Bone marrow-targeted delivery of canonical lipolytic receptor agonists via neutrophil hitchhiking reverses osteoporosis

Bone marrow-targeted delivery of canonical lipolytic receptor agonists via neutrophil hitchhiking reverses osteoporosis | 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 Bone marrow-targeted delivery of canonical lipolytic receptor agonists via neutrophil hitchhiking reverses osteoporosis Changhua Wu, Jiu Zhao, Qibo Li, Weihao Zhang, Shuhao Meng, Huatao Liu, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7569697/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Current osteoporosis therapies fail to fully restore bone mass because of insufficient targeting of the bone marrow microenvironment. Here, we report a cell-mediated delivery platform that leverages senescent neutrophils to specifically transport canonical lipolytic receptor (CLR) agonists to bone marrow adipose tissue (BMAT). Using both ovariectomy-induced and aged osteoporosis mouse models, we demonstrated that systemic CLR activation significantly reduces BMAT volume while improving trabecular bone structure but at the cost of inducing systemic lipolysis and metabolic disturbances. To overcome these limitations, we developed CL316243(CL)-loaded nanoparticles delivered by senescent neutrophils (CL-NPs@NEs), which exhibited greater bone marrow accumulation than free drug. CL-NPs@NEs treatment led to remarkable bone mass recovery without causing peripheral fat loss or metabolic complications. Combining neutrophil-delivered CL and parathyroid hormone further enhanced therapeutic efficacy. Our findings establish senescent neutrophils as effective drug carriers for bone marrow-targeted therapy and reveal that CLR agonism is a viable strategy to remodel the adipocyte-rich bone marrow microenvironment. Targeted modulation of marrow adipose tissue combined with the osteoanabolic agent teriparatide holds promise for superior bone microarchitecture reconstruction and bone quality improvement in osteoporosis. Ostoporosis CL316243 canonical lipolytic receptor neutrophil drug targeting Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Osteoporosis has emerged as an increasingly severe global public health challenge. As the world's population ages more rapidly, the incidence of fragility fractures is increasing significantly[ 1 , 2 ]. Therapeutic strategies for osteoporosis have remained largely unchanged for decades, with a focus on either inhibiting osteoclast activity or intermittently stimulating osteoblast function[ 3 , 4 ]. While bisphosphonates and RANKL inhibitors prevent further bone loss, they fail to reverse established microarchitectural damage, which is the principal determinant of fracture risk[ 5 ]. In recent years, the relationship between lipid metabolism and bone metabolism has attracted increasing attention. Lipid metabolism dysregulation, through its impact on the homeostasis of the bone marrow microenvironment, has been identified as a significant factor in the development of osteoporosis[ 6 , 7 ]. Bone marrow adipocytes (BMAds) are a crucial cell type in the bone marrow microenvironment; they regulate bone remodeling by secreting various cytokines and adipokines and directly affect the function of other cells in the bone marrow[ 8 ]. Emerging evidence reveals that bone marrow adipocytes actively suppress osteogenesis through paracrine signaling, establishing marrow adipose tissue (BMAT) as a previously overlooked but critical therapeutic target[ 9 ]. This paradigm shift calls for innovative approaches that can address both the cellular origin of impaired bone formation and the pharmacological challenges of targeting marrow compartments simultaneously. β3 Adrenergic receptor (β3AR), a canonical lipolytic receptor (CLR), has emerged as a potential target for modulating bone marrow adiposity because of its specific expression pattern and metabolic effects[ 10 ]. Unlike other canonical lipolytic receptor subtypes (β1 and β2), β3AR is preferentially expressed in adipocytes[ 11 ]. CL316243 (CL) is a CLR agonist in phase II clinical trials that primarily targets β3-AR[ 12 ] and shows therapeutic promise for obesity and type II diabetes through enhanced insulin sensitivity and fat oxidation[ 13 , 14 ]. Notably, the CL remodels bone marrow adipose tissue via CLR activation, although its potential to concomitantly improve osteogenesis and the osteoporotic microenvironment requires further investigation. While effective for weight loss, the systemic administration of CLs faces clinical limitations, including cardiovascular/metabolic side effects and poor oral bioavailability, highlighting the need for targeted delivery approaches[ 15 ]. Targeted drug delivery to the bone marrow faces persistent challenges due to physiological barriers such as the marrow‒blood barrier, which restricts passive diffusion[ 16 ], whereas conventional bisphosphonate-conjugated nanoparticles exhibit limited efficacy[ 17 ]. Neutrophils provide an effective alternative through their natural vascular penetration and bone marrow homing abilities[ 18 ]. These phagocytes exploit chemotactic signals and constitutive marrow tropism, enabling efficient drug transport[ 19 ]. Recent neutrophil-mediated delivery strategies, such as nanoparticle hitchhiking via phagocytosed PLGA carriers, leverage these natural migratory pathways without artificial modification. This approach has proven effective in osseous diseases, including osteoporosis and bone metastasis, through targeted parathyroid hormone and chemotherapy delivery[ 20 ]. By merging neutrophil biology with nanomedicine, this strategy overcomes critical barriers-penetration and clearance-ushering in biologically inspired, precision therapies for bone disorders. Here, we present a therapeutic platform that integrates three recent innovations: first, the discovery that CLR activation reprograms marrow adipocyte metabolism to create a pro-osteogenic microenvironment; second, the development of an autologous neutrophil vehicle capable of bypassing physiological barriers to deliver payloads specifically to the bone marrow; and third, the dual-drug combination demonstrates significantly greater therapeutic efficacy than either agent alone, achieving synergistic effects that transcend conventional “antiresorptive versus anabolic” paradigms. This combinatorial approach uniquely restores bone marrow homeostasis through concurrent modulation of osteoblast‒osteoclast crosstalk and adipocyte signaling pathways. Results CLR activation reduces marrow adiposity and improves bone microstructure in osteoporotic mice To investigate the effects of CLR agonists (CL316243, CL) on bone marrow adipocyte lipolysis and bone formation, we first performed immunohistochemical analysis on femoral sections from ovariectomized (OVX) and aged (24-month-old, SENILE) mice, which revealed CLR-β3AR expression on bone marrow adipocyte membranes in OVX-B3 and SENILE-B3 mice relative to isotype control (Fig. 1 a). We subsequently established osteoporosis models in 8-week-old OVX C57BL/6 female mice and SENILE mice. The animals received daily intraperitoneal CL injections (1 mg kg − 1 ) for 30 days before euthanasia (Fig. 1 b). HE staining revealed significant decreases in brown adipose tissue lipid droplet and bone marrow adipocyte parameters in the CL-treated groups compared with those in the controls (Fig. 1 c, d). Specifically, the adipocyte lipid droplet volume (AV/NA) was reduced by 23.3% and 26.1% in the respective groups ( p < 0.05 ), whereas the marrow adipocyte proportion (AV/MV) was decreased by 53.3% in the OVX-CL groups ( p < 0.001 ) and 47.8% in the SENILE-CL groups ( p < 0.001 ) (Fig. 1 e-g). Micro-CT analysis revealed substantial improvements in the trabecular bone parameters in the OVX-CL group compared with those in the OVX-NC group, including an 82.4% increase in the BV/TV ( p < 0.001 ), a 74.5% increase in the BS/TV ( p < 0.001 ), a 57.0% increase in the Tb.N ( p < 0.01 ), a 16.9% increase in the Tb.Th ( p < 0.01 ), and a 23.6% decrease in the Tb.Pf ( p 0.05 ), likely due to baseline physiological differences, whereas the SENILE-CL group exhibited improvements comparable to those of the OVX-CL group ( p < 0.05 vs SENILE-NC). Collectively, our data demonstrate that CL not only diminishes marrow fat content but also augments bone formation, suggesting a β3-AR-dependent lipolytic mechanism within marrow adipocytes. Effects of CL on Bone Metabolism Serological and histological analyses revealed significant changes in the levels of bone metabolism markers following CL treatment. Moreover, peripheral blood analysis revealed elevated levels of both the bone resorption marker CTX-1 (OVX-CL: 12.900 ± 1.295 vs OVX-NC: 10.300 ± 0.707 ng mL − 1 , p < 0.01 ; SENILE-CL: 13.043 ± 1.031 vs SENILE-NC: 10.600 ± 1.409 ng mL − 1 , p < 0.01 ) and the bone formation marker P1NP (OVX-CL: 17.027 ± 1.500 vs OVX-NC: 10.417 ± 1.726 ng mL − 1 , p < 0.001 ; SENILE-CL: 17.183 ± 1.420 vs SENILE-NC: 11.100 ± 1.556 ng mL − 1 , p < 0.001 ) after CL treatment, indicating enhanced bone remodeling activity (Fig. 2 a). Histomorphometric analysis revealed (1) increased osteoclast numbers via TRAP staining (SH-CL: 19.567 ± 1.494 vs SH-NC: 15.500 ± 1.747 cells mm − 2 , p < 0.05 ; OVX-CL: 26.067 ± 1.098 vs OVX-NC: 21.567 ± 1.592 cells mm − 2 , p < 0.01 ; SENILE-CL: 29.200 ± 1.539 vs SENILE-NC: 14.400 ± 1.464 cells mm − 2 , p < 0.001 ) and (2) enhanced bone matrix formation via Masson's trichrome staining (Fig. 2 b,c). Angiogenesis analysis revealed CL-induced increases in CD31 + Emcn + H-type vessel density (OVX-CL: 0.980 ± 0.118 vs OVX-NC: 0.435 ± 0.121 vessel intensity, p < 0.001 ; SENILE-CL: 0.892 ± 0.134 vs SENILE-NC: 0.492 ± 0.092 vessel intensity, p < 0.01 ) in the femoral metaphysis (Fig. 2 d, e). In vitro experiments using adipocytes derived from human bone marrow in CL-conditioned media at different concentrations revealed that 5 µM adipocyte-CL-conditioned supernatants significantly enhanced MSC osteogenesis (ALP activity: 1.35-fold increase on day 21, p < 0.001 ) (Fig. 2 g, i) and promoted osteoclastogenesis from CD14 + cells (TRAP + multinucleated cells: 1.42-fold increase vs control, p < 0.001 ) (Fig. 2 h, j). Additionally, within 4h in the in vitro H-type vessel formation assay, 5 µM adipocyte-CL-conditioned supernatants significantly increased both the total length and total branching length of the capillary networks formed by HUVECs compared with those formed by the other concentrations (Fig. 2 k, l) (Supplementary Fig. 1). These results demonstrate that CL modulates bone metabolism through the coordinated regulation of osteoblast‒osteoclast coupling and angiogenic‒osteogenic coupling, which is mediated by adipocyte-derived factors. Adverse metabolic and cardiovascular effects of systemic CL administration Although CL promotes bone formation in OVX mice, systemic CLR activation can trigger detrimental effects across multiple physiological systems. Cardiac dysfunction may arise from disrupted calcium homeostasis, including sarcoplasmic reticulum (Ca²⁺) leakage, which impairs myocardial contractility (negative inotropy)[ 21 ]. Additionally, CL-induced nitric oxide (NO) overproduction and excessive lipolysis may contribute to cardiac lipotoxicity, particularly under pathological conditions such as sepsis[ 22 ]. In our experiments, CL administration led to significant weight loss and fat depletion. Specifically, compared with the OVX-NC controls, the OVX-CL mice lost 4.007 ± 0.52 g ( p < 0.001 ) by day 30, whereas the aged SENILE-CL mice exhibited a reduction of 2.491 ± 0.41 g ( p < 0.001 ) (Fig. 3 a). Histological analysis confirmed substantial decreases in abdominal and inguinal adipose tissue mass (approximately 38–41%, p < 0.001 ) (Fig. 3 b-d), which may negatively impact skeletal health, as peripheral fat reserves are inversely correlated with osteoporosis risk[ 23 ]. Moreover, rapid adipose tissue loss, particularly subcutaneous fat loss, has been linked to impaired wound healing, raising concerns for elderly and diabetic populations[ 24 ]. Systemic metabolic disturbances were evident within just 4 hours of CL exposure. Blood glucose levels declined sharply (SH-CL: − 11.81%, p < 0.001 ; OVX-CL: − 9.83%, p < 0.01 ; SENILE-CL: − 12.58%, p < 0.05 ), concurrent with a 20–40% reduction in leptin ( p < 0.001 ). In contrast, circulating nonesterified fatty acids (NEFAs, 1.38- to 2.10-fold increase, p < 0.01 ), insulin (1.50- to 1.96-fold, p < 0.05 ), and interleukin-6 (IL-6, 1.66- to 2.00-fold increase; all p < 0.05 ) were markedly elevated, indicating dysregulated lipolysis and a proinflammatory response (Fig. 3 d-i). Taken together, these findings reveal that while CL exerts anabolic effects on bone, its systemic actions, which involve cardiac stress, adipose tissue atrophy, and metabolic disarray, impose significant limitations for therapeutic use in osteoporosis and aging-related conditions. Targeted Bone Marrow Delivery of CL to Bone Marrow via Senescent Neutrophils Recent studies have demonstrated that senescent neutrophils possess a natural ability to migrate back to the bone marrow, presenting a unique opportunity for targeted delivery[ 25 ]. Capitalizing on this intrinsic homing mechanism, we developed a neutrophil-mediated delivery system for CLs designed to minimize peripheral tissue exposure. The CL-loaded PLGA nanoparticles (CL-NPs) were engineered to exhibit optimal characteristics, including an average diameter of 230 nm (Fig. 4 a), uniform morphology (Fig. 4 b), and a sustained drug release profile that delivered 28% of the payload within 48 hours, with nearly complete release achieved over 10 days (Fig. 4 c) (Supplementary Fig. 2). After neutrophils were cocultured with 3 mg/L CL-NPs, validation studies confirmed the rapid and efficient uptake of FITC-labeled CL-NPs by neutrophils (Fig. 4 d), with complete internalization within 2 h to form CL-NPs@NEs complexes (Fig. 4 e). HPLC analysis confirmed an effective drug payload of 2.5 µg CL per 10^6 cells, ensuring therapeutic efficacy. Functional characterization revealed time-dependent upregulation of CXCR4 on CL-NPs@NEs (Supplementary Fig. 3) while maintaining robust chemotactic responses to CXCL12 (SDF-1α) beyond 6 h in culture. Crucially, the CXCR4 antagonist AMD3100 abrogated this chemotaxis, confirming an uncompromised bone marrow homing capacity (Fig. 4 f, g) (Supplementary Fig. 4). In vivo biodistribution studies using DiD-labeled NEs (6 h) revealed remarkable targeting efficiency, with detectable bone marrow accumulation within 2 h that progressively increased through 6 h postinjection compared with that of free DiD, which was absorbed mainly by the liver and spleen, with minimal accumulation in the bone marrow (Fig. 4 h-j). However, compared with DiD-labeled NEs (6 h), DiD-labeled NEs (24 h) in the bone marrow were reduced through 6 h postinjection, likely due to neutrophil apoptosis in the spleen and liver. Additionally, compared with CL-NPs, CL-NPs@NEs exhibited superior targeting to the bone marrow. Compared with CL-NPs, CL-NPs@NEs demonstrated approximately 1.80-fold greater bone marrow accumulation while significantly reducing off-target deposition in adipose tissues and major organs (Fig. 4 k-m). Notably, we observed prolonged retention in the bone marrow associated with delayed neutrophil apoptosis, in contrast to the rapid clearance observed with free formulations through hepatic and splenic pathways. These results suggest that CL-NPs@NEs effectively target the bone marrow while minimizing peripheral tissue exposure. In Vivo Evaluation of CL-NPs@NEs in Osteoporotic Mice To evaluate the in vivo regulatory effects of neutrophil-mediated CL delivery (CL-NPs@NEs), an osteoporosis model was established in 8-week-old female C57BL/6 mice subjected to OVX, followed by a 30-day recovery period. The mice were then randomly assigned to five groups (Fig. 5 a). Our preclinical evaluation in this model demonstrated the superior therapeutic profile of neutrophil-derived CL-NPs@NEs. The results of HE staining revealed that, compared with that in the OVX groups, the number of bone marrow adipocytes in the CL, CL-NPs and CL-NPs@NEs groups was significantly lower (Fig. 5 b). Compared with OVX, CL-NPs@NEs treatment reduced the adipocyte lipid droplet volume per unit area (AV/NA) by approximately 30.87% ( p < 0.01 ), outperforming both free CL (20.87% reduction) and CL-NPs (19.35% reduction) (Fig. 5 c). Similarly, the adipocyte volume fraction (AV/MV) was decreased by 35.37% in the CL-NPs@NEs group relative to that in the OVX group ( p < 0.001 ), representing a 6.90–9.57% greater reduction than that of the alternative CL formulations (Fig. 5 c). Compared with OVX, CL-NPs@NEs treatment markedly improved the following trabecular bone parameters: the bone volume fraction (BV/TV) increased by 101.12% ( p < 0.001 ), the trabecular thickness (Tb.Th) increased by 39.84% ( p < 0.01 ), and the trabecular number (Tb.N) increased by 62.46% ( p < 0.01 ). Trabecular spacing (Tb.Pf) decreased by 37.28% ( p < 0.001 ) (Fig. 5 d-j). Notably, this targeted delivery approach largely avoids the adverse metabolic effects observed with systemic CL administration. CL-NPs@NEs maintained the peripheral adipose tissue mass at OVX control levels, in contrast to the 28.41–43.06% fat loss observed with conventional CL delivery (Fig. 5 k‒m). Corresponding serum analyses revealed that the neutrophil-mediated strategy significantly attenuated CL-induced metabolic dysregulation, resulting in a 39.89% reduction in proinflammatory IL-6 levels, a 52.68% decrease in NEFAs, and preservation of basically normal insulin sensitivity and leptin regulation (Fig. 5 n-r). These data collectively demonstrate that neutrophil-mediated delivery achieves superior bone-targeting specificity while preventing the characteristic systemic side effects of CLR agonist therapy. Synergistic Combination Therapy with CL and Teriparatide via Neutrophil-Mediated Delivery Enhances Osteoporosis Treatment Despite its osteogenic potential, CL also promotes osteoclast activity, which limits its monotherapeutic efficacy. To address this, we strategically combined CL with teriparatide (PTH) to foster a synergistic effect. Unlike purely antiresorptive agents (e.g., bisphosphonates or denosumab), PTH has dual-action properties: it stimulates osteoblast activity while modulating osteoclast function through RANKL/OPG regulation[ 26 ]. This combination therapy may regulate both osteoblastic and osteoclastic activities, enabling balanced bone remodeling and improved therapeutic efficacy (Fig. 6 a). Our neutrophil-mediated nanodelivery system (CL + PTH-NPs@NEs) demonstrated superior therapeutic effects in OVX mice compared with either agent alone. In the adipose tissue analysis, CL + PTH-NPs@NEs reduced AV/NA by 54.55% (0.025 ± 0.004 vs OVX 0.056 ± 0.016, p < 0.01), and CL + PTH-NPs@NEs decreased AV/MV by 48.52% (0.089 ± 0.008 vs OVX 0.173 ± 0.018, p < 0.001 ) (Fig. 6 b-d). These reductions were significantly greater than those resulting from either CL-NPs@NEs or PTH-NPs@NEs monotherapy ( p < 0.05 ). Micro-CT analysis revealed that the combination therapy enhanced osteogenic effects: BV/TV increased 31.02% more than did PTH alone, Tb.N improved 23.72% more than did CL alone, and Tb.Pf was reduced by 18.55% compared with that of the monotherapies (Fig. 6 e-j). These findings position CL + PTH-NPs@NEs as a promising next-generation therapy that may overcome current limitations in osteoporosis treatment by simultaneously enhancing bone formation while maintaining balanced remodeling activity. Discussion Our findings significantly advance the understanding of canonical lipolytic signaling in bone metabolism by elucidating its spatial regulation within marrow microenvironments (Fig. 7 ). While the lipolytic effects of CLR agonists are well characterized in metabolic tissues, their specific effects on bone marrow adipocytes remain unclear. Clinically, chronic CLR agonist administration may benefit bone metabolism through lipid depletion, glucose modulation, and altered adipokine secretion[ 27 ]. In our study, CL treatment increased nonesterified fatty acid (NEFA) levels. These NEFAs, including palmitoleic acid (PLA) and oleic acid (OA), which increase osteoblast activity via FFAR4/β-arrestin2 signaling, may be produced via lipolysis and play pivotal roles in bone remodeling[ 28 ]. Notably, ω-3 polyunsaturated fatty acids such as docosahexaenoic acid (DHA) activate the Wnt/β-catenin and Akt pathways to stimulate bone formation[ 29 ]. Similarly, Zhang et al. demonstrated that DHA-containing lipids increase bone mineral density in ovariectomized mice by promoting chondrocyte-to-osteoblast transdifferentiation[ 30 ]. In addition, NEFAs exhibit dual regulation of bone remodeling. While PLA/OA/DHA promote osteogenesis, arachidonic acid-derived prostaglandin E2 (PGE2) enhances osteoclastogenesis via EP2/EP4 receptor-mediated cAMP signaling and RANKL/OPG axis activation[ 31 , 32 ]. Conversely, palmitic acid both potentiates RANKL-induced osteoclast differentiation and independently triggers osteoclastogenesis[ 15 ]. Additionally, β3-adrenergic-stimulated adipocytes secrete zinc, which orchestrates bone homeostasis through various mechanisms: promoting osteogenesis via PI3K/Akt activation while suppressing osteoclast differentiation through GRB2/ERK inhibition[ 33 , 34 ]. Collectively, these mechanisms explain the CL-induced elevation of bone turnover markers, demonstrating balanced stimulation of both osteoblastic and osteoclastic activity that ultimately improves bone quality in osteoporosis. While CL shows promise for osteoporosis treatment, systemic administration poses significant clinical challenges because of its multifaceted off-target effects. The drug’s potent lipolytic action risks compromising dermal adipose tissue function, a specialized fat depot essential for wound repair[ 35 ] and antimicrobial defense[ 36 ]. In our models, both ovariectomized and aged mice exhibited substantial weight loss and peripheral fat depletion changes that may impair wound healing and increase infection susceptibility. Such adipose reduction becomes particularly concerning in elderly populations with already diminished fat reserves, where further depletion may precipitate cell dysfunction through lipotoxicity and insulin resistance[ 37 ]. Concurrent metabolic disturbances emerged in our studies, including significantly elevated IL-6 and insulin alongside sharp decreases in leptin and glucose levels, mirroring previous clinical observations[ 38 – 40 ]. These metabolic shifts may have dual negative consequences: potentiating osteoclast activity through increased CTX-1 while simultaneously suppressing appetite via leptin reduction[ 41 ], thereby potentially undermining the desired anabolic bone effects. Clinical translation faces additional pharmacological hurdles, as evidenced by phase II trials revealing poor oral bioavailability and problematic β1-adrenergic receptor cross-reactivity[ 42 ] factors that help explain Kurabayashi’s paradoxical observation of reduced bone volume with BRL35135 treatment[ 43 ]. Taken together, these findings highlight the critical need for the development of novel strategies that can preserve the osteoprotective benefits of CL while circumventing its systemic liabilities. To overcome the systemic side effects of conventional drug delivery, we developed a neutrophil-engineered (NE) nanoparticle system for bone marrow-specific CL delivery. Building upon emerging neutrophil-based drug delivery platforms[ 18 ], our approach leverages the unique biological properties of aged neutrophils, including their strong phagocytic capacity, CXCR4-mediated chemotaxis, and intrinsic immune evasion capabilities, to increase drug targeting while extending the circulation half-life. Previous studies have demonstrated the potential of neutrophil-mediated delivery: Xue et al. reported that neutrophil-carried liposomes could target invasive tumor cells[ 44 ], whereas Luo et al. utilized aged neutrophils to achieve high bone marrow drug accumulation in both metastatic cancer and osteoporosis models[ 20 ]. The efficacy of this platform stems from the CXCR4/CXCL12 chemokine axis, where CXCR4-expressing neutrophils home to CXCL12-rich bone marrow niches, effectively crossing the marrow‒blood barrier. Our PET/CT and fluorescence imaging studies confirmed efficient marrow accumulation of NE-delivered nanoparticles in osteoporotic mice. Importantly, the CL-NPs@NEs system demonstrated dual therapeutic advantages: compared with free CL administration, quantitative imaging revealed enhanced bone-specific drug retention, whereas biochemical analyses revealed marked attenuation of systemic effects. The targeted delivery approach significantly reduced peripheral adipose lipolysis and mitigated metabolic disturbances. These findings establish neutrophil-mediated targeting as an effective strategy to simultaneously increase bone anabolism while minimizing the off-target effects of CLR agonism. Contemporary clinical management of osteoporosis employs sequential or concurrent administration of antiresorptive and anabolic agents to maximize therapeutic benefits[ 1 ]. While short-term combination therapy has synergistic effects on bone mineral density, prolonged coadministration may induce hypercalcemia and paradoxical antagonism of therapeutic mechanisms, as observed between zoledronic acid and teriparatide[ 45 ]. CLR agonists have emerged as promising therapeutic alternatives because of their unique signaling cascades, which bypass pharmacodynamic interference while enabling balanced bone remodeling via coupled regulation of osteoanabolic and osteocatabolic activities. This dual-action mechanism presents a distinct advantage over conventional antiresorptives (e.g., bisphosphonates or denosumab) that solely suppress osteoclast function. Furthermore, unlike the demanding daily subcutaneous injections required for teriparatide monotherapy or the extended dosing intervals of antiresorptive agents, our neutrophil-mediated nanoparticle delivery system (CL + PTH-NPs@NEs) synergistically combines the therapeutic benefits of both drug classes while potentially improving administration frequency. Our studies in OVX mice demonstrate that this combinatorial approach yields superior outcomes compared with those of monotherapy, with microCT analyses revealing significantly increased bone density and trabecular microarchitecture. Importantly, neutrophil-directed delivery maintains osteogenic amplification from CL-induced lipolysis while simultaneously addressing the pharmacokinetic limitations of teriparatide, effectively translating the theoretical compatibility of these pathways into a therapeutic advantage. While our findings demonstrate promising osteoprotective effects, important pharmacological and translational challenges must be addressed before clinical implementation. In addition to the aforementioned adverse effects, preclinical evidence suggests that chronic CLR agonism may impair additional off-target effects on bladder smooth muscle, which could influence micturition function[ 46 , 47 ]. Although our current study specifically evaluated skeletal outcomes, a rigorous assessment of these organ-specific effects—particularly under targeted delivery conditions—remains imperative. The age-dependent properties of neutrophil carriers introduce additional biological complexity, while their intrinsic bone marrow homing capability facilitates drug delivery, the immunological alterations characteristic of aged neutrophils[ 48 ] and their potential to modulate inflammatory responses or immune suppression[ 49 , 50 ] warrant thorough investigation in chronic treatment settings. From a translational standpoint, the clinical scalability of autologous neutrophil-based delivery systems faces practical limitations related to cell harvesting costs and patient compliance. Future research directions will focus on engineering next-generation nanoparticles with enhanced bone-specific targeting and controlled release kinetics to minimize systemic exposure, coupled with comprehensive long-term safety evaluations across multiple osteoporotic models to systematically characterize cardiovascular, urological, and immunological impact-critical steps toward establishing the clinical viability of this therapeutic strategy. In conclusion, by leveraging neutrophil-mediated hitchhiking as a bone marrow-targeted delivery platform, we demonstrate that localized CLR agonism effectively reverses skeletal deterioration in osteoporosis while circumventing the systemic metabolic perturbations typically associated with CLR activation. This neutrophil-based delivery system achieves spatiotemporally controlled release of CL316243 and teriparatide specifically within the bone marrow microenvironment, where it simultaneously attenuates marrow adipogenesis and promotes osteoblast differentiation, thereby addressing two key pathological features of osteoporosis. Importantly, our approach maintains the metabolic benefits of CLR signaling confined to bone tissue, avoiding undesirable peripheral effects. These findings establish neutrophil-assisted drug delivery as a promising therapeutic paradigm for precise modulation of bone remodeling with minimal off-target consequences, offering a clinically translatable solution for osteoporosis treatment. Methods Study approval This study conforms to the Declaration of Helsinki and was approved by the Ethics Committee of the Eighth Affiliated Hospital, Sun Yat-Sen University, Guangzhou, China. The experiments involving mice were approved by the Institutional Animal Care and Use Committee of Sun Yat-Sen University, Guangzhou, China. All experimental procedures involving mice were carried out in strict adherence to the rules and guidelines for the ethical use of animals in research. Animal experiments Three-month-old mature female C57BL/6 mice underwent bilateral ovariectomy (OVX) or sham surgery (SH). After anesthesia, a lateral lumbar incision was made at the midpoint between the free rib and the iliac crest to remove the ovaries. The incision was sealed, and the skin was sutured. Thirty days after surgery, the sham, OVX, and 24-month-old elderly (SENILE) mice were randomly divided into six groups (n = 6): the SH-NC (saline, intraperitoneal injection), SH-CL (CL316243, intraperitoneal injection), OVX-NC (saline, intraperitoneal injection), OVX-CL (CL316243, intraperitoneal injection), SENILE-NC (saline, intraperitoneal injection), and SENILE-CL (CL316243, intraperitoneal injection) groups. Body weight changes were recorded throughout the experiment. After 30 days of treatment, the mice were euthanized. Peripheral adipose tissue was collected, and femurs and tibiae were harvested for micro-CT analysis. Blood samples were collected to measure the serum levels of P1NP (Macklin, P771527), CTX-1 (IDS, AC-06F1), IL-6 (R&D, VAL604G), leptin (Elabscience, E-EL-M3008), and insulin (Elabscience, E-EL-M2614) via ELISA kits. Free fatty acid (FFA) and glucose levels were assessed via a colorimetric assay kit (Elabscience, E-BC-K792-M) and a glucose assay kit (Elabscience, E-BC-K234-M), respectively. The additional batches used for OVX modeling and sample collection followed the same procedures described above. Extraction and stimulation of bone marrow adipocytes Bone marrow aspirates from NC patients were processed via density gradient centrifugation (150 × g min − 1 , 10 min, Invitrogen) to isolate bone marrow adipose tissue. The isolated adipocytes were cultured in osteogenic medium or osteoclastogenic medium containing 0 µM, 1 µM, or 5 µM CL. The osteogenic medium consisted of 10% FBS DMEM supplemented with 100 IU mL − 1 penicillin, 100 IU mL − 1 streptomycin, 0.1 µmol L − 1 dexamethasone, 10 mmol L − 1 β-glycerophosphate, and 50 µmol L − 1 ascorbic acid (Sigma‒Aldrich). The osteoclastogenic medium consisted of 10% FBS α-MEM supplemented with 100 IU mL − 1 penicillin, 100 IU mL − 1 streptomycin, 50 µg mL − 1 RANKL (R&D, 462-TEC-010), and 25 µg mL − 1 M-CSF (R&D, 216-MC-010). After 6 hours of culture, the adipocytes were removed via centrifugation (150 g·min − 1 ), and the fatty acid-induced supernatant was collected. MSC isolation and culture Bone marrow aspirates from NC patients were processed via density gradient centrifugation (12,000 r min − 1 , 30 min, Invitrogen) to isolate mesenchymal stem cells (MSCs). The extracted MSCs were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco) supplemented with 10% fetal bovine serum (FBS; Hangzhou Sijiquan Biotechnology). For osteogenic induction and CL stimulation, the MSCs were divided into four groups: the NC group (osteogenic medium containing 5 µM CL-primed cell-free medium), the 0 µM group (osteogenic medium containing adipocyte-0 µM CL-conditioned culture), the 1 µM group (osteogenic medium containing adipocyte-1 µM CL-conditioned culture), and the 5 µM group (osteogenic medium containing adipocyte-5 µM CL-conditioned culture). The osteogenic medium was replaced every three days. Osteoclast culture and induction Peripheral blood mononuclear cells (PBMCs) were isolated from the anticoagulated peripheral blood of volunteers via density gradient centrifugation (400 g min − 1 , 30 min, Invitrogen). CD14 + cells were subsequently isolated via magnetic bead sorting. For osteoclast induction, CD14 + cells were divided into four groups: the NC group (osteoclastogenic medium containing 5 µM CL-primed cell-free medium), the 0 µM group (osteoclastogenic medium containing adipocyte-0 µM CL-conditioned supernatant), the 1 µM group (osteoclastogenic medium containing adipocyte-1 µM CL-conditioned supernatant), and the 5 µM group (osteoclastogenic medium containing adipocyte-5 µM CL-conditioned supernatant). CD14 + cells were cultured in fatty acid-induced supernatants with different concentrations of CL. After 8 days of induction, tartrate-resistant acid phosphatase (TRAP) staining was performed via a TRAP staining kit. Endothelial tube formation assay Human umbilical vein endothelial cells (HUVECs, iCell) were used for the endothelial tube formation experiments. HUVECs were cultured on plates precoated with 1% (w/v) gelatin and maintained in endothelial cell medium (ECM, ScienCell) supplemented with 20% fetal bovine serum, 100 IU mL − 1 penicillin and 100 IU mL − 1 streptomycin. For tube formation induction and CL stimulation, HUVECs were divided into four groups: the NC group (endothelial medium containing 5 µM CL-primed cell-free medium), the 0 µM group (endothelial medium containing adipocyte-0 µM CL-conditioned culture), the 1 µM group (endothelial medium containing adipocyte-1 µM CL-conditioned culture), and the 5 µM group (endothelial medium containing adipocyte-5 µM CL-conditioned culture). After 4 hours of induction, samples were collected for quantification of tube formation metrics. Neutrophil isolation Femurs and tibiae were harvested from euthanized mice under sterile conditions. The ends of the bones were cut off and placed in a 1.5 mL Eppendorf tube containing 200 µL of PBS and a 1 µL pipette tip with the tip removed. The samples were centrifuged at 12,000 × g min − 1 for 15 seconds, and the bone marrow was resuspended in 500 µL of PBS. A density gradient was prepared by carefully layering 2 mL of 75%, 65%, and 55% (vol/vol) Percoll (Pharmacia) solutions, followed by 500 µL of the single-cell suspension on top. The gradient was centrifuged at 700 × g min − 1 for 30 minutes, and neutrophils were collected from the interface between the 75% and 65% fractions. The collected neutrophils were washed three times with ice-cold PBS to remove residual Percoll and subsequently lysed with red blood cell lysis buffer for 15 minutes. The isolated neutrophils were cultured in RPMI 1640 medium supplemented with 1% penicillin/streptomycin at 37°C in a humidified atmosphere containing 5% CO₂. Typically, senescent neutrophils were obtained after 6 hours of in vitro culture. Proportion of CXCR4-expressing Neutrophils Mouse bone marrow cells were cultured in an incubator, and the cells were collected at 0 h, 6 h, and 24 h. The cells were stained with APC-conjugated anti-mouse Ly-6G and PE-conjugated anti-mouse CD184 (CXCR4) antibodies (diluted 1:200 in PBS) for 25 minutes. After the unbound antibodies were removed, the proportion of CXCR4-expressing neutrophils was analyzed via flow cytometry. Preparation of CL-NPs CL-NPs were synthesized via a double emulsion (W/O/W) method. Briefly, 1 mg of CL316243 was dissolved in 50 µL of cold water as the inner aqueous phase. Concurrently, 29 mg of PLGA (65:35 wt/wt, Mw: 24,000–38,000, Sigma‒Aldrich) was dissolved in 450 µL of dichloromethane (DCM) as the oil phase. The inner aqueous phase (W) was quickly injected into the oil phase (O) and sonicated for 3 minutes to form a primary W/O emulsion. This emulsion was then added to 3 mL of 2.0% polyvinyl alcohol (PVA, Mw: 38 kDa, Sigma‒Aldrich) solution and further sonicated for 3 minutes to form the W/O/W emulsion. The resulting emulsion was poured into 20 mL of 0.2% PVA solution and stirred overnight to solidify the nanoparticles. The CL-NPs were collected by centrifugation at 18,000 × g min − 1 for 20 minutes and washed three times with Milli-Q water. Drug encapsulation efficiency was determined via a titration method. Lyophilized CL-NPs (5 mg) were dissolved in 2 mL of acetonitrile, and the sample was analyzed via high-performance liquid chromatography (HPLC). For cumulative drug release analysis, 5 mg of lyophilized CL-NPs were suspended in 10 mL of PBS containing 0.2% Tween 80 and incubated at 37°C in a shaking water bath (SHY-2A, Guoyu Instruments). At specific time points (0 h, 1 h, 3 h, 6 h, 1 day, 2 days, 4 days, 7 days, and 10 days), 1 mL of the supernatant was collected via centrifugation (20,000 × g min − 1 , 20 minutes) and replaced with 1 mL of fresh PBS containing 0.2% Tween 80. The cumulative drug content in the supernatant was determined via HPLC (HPLC 1200 series, Agilent Technologies). The particle size of the PLGA nanoparticles was measured via dynamic light scattering (Malvern Zetasizer Nano-ZS), and their morphology was observed via transmission electron microscopy (TEM, JEOL JEM-1400). PTH-NPs (PTH, MedChemExpress, HY-P0059) and CL-PTH-NPs were prepared via the same method as CL-NPs. Assessment of the Migration of CL-NPs@NEs The ability of CL-NPs@NEs to migrate toward CXCL12 (SDF-1α, PeproTech) was evaluated via Transwell inserts with 3 µm pore polyester membranes. DiD-labeled neutrophils or CL-NPs@NEs (10⁶ cells mL − 1 ) were seeded into the upper chamber, and 10 ng of CXCL12 (final concentration: 20 ng mL − 1 ) was added to the lower chamber. After 1 h, fluorescence images of the lower chamber were captured via an inverted fluorescence microscope (AIR, Nikon). The fluorescence intensity was analyzed via ImageJ software. For wells containing the CXCR4 antagonist plerixafor (AMD3100, PeproTech), neutrophils were preincubated with 1 mg mL − 1 AMD3100 for 15 minutes before performing the migration assay, following the same procedure as described above. Immunohistochemistry Femurs from OVX and SENILE mice were collected for immunohistochemical analysis. The samples were assigned to four groups: OVX-NC, OVX-B3, SENILE-NC, and SENILE-B3. The bone tissue sections were dewaxed and rehydrated. The sections were immersed in EDTA buffer and incubated overnight at 60°C for antigen retrieval. Following antigen retrieval, sections from the OVX-B3 and SENILE-B3 groups were incubated overnight at 4°C with a primary antibody against the β3-adrenergic receptor (Abcam, ab94506). Sections from the OVX-NC and SENILE-NC groups were incubated with an isotype control rabbit IgG polyclonal antibody (Abcam, ab37415) under the same conditions. All the sections were subsequently incubated with the corresponding secondary antibodies at room temperature for 1 hour. Images were finally captured via a light microscope. Immunofluorescence Femurs from the mice were collected for immunofluorescence analysis. Bone tissue sections were deparaffinized and rehydrated, followed by antigen retrieval via EDTA buffer. The sections were immersed in EDTA buffer and incubated overnight at 60°C for antigen retrieval. The samples were then incubated with primary antibodies against CD31 (Abcam, ab104854) and Emcn (Proteintech, 13440S) at 4°C overnight. Afterward, the sections were incubated at room temperature for 1 hour with the secondary antibodies anti-mouse Alexa 488 (Cell Signaling Technology, 4408) and anti-rabbit Alexa 555 (Cell Signaling Technology, 4413). DAPI antifade mounting medium (Beyotime, P0131) was used for mounting. Images were captured via a Zeiss LSM 880 confocal microscope. Histological Staining Bone tissues were collected and fixed in 4% paraformaldehyde (PFA) at 4°C overnight, followed by decalcification and paraffin embedding. The tissue sections were subjected to hematoxylin and eosin (H&E, Boster, AR1180), Masson's trichrome (Solarbio, G1340-100), and tartrate-resistant acid phosphatase (TRAP) staining. IVIS Isolated neutrophils were cultured in RPMI 1640 medium, followed by the addition of 5 µM DiD (T15118, Targetmol) at a concentration of 10 6 cells mL − 1 . The cells were incubated for 15 minutes, and unbound DiD was removed by washing three times with PBS. The resulting DiD-labeled neutrophils were injected into the mice via the tail vein. Two groups of neutrophils were used in the experiment: one group was isolated and cultured for 6 hours (age: 6 hours), and the other was cultured for 24 hours (age: 24 hours). The in vivo distributions of labeled neutrophils and free DiD were examined via an in vivo imaging system (IVIS) at 2 hours and 6 hours postinjection. Micro-CT The femurs were analyzed via a micro-CT scanner (SKYSCAN 1276). Images and key bone parameters, such as the bone volume fraction (BV/TV) and trabecular separation (Tb.Sp), were obtained via CTAn and DataViewer software. The data were analyzed with GraphPad Prism 8 software. Statistical analysis All the data are presented as representative results from multiple independent experiments. Data comparisons were made via unpaired two-tailed t tests and one-way analysis of variance tests in GraphPad Prism. The outcomes are presented as the means ± SDs. ns = statistically nonsignificant, *p < 0.05, **p < 0.01 and ***p < 0.001 . Declarations Ethics Approval and Patient Consent Statement: This work had complied with all relevant ethical regulations for clinical samples and animalresearch. Bone marrow and cellular specimens were obtained from patients undergoing orthopedic surgery with or without osteoporosis. The human study of this research was conducted in accordance with theprinciples expressed in the Declaration of Helsinki and was approved by the ethical committee of The Eighth Affiliated Hospital of SunYat-Sen University (approval number: 2021r208). Written informedconsent was obtained from each enrolled patient. All animal experimentswere performed in the Central of Experimental Animal SunYat-Sen University. The animal experiments were conducted accordingto the protocol (approval number: 2023000937, 2023002888, 2024001423) authorized by the Experi-mental Animal Welfare Ethics Committee, SunYat-Sen University. Acknowledgments C. Wu, J. Zhao and Q. Li contributed equally to this work. G. Zheng, Z. Xie and W. Yu conceived designed and supervised the study. C. Wu, J. Zhao and Q. Li performed the experiments and analyzed the data. W. Zhang, S Meng, H. Liu, Y. Zeng, X. Ning, P. Wang and Y. Wu provided suggestions. C. Wu, J. Zhao, Q. Li and W. Zhang wrote the manuscript, and G. Zheng, Z. Xie, W. Yu and Y. Wu revised the manuscript. This work was supported by the Shenzhen Science and Technology Program (RCBS20221008093103013, JCYJ20220530144016039), the National Natural Science Foundation of China (82102529, 82402764), the Guangdong Natural Science Foundation (2023A1515010226, 2023A1515111078) and the Shenzhen Medical Research Fund (A2403047). Corresponding author Correspondence and requests for materials should be addressed to WenHui Yu, Zhongyu Xie or Guan Zheng. Conflict of interest The authors declare no conflict of interest. Data Availability Statement The data that support the findings of this study are available from the corresponding author upon reasonable request. Supporting Information Supporting Information is available from the Wiley Online Library or from the author. References Ayers C, Kansagara D, Lazur B et al (2023) Effectiveness and safety of treatments to prevent fractures in people with low bone mass or primary osteoporosis: A living systematic review and network meta-analysis for the american college of physicians. Ann Intern Med 176:182-195. https://doi.org/10.7326/M22-0684. Libman H, Yu EW, Malabanan AO et al (2025) How would you manage this patient with decreased bone density? Grand rounds discussion from beth israel deaconess medical center. 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10:51:00","extension":"html","order_by":19,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":163082,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7569697/v1/bb5fcc2aeb5ddc1c9c039479.html"},{"id":92165849,"identity":"4dc4cb5f-e251-4355-a7ab-3398c9f13598","added_by":"auto","created_at":"2025-09-25 10:58:59","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":597303,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCLR activation reduces marrow adiposity and improves bone microstructure in osteoporotic mice a\u003c/strong\u003e, Immunohistochemical staining of femoral bone marrow adipocytes in ovariectomized (OVX) and aged (SENILE) mouse models. Scale bar: 50 μm. \u003cstrong\u003eb\u003c/strong\u003e, Schematic representation of the treatment regimens applied to the OVX and SENILEC57BL/6 mouse models. \u003cstrong\u003ec-d\u003c/strong\u003e, Representative hematoxylin and eosin (H\u0026amp;E)-stained images of brown adipose tissue (\u003cstrong\u003ec\u003c/strong\u003e) and inguinal white adipose tissue (\u003cstrong\u003ed\u003c/strong\u003e) collected at the end of the experimental period. Scale bars: 100 μm(c) and 200 μm(d). \u003cstrong\u003ee\u003c/strong\u003e, H\u0026amp;E staining of distal femoral bone sections at the endpoint, illustrating the bone microarchitecture and marrow adiposity. Scale bar: 500 μm. \u003cstrong\u003ef-g\u003c/strong\u003e, Quantitative analysis of adipocyte lipid droplet marrow occupancy (AV/MV, \u003cstrong\u003ef\u003c/strong\u003e) and the mean lipid droplet volume per adipocyte (AV/NA, \u003cstrong\u003eg\u003c/strong\u003e) (n = 6). \u003cstrong\u003eh-i\u003c/strong\u003e, Representative microcomputed tomography (micro-CT) images and three-dimensional reconstructions of distal femoral bones from the experimental groups. \u003cstrong\u003ej-n\u003c/strong\u003e, Quantitative evaluation of bone morphometric parameters, including the bone volume fraction (BV/TV, \u003cstrong\u003ej\u003c/strong\u003e), the bone surface-to-volume ratio (BS/BV, \u003cstrong\u003ek\u003c/strong\u003e), the trabecular thickness (Tb.Th, \u003cstrong\u003el\u003c/strong\u003e), the trabecular number (Tb.N, \u003cstrong\u003em\u003c/strong\u003e), and the trabecular pattern factor (Tb.Pf, \u003cstrong\u003en\u003c/strong\u003e) (n = 6). The outcomes are presented as the means ± SDs. ns = statistically nonsignificant, \u003cem\u003e*p\u0026lt;0.05, **p\u0026lt;0.01 and ***p\u0026lt;0.001\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7569697/v1/38e78c1c27e847897589e907.png"},{"id":92164942,"identity":"4eadd6a0-5e3e-4105-8000-b4a5eea3c1a0","added_by":"auto","created_at":"2025-09-25 10:50:59","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":733987,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of CL on Bone Metabolism.\u003c/strong\u003e \u003cstrong\u003ea\u003c/strong\u003e, Serum samples were collected at designated time points before the end of the experiment to evaluate the expression of bone turnover markers, including the C-terminal telopeptide of type I collagen (CTX-1) and procollagen type I N-terminal propeptide (P1NP) (n = 6). \u003cstrong\u003eb\u003c/strong\u003e, Representative images of tartrate-resistant acid phosphatase (TRAP) staining and Masson's trichrome staining of femoral sections to assess osteoclastic activity and collagen deposition. Scale bar: 500 μm. \u003cstrong\u003ec\u003c/strong\u003e, Quantification of osteoclasts within the specificallymarked areas (black circles) in representative TRAP-stained images (n=6). \u003cstrong\u003ed\u003c/strong\u003e, Immunofluorescence staining of H-type vessels in the femur with CD31 (green), endomucin (EMCN, red), and DAPI (blue) to assess the angiogenic response. Scale bar: 500 μm. \u003cstrong\u003ee\u003c/strong\u003e, Quantification of H-type vessels in the femur on the basis of colocalization of CD31 and EMCN signals (n = 6). \u003cstrong\u003ef\u003c/strong\u003e, Schematic workflow for evaluating the effects of CL-stimulated bone marrow adipocytes on osteogenic and osteoclastic differentiation in vitro. \u003cstrong\u003eg-h\u003c/strong\u003e, Alizarin Red S staining of mineralized nodules (g) following osteogenic induction in bone marrow adipocytes pretreated with various concentrations of CL. Quantitative analysis of Alizarin Red S staining (h) was performed to assess the impact of CL-preconditioned adipocytes on osteogenic differentiation (n = 6). Scale bar: 200 μm. \u003cstrong\u003ei-j\u003c/strong\u003e, TRAP staining of osteoclasts (i) following osteoclastogenic induction in bone marrow adipocytes pretreated with various concentrations of CL. TRAP staining (j) was used to assess the impact of CL-preconditioned adipocytes on osteoclastic differentiation (n = 6). Scale bar: 500 μm. \u003cstrong\u003ek‒l\u003c/strong\u003e A tube formation assay was performed using HUVECs cultured with conditioned media from bone marrow adipocytes pretreated with different concentrations of CL to assess angiogenic parameters (n = 6). Scale bar: 500 μm. The outcomes are presented as the means ± SDs. ns = statistically nonsignificant, \u003cem\u003e*p\u0026lt;0.05, **p\u0026lt;0.01 and ***p\u0026lt;0.001\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7569697/v1/6030adf956d1be9bd026e781.png"},{"id":92164943,"identity":"6ebafd3d-0c59-40a2-ac10-87993aecb59d","added_by":"auto","created_at":"2025-09-25 10:50:59","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":304327,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSystemic metabolic effects of CL treatment in osteoporotic and aged mice.\u003c/strong\u003e \u003cstrong\u003ea,\u003c/strong\u003eRepresentative images comparing body weight changes among groups after 30 days of drug administration (n = 6). \u003cstrong\u003eb,\u003c/strong\u003e Photographs of abdominal and inguinal adipose tissues collected at the end of the experiment to assess adipose tissue weights. \u003cstrong\u003ec-d,\u003c/strong\u003e Quantitative analysis of the weights of inguinal (c) and abdominal (d) adipose tissues (n = 6). \u003cstrong\u003ee-i,\u003c/strong\u003e Serum samples were collected at designated time points before the end of the experiment to evaluate systemic metabolic indicators. Enzyme-linked immunosorbent assay (ELISA) was used to measure the circulating levels of interleukin-6 (IL-6) (e), nonesterified fatty acids (NEFAs) (f), insulin (g), leptin (h), and glucose (i) (n = 6). The outcomes are presented as the means ± SDs. ns = statistically nonsignificant, \u003cem\u003e*p\u0026lt;0.05, **p\u0026lt;0.01 and ***p\u0026lt;0.001\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7569697/v1/06ceb041ff7ddea57d419add.png"},{"id":92164946,"identity":"eccb9a7a-7f36-44d2-bb45-6e1c68e8f0f2","added_by":"auto","created_at":"2025-09-25 10:50:59","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":499833,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCharacterization and biodistribution of CL-NPs@NEs. a\u003c/strong\u003e, Dynamic light scattering (DLS) analysis showing the hydrodynamic size distribution of CL-loaded PLGA nanoparticles (CL-NPs). \u003cstrong\u003eb\u003c/strong\u003e, Transmission electron microscopy (TEM) image of CL-NPs, revealing a uniform spherical morphology. Scale bars: 500 μm and 100 μm. \u003cstrong\u003ec\u003c/strong\u003e, Cumulative in vitro release profile of CL from PLGA nanoparticles over 10 days, as measured by high-performance liquid chromatography (HPLC). \u003cstrong\u003ed\u003c/strong\u003e, Confocal laser scanning microscopy image of CL-NPs@NEs. DAPI was used to stain the nuclei (blue), DiD was used to label the neutrophil membranes (red), and FITC was used to stain the CL-NPs (green). Scale bar: 5 μm. \u003cstrong\u003ee\u003c/strong\u003e, TEM image of CL-NPs@NEs, with arrows indicating encapsulated CL-NPs within neutrophils. Scale bar: 500 μm. \u003cstrong\u003ef-g\u003c/strong\u003e, Quantitative analysis of neutrophil or CL-NPs@NEs (labeled with DiD) migration toward the lower chamber in the presence or absence of CXCL12 (20 ng mL\u003csup\u003e−1\u003c/sup\u003e) and AMD3100 (1 mg mL\u003csup\u003e−1\u003c/sup\u003e) (n = 6). \u003cstrong\u003eh\u003c/strong\u003e, In vivo imaging system (IVIS) images demonstrating the biodistribution of free DiD-labeled neutrophils at 2 and 6 hours postinjection (6 and 24 hours postisolation). \u003cstrong\u003ei\u003c/strong\u003e, Ex vivo fluorescence images of major organs (bone, liver, and spleen) from panel h. \u003cstrong\u003ej\u003c/strong\u003e, Quantitative comparison of the relative radiotracer intensities in the bone, liver, and spleen shown in i (n = 6). \u003cstrong\u003ek\u003c/strong\u003e, IVIS imaging showing the biodistribution of DiD-labeled CL-NPs@NEs or free DiD-CL-NPs at 2 h postinjection. \u003cstrong\u003el\u003c/strong\u003e, Corresponding ex vivo fluorescence images of bone, liver, and spleen from panel k. \u003cstrong\u003em\u003c/strong\u003e, Quantitative analysis of relative fluorescence intensity in bone, liver, and spleen from l (n = 6). All experiments were independently repeated at least six times with consistent results. The outcomes are presented as the means ± SDs. ns = statistically nonsignificant, \u003cem\u003e*p\u0026lt;0.05, **p\u0026lt;0.01 and ***p\u0026lt;0.001\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7569697/v1/b6fa53155b62ce978674c922.png"},{"id":92166234,"identity":"84f8f69f-2b45-4067-ad89-00ffd44f4d7c","added_by":"auto","created_at":"2025-09-25 11:06:59","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":533172,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIn vivo therapeutic evaluation of CL-NPs@NEs. a\u003c/strong\u003e, Treatment schemeof CL-NPs@NEs in OVX C57BL/6 mice. \u003cstrong\u003eb\u003c/strong\u003e, Representative hematoxylin and eosin (H\u0026amp;E) staining images of distal femoral bone sections collected at the endpoint of treatment. Scale bar: 500 μm. \u003cstrong\u003ec\u003c/strong\u003e, Quantitative analysis of the adipocyte lipid droplet volume per adipocyte (AV/NA) and bone marrow occupancy (AV/MV) in the femoral bone marrow (n = 6). \u003cstrong\u003ed-e\u003c/strong\u003e, Representative microcomputed tomography (micro-CT) images and corresponding 3D reconstructions of femoral trabecular bone architecture. \u003cstrong\u003ef-j\u003c/strong\u003e, Quantitative micro-CT-based evaluation of bone parameters, including the bone volume fraction (BV/TV, f), bone surface-to-volume ratio (BS/BV, g), trabecular thickness (Tb.Th, h), trabecular number (Tb.N, i), and trabecular pattern factor (Tb.Pf, j) (n = 6). \u003cstrong\u003ek\u003c/strong\u003e, Representative images of inguinal and visceral adipose tissues harvested at the end of the experiment. \u003cstrong\u003el-m\u003c/strong\u003e, Quantification of inguinal (l) and visceral (m) adipose tissue weights (n = 6). \u003cstrong\u003en-r\u003c/strong\u003e, Serum levels of inflammatory and metabolic indicators, including IL-6 (n), leptin (o), nonesterified fatty acids (NEFAs, p), insulin (q), and glucose (r), were measured at designated time points before the endpoint (n = 6). The outcomes are presented as the means ± SDs. ns = statistically nonsignificant, \u003cem\u003e*p\u0026lt;0.05, **p\u0026lt;0.01and ***p\u0026lt;0.001.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-7569697/v1/5597f7698a532c853706fbbd.png"},{"id":92165850,"identity":"70781a1a-361c-47ec-b4ec-f6487cea719d","added_by":"auto","created_at":"2025-09-25 10:58:59","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":410284,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCombination Therapy with CL and Teriparatide\u003c/strong\u003e. \u003cstrong\u003ea\u003c/strong\u003e, Schematic diagram of the treatment regimen applied to C57BL/6 mice in the OVX model. \u003cstrong\u003eb\u003c/strong\u003e, Representative hematoxylin and eosin (H\u0026amp;E) staining images of distal femoral sections collected at the end of treatment. Scale bar: 500 μm. \u003cstrong\u003ec-d\u003c/strong\u003e, Quantitative analysis of lipid droplet volume per adipocyte (AV/NA) and the percentage of bone marrow volume occupied by adipocyte lipid droplets (AV/MV) (n = 6). \u003cstrong\u003ee\u003c/strong\u003e, Representative microcomputed tomography (micro-CT) images and three-dimensional (3D) reconstructions of femoral trabecular bone. \u003cstrong\u003ef-j\u003c/strong\u003e, Quantitative evaluation of bone morphometric parameters, including the bone volume fraction (BV/TV, f), bone surface-to-volume ratio (BS/BV, g), trabecular number (Tb.N, h), trabecular thickness (Tb.Th, i), and trabecular pattern factor (Tb.Pf, j) (n = 6). The outcomes are presented as the means ± SDs. ns = statistically nonsignificant, \u003cem\u003e*p\u0026lt;0.05, **p\u0026lt;0.01 and ***p\u0026lt;0.001\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-7569697/v1/b8de2128e9ec1e035f30be17.png"},{"id":92165853,"identity":"1ec64f83-b859-47af-b185-5edd717f3aa9","added_by":"auto","created_at":"2025-09-25 10:58:59","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":176223,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSchematic diagram of CLR agonist therapy and targeted therapy for osteoporosis. \u003c/strong\u003eBy leveraging neutrophil-mediated hitchhiking as a bone marrow-targeted delivery platform, we demonstrate that localized \u003cstrong\u003eCLR\u003c/strong\u003e agonism effectively reverses skeletal deterioration in osteoporosis by promoting lipolysis, improving bone remodeling and promoting angiogenesis. Critically, this targeted approach circumvents systemic metabolic perturbations typically reduced by global CLR activation.\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-7569697/v1/6fad648b95f8890018a17b04.png"},{"id":92756078,"identity":"d82808db-90ec-4cfe-b0cb-379e03c47417","added_by":"auto","created_at":"2025-10-04 03:16:39","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4489545,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7569697/v1/88e08d7b-df0e-4379-be6c-1f3d64720a31.pdf"},{"id":92164941,"identity":"899d48aa-4be6-4599-acd8-b5c7dab7983c","added_by":"auto","created_at":"2025-09-25 10:50:59","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":438560,"visible":true,"origin":"","legend":"","description":"","filename":"SupportingInformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-7569697/v1/6945f00c0c37a705ef1e5216.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Bone marrow-targeted delivery of canonical lipolytic receptor agonists via neutrophil hitchhiking reverses osteoporosis","fulltext":[{"header":"Introduction","content":"\u003cp\u003eOsteoporosis has emerged as an increasingly severe global public health challenge. As the world's population ages more rapidly, the incidence of fragility fractures is increasing significantly[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Therapeutic strategies for osteoporosis have remained largely unchanged for decades, with a focus on either inhibiting osteoclast activity or intermittently stimulating osteoblast function[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. While bisphosphonates and RANKL inhibitors prevent further bone loss, they fail to reverse established microarchitectural damage, which is the principal determinant of fracture risk[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. In recent years, the relationship between lipid metabolism and bone metabolism has attracted increasing attention. Lipid metabolism dysregulation, through its impact on the homeostasis of the bone marrow microenvironment, has been identified as a significant factor in the development of osteoporosis[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Bone marrow adipocytes (BMAds) are a crucial cell type in the bone marrow microenvironment; they regulate bone remodeling by secreting various cytokines and adipokines and directly affect the function of other cells in the bone marrow[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Emerging evidence reveals that bone marrow adipocytes actively suppress osteogenesis through paracrine signaling, establishing marrow adipose tissue (BMAT) as a previously overlooked but critical therapeutic target[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. This paradigm shift calls for innovative approaches that can address both the cellular origin of impaired bone formation and the pharmacological challenges of targeting marrow compartments simultaneously.\u003c/p\u003e\u003cp\u003eβ3 Adrenergic receptor (β3AR), a canonical lipolytic receptor (CLR), has emerged as a potential target for modulating bone marrow adiposity because of its specific expression pattern and metabolic effects[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Unlike other canonical lipolytic receptor subtypes (β1 and β2), β3AR is preferentially expressed in adipocytes[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. CL316243 (CL) is a CLR agonist in phase II clinical trials that primarily targets β3-AR[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e] and shows therapeutic promise for obesity and type II diabetes through enhanced insulin sensitivity and fat oxidation[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Notably, the CL remodels bone marrow adipose tissue via CLR activation, although its potential to concomitantly improve osteogenesis and the osteoporotic microenvironment requires further investigation. While effective for weight loss, the systemic administration of CLs faces clinical limitations, including cardiovascular/metabolic side effects and poor oral bioavailability, highlighting the need for targeted delivery approaches[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eTargeted drug delivery to the bone marrow faces persistent challenges due to physiological barriers such as the marrow‒blood barrier, which restricts passive diffusion[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], whereas conventional bisphosphonate-conjugated nanoparticles exhibit limited efficacy[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Neutrophils provide an effective alternative through their natural vascular penetration and bone marrow homing abilities[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. These phagocytes exploit chemotactic signals and constitutive marrow tropism, enabling efficient drug transport[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Recent neutrophil-mediated delivery strategies, such as nanoparticle hitchhiking via phagocytosed PLGA carriers, leverage these natural migratory pathways without artificial modification. This approach has proven effective in osseous diseases, including osteoporosis and bone metastasis, through targeted parathyroid hormone and chemotherapy delivery[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. By merging neutrophil biology with nanomedicine, this strategy overcomes critical barriers-penetration and clearance-ushering in biologically inspired, precision therapies for bone disorders.\u003c/p\u003e\u003cp\u003eHere, we present a therapeutic platform that integrates three recent innovations: first, the discovery that CLR activation reprograms marrow adipocyte metabolism to create a pro-osteogenic microenvironment; second, the development of an autologous neutrophil vehicle capable of bypassing physiological barriers to deliver payloads specifically to the bone marrow; and third, the dual-drug combination demonstrates significantly greater therapeutic efficacy than either agent alone, achieving synergistic effects that transcend conventional \u0026ldquo;antiresorptive versus anabolic\u0026rdquo; paradigms. This combinatorial approach uniquely restores bone marrow homeostasis through concurrent modulation of osteoblast‒osteoclast crosstalk and adipocyte signaling pathways.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eCLR activation reduces marrow adiposity and improves bone microstructure in osteoporotic mice\u003c/h2\u003e\u003cp\u003eTo investigate the effects of CLR agonists (CL316243, CL) on bone marrow adipocyte lipolysis and bone formation, we first performed immunohistochemical analysis on femoral sections from ovariectomized (OVX) and aged (24-month-old, SENILE) mice, which revealed CLR-β3AR expression on bone marrow adipocyte membranes in OVX-B3 and SENILE-B3 mice relative to isotype control (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). We subsequently established osteoporosis models in 8-week-old OVX C57BL/6 female mice and SENILE mice. The animals received daily intraperitoneal CL injections (1 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) for 30 days before euthanasia (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). HE staining revealed significant decreases in brown adipose tissue lipid droplet and bone marrow adipocyte parameters in the CL-treated groups compared with those in the controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec, d). Specifically, the adipocyte lipid droplet volume (AV/NA) was reduced by 23.3% and 26.1% in the respective groups (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e), whereas the marrow adipocyte proportion (AV/MV) was decreased by 53.3% in the OVX-CL groups (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e) and 47.8% in the SENILE-CL groups (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee-g). Micro-CT analysis revealed substantial improvements in the trabecular bone parameters in the OVX-CL group compared with those in the OVX-NC group, including an 82.4% increase in the BV/TV (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e), a 74.5% increase in the BS/TV (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e), a 57.0% increase in the Tb.N (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.01\u003c/em\u003e), a 16.9% increase in the Tb.Th (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.01\u003c/em\u003e), and a 23.6% decrease in the Tb.Pf (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.01\u003c/em\u003e) (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eh-n). The sham group showed no significant changes (\u003cem\u003ep\u0026thinsp;\u0026gt;\u0026thinsp;0.05\u003c/em\u003e), likely due to baseline physiological differences, whereas the SENILE-CL group exhibited improvements comparable to those of the OVX-CL group (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e vs SENILE-NC). Collectively, our data demonstrate that CL not only diminishes marrow fat content but also augments bone formation, suggesting a β3-AR-dependent lipolytic mechanism within marrow adipocytes.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eEffects of CL on Bone Metabolism\u003c/h3\u003e\n\u003cp\u003eSerological and histological analyses revealed significant changes in the levels of bone metabolism markers following CL treatment. Moreover, peripheral blood analysis revealed elevated levels of both the bone resorption marker CTX-1 (OVX-CL: 12.900\u0026thinsp;\u0026plusmn;\u0026thinsp;1.295 vs OVX-NC: 10.300\u0026thinsp;\u0026plusmn;\u0026thinsp;0.707 ng mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, \u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.01\u003c/em\u003e; SENILE-CL: 13.043\u0026thinsp;\u0026plusmn;\u0026thinsp;1.031 vs SENILE-NC: 10.600\u0026thinsp;\u0026plusmn;\u0026thinsp;1.409 ng mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, \u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.01\u003c/em\u003e) and the bone formation marker P1NP (OVX-CL: 17.027\u0026thinsp;\u0026plusmn;\u0026thinsp;1.500 vs OVX-NC: 10.417\u0026thinsp;\u0026plusmn;\u0026thinsp;1.726 ng mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, \u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e; SENILE-CL: 17.183\u0026thinsp;\u0026plusmn;\u0026thinsp;1.420 vs SENILE-NC: 11.100\u0026thinsp;\u0026plusmn;\u0026thinsp;1.556 ng mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, \u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e) after CL treatment, indicating enhanced bone remodeling activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). Histomorphometric analysis revealed (1) increased osteoclast numbers via TRAP staining (SH-CL: 19.567\u0026thinsp;\u0026plusmn;\u0026thinsp;1.494 vs SH-NC: 15.500\u0026thinsp;\u0026plusmn;\u0026thinsp;1.747 cells mm\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e, \u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e; OVX-CL: 26.067\u0026thinsp;\u0026plusmn;\u0026thinsp;1.098 vs OVX-NC: 21.567\u0026thinsp;\u0026plusmn;\u0026thinsp;1.592 cells mm\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e, \u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.01\u003c/em\u003e; SENILE-CL: 29.200\u0026thinsp;\u0026plusmn;\u0026thinsp;1.539 vs SENILE-NC: 14.400\u0026thinsp;\u0026plusmn;\u0026thinsp;1.464 cells mm\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e, \u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e) and (2) enhanced bone matrix formation via Masson's trichrome staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb,c). Angiogenesis analysis revealed CL-induced increases in CD31\u003csup\u003e+\u003c/sup\u003eEmcn\u003csup\u003e+\u003c/sup\u003e H-type vessel density (OVX-CL: 0.980\u0026thinsp;\u0026plusmn;\u0026thinsp;0.118 vs OVX-NC: 0.435\u0026thinsp;\u0026plusmn;\u0026thinsp;0.121 vessel intensity, \u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e; SENILE-CL: 0.892\u0026thinsp;\u0026plusmn;\u0026thinsp;0.134 vs SENILE-NC: 0.492\u0026thinsp;\u0026plusmn;\u0026thinsp;0.092 vessel intensity, \u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.01\u003c/em\u003e) in the femoral metaphysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed, e). In vitro experiments using adipocytes derived from human bone marrow in CL-conditioned media at different concentrations revealed that 5 \u0026micro;M adipocyte-CL-conditioned supernatants significantly enhanced MSC osteogenesis (ALP activity: 1.35-fold increase on day 21, \u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eg, i) and promoted osteoclastogenesis from CD14\u003csup\u003e+\u003c/sup\u003e cells (TRAP\u003csup\u003e+\u003c/sup\u003e multinucleated cells: 1.42-fold increase vs control, \u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eh, j). Additionally, within 4h in the in vitro H-type vessel formation assay, 5 \u0026micro;M adipocyte-CL-conditioned supernatants significantly increased both the total length and total branching length of the capillary networks formed by HUVECs compared with those formed by the other concentrations (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ek, l) (Supplementary Fig.\u0026nbsp;1). These results demonstrate that CL modulates bone metabolism through the coordinated regulation of osteoblast‒osteoclast coupling and angiogenic‒osteogenic coupling, which is mediated by adipocyte-derived factors.\u003c/p\u003e\n\u003ch3\u003eAdverse metabolic and cardiovascular effects of systemic CL administration\u003c/h3\u003e\n\u003cp\u003eAlthough CL promotes bone formation in OVX mice, systemic CLR activation can trigger detrimental effects across multiple physiological systems. Cardiac dysfunction may arise from disrupted calcium homeostasis, including sarcoplasmic reticulum (Ca\u0026sup2;⁺) leakage, which impairs myocardial contractility (negative inotropy)[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Additionally, CL-induced nitric oxide (NO) overproduction and excessive lipolysis may contribute to cardiac lipotoxicity, particularly under pathological conditions such as sepsis[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn our experiments, CL administration led to significant weight loss and fat depletion. Specifically, compared with the OVX-NC controls, the OVX-CL mice lost 4.007\u0026thinsp;\u0026plusmn;\u0026thinsp;0.52 g (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e) by day 30, whereas the aged SENILE-CL mice exhibited a reduction of 2.491\u0026thinsp;\u0026plusmn;\u0026thinsp;0.41 g (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). Histological analysis confirmed substantial decreases in abdominal and inguinal adipose tissue mass (approximately 38\u0026ndash;41%, \u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb-d), which may negatively impact skeletal health, as peripheral fat reserves are inversely correlated with osteoporosis risk[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Moreover, rapid adipose tissue loss, particularly subcutaneous fat loss, has been linked to impaired wound healing, raising concerns for elderly and diabetic populations[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eSystemic metabolic disturbances were evident within just 4 hours of CL exposure. Blood glucose levels declined sharply (SH-CL: \u0026minus;\u0026thinsp;11.81%, \u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e; OVX-CL: \u0026minus;\u0026thinsp;9.83%, \u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.01\u003c/em\u003e; SENILE-CL: \u0026minus;\u0026thinsp;12.58%, \u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e), concurrent with a 20\u0026ndash;40% reduction in leptin (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e). In contrast, circulating nonesterified fatty acids (NEFAs, 1.38- to 2.10-fold increase, \u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.01\u003c/em\u003e), insulin (1.50- to 1.96-fold, \u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e), and interleukin-6 (IL-6, 1.66- to 2.00-fold increase; all \u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e) were markedly elevated, indicating dysregulated lipolysis and a proinflammatory response (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed-i).\u003c/p\u003e\u003cp\u003eTaken together, these findings reveal that while CL exerts anabolic effects on bone, its systemic actions, which involve cardiac stress, adipose tissue atrophy, and metabolic disarray, impose significant limitations for therapeutic use in osteoporosis and aging-related conditions.\u003c/p\u003e\n\u003ch3\u003eTargeted Bone Marrow Delivery of CL to Bone Marrow via Senescent Neutrophils\u003c/h3\u003e\n\u003cp\u003eRecent studies have demonstrated that senescent neutrophils possess a natural ability to migrate back to the bone marrow, presenting a unique opportunity for targeted delivery[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Capitalizing on this intrinsic homing mechanism, we developed a neutrophil-mediated delivery system for CLs designed to minimize peripheral tissue exposure. The CL-loaded PLGA nanoparticles (CL-NPs) were engineered to exhibit optimal characteristics, including an average diameter of 230 nm (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea), uniform morphology (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb), and a sustained drug release profile that delivered 28% of the payload within 48 hours, with nearly complete release achieved over 10 days (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec) (Supplementary Fig.\u0026nbsp;2). After neutrophils were cocultured with 3 mg/L CL-NPs, validation studies confirmed the rapid and efficient uptake of FITC-labeled CL-NPs by neutrophils (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed), with complete internalization within 2 h to form CL-NPs@NEs complexes (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee). HPLC analysis confirmed an effective drug payload of 2.5 \u0026micro;g CL per 10^6 cells, ensuring therapeutic efficacy. Functional characterization revealed time-dependent upregulation of CXCR4 on CL-NPs@NEs (Supplementary Fig.\u0026nbsp;3) while maintaining robust chemotactic responses to CXCL12 (SDF-1α) beyond 6 h in culture. Crucially, the CXCR4 antagonist AMD3100 abrogated this chemotaxis, confirming an uncompromised bone marrow homing capacity (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ef, g) (Supplementary Fig.\u0026nbsp;4).\u003c/p\u003e\u003cp\u003eIn vivo biodistribution studies using DiD-labeled NEs (6 h) revealed remarkable targeting efficiency, with detectable bone marrow accumulation within 2 h that progressively increased through 6 h postinjection compared with that of free DiD, which was absorbed mainly by the liver and spleen, with minimal accumulation in the bone marrow (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eh-j). However, compared with DiD-labeled NEs (6 h), DiD-labeled NEs (24 h) in the bone marrow were reduced through 6 h postinjection, likely due to neutrophil apoptosis in the spleen and liver. Additionally, compared with CL-NPs, CL-NPs@NEs exhibited superior targeting to the bone marrow. Compared with CL-NPs, CL-NPs@NEs demonstrated approximately 1.80-fold greater bone marrow accumulation while significantly reducing off-target deposition in adipose tissues and major organs (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ek-m). Notably, we observed prolonged retention in the bone marrow associated with delayed neutrophil apoptosis, in contrast to the rapid clearance observed with free formulations through hepatic and splenic pathways. These results suggest that CL-NPs@NEs effectively target the bone marrow while minimizing peripheral tissue exposure.\u003c/p\u003e\n\u003ch3\u003eIn Vivo Evaluation of CL-NPs@NEs in Osteoporotic Mice\u003c/h3\u003e\n\u003cp\u003eTo evaluate the in vivo regulatory effects of neutrophil-mediated CL delivery (CL-NPs@NEs), an osteoporosis model was established in 8-week-old female C57BL/6 mice subjected to OVX, followed by a 30-day recovery period. The mice were then randomly assigned to five groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). Our preclinical evaluation in this model demonstrated the superior therapeutic profile of neutrophil-derived CL-NPs@NEs. The results of HE staining revealed that, compared with that in the OVX groups, the number of bone marrow adipocytes in the CL, CL-NPs and CL-NPs@NEs groups was significantly lower (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). Compared with OVX, CL-NPs@NEs treatment reduced the adipocyte lipid droplet volume per unit area (AV/NA) by approximately 30.87% (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.01\u003c/em\u003e), outperforming both free CL (20.87% reduction) and CL-NPs (19.35% reduction) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec). Similarly, the adipocyte volume fraction (AV/MV) was decreased by 35.37% in the CL-NPs@NEs group relative to that in the OVX group (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e), representing a 6.90\u0026ndash;9.57% greater reduction than that of the alternative CL formulations (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec). Compared with OVX, CL-NPs@NEs treatment markedly improved the following trabecular bone parameters: the bone volume fraction (BV/TV) increased by 101.12% (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e), the trabecular thickness (Tb.Th) increased by 39.84% (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.01\u003c/em\u003e), and the trabecular number (Tb.N) increased by 62.46% (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.01\u003c/em\u003e). Trabecular spacing (Tb.Pf) decreased by 37.28% (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed-j).\u003c/p\u003e\u003cp\u003eNotably, this targeted delivery approach largely avoids the adverse metabolic effects observed with systemic CL administration. CL-NPs@NEs maintained the peripheral adipose tissue mass at OVX control levels, in contrast to the 28.41\u0026ndash;43.06% fat loss observed with conventional CL delivery (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ek‒m). Corresponding serum analyses revealed that the neutrophil-mediated strategy significantly attenuated CL-induced metabolic dysregulation, resulting in a 39.89% reduction in proinflammatory IL-6 levels, a 52.68% decrease in NEFAs, and preservation of basically normal insulin sensitivity and leptin regulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003en-r). These data collectively demonstrate that neutrophil-mediated delivery achieves superior bone-targeting specificity while preventing the characteristic systemic side effects of CLR agonist therapy.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eSynergistic Combination Therapy with CL and Teriparatide via Neutrophil-Mediated Delivery Enhances Osteoporosis Treatment\u003c/h2\u003e\u003cp\u003eDespite its osteogenic potential, CL also promotes osteoclast activity, which limits its monotherapeutic efficacy. To address this, we strategically combined CL with teriparatide (PTH) to foster a synergistic effect. Unlike purely antiresorptive agents (e.g., bisphosphonates or denosumab), PTH has dual-action properties: it stimulates osteoblast activity while modulating osteoclast function through RANKL/OPG regulation[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. This combination therapy may regulate both osteoblastic and osteoclastic activities, enabling balanced bone remodeling and improved therapeutic efficacy (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea).\u003c/p\u003e\u003cp\u003eOur neutrophil-mediated nanodelivery system (CL\u0026thinsp;+\u0026thinsp;PTH-NPs@NEs) demonstrated superior therapeutic effects in OVX mice compared with either agent alone. In the adipose tissue analysis, CL\u0026thinsp;+\u0026thinsp;PTH-NPs@NEs reduced AV/NA by 54.55% (0.025\u0026thinsp;\u0026plusmn;\u0026thinsp;0.004 vs OVX 0.056\u0026thinsp;\u0026plusmn;\u0026thinsp;0.016, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01), and CL\u0026thinsp;+\u0026thinsp;PTH-NPs@NEs decreased AV/MV by 48.52% (0.089\u0026thinsp;\u0026plusmn;\u0026thinsp;0.008 vs OVX 0.173\u0026thinsp;\u0026plusmn;\u0026thinsp;0.018, \u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb-d). These reductions were significantly greater than those resulting from either CL-NPs@NEs or PTH-NPs@NEs monotherapy (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e). Micro-CT analysis revealed that the combination therapy enhanced osteogenic effects: BV/TV increased 31.02% more than did PTH alone, Tb.N improved 23.72% more than did CL alone, and Tb.Pf was reduced by 18.55% compared with that of the monotherapies (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ee-j). These findings position CL\u0026thinsp;+\u0026thinsp;PTH-NPs@NEs as a promising next-generation therapy that may overcome current limitations in osteoporosis treatment by simultaneously enhancing bone formation while maintaining balanced remodeling activity.\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eOur findings significantly advance the understanding of canonical lipolytic signaling in bone metabolism by elucidating its spatial regulation within marrow microenvironments (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). While the lipolytic effects of CLR agonists are well characterized in metabolic tissues, their specific effects on bone marrow adipocytes remain unclear. Clinically, chronic CLR agonist administration may benefit bone metabolism through lipid depletion, glucose modulation, and altered adipokine secretion[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. In our study, CL treatment increased nonesterified fatty acid (NEFA) levels. These NEFAs, including palmitoleic acid (PLA) and oleic acid (OA), which increase osteoblast activity via FFAR4/β-arrestin2 signaling, may be produced via lipolysis and play pivotal roles in bone remodeling[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Notably, ω-3 polyunsaturated fatty acids such as docosahexaenoic acid (DHA) activate the Wnt/β-catenin and Akt pathways to stimulate bone formation[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Similarly, Zhang et al. demonstrated that DHA-containing lipids increase bone mineral density in ovariectomized mice by promoting chondrocyte-to-osteoblast transdifferentiation[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. In addition, NEFAs exhibit dual regulation of bone remodeling. While PLA/OA/DHA promote osteogenesis, arachidonic acid-derived prostaglandin E2 (PGE2) enhances osteoclastogenesis via EP2/EP4 receptor-mediated cAMP signaling and RANKL/OPG axis activation[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Conversely, palmitic acid both potentiates RANKL-induced osteoclast differentiation and independently triggers osteoclastogenesis[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Additionally, β3-adrenergic-stimulated adipocytes secrete zinc, which orchestrates bone homeostasis through various mechanisms: promoting osteogenesis via PI3K/Akt activation while suppressing osteoclast differentiation through GRB2/ERK inhibition[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Collectively, these mechanisms explain the CL-induced elevation of bone turnover markers, demonstrating balanced stimulation of both osteoblastic and osteoclastic activity that ultimately improves bone quality in osteoporosis.\u003c/p\u003e\u003cp\u003eWhile CL shows promise for osteoporosis treatment, systemic administration poses significant clinical challenges because of its multifaceted off-target effects. The drug\u0026rsquo;s potent lipolytic action risks compromising dermal adipose tissue function, a specialized fat depot essential for wound repair[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e] and antimicrobial defense[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. In our models, both ovariectomized and aged mice exhibited substantial weight loss and peripheral fat depletion changes that may impair wound healing and increase infection susceptibility. Such adipose reduction becomes particularly concerning in elderly populations with already diminished fat reserves, where further depletion may precipitate cell dysfunction through lipotoxicity and insulin resistance[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Concurrent metabolic disturbances emerged in our studies, including significantly elevated IL-6 and insulin alongside sharp decreases in leptin and glucose levels, mirroring previous clinical observations[\u003cspan additionalcitationids=\"CR39\" citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. These metabolic shifts may have dual negative consequences: potentiating osteoclast activity through increased CTX-1 while simultaneously suppressing appetite via leptin reduction[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e], thereby potentially undermining the desired anabolic bone effects. Clinical translation faces additional pharmacological hurdles, as evidenced by phase II trials revealing poor oral bioavailability and problematic β1-adrenergic receptor cross-reactivity[\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e] factors that help explain Kurabayashi\u0026rsquo;s paradoxical observation of reduced bone volume with BRL35135 treatment[\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Taken together, these findings highlight the critical need for the development of novel strategies that can preserve the osteoprotective benefits of CL while circumventing its systemic liabilities.\u003c/p\u003e\u003cp\u003eTo overcome the systemic side effects of conventional drug delivery, we developed a neutrophil-engineered (NE) nanoparticle system for bone marrow-specific CL delivery. Building upon emerging neutrophil-based drug delivery platforms[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], our approach leverages the unique biological properties of aged neutrophils, including their strong phagocytic capacity, CXCR4-mediated chemotaxis, and intrinsic immune evasion capabilities, to increase drug targeting while extending the circulation half-life. Previous studies have demonstrated the potential of neutrophil-mediated delivery: Xue et al. reported that neutrophil-carried liposomes could target invasive tumor cells[\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e], whereas Luo et al. utilized aged neutrophils to achieve high bone marrow drug accumulation in both metastatic cancer and osteoporosis models[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. The efficacy of this platform stems from the CXCR4/CXCL12 chemokine axis, where CXCR4-expressing neutrophils home to CXCL12-rich bone marrow niches, effectively crossing the marrow‒blood barrier. Our PET/CT and fluorescence imaging studies confirmed efficient marrow accumulation of NE-delivered nanoparticles in osteoporotic mice. Importantly, the CL-NPs@NEs system demonstrated dual therapeutic advantages: compared with free CL administration, quantitative imaging revealed enhanced bone-specific drug retention, whereas biochemical analyses revealed marked attenuation of systemic effects. The targeted delivery approach significantly reduced peripheral adipose lipolysis and mitigated metabolic disturbances. These findings establish neutrophil-mediated targeting as an effective strategy to simultaneously increase bone anabolism while minimizing the off-target effects of CLR agonism.\u003c/p\u003e\u003cp\u003eContemporary clinical management of osteoporosis employs sequential or concurrent administration of antiresorptive and anabolic agents to maximize therapeutic benefits[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. While short-term combination therapy has synergistic effects on bone mineral density, prolonged coadministration may induce hypercalcemia and paradoxical antagonism of therapeutic mechanisms, as observed between zoledronic acid and teriparatide[\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. CLR agonists have emerged as promising therapeutic alternatives because of their unique signaling cascades, which bypass pharmacodynamic interference while enabling balanced bone remodeling via coupled regulation of osteoanabolic and osteocatabolic activities. This dual-action mechanism presents a distinct advantage over conventional antiresorptives (e.g., bisphosphonates or denosumab) that solely suppress osteoclast function. Furthermore, unlike the demanding daily subcutaneous injections required for teriparatide monotherapy or the extended dosing intervals of antiresorptive agents, our neutrophil-mediated nanoparticle delivery system (CL\u0026thinsp;+\u0026thinsp;PTH-NPs@NEs) synergistically combines the therapeutic benefits of both drug classes while potentially improving administration frequency. Our studies in OVX mice demonstrate that this combinatorial approach yields superior outcomes compared with those of monotherapy, with microCT analyses revealing significantly increased bone density and trabecular microarchitecture. Importantly, neutrophil-directed delivery maintains osteogenic amplification from CL-induced lipolysis while simultaneously addressing the pharmacokinetic limitations of teriparatide, effectively translating the theoretical compatibility of these pathways into a therapeutic advantage.\u003c/p\u003e\u003cp\u003eWhile our findings demonstrate promising osteoprotective effects, important pharmacological and translational challenges must be addressed before clinical implementation. In addition to the aforementioned adverse effects, preclinical evidence suggests that chronic CLR agonism may impair additional off-target effects on bladder smooth muscle, which could influence micturition function[\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. Although our current study specifically evaluated skeletal outcomes, a rigorous assessment of these organ-specific effects\u0026mdash;particularly under targeted delivery conditions\u0026mdash;remains imperative. The age-dependent properties of neutrophil carriers introduce additional biological complexity, while their intrinsic bone marrow homing capability facilitates drug delivery, the immunological alterations characteristic of aged neutrophils[\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e] and their potential to modulate inflammatory responses or immune suppression[\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e] warrant thorough investigation in chronic treatment settings. From a translational standpoint, the clinical scalability of autologous neutrophil-based delivery systems faces practical limitations related to cell harvesting costs and patient compliance. Future research directions will focus on engineering next-generation nanoparticles with enhanced bone-specific targeting and controlled release kinetics to minimize systemic exposure, coupled with comprehensive long-term safety evaluations across multiple osteoporotic models to systematically characterize cardiovascular, urological, and immunological impact-critical steps toward establishing the clinical viability of this therapeutic strategy.\u003c/p\u003e\u003cp\u003eIn conclusion, by leveraging neutrophil-mediated hitchhiking as a bone marrow-targeted delivery platform, we demonstrate that localized CLR agonism effectively reverses skeletal deterioration in osteoporosis while circumventing the systemic metabolic perturbations typically associated with CLR activation. This neutrophil-based delivery system achieves spatiotemporally controlled release of CL316243 and teriparatide specifically within the bone marrow microenvironment, where it simultaneously attenuates marrow adipogenesis and promotes osteoblast differentiation, thereby addressing two key pathological features of osteoporosis. Importantly, our approach maintains the metabolic benefits of CLR signaling confined to bone tissue, avoiding undesirable peripheral effects. These findings establish neutrophil-assisted drug delivery as a promising therapeutic paradigm for precise modulation of bone remodeling with minimal off-target consequences, offering a clinically translatable solution for osteoporosis treatment.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eStudy approval\u003c/h2\u003e\u003cp\u003e This study conforms to the Declaration of Helsinki and was approved by the Ethics Committee of the Eighth Affiliated Hospital, Sun Yat-Sen University, Guangzhou, China. The experiments involving mice were approved by the Institutional Animal Care and Use Committee of Sun Yat-Sen University, Guangzhou, China. All experimental procedures involving mice were carried out in strict adherence to the rules and guidelines for the ethical use of animals in research.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eAnimal experiments\u003c/h2\u003e\u003cp\u003eThree-month-old mature female C57BL/6 mice underwent bilateral ovariectomy (OVX) or sham surgery (SH). After anesthesia, a lateral lumbar incision was made at the midpoint between the free rib and the iliac crest to remove the ovaries. The incision was sealed, and the skin was sutured. Thirty days after surgery, the sham, OVX, and 24-month-old elderly (SENILE) mice were randomly divided into six groups (n\u0026thinsp;=\u0026thinsp;6): the SH-NC (saline, intraperitoneal injection), SH-CL (CL316243, intraperitoneal injection), OVX-NC (saline, intraperitoneal injection), OVX-CL (CL316243, intraperitoneal injection), SENILE-NC (saline, intraperitoneal injection), and SENILE-CL (CL316243, intraperitoneal injection) groups. Body weight changes were recorded throughout the experiment. After 30 days of treatment, the mice were euthanized. Peripheral adipose tissue was collected, and femurs and tibiae were harvested for micro-CT analysis. Blood samples were collected to measure the serum levels of P1NP (Macklin, P771527), CTX-1 (IDS, AC-06F1), IL-6 (R\u0026amp;D, VAL604G), leptin (Elabscience, E-EL-M3008), and insulin (Elabscience, E-EL-M2614) via ELISA kits. Free fatty acid (FFA) and glucose levels were assessed via a colorimetric assay kit (Elabscience, E-BC-K792-M) and a glucose assay kit (Elabscience, E-BC-K234-M), respectively. The additional batches used for OVX modeling and sample collection followed the same procedures described above.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eExtraction and stimulation of bone marrow adipocytes\u003c/h2\u003e\u003cp\u003eBone marrow aspirates from NC patients were processed via density gradient centrifugation (150 \u0026times; g min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, 10 min, Invitrogen) to isolate bone marrow adipose tissue. The isolated adipocytes were cultured in osteogenic medium or osteoclastogenic medium containing 0 \u0026micro;M, 1 \u0026micro;M, or 5 \u0026micro;M CL. The osteogenic medium consisted of 10% FBS DMEM supplemented with 100 IU mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e penicillin, 100 IU mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e streptomycin, 0.1 \u0026micro;mol L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e dexamethasone, 10 mmol L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e β-glycerophosphate, and 50 \u0026micro;mol L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e ascorbic acid (Sigma‒Aldrich). The osteoclastogenic medium consisted of 10% FBS α-MEM supplemented with 100 IU mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e penicillin, 100 IU mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e streptomycin, 50 \u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e RANKL (R\u0026amp;D, 462-TEC-010), and 25 \u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e M-CSF (R\u0026amp;D, 216-MC-010). After 6 hours of culture, the adipocytes were removed via centrifugation (150 g\u0026middot;min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), and the fatty acid-induced supernatant was collected.\u003c/p\u003e\u003cp\u003e\u003cb\u003eMSC isolation and culture\u003c/b\u003e\u003c/p\u003e\u003cp\u003eBone marrow aspirates from NC patients were processed via density gradient centrifugation (12,000 r min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, 30 min, Invitrogen) to isolate mesenchymal stem cells (MSCs). The extracted MSCs were cultured in Dulbecco\u0026rsquo;s modified Eagle\u0026rsquo;s medium (DMEM, Gibco) supplemented with 10% fetal bovine serum (FBS; Hangzhou Sijiquan Biotechnology). For osteogenic induction and CL stimulation, the MSCs were divided into four groups: the NC group (osteogenic medium containing 5 \u0026micro;M CL-primed cell-free medium), the 0 \u0026micro;M group (osteogenic medium containing adipocyte-0 \u0026micro;M CL-conditioned culture), the 1 \u0026micro;M group (osteogenic medium containing adipocyte-1 \u0026micro;M CL-conditioned culture), and the 5 \u0026micro;M group (osteogenic medium containing adipocyte-5 \u0026micro;M CL-conditioned culture). The osteogenic medium was replaced every three days.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eOsteoclast culture and induction\u003c/h2\u003e\u003cp\u003ePeripheral blood mononuclear cells (PBMCs) were isolated from the anticoagulated peripheral blood of volunteers via density gradient centrifugation (400 g min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, 30 min, Invitrogen). CD14\u003csup\u003e+\u003c/sup\u003e cells were subsequently isolated via magnetic bead sorting. For osteoclast induction, CD14\u003csup\u003e+\u003c/sup\u003e cells were divided into four groups: the NC group (osteoclastogenic medium containing 5 \u0026micro;M CL-primed cell-free medium), the 0 \u0026micro;M group (osteoclastogenic medium containing adipocyte-0 \u0026micro;M CL-conditioned supernatant), the 1 \u0026micro;M group (osteoclastogenic medium containing adipocyte-1 \u0026micro;M CL-conditioned supernatant), and the 5 \u0026micro;M group (osteoclastogenic medium containing adipocyte-5 \u0026micro;M CL-conditioned supernatant). CD14\u003csup\u003e+\u003c/sup\u003e cells were cultured in fatty acid-induced supernatants with different concentrations of CL. After 8 days of induction, tartrate-resistant acid phosphatase (TRAP) staining was performed via a TRAP staining kit.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003eEndothelial tube formation assay\u003c/h2\u003e\u003cp\u003eHuman umbilical vein endothelial cells (HUVECs, iCell) were used for the endothelial tube formation experiments. HUVECs were cultured on plates precoated with 1% (w/v) gelatin and maintained in endothelial cell medium (ECM, ScienCell) supplemented with 20% fetal bovine serum, 100 IU mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e penicillin and 100 IU mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e streptomycin. For tube formation induction and CL stimulation, HUVECs were divided into four groups: the NC group (endothelial medium containing 5 \u0026micro;M CL-primed cell-free medium), the 0 \u0026micro;M group (endothelial medium containing adipocyte-0 \u0026micro;M CL-conditioned culture), the 1 \u0026micro;M group (endothelial medium containing adipocyte-1 \u0026micro;M CL-conditioned culture), and the 5 \u0026micro;M group (endothelial medium containing adipocyte-5 \u0026micro;M CL-conditioned culture). After 4 hours of induction, samples were collected for quantification of tube formation metrics.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003eNeutrophil isolation\u003c/h2\u003e\u003cp\u003eFemurs and tibiae were harvested from euthanized mice under sterile conditions. The ends of the bones were cut off and placed in a 1.5 mL Eppendorf tube containing 200 \u0026micro;L of PBS and a 1 \u0026micro;L pipette tip with the tip removed. The samples were centrifuged at 12,000 \u0026times; g min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e for 15 seconds, and the bone marrow was resuspended in 500 \u0026micro;L of PBS. A density gradient was prepared by carefully layering 2 mL of 75%, 65%, and 55% (vol/vol) Percoll (Pharmacia) solutions, followed by 500 \u0026micro;L of the single-cell suspension on top. The gradient was centrifuged at 700 \u0026times; g min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e for 30 minutes, and neutrophils were collected from the interface between the 75% and 65% fractions. The collected neutrophils were washed three times with ice-cold PBS to remove residual Percoll and subsequently lysed with red blood cell lysis buffer for 15 minutes. The isolated neutrophils were cultured in RPMI 1640 medium supplemented with 1% penicillin/streptomycin at 37\u0026deg;C in a humidified atmosphere containing 5% CO₂. Typically, senescent neutrophils were obtained after 6 hours of in vitro culture.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003eProportion of CXCR4-expressing Neutrophils\u003c/h2\u003e\u003cp\u003eMouse bone marrow cells were cultured in an incubator, and the cells were collected at 0 h, 6 h, and 24 h. The cells were stained with APC-conjugated anti-mouse Ly-6G and PE-conjugated anti-mouse CD184 (CXCR4) antibodies (diluted 1:200 in PBS) for 25 minutes. After the unbound antibodies were removed, the proportion of CXCR4-expressing neutrophils was analyzed via flow cytometry.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003ePreparation of CL-NPs\u003c/h2\u003e\u003cp\u003eCL-NPs were synthesized via a double emulsion (W/O/W) method. Briefly, 1 mg of CL316243 was dissolved in 50 \u0026micro;L of cold water as the inner aqueous phase. Concurrently, 29 mg of PLGA (65:35 wt/wt, Mw: 24,000\u0026ndash;38,000, Sigma‒Aldrich) was dissolved in 450 \u0026micro;L of dichloromethane (DCM) as the oil phase. The inner aqueous phase (W) was quickly injected into the oil phase (O) and sonicated for 3 minutes to form a primary W/O emulsion. This emulsion was then added to 3 mL of 2.0% polyvinyl alcohol (PVA, Mw: 38 kDa, Sigma‒Aldrich) solution and further sonicated for 3 minutes to form the W/O/W emulsion. The resulting emulsion was poured into 20 mL of 0.2% PVA solution and stirred overnight to solidify the nanoparticles. The CL-NPs were collected by centrifugation at 18,000 \u0026times; g min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e for 20 minutes and washed three times with Milli-Q water.\u003c/p\u003e\u003cp\u003eDrug encapsulation efficiency was determined via a titration method. Lyophilized CL-NPs (5 mg) were dissolved in 2 mL of acetonitrile, and the sample was analyzed via high-performance liquid chromatography (HPLC). For cumulative drug release analysis, 5 mg of lyophilized CL-NPs were suspended in 10 mL of PBS containing 0.2% Tween 80 and incubated at 37\u0026deg;C in a shaking water bath (SHY-2A, Guoyu Instruments). At specific time points (0 h, 1 h, 3 h, 6 h, 1 day, 2 days, 4 days, 7 days, and 10 days), 1 mL of the supernatant was collected via centrifugation (20,000 \u0026times; g min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, 20 minutes) and replaced with 1 mL of fresh PBS containing 0.2% Tween 80. The cumulative drug content in the supernatant was determined via HPLC (HPLC 1200 series, Agilent Technologies).\u003c/p\u003e\u003cp\u003eThe particle size of the PLGA nanoparticles was measured via dynamic light scattering (Malvern Zetasizer Nano-ZS), and their morphology was observed via transmission electron microscopy (TEM, JEOL JEM-1400). PTH-NPs (PTH, MedChemExpress, HY-P0059) and CL-PTH-NPs were prepared via the same method as CL-NPs.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003eAssessment of the Migration of CL-NPs@NEs\u003c/h2\u003e\u003cp\u003eThe ability of CL-NPs@NEs to migrate toward CXCL12 (SDF-1α, PeproTech) was evaluated via Transwell inserts with 3 \u0026micro;m pore polyester membranes. DiD-labeled neutrophils or CL-NPs@NEs (10⁶ cells mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) were seeded into the upper chamber, and 10 ng of CXCL12 (final concentration: 20 ng mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) was added to the lower chamber. After 1 h, fluorescence images of the lower chamber were captured via an inverted fluorescence microscope (AIR, Nikon). The fluorescence intensity was analyzed via ImageJ software. For wells containing the CXCR4 antagonist plerixafor (AMD3100, PeproTech), neutrophils were preincubated with 1 mg mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e AMD3100 for 15 minutes before performing the migration assay, following the same procedure as described above.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003eImmunohistochemistry\u003c/h2\u003e\u003cp\u003eFemurs from OVX and SENILE mice were collected for immunohistochemical analysis. The samples were assigned to four groups: OVX-NC, OVX-B3, SENILE-NC, and SENILE-B3. The bone tissue sections were dewaxed and rehydrated. The sections were immersed in EDTA buffer and incubated overnight at 60\u0026deg;C for antigen retrieval. Following antigen retrieval, sections from the OVX-B3 and SENILE-B3 groups were incubated overnight at 4\u0026deg;C with a primary antibody against the β3-adrenergic receptor (Abcam, ab94506). Sections from the OVX-NC and SENILE-NC groups were incubated with an isotype control rabbit IgG polyclonal antibody (Abcam, ab37415) under the same conditions. All the sections were subsequently incubated with the corresponding secondary antibodies at room temperature for 1 hour. Images were finally captured via a light microscope.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\u003ch2\u003eImmunofluorescence\u003c/h2\u003e\u003cp\u003eFemurs from the mice were collected for immunofluorescence analysis. Bone tissue sections were deparaffinized and rehydrated, followed by antigen retrieval via EDTA buffer. The sections were immersed in EDTA buffer and incubated overnight at 60\u0026deg;C for antigen retrieval. The samples were then incubated with primary antibodies against CD31 (Abcam, ab104854) and Emcn (Proteintech, 13440S) at 4\u0026deg;C overnight. Afterward, the sections were incubated at room temperature for 1 hour with the secondary antibodies anti-mouse Alexa 488 (Cell Signaling Technology, 4408) and anti-rabbit Alexa 555 (Cell Signaling Technology, 4413). DAPI antifade mounting medium (Beyotime, P0131) was used for mounting. Images were captured via a Zeiss LSM 880 confocal microscope.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\u003ch2\u003eHistological Staining\u003c/h2\u003e\u003cp\u003eBone tissues were collected and fixed in 4% paraformaldehyde (PFA) at 4\u0026deg;C overnight, followed by decalcification and paraffin embedding. The tissue sections were subjected to hematoxylin and eosin (H\u0026amp;E, Boster, AR1180), Masson's trichrome (Solarbio, G1340-100), and tartrate-resistant acid phosphatase (TRAP) staining.\u003c/p\u003e\u003cdiv id=\"Sec23\" class=\"Section3\"\u003e\u003ch2\u003eIVIS\u003c/h2\u003e\u003cp\u003eIsolated neutrophils were cultured in RPMI 1640 medium, followed by the addition of 5 \u0026micro;M DiD (T15118, Targetmol) at a concentration of 10\u003csup\u003e6\u003c/sup\u003e cells mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. The cells were incubated for 15 minutes, and unbound DiD was removed by washing three times with PBS. The resulting DiD-labeled neutrophils were injected into the mice via the tail vein. Two groups of neutrophils were used in the experiment: one group was isolated and cultured for 6 hours (age: 6 hours), and the other was cultured for 24 hours (age: 24 hours). The in vivo distributions of labeled neutrophils and free DiD were examined via an in vivo imaging system (IVIS) at 2 hours and 6 hours postinjection.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec24\" class=\"Section2\"\u003e\u003ch2\u003eMicro-CT\u003c/h2\u003e\u003cp\u003eThe femurs were analyzed via a micro-CT scanner (SKYSCAN 1276). Images and key bone parameters, such as the bone volume fraction (BV/TV) and trabecular separation (Tb.Sp), were obtained via CTAn and DataViewer software. The data were analyzed with GraphPad Prism 8 software.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec25\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eAll the data are presented as representative results from multiple independent experiments. Data comparisons were made via unpaired two-tailed \u003cem\u003et\u003c/em\u003e tests and one-way analysis of variance tests in GraphPad Prism. The outcomes are presented as the means\u0026thinsp;\u0026plusmn;\u0026thinsp;SDs. ns\u0026thinsp;=\u0026thinsp;statistically nonsignificant, \u003cem\u003e*p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, **p\u0026thinsp;\u0026lt;\u0026thinsp;0.01 and ***p\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics Approval and Patient Consent Statement:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work had complied with all relevant ethical regulations for clinical samples and animalresearch. Bone marrow and cellular specimens were obtained from patients undergoing orthopedic surgery with or without osteoporosis. The human study of this research was conducted in accordance with theprinciples expressed in the Declaration of Helsinki and was approved by the ethical committee of \u0026nbsp;The Eighth Affiliated Hospital of SunYat-Sen University (approval number: 2021r208). Written informedconsent was obtained from each enrolled patient. All animal experimentswere performed in the Central of Experimental Animal SunYat-Sen University. The animal experiments were conducted accordingto the protocol (approval number: 2023000937, 2023002888, 2024001423) authorized by the Experi-mental Animal Welfare Ethics Committee, SunYat-Sen University.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eC. Wu, J. Zhao and Q. Li\u0026nbsp;contributed equally to this work.\u0026nbsp;G.\u0026nbsp;Zheng,\u0026nbsp;Z.\u0026nbsp;Xie\u0026nbsp;and\u0026nbsp;W.\u0026nbsp;Yu\u0026nbsp;conceived\u0026nbsp;designed and supervised the study.\u0026nbsp;C.\u0026nbsp;Wu, J. Zhao and\u0026nbsp;Q.\u0026nbsp;Li\u0026nbsp;performed the experiments and analyzed the data.\u0026nbsp;W.\u0026nbsp;Zhang, S Meng, H.\u0026nbsp;Liu,\u0026nbsp;Y.\u0026nbsp;Zeng, X.\u0026nbsp;Ning, P.\u0026nbsp;Wang\u0026nbsp;and Y. Wu\u0026nbsp;provided\u0026nbsp;suggestions.\u0026nbsp;C.\u0026nbsp;Wu, J. Zhao,\u0026nbsp;Q.\u0026nbsp;Li\u0026nbsp;and W. Zhang\u0026nbsp;wrote\u0026nbsp;the manuscript, and\u0026nbsp;G.\u0026nbsp;Zheng,\u0026nbsp;Z.\u0026nbsp;Xie,\u0026nbsp;W.\u0026nbsp;Yu\u0026nbsp;and Y. Wu\u0026nbsp;revised the manuscript.\u0026nbsp;This work was supported by the Shenzhen Science and Technology Program (RCBS20221008093103013, JCYJ20220530144016039),\u0026nbsp;the National Natural Science Foundation of China (82102529, 82402764), the Guangdong Natural Science Foundation (2023A1515010226, 2023A1515111078)\u0026nbsp;and the Shenzhen Medical Research Fund (A2403047).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCorresponding author\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCorrespondence and requests for materials should be addressed to WenHui Yu, Zhongyu Xie or Guan Zheng.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003einterest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data that support the findings of this study are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupporting Information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSupporting Information is available from the Wiley Online Library or from the author.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAyers C, Kansagara D, Lazur B et al (2023) Effectiveness and safety of treatments to prevent fractures in people with low bone mass or primary osteoporosis: A living systematic review and network meta-analysis for the american college of physicians. 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Front Immunol 13:866747. https://doi.org/10.3389/fimmu.2022.866747.\u003c/li\u003e\n\u003cli\u003eHerrero-Cervera A, Soehnlein O, Kenne E (2022) Neutrophils in chronic inflammatory diseases. Cell Mol Immunol 19:177-191. https://doi.org/10.1038/s41423-021-00832-3.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Ostoporosis, CL316243, canonical lipolytic receptor, neutrophil, drug targeting","lastPublishedDoi":"10.21203/rs.3.rs-7569697/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7569697/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eCurrent osteoporosis therapies fail to fully restore bone mass because of insufficient targeting of the bone marrow microenvironment. Here, we report a cell-mediated delivery platform that leverages senescent neutrophils to specifically transport canonical lipolytic receptor (CLR) agonists to bone marrow adipose tissue (BMAT). Using both ovariectomy-induced and aged osteoporosis mouse models, we demonstrated that systemic CLR activation significantly reduces BMAT volume while improving trabecular bone structure but at the cost of inducing systemic lipolysis and metabolic disturbances. To overcome these limitations, we developed CL316243(CL)-loaded nanoparticles delivered by senescent neutrophils (CL-NPs@NEs), which exhibited greater bone marrow accumulation than free drug. CL-NPs@NEs treatment led to remarkable bone mass recovery without causing peripheral fat loss or metabolic complications. Combining neutrophil-delivered CL and parathyroid hormone further enhanced therapeutic efficacy. Our findings establish senescent neutrophils as effective drug carriers for bone marrow-targeted therapy and reveal that CLR agonism is a viable strategy to remodel the adipocyte-rich bone marrow microenvironment. Targeted modulation of marrow adipose tissue combined with the osteoanabolic agent teriparatide holds promise for superior bone microarchitecture reconstruction and bone quality improvement in osteoporosis.\u003c/p\u003e","manuscriptTitle":"Bone marrow-targeted delivery of canonical lipolytic receptor agonists via neutrophil hitchhiking reverses osteoporosis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-25 10:50:54","doi":"10.21203/rs.3.rs-7569697/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"6cfbdcfe-6e7e-4c17-9452-675c39157b07","owner":[],"postedDate":"September 25th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-10-04T03:08:30+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-25 10:50:54","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7569697","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7569697","identity":"rs-7569697","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}