, 30\u0026deg; forward-oblique arthroscope (Arthrex Inc., Florida, USA). Joint distension was maintained by pressurizing 2 L of lactated Ringer\u0026rsquo;s solution to 30\u0026ndash;50 mm Hg, which was controlled using arthroscopic equipment. The trochlear groove was centered on the monitor, and the stifle joint was gradually flexed to an angle of 30\u0026ndash;45\u0026deg; with the scope directed distally for a comprehensive examination. The medial and lateral sides of the joint capsule in the suprapatellar pouch were imaged and scored by two blinded observers using the OARSI scoring system by two blinded observers (Cook, Kuroki et al. 2010). In addition, the femorotibial joint surfaces were imaged to indirectly observe the cartilage and meniscus.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eRadiographic assessment\u003c/h2\u003e\u003cp\u003eSix weeks post-surgery, rabbit knee joints were scanned using an X-ray imager (DRGEM Co., Korea). The articular surface and joint space widths were analyzed across the different groups. Six weeks post-surgery, the knee joints were harvested and assessed using high-resolution micro-CT (Quantum GX2 MicroCT, PerkinElmer, Inc., USA) with an isometric resolution of 76 \u0026micro;m. The scanning parameters included a voltage of 90 kV and a current of 88 \u0026micro;A. All samples were analyzed using Analyze 14.0 software. Based on the division of the knee joint into four major regions (i.e., medial femoral condyle, lateral femoral condyle, medial tibial plateau, and lateral tibial plateau), the medial tibial plateau was broadly defined as the osteochondral unit that includes the subchondral bone, spanning from the medial end of the tibial articular surface to its midline. The bone volume fraction (BV/TV) and trabecular thickness (Tb.Th) were obtained through software analysis.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eHistological examination and OARSI score\u003c/h2\u003e\u003cp\u003eSynovial specimens were fixed in 10% formaldehyde for 24 h, subjected to conventional dehydration, and embedded in paraffin. Serial 5-\u0026micro;m sections were prepared. Cartilage specimens were similarly fixed in 10% formaldehyde, decalcified, and embedded in paraffin, followed by the preparation of serial 5-\u0026micro;m sections. Synovial and cartilage sections were stained with hematoxylin-eosin (H\u0026amp;E). Additionally, the cartilage sections were subjected to Safranin O-fast green staining. The OA damage observed in the Safranin O-fast green-stained cartilage sections and H\u0026amp;E-stained synovial sections was evaluated according to the widely accepted OARSI scoring system [46,47] (Pritzker, Gay et al. 2006, Laverty, Girard et al. 2010). For cartilage assessment, the OARSI score considers four stages (extent of involvement) and six grades (depth of lesion), assigning scores from 0 (normal) to 24 (severe). For synovial assessment, the degree of synoviocyte proliferation and hypertrophy observed in H\u0026amp;E-stained sections was evaluated using the OARSI scoring system. All assessments were performed by two blinded observers.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eAll data are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD. The statistical software package SPSS v29 (IBM Corp., Armonk, NY, USA) was used for all analyses. An independent-samples t-test was used to compare the two group-design experiments. One-way analysis of variance was used to compare multiple group experiments. OARSI scores were compared using a chi-square test. P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003eMicrofluidic fabrication and characterization of liposome\u003c/h2\u003e\u003cp\u003eLiposomes were synthesized using a microfluidic system (Dolomite, Royston, UK) equipped with a five-input microfluidic chip. Transmission electron microscopy (TEM) revealed uniform spherical particles with smooth surfaces (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). When the lipid solution contained rebastinib concentrations of 5 mg/mL and 10 mg/mL, the encapsulation efficiencies were comparable, yielding 88.64\u0026thinsp;\u0026plusmn;\u0026thinsp;4.23% and 87.95\u0026thinsp;\u0026plusmn;\u0026thinsp;1.51%, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). The size of the liposomes decreased as the flow rate ratio of the lipid phase to the aqueous phase increased, with mean sizes of 192.26\u0026thinsp;\u0026plusmn;\u0026thinsp;2.65 nm (1:2), 159.6\u0026thinsp;\u0026plusmn;\u0026thinsp;1.41 nm (1:3), and 147.46\u0026thinsp;\u0026plusmn;\u0026thinsp;2.87 nm (1:4) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). The rebastinib release profile demonstrated an initial rapid phase, releasing approximately 80% of the drug within the first 24 h, followed by sustained release over the subsequent 96-hour period, as quantified by HPLC analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003eSafety test of rebastinib\u003c/h2\u003e\u003cp\u003eSix weeks post-treatment, hematological parameters and biochemical markers showed no statistically significant differences between the rebastinib group and PBS group (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05) (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Animals from both groups exhibited normal physiological behavior, including good physical activity, stable appetite, and absence of adverse effects such as alopecia, skin infections, or diarrhea. Body weight remained consistent between groups throughout the study period (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). Histological evaluation of the synovial tissue using H\u0026amp;E staining revealed that both groups had synovial layers consisting of 1\u0026ndash;2 cells, with no evidence of synovial or vascular hyperplasia. Cartilage sections stained with H\u0026amp;E and safranin O-Fast Green were intact and hyaline cartilage structure, clearly defined tidal lines, and normal chondrocyte morphology with no significant structural abnormalities in either group were apparent (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eBlood cell count and biochemical test results of the experimental and control groups (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD)\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"7\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGroups\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eNum\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eWhite blood cell (WBC)\u003c/p\u003e\u003cp\u003e(K/\u0026micro;L)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eRed blood cell (RBC)\u003c/p\u003e\u003cp\u003e(M/\u0026micro;L)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003ePlatelet\u003c/p\u003e\u003cp\u003e(PLT)\u003c/p\u003e\u003cp\u003e(K/\u0026micro;L)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eAspartate\u003c/p\u003e\u003cp\u003eaminotransferase (AST)\u003c/p\u003e\u003cp\u003e(U/L)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003eBlood urea nitrogen (BUN)\u003c/p\u003e\u003cp\u003e(mg/dL)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePBS group\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e9.96\u0026thinsp;\u0026plusmn;\u0026thinsp;3.41\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e6.30\u0026thinsp;\u0026plusmn;\u0026thinsp;0.80\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e342\u0026thinsp;\u0026plusmn;\u0026thinsp;116\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e\u003cp\u003e24.67\u0026thinsp;\u0026plusmn;\u0026thinsp;14.22\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e\u003cp\u003e17.61\u0026thinsp;\u0026plusmn;\u0026thinsp;3.12\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eRebastinib group\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e9.27\u0026thinsp;\u0026plusmn;\u0026thinsp;2.54\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e6.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.42\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e392\u0026thinsp;\u0026plusmn;\u0026thinsp;90\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e\u003cp\u003e17.33\u0026thinsp;\u0026plusmn;\u0026thinsp;3.67\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e\u003cp\u003e15.64\u0026thinsp;\u0026plusmn;\u0026thinsp;1.30\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"7\"\u003eNo statistical signification was detected among all parameters\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003eRadiographic assessment\u003c/h2\u003e\u003cp\u003eThe \"fat pad sign,\" indicative of synovial effusion compressing the fat pad within the stifle joint, was evident in radiographic assessments of the ACLT and PBS groups (Fuller et al. 2014). The Reba group exhibited reduced effusion, whereas the Lipo@Reba group exhibited minimal effusion (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). Micro-CT imaging performed with coronal plane alignment revealed substantial osteophyte formation in both ACLT and PBS groups. In contrast, the presence of osteophytes was negligible in both Reba and Lipo@Reba groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). Quantitative analysis of the medial tibial plateau bone volume fraction (BV/TV) indicated no significant difference between the ACLT and PBS groups (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05), with both exhibiting similar trends. However, the Reba and Lipo@Reba groups displayed significantly lower BV/TV values than the ACLT and PBS groups, with the Lipo@Reba group exhibiting the lowest values (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05)(Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec). Trabecular thickness (Tb.Th) followed a similar pattern, although statistical significance was observed only between the Lipo@Reba group and the ACLT and PBS groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05)(Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003eArthroscopic examination\u003c/h2\u003e\u003cp\u003eAll experimental rabbits were arthroscopically evaluated prior to euthanasia. Examination of the femorotibial joint surfaces revealed no significant macroscopic differences across groups; however, fissure lines were observed on the medial tibial plateau in both the ACLT (no treatment) and PBS (intra-articular PBS injection) groups, along with partial fibrillation of the meniscus. In contrast, the Reba group (rebastinib intra-articular injection) and Lipo@Reba group (liposome-encapsulated rebastinib injection) demonstrated relatively smooth cartilage surfaces with reduced evidence of damage (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). Examination of synovial tissue revealed pronounced differences between groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). The ACLT and PBS groups exhibited severe pathological changes including thickened synovial tissue, fibrillation, and discoloration. In contrast, the synovium in the Reba and Lipo@Reba groups displayed substantially improved morphology, retaining an opalescent white appearance with minimal discoloration. OARSI gross scoring indicated significant differences between the groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec); the Reba group showed a notable reduction in pathological changes compared to the ACLT and PBS groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), while the Lipo@Reba group exhibited the lowest scores, significantly differing from all other groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003eHistological examination and OARSI scores\u003c/h2\u003e\u003cp\u003eHistological analysis of the synovial tissue in the Reba group revealed a synovial lining comprising two to three layers of synovial cells accompanied by abundant loose connective tissue, minimal fibrous tissue, and slight vascular hyperplasia within the subsynovial layer. The Lipo@Reba group exhibited similar histological features, but with a thinner synovial lining (1\u0026ndash;2 layers) and more prominent loose subsynovial connective tissue. Conversely, the ACLT and PBS groups displayed a marked increases in synovial cell proliferation, resulting in the formation of multiple synovial layers. Cartilage analysis using H\u0026amp;E and safranin O-fast green staining revealed distinct differences between groups. The Reba group showed mild superficial fibrillation, a well-preserved proteoglycan layer, a prominent tidal line, and intact cartilage structure. The Lipo@Reba group demonstrated an even smoother cartilage surface, superior chondrocyte organization, and a dense and vividly stained proteoglycan layer. In contrast, the ACLT and PBS groups showed reduced proteoglycan staining, irregular cartilage surfaces, and Safranin O staining.\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn the early stages of OA, subchondral bone undergoes significant pathological remodeling, driven in part by increased angiogenesis. This process facilitates vascular invasion at the osteochondral junction, contributing to cartilage degradation through inflammatory cell infiltration and matrix remodeling. Imaging techniques such as micro-computed tomography (\u0026micro;CT) and radiography commonly reveal increased subchondral bone density and sclerosis, indicative of early disease progression. Histologically, vascular channels penetrating the calcified cartilage layer often co-occur with sensory nerve fibers, which are implicated in OA-associated pain. Over time, these changes compromise the structural integrity of the osteochondral unit, exacerbating disease symptoms and progression (Goldring and Goldring, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Saito et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Stewart and Kawcak, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe synovium is similarly affected by pathological changes during early OA that are largely driven by increased angiogenesis. Elevated levels of pro-inflammatory cytokines and angiogenic mediators, such as VEGF, promote neovascularization and immune cell recruitment, and increase vascular permeability. Histological findings in early OA often include synovial lining hyperplasia, mononuclear cell infiltration, and microvessel proliferation within the sublining layer. These changes contribute to chronic inflammation and synovial thickening, thereby perpetuating OA progression by facilitating sustained cytokine infiltration and immune cell activity (Mathiessen and Conaghan, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Tak, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2001\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eDespite advances in our understanding of osteochondral and synovial pathologies in OA, the precise mechanisms and initiating factors of early OA remain unclear. Previous studies have demonstrated that inhibition of VEGF and PDGF signaling reduces angiogenesis in the subchondral bone and synovium, thereby mitigating OA progression (Goldring and Goldring, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Li et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Su et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Given the close interplay between VEGF/PDGF-mediated angiogenesis and the Ang-Tie-2 pathway, targeting Tie-2 is a promising therapeutic strategy (Enholm et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e1997\u003c/span\u003e; Fagiani and Christofori, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Peng et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Moreover, anti-angiogenic therapies targeting VEGF alone can induce hypoxic conditions and inadvertently promote pro-angiogenic factors and resistance mechanisms in diseased tissues (Bergers and Hanahan, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Rigamonti et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). These findings underscore the rationale for targeting the Ang-Tie-2 pathway using rebastinib.\u003c/p\u003e\u003cp\u003eIn this study, we demonstrated that rebastinib effectively mitigated early osteoarthritic changes. Although radiographic evaluations have limited reliability for objectively assessing arthritis, differences in the fat pad sign provide indirect indicators of joint pathology (Fife et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e1991\u003c/span\u003e). Micro-CT analysis revealed significantly fewer osteophytes in the rebastinib-treated group, indicating reduced pathological remodeling in the subchondral bone. Quantitative metrics, such as BV/TV and Tb.Th, further support the efficacy of rebastinib in alleviating OA-associated bone changes. Additionally, arthroscopic evaluation highlighted smoother cartilage surfaces and reduced fibrillation in the rebastinib-treated groups. Improvements in synovial morphology, supported by the OARSI gross scores and histological analyses, further confirmed the therapeutic benefits of rebastinib in targeting angiogenesis-driven joint degeneration.\u003c/p\u003e\u003cp\u003eThe encapsulation of rebastinib in liposomes (Lipo@Reba) further enhanced its therapeutic efficacy. The Lipo@Reba group exhibited significantly improved metrics across multiple parameters, including BV/TV, Tb.Th, and synovial morphology. Histological examination revealed that liposome-encapsulated rebastinib provided superior preservation of cartilage and synovial integrity, outperforming free rebastinib. These findings highlight the potential of liposomal delivery systems to maximize drug efficacy and extend therapeutic benefits.\u003c/p\u003e\u003cp\u003eThis study had several limitations. First, the relatively small sample size may have limited the generalizability of our findings. Second, a comparative evaluation of rebastinib with other antiangiogenic agents was not conducted. Third, the effects of varying rebastinib concentrations were not explored. Fourth, additional optimization of the liposome preparation conditions could provide more comprehensive insights. Lastly, \u003cem\u003ein vitro\u003c/em\u003e analyses of the cellular effects of rebastinib in joint tissues were not performed. Future research should address these limitations by employing diverse experimental conditions and evaluating the effects of rebastinib in different models.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn conclusion, rebastinib, with its selective Tie-2 inhibition and favorable pharmacokinetics, demonstrated significant potential for the modification of early osteoarthritic progression. The integration of liposomal delivery further amplified its therapeutic effect, positioning liposome-encapsulated Tie-2 inhibitors as promising options for OA treatment. With further validation, this approach could offer a transformative alternative to conventional OA therapies.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003ch2\u003eConflict of interests:\u003c/h2\u003e\u003cp\u003eThe authors declare no conflicts of interest.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003ch2\u003eEthic approval\u003c/h2\u003e\u003cp\u003e This research was approved by the Institutional Animal Care and Use Committee of Kangwon National University (Approval No. KW-240202-1).\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eInformed Consent Statement\u003c/strong\u003e: Not applicable\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding:\u003c/h2\u003e\u003cp\u003eThis research was funded by the Basic Science Research Program through the National Research Foundation of Korea (NRF), funded by the Ministry of Education (RS-2023-00210585), and by a 2023 Research Grant from Kangwon National University(202305040001).\u003c/p\u003e\u003ch2\u003eAuthor Contributions:\u003c/h2\u003e\u003cp\u003eConceptualization, M.J. and J.K.; methodology, M.J. and J.H.Y.; software, M.J.; validation, M.J.; formal analysis, M.J.; investigation, M.J.; resources: M.J., data curation, M.J..; writing\u0026mdash;original draft preparation, M.J.; writing\u0026mdash;review and editing, J.K.; visualization, M.J. and H.W.; supervision, J.K.; project administration: J.K. All authors have read and agreed to the published version of the manuscript\u003c/p\u003e\u003ch2\u003eData availability:\u003c/h2\u003e\u003cp\u003eAll the data are discussed in the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAnderson KL, Zulch H, O'Neill DG, Meeson RL, Collins LM (2020) Risk factors for canine osteoarthritis and its predisposing arthropathies: a systematic review. Front veterinary Sci 7:220. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3389/fvets.2020.00220\u003c/span\u003e\u003cspan address=\"10.3389/fvets.2020.00220\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBergers G, Hanahan D (2008) Modes of resistance to anti-angiogenic therapy. 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Genes 9(6):283. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/genes9060283\u003c/span\u003e\u003cspan address=\"10.3390/genes9060283\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"inflammopharmacology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"iphm","sideBox":"Learn more about [Inflammopharmacology](https://www.springer.com/journal/10787)","snPcode":"10787","submissionUrl":"https://submission.nature.com/new-submission/10787/3","title":"Inflammopharmacology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Early-stage arthritis, inflammation, liposomes, drug delivery, tie-2 receptor inhibitor, angiogenesis","lastPublishedDoi":"10.21203/rs.3.rs-7380099/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7380099/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe prevalence of arthritis continues to increase, which has driven research on new therapeutic approaches. However, existing treatments often have limitations. Angiogenesis and pathological changes in the synovium are the key contributors to the early development of arthritis. Rebastinib, a tie-2 receptor inhibitor, blocks the activation of tie2-expressing macrophages, which are involved in angiogenesis. Although previous studies have highlighted the importance of angiogenesis in early arthritis, few have focused on targeting the tie-2 receptor to slow disease progression. In this study, we evaluated the effects of rebastinib encapsulated in pH-dependent liposomes in a rabbit model of surgically induced arthritis. Additionally, we investigated the efficacy of a pH-dependent liposomal formulation, developed using microfluidic technology for sustained drug release. The results demonstrated that rebastinib-loaded pH-dependent liposomes were stable and provided controlled release and rebastinib effectively inhibited the progression of early stage arthritis in this model. Statistical analyses were performed using SPSS software (IBM Corp., Armonk, NY, USA), and significance was assessed using one-way ANOVA. In conclusion, rebastinib encapsulated in pH-dependent liposomes holds promise as a potential therapeutic strategy for the treatment of early arthritis, offering both stability and efficacy in disease suppression.\u003c/p\u003e","manuscriptTitle":"TitleTargeting tie-2 receptor with rebastinib (DCC-2036) for angiogenesis inhibition in early-stage arthritis: enhanced efficacy through liposomal sustained release","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-01 09:56:27","doi":"10.21203/rs.3.rs-7380099/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revisions","date":"2025-09-08T07:14:17+00:00","index":"","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-08-22T09:44:44+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-08-20T07:52:14+00:00","index":"","fulltext":""},{"type":"submitted","content":"Inflammopharmacology","date":"2025-08-15T05:21:45+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"inflammopharmacology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"iphm","sideBox":"Learn more about [Inflammopharmacology](https://www.springer.com/journal/10787)","snPcode":"10787","submissionUrl":"https://submission.nature.com/new-submission/10787/3","title":"Inflammopharmacology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"30f81833-2c7d-4add-9aa1-3f5f02d5ed5c","owner":[],"postedDate":"September 1st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-12-08T16:05:10+00:00","versionOfRecord":{"articleIdentity":"rs-7380099","link":"https://doi.org/10.1007/s10787-025-02081-6","journal":{"identity":"inflammopharmacology","isVorOnly":false,"title":"Inflammopharmacology"},"publishedOn":"2025-12-02 15:57:31","publishedOnDateReadable":"December 2nd, 2025"},"versionCreatedAt":"2025-09-01 09:56:27","video":"","vorDoi":"10.1007/s10787-025-02081-6","vorDoiUrl":"https://doi.org/10.1007/s10787-025-02081-6","workflowStages":[]},"version":"v1","identity":"rs-7380099","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7380099","identity":"rs-7380099","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}