Chylomicron-biomimetic nanoparticles promote intestinal lymphatic transport by fast fusion with chylomicrons

Chylomicron-biomimetic nanoparticles promote intestinal lymphatic transport by fast fusion with chylomicrons | 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 Article Chylomicron-biomimetic nanoparticles promote intestinal lymphatic transport by fast fusion with chylomicrons Wei Xiong, Yawen Yao, Cuihua Zhang, Jieying Zhou, Shihao Xu, Ying Li, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6301196/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 Intestinal lymphatic transport (ILT) represents a promising pathway for the oral absorption of macromolecular drugs, but the formation of lipid droplet is a rate-limiting step during ILT. In this study, we developed a chylomicron (CM)-biomimetic nanoparticle constructed from CM components, characterized by high lipoprotein affinity, to enable efficient oral delivery of the anti-fibrotic protein klotho via ILT. This approach demonstrated potent therapeutic efficacy in the treatment of renal fibrosis. The nanoparticle exhibited size stability and retained 78.3% enzyme activity after 12 hours of incubation in simulated digestive fluid, while facilitating rapid diffusion through the mucus layer. Within enterocytes, the nanoparticle underwent a CM-like transcytosis process and showed a preference for ILT, as evidenced by cannulation into the main mesenteric lymphatic duct in rats. Notably, this biocompatible oral nanoparticle achieved an absolute bioavailability of 2.7%, delivering superior anti-fibrotic activity in a mouse disease model compared to a 125-fold higher dose of intraperitoneally administered captopril, a first-line anti-fibrotic drug. Our innovative nanoparticle design based on high lipoprotein affinity enables enhanced oral absorption of macromolecular drugs via ILT. Health sciences/Nephrology/Kidney diseases/Renal fibrosis Biological sciences/Biotechnology/Protein delivery Physical sciences/Nanoscience and technology/Nanobiotechnology/Nanoparticles Intestinal lymphatic transport Chylomicron Klotho Oral Renal fibrosis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Oral administration of macromolecular drugs is the preferred option for patients requiring daily and long-term medication 1 , 2 . Most oral macromolecular drugs, however, fail to survive in the gastrointestinal tract 3 , penetrate the mucus layer 4 , cross the intestinal epithelial barrier 5 , and reach target sites at therapeutic concentration 6 . While encapsulation 7 , 8 and PEGylation technologies 9 , 10 have successfully addressed the first two challenges, the latter two remain significant hurdles, resulting in low bioavailability and poor therapeutic efficacy for oral macromolecular drugs. Inspired by the endogenous transport of macromolecules from the intestinal epithelium into lymphatic vessels rather than the bloodstream 11 , 12 , rerouting drug absorption to intestinal lymph transport (ILT) has attracted widespread attention 13 . ILT is the primary pathway for dietary lipid transport, such as triglycerides (TGs), following absorption by enterocytes 14 , 15 . During this process, orally administered TGs are digested into fatty acids and 2-monoglycerides (MGs) in the intestinal lumen, absorbed by enterocytes, and re-esterified to TGs. These TG droplets then bind with lipoproteins, including L-FABP and ApoB-48, to form chylomicrons (CMs) in the endoplasmic reticulum (ER) and Golgi apparatus. CMs are subsequently secreted into mesenteric lymphatic vessels 16 , which drain directly into the systemic circulation via the left subclavian vein, bypassing first-pass hepatic metabolism 17 . Recent studies have suggested that TG/MG-conjugated prodrugs 18 and nanoparticles 19 penetrated the intestinal epithelial barrier through ILT, enhancing oral bioavailability by 6.1-fold and 10.6-fold, respectively. The extent of ILT for these prodrugs and nanoparticles, however, remains unclear. Most studies have primarily assessed plasma drug levels with and without cycloheximide (an inhibitor of lipoprotein secretion), without directly quantifying lymphatic transport. Here, we report the significant finding that chylomicron-biomimetic nanoparticles (CMB-NPs) with high lipoprotein affinity effectively cross the intestinal epithelial barrier intact via ILT. Basing on the components of CMs 20 , we constructed CMB-NPs with a TG-rich core surrounded by phospholipids, cholesterol, and Brij-O10. Since TG resynthesis and lipid droplet (LD) formation represent rate-limiting steps in ILT 21 , we screened 319 pharmaceutical excipients for lipoprotein affinity and identified Brij-O10 as a key component that enhances lymphatic transport by facilitating rapid fusion with CMs (Fig. 1 d). Moreover, we established a novel animal model by cannulating the main mesenteric lymphatic duct of a rat, enabling direct quantification of ILT. To explore the therapeutic applications, we encapsulated the anti-fibrotic macromolecular drug klotho (KLO) within zwitterionic chitosan nanoparticles (KLO-ZWC-NPs) to facilitate oral delivery for the treatment of renal fibrosis (RF). Due to the overexpression of the OCTN2 transporter in the kidney, ZWC-NPs demonstrated kidney-specific distribution. Accordingly, KLO-loaded ZWC-NPs (KLO-ZWC-NPs) were further encapsulated within CMB-NPs (KLO-CMB-NPs) using a modified film dispersion method (Fig. 1 b). This dual-encapsulation strategy enabled the transport of KLO-CMB-NPs into systemic circulation via ILT, bypassing first-pass hepatic metabolism and achieving an absolute oral bioavailability of up to 2.7% (Fig. 1 c). In the bloodstream, KLO-CMB-NPs are hydrolyzed by lipoprotein lipase, releasing KLO-ZWC-NPs for kidney-targeted delivery (Fig. 1 e). Both in vitro and in vivo studies confirmed kidney-specific accumulation and demonstrated robust therapeutic efficacy against RF following oral administration of KLO-CMB-NPs. Results Construction and characterization of KLO-CMB-NPs To develop CMB-NPs with high lipoprotein affinity, we first obtain natural chylomicrons (nCMs) from the main mesenteric lymphatic duct of a rat by cannulation (Supplementary Fig. 1). From a single rat, only small amounts of nCMs (80–100 µL) were collected, with an average particle size of approximately 93.6 nm (Fig. 2 a). To address the limited availability of nCMs, we developed artificial chylomicrons (aCMs) using a film dispersion method 22 . These aCMs were formulated with components mimicking nCMs, including olive oil, l-α-phosphatidylcholine, lysophosphatidylcholine, cholesteryl oleate, and cholesterol, in a molar ratio of 70:22.7:2.3:3.0:2.0 22 . The resulting aCMs formed an emulsion with an average particle size of approximately 160.3 nm (Fig. 2 b). As TGs are the primary component of CMB-NPs, we initially screened different TG types for their binding affinity with aCMs. Interestingly, CMB-NPs prepared using triolein (TO) exhibited a significantly higher binding rate with aCMs (70.7%) compared to trimyristin (TM), tripalmitin (TP), and tristearin (TS) (Fig. 2 c). To further enhance the binding rate with aCMs, we evaluated 319 pharmaceutical excipients for their lipoprotein affinity using a cluster analysis method. We identified Brij-C10, Brij-S10, Brij-O10, TPGS, Span-40, Span-60, and Span-80 as candidates with potential to improve lipoprotein affinity (Supplementary Fig. 2). These excipients were subsequently incorporated into the aqueous phase during the preparation of CMB-NPs. Among them, Brij-O10 significantly increased the binding rate from 70.7–84.8% (Fig. 2 d). The optimized CMB-NP formulation, comprising a TG-rich core surrounded by Brij-O10, demonstrated a binding rate comparable to that of nCMs (Fig. 2 e). Binding affinity between CMB-NPs and nCMs was further quantified using surface plasmon resonance (SPR) (Fig. 2 f). Consistent with the binding rate results, CMB-NPs containing Brij-O10 exhibited a higher association rate constant (ka = 7.8 × 10⁶ a/Ms) and a lower dissociation constant (KD = 1.4 × 10⁻⁹ M) than CMB-NPs without Brij-O10. These findings highlight the role of Brij-O10 in enhancing the binding affinity of CMB-NPs with nCMs. Next, we prepared KLO-ZWC-NPs and further encapsulated within CMB-NPs using the optimized formulation. Both KLO-ZWC-NPs and KLO-CMB-NPs exhibited similar particle sizes, measuring 172.1 nm and 176.3 nm, respectively, with a smooth spherical structure and neutral charge (Fig. 2 h). The particle size of KLO-CMB-NPs remained stable after storage at 4°C for 5 days, indicating good storage stability (Fig. 2 g). Furthermore, the encapsulation efficiency (EE) and loading capacity (LC) of KLO-CMB-NPs were 98.6% and 6.0%, respectively (Fig. 2 i). To investigate the drug release behavior of CMB-NPs, KLO was replaced with bovine serum albumin (BSA) as a model protein and subjected to dialysis in release media at pH 1.2 and 6.8. BSA-CMB-NPs displayed similar particle size, zeta potential, and LC to those of KLO-CMB-NPs (Supplementary Table 1). Free BSA, as a water-soluble macromolecule, exhibited rapid release in the release media, with 65.7% released. By contrast, BSA-CMB-NPs released only 18.4% in the pH 1.2 medium and 27.5% in the pH 6.8 medium (Fig. 2 j). The controlled release profile of CMB-NPs suggests that the encapsulated macromolecules are likely to survive in the harsh gastrointestinal environment. KLO-CMB-NPs survive in the gastrointestinal tract To evaluate the protective effects of KLO-CMB-NPs, we used Förster resonance energy transfer (FRET) to validate nanoparticle integrity in vitro . FRET-CMB-NPs were developed by co-loading fluorescein isothiocyanate (FITC, FRET donor) and rhodamine B (RhoB, FRET acceptor). A FRET signal was generated when FRET-CMB-NPs remained intact, indicating nanoparticle stability (Fig. 3 a). To assess the integrity of FRET-CMB-NPs in the gastrointestinal environment, they were incubated in 0.1M HCl (pH 1.2) and PBS (pH 6.8) for 12 hours. The FRET signal was observed at the indicated time points, demonstrating that FRET-CMB-NPs retained their structural integrity under harsh pH conditions for up to 12 hours (Fig. 3 b). These findings confirm the stability of CMB-NPs in gastrointestinal pH environments. KLO specifically hydrolyzes β-D-glucuronide into fluorescent 4-methylumbelliferone, enabling assessment of its enzymatic activity 23 . To further confirm the protective effects of KLO-CMB-NPs, we therefore evaluated the β-glucuronidase activity of the encapsulated KLO. After incubation in simulated gastric fluid and simulated intestinal fluid for 12 hours, KLO-CMB-NPs retained 78.3% of their β-glucuronidase activity compared to freshly prepared KLO-CMB-NPs (Fig. 3 c). These findings demonstrate that encapsulation within CMB-NPs effectively protects KLO, allowing it to survive in the gastrointestinal tract. KLO-CMB-NPs increase transport through mucus layer and epithelium Despite KLO-CMB-NPs survive in the gastrointestinal tract, they need to penetrate the mucus layers covering the apical side of the intestinal epithelium. To evaluate the mucus-penetrating capacity, we synthesized FITC-labelled ZWC (FITC-ZWC), which was used to produce FITC-ZWC-NPs and FITC-CMB-NPs for fluorescent tracing in the mucus layer. Compared to FITC-ZWC-NPs, FITC-CMB-NPs exhibited significantly stronger fluorescence intensity in the lower side of the jejunum of a rat within 1 hour of incubation (Fig. 3 d), suggesting FITC-CMB-NPs penetrated the mucus layer more effectively than FITC-ZWC-NPs. Semiquantitative analysis of each slice further demonstrated that FITC-CMB-NPs increased the mucus permeability by 1.9-fold compared to FITC-ZWC-NPs (Fig. 3 e, f). Subsequently, an in vitro cellular uptake assay was performed using confocal laser scanning microscopy and flow cytometry. After 2 hours of incubation with Caco-2 cells, FITC-CMB-NPs demonstrated strong fluorescence intensity and increased cellular uptake by 11.2- and 1.7-fold compared to FITC-ZWC and FITC-ZWC-NPs, respectively (Fig. 4 a-c). To investigate the endocytosis pathways of FITC-CMB-NPs, cells were pretreated with specific endocytosis inhibitors. Cellular uptake was reduced to 38.6% and 44.4% of the original levels following treatment with chlorpromazine and indomethacin, respectively (Fig. 4 d). These results indicate that FITC-CMB-NPs are primarily internalized via clathrin- and caveolae-mediated pathways. Next, we constructed a Caco-2 cell monolayer to evaluate the permeation capability of FITC-CMB-NPs. FITC-CMB-NPs again exhibited a strong permeability coefficient (3.81 ± 0.04 × 10⁻⁶ cm/s), which was 1.7 times higher than that of FITC-ZWC-NPs (2.31 ± 0.04 × 10⁻⁶ cm/s) (Fig. 4 e). Three-dimensional visualization further confirmed that FITC-CMB-NPs successfully crossed the cell monolayer (Fig. 4 f, g). The permeation capability of FITC-CMB-NPs was significantly reduced to 26.0% of the original level in the presence of Pluronic-L81, a known chylomicron (CM) secretion inhibitor (Supplementary Fig. 3). Conversely, the addition of oleic acid, a CM secretion stimulator 24 , 25 , enhanced the transmembrane transport of FITC-CMB-NPs by 69.8% of the original level. These findings indicate that the transmembrane transport of CMB-NPs is mediated by CMs within the cells. In addition, we evaluated the integrity of CMB-NPs after transmembrane transport through the Caco-2 cell monolayer using FRET. The transmembrane FRET-CMB-NPs demonstrated an increasing FRET signal over incubation time (Supplementary Fig. 4), indicating that CMB-NPs remained intact after penetrating the cell monolayer. CM-like lymphatic transport within enterocytes Building on the finding that the enhanced transmembrane transport of FITC-CMB-NPs was mediated by CMs in Caco-2 cells, we hypothesized that FITC-CMB-NPs traverse cells via the lymphatic transport pathway. To test this hypothesis, lymphatic transport-associated organelles within the cells were labelled, and their colocalization of FITC-CMB-NPs was examined. Merged confocal micrographs revealed substantial colocalization of FITC-CMB-NPs with the ER and Golgi apparatus (Fig. 5 a), with high colocalization coefficient of 0.745 ± 0.048 and 0.668 ± 0.041 respectively. By contrast, FITC-ZWC-NPs did not show obvious colocalization with these labelled organelles. Transmission electron microscopy (TEM) provided more precise evidence of FITC-CMB-NPs colocalization with the ER and Golgi apparatus. CMB-NPs were observed within the ER and Golgi apparatus (Fig. 5 b). These confocal and TEM observations confirm that the transport of CMB-NPs within cells depend on lymphatic transport pathway. To further elucidate the CM-like lymphatic transport mechanism of CMB-NPs, we established an in vitro CM-assembly model using Caco-2 cells. CMB-NPs, TGs (positive control), and ZWC-NPs (negative control) were incubated with Caco-2 cells for 24 hours. L-FABP and ApoB-48 are two key proteins involved in the CM assembly process 26 , 27 . As anticipated, CMB-NPs and TGs significantly increased cellular L-FABP expression by 2.6-fold and 2.9-fold, respectively, compared to the control group, while treatment with ZWC-NPs did not show a statistically significant difference (Fig. 5 c). Additionally, ApoB-48 presence, which adheres to the surface of CM particles after exocytosis, was evaluated in exocytosed CMB-NPs collected and concentrated from the basolateral chamber after 24-hour incubation. Following separation and incubation with an ApoB-48 antibody, exocytosed CMB-NPs were found to be anchored with ApoB-48 (Fig. 5 d). Furthermore, TEM analysis revealed that exocytosed CMB-NPs had a garland-like layer surrounding the particle (Fig. 5 d), with a size of 298 nm, which was slightly larger than ZWC-NPs (280 nm). In summary, western blotting and TEM results demonstrated that CMB-NPs enhance L-FABP expression and are covered with ApoB-48 during lymphatic transport within cells, mimicking the CM assembly process 28 (Fig. 5 e). Having successfully verified the in vitro lymphatic transport of CMB-NPs, we next demonstrated their in vivo ILT. Compared to FITC-ZWC-NPs, FITC-CMB-NPs exhibited significantly higher fluorescence intensity in the rat mesentery, 1 hour after oral administration (Fig. 5 f). This mesenteric absorption was significantly inhibited by cycloheximide (CHX), confirming the involvement of ILT in the oral absorption process. To further quantify ILT, we measured KLO concentrations in the lymphatic vessels and blood using a novel rat model. In this model, an overnight-fasted rat underwent in situ single-pass intestinal perfusion, followed by collection of absorbed KLO-CMB-NPs from the lymphatic ducts and blood vessels via a cannulated catheter (Supplementary Fig. 5). Notably, KLO concentrations in the lymph were higher than in the serum, with the area under the curve (AUC) in the lymph being 1.2-fold greater than that in the serum (Fig. 5 g). These findings indicate that lymphatic transport accounted for most of the intestinal absorption of KLO-CMB-NPs. Consistent with these results, KLO-CMB-NPs achieved an absolute oral bioavailability of 2.7% (Fig. 5 h and Supplementary Table 2). These data confirm that KLO-CMB-NPs significantly enhance the oral bioavailability of KLO via ILT, providing evidence to support further studies on biodistribution and therapeutic efficacy. KLO-CMB-NPs enhance kidney-targeted distribution We next performed ex vivo fluorescence imaging to visualize the biodistribution of FITC-CMB-NPs in normal and unilateral ureteral obstruction (UUO) mice. As expected, normal mice treated with FITC-CMB-NPs exhibited significant fluorescence intensity in the kidneys up to 8 hours post-oral administration, along with strong signals in the liver (Fig. 6 a). The biodistribution of FITC-CMB-NPs in UUO mice was markedly different compared to normal mice. Specifically, FITC-CMB-NPs displayed stronger fluorescence signals in the kidneys, particularly in the obstructed kidney of UUO mice, rather than the liver. At 12 hours post-oral administration, the average fluorescence intensity in the obstructed kidney was 2.2-fold higher than that in the normal kidney (Fig. 6 b). To further investigate this phenomenon, we collected the obstructed kidneys of UUO mice were collected 12 hours post-oral administration for renal sectioning and immunofluorescent observation. Immunofluorescence analysis revealed colocalization of OCTN2 transporter and FITC-CMB-NPs in the renal tubules (Fig. 6 c), indicating OCTN2-mediated kidney accumulation. These findings demonstrate FITC-CMB-NPs enhance kidney-targeted distribution, which is essential for their potential therapeutic application in treating RF. KLO-CMB-NPs improve therapeutic effects against RF In our final set of analyses, we used the UUO-induced RF mouse model to evaluate the anti-fibrotic efficacy of KLO-CMB-NPs. Three days before UUO surgery, mice were orally administered KLO-CMB-NPs (80 µg/kg/day) once daily. In control animals, free KLO (10 µg/kg/day) and captopril (10 mg/kg/day) were intraperitoneally injected once daily (Fig. 6 d). On day 8 post-surgery, the kidneys from all groups were collected for observation. In the UUO mice, the obstructed (right) kidneys exhibited typical pathological signs of RF, including atrophy, paleness, and reduced elasticity, compared to the healthy (left) kidneys (Fig. 6 e). However, the obstructed kidneys in the KLO-CMB-NPs-treated group appeared nearly identical to the healthy kidneys, indicating a strong anti-fibrotic effect. Furthermore, treatment with KLO-CMB-NPs significantly reduced serum creatinine and blood urea nitrogen levels (Fig. 6 f, g), demonstrating effective restoration of renal function. Consistent with the kidney function results, histopathological analysis confirmed that KLO-CMB-NPs significantly inhibited RF (Fig. 6 i). Mice treated with KLO-CMB-NPs exhibited reduced tubulointerstitial injury in H&E-stained kidney sections, including less tubular epithelial cell apoptosis and inflammatory cell infiltration, compared to mice injected with free KLO and captopril groups. Masson's trichrome staining further demonstrated significantly less fibrous tissue in the kidneys of KLO-CMB-NPs-treated mice compared to UUO mice, indicating reduced collagen accumulation and deposition. Additionally, immunohistochemical staining of kidney sections revealed significantly fewer brown granules representing fibronectin and collagen-3 in KLO-CMB-NPs-treated mice compared to UUO mice (Supplementary Fig. 6). These findings suggest that KLO-CMB-NPs effectively alleviated extracellular matrix accumulation. To further elucidate the therapeutic effects of KLO-CMB-NPs against RF, we examined the protein expression of the TGF-β1 signaling pathway in obstructed kidneys. TGF-β1 activation was pronounced in the obstructed kidneys, particularly in the untreated UUO group (Fig. 6 h and Supplementary Fig. S7). Elevated TGF-β1 levels triggered epithelial-mesenchymal transition (EMT), a critical event in RF development, characterized by increased α-smooth muscle actin (α-SMA) expression and reduced E-cadherin expression. However, treatment with KLO-CMB-NPs significantly suppressed TGF-β1 and α-SMA expression, while enhancing E-cadherin expression in the obstructed kidneys. Additionally, KLO expression was markedly increased in the obstructed kidneys of KLO-CMB-NPs-treated mice. These findings demonstrate the efficient kidney-targeted delivery of KLO by KLO-CMB-NPs and underscore their therapeutic potential in mitigating RF through modulation of the TGF-β1 signalling pathway and inhibition of EMT. Discussion ILT is a promising pathway for the efficient oral delivery of macromolecular drugs. Recent studies have focused on TG/MG-conjugated prodrugs or nanoparticles 18 , 19 , achieving satisfactory oral bioavailability. However, since TG resynthesis and LD formation represent rate-limiting steps in ILT (Fig. 1 a), we directly developed CMB-NPs based on the natural components of CMs. To enhance the lipoprotein affinity of CMB-NPs, we conducted a comprehensive formulation study. While the binding affinity to aCMs reached 70.7% by selecting TO as the TG core of CMB-NPs (Fig. 2 c), further screening of 319 pharmaceutical excipients identified Brij-O10 as a key additive. Incorporating Brij-O10 into the CMB-NP formulation significantly increased the binding rate to 84.8% with aCMs and 88.1% with nCMs (Fig. 2 e). SPR analysis confirmed rapid fusion between CMB-NPs and nCMs (Fig. 2 f). After loading with the anti-fibrotic protein KLO 29 , the optimized KLO-CMB-NPs were shown to be an effective drug carrier for daily oral delivery of KLO in the treatment of RF. To overcome the intestinal epithelial barrier, KLO-CMB-NPs must remain stable in the harsh gastrointestinal environment and effectively penetrate the mucus layer. Using the FRET technique, we confirmed that CMB-NPs remained intact for 12 hours under gastrointestinal pH conditions (Fig. 3 b). Additionally, KLO-CMB-NPs retained 78.3% of their β-glucuronidase activity after incubation in simulated digestive fluids, demonstrating enzymatic stability (Fig. 3 c). Furthermore, mucus penetration studies showed that FITC-CMB-NPs rapidly diffused through the mucus layer (Fig. 3 d-f), highlighting their ability to traverse this critical barrier efficiently. Next, we demonstrated that KLO-CMB-NPs enhance intestinal permeability via lymphatic transport in vitro and in vivo and elucidated the underlying mechanism. To better quantify the transcytosis pathway of CMB-NPs, we used fluorescent FITC-CMB-NPs in confocal imaging experiments, with FITC-ZWC-NPs serving as a control. Compared to FITC-ZWC-NPs, FITC-CMB-NPs significantly increased cellular uptake and the apparent permeability coefficient in Caco-2 cells and cell monolayers (Fig. 4 c, g). Additionally, the enhanced transmembrane transport of CMB-NPs was mediated by CMs within the cells (Fig. 4 e). Notably, CMB-NPs exhibited CM-like lymphatic transport. Confocal and TEM imaging revealed colocalization of CMB-NPs with the ER and Golgi apparatus (Fig. 5 a, b), indicating that CMB-NPs were primarily absorbed via lymphatic transport within cells. Furthermore, two key proteins, L-FABP and ApoB-48, were successfully identified and coated the surface of CMB-NPs (Fig. 5 c, d), achieving the intended design of the nanoparticles. Taken together, these findings confirm that the constructed CMB-NPs traffic across enterocytes via lymphatic transport in a manner resembling CMs (Fig. 5 e). Subsequently, we observed that ILT was involved in the oral absorption of FITC-CMB-NPs (Fig. 5 f). To further quantify this process, we performed cannulation into the main mesenteric lymphatic duct of a rat (Supplementary Fig. 5). The results indicated that lymphatic transport predominantly facilitated the intestinal absorption of KLO-CMB-NPs (Fig. 5 g). Although the KLO ELISA measurement presented challenges in the pharmacokinetic study 29 , we successfully quantified the administered KLO using an ELISA kit from Tongwei Biotechnology. This allowed us to report the pharmacokinetic parameters of KLO for the first time (Fig. 5 h and Supplementary Table 2). The findings demonstrated that KLO-CMB-NPs achieved an absolute oral bioavailability of up to 2.7%, confirming their potential as an effective oral delivery system for KLO. In addition to enhancing oral absorption, the absorbed FITC-CMB-NPs demonstrated kidney-targeted distribution, particularly in the obstructed kidneys of UUO mice (Fig. 7a, b). This kidney-specific accumulation was attributed to the overexpression of the OCTN2 transporter in renal tubules, with ZWC-NPs serving as a substrate for OCTN2. In the anti-fibrosis study, daily oral administration of KLO-CMB-NPs (80 µg/kg/day) produced superior therapeutic effects compared to intraperitoneal injection of free KLO (10 µg/kg/day) in UUO mice. Improvements were observed in kidney appearance, renal function, reduced extracellular matrix accumulation, and alleviated pathological changes (Fig. 7e, f, and i). Furthermore, KLO-CMB-NPs preserved KLO expression in the obstructed kidneys of UUO mice (Fig. 7h), which is critical for delaying the progression of RF. Our comprehensive studies have demonstrated significant enhancements in the oral bioavailability and anti-fibrotic effects of KLO-CMB-NPs; however, certain limitations should be acknowledged. First, while KLO-CMB-NPs were shown to be safe and biocompatible during the 7-day observation period (Supplementary Fig. 8), long-term biosafety evaluations are necessary before considering potential clinical applications. Second, although the anti-fibrotic efficacy of KLO-CMB-NPs was well-validated in a 10-day UUO mouse model, further investigations are warranted in chronic kidney disease (CKD) models over extended treatment periods to assess their efficacy in more clinically relevant conditions. Third, this study identified OCTN2-mediated kidney-targeted accumulation of KLO-CMB-NPs; however, the underlying mechanisms for the overexpression of OCTN2 in the obstructed kidneys of UUO mice remain unclear and should be explored in future research. In summary, we developed KLO-CMB-NPs as an efficient oral delivery system utilizing ILT for the treatment of RF. Our findings demonstrated that KLO-CMB-NPs effectively protected encapsulated KLO in the gastrointestinal environment, facilitated penetration through the mucus layer, enhanced oral bioavailability via lymphatic transport, and ultimately exerted significant therapeutic effects on RF. Overall, this study provides an innovative design approach for oral drug carriers targeting ILT. Methods Chemicals, reagents and animals The chemicals and reagents used in this study included phosphatidylcholine, lysophosphatidylcholine, cholesteryl oleate, and cholesterol (AVT, Shanghai, China); olive oil, sodium tripolyphosphate, pluronic-L81, oleic acid, chlorpromazine, indomethacin, colchicine, quercetin, dichloromethane, and triglycerides (TGs) including triolein, trimyristin, tripalmitin, and tristearin (Aladdin, Shanghai, China); Brij-C10, Brij-S10, Brij-O10, TPGS, and Span-40/60/80 (Sigma-Aldrich, St. Louis, MO, USA); and recombinant mouse Klotho (aa 35–982, R&D Systems, Minneapolis, MN, USA). ZWC and FITC-ZWC were synthesized following previously reported methods 30 . All reagents and chemicals were of analytical grade. Caco-2 cells (human colon epithelial cell line, passage number 10–15) were obtained from Procell Life Science & Technology Co., Ltd. (Wuhan, China) and cultured in DMEM (Gibco, Beijing, China) supplemented with 20% FBS (TransGen Biotech, China), 1% penicillin/streptomycin (Gibco, USA), and 1% non-essential amino acids (Gibco, USA) at 37°C in a humidified incubator with 5% CO₂ (Thermo, USA). Cells were collected upon reaching 80% confluence, with the medium changed every 48 hours. Male C57BL/6 mice (8 weeks old, 25–28 g) and Sprague Dawley rats (12 weeks old, 260–280 g) were obtained from Guangdong Medical Laboratory Animal Center (Foshan, China). The animals were maintained under standard conditions at 22°C with 60 ± 10% humidity and a 12-hour light/dark cycle, with free access to fresh water and rodent chow. All experiments were conducted after a 1-week acclimation period to the new environment. Experimental protocols adhered to the National Research Council's Guide for the Care and Use of Laboratory Animals and were approved by the Animal Research Ethics Committee (IACUC-202300106) of Shenzhen University, Shenzhen, China. Collection of nCMs An overnight-fasted Sprague Dawley rat was administered olive oil (2 mL) to stimulate nCM secretion. After 1 hour, the rat was anesthetized and opened via a midline incision, with a catheter inserted into the main mesenteric lymphatic duct. Additionally, fresh blood was supplied through the right jugular vein. The milky white nCMs (approximately 80–100 µL) were collected as they flowed out into a centrifuge tube (Supplementary Fig. 1). Development of aCMs As the quantity of collected nCMs was insufficient to support the formulation optimization study of CMB-NPs, artificial chylomicrons (aCMs) were prepared using a thin-film dispersion method, as previously described 22 . In brief, 0.77 mL olive oil, 227 mg phosphatidylcholine, 23 mg lysophosphatidylcholine, and 20 mg cholesterol were dissolved in 20 mL dichloromethane. The solvent was evaporated to form a lipid film using a rotary evaporator (Buchi, Switzerland) under gentle stirring (30 rpm) for 2 hours.The lipid film was further dehydrated under nitrogen and hydrated overnight at 4°C with 25 mL deionized water. The resulting suspension was vortex-mixed vigorously for 60 seconds and then sonicated using a probe ultrasound (Scientz, China) at 400 W for 15 minutes. The milky white emulsion obtained was sequentially extruded through 0.45 µm and 0.22 µm PVDF filters (Millipore) and characterized by dynamic light scattering (DLS, Malvern Instruments, UK). Preparation of KLO-CMB-NPs KLO-ZWC-NPs were prepared using a cross-linking method as previously described 31 . Specifically, ZWC was dissolved in 0.2% (v/v) acetic acid at a concentration of 3 mg/mL. After filtration through a 0.45 µm PVDF filter (Millipore), 8 mL of sodium tripolyphosphate (0.5 mg/mL, dissolved in deionized water) was added dropwise to 8 mL of ZWC solution containing 20 µg of KLO, with stirring at 300 rpm for 30 minutes. The mixture was then processed using probe ultrasound (200 W, 10 minutes) under an ice bath and filtered through a 0.22 µm filter to produce uniformly dispersed KLO-ZWC-NPs. KLO-CMB-NPs were prepared using the thin-film dispersion method with minor modifications 32 . Specifically, 70 mg of triglyceride (triolein, trimyristin, tripalmitin, or tristearin), 23 mg of phospholipids, 2 mg of lysophosphatidylcholine, and 3 mg of cholesterol were dissolved in 20 mL dichloromethane. The mixture was evaporated to form a lipid film using a rotary evaporator. Any residual dichloromethane in the lipid film was removed under vacuum in a desiccator for 2 hours. Subsequently, 2 mg of a surfactant (Brij-C10, Brij-S10, Brij-O10, TPGS, or Span-40/60/80) was added to freshly prepared KLO-ZWC-NPs (15 mL), which were used to hydrate the lipid film with vigorous vortex mixing and stirring at 70 rpm for 60 minutes. The resulting nanosuspension was sonicated using probe ultrasound and sequentially extruded through 0.45 µm and 0.22 µm filters. Additionally, unloaded nanoparticles (CMB-NPs) were prepared following the same protocol in the absence of KLO. FITC-CMB-NPs were developed similarly, with ZWC replaced by FITC-ZWC. Characterization of KLO-CMB-NPs As CMB-NPs undergo fusion and increase in size after binding with aCMs, unbound CMB-NPs can be quantified using ultracentrifugation. Briefly, 2 mL of CMB-NPs were mixed with an equal volume of aCMs and stirred for 1 hour. The mixture was then centrifuged at 17,000 × g for 30 minutes. This process was repeated twice to ensure complete separation. The unbound CMB-NPs in the supernatant were quantified using a commercial triglyceride (TG) colorimetric assay kit (Nanjing Jiancheng, China). The binding rate of CMB-NPs with aCMs was calculated using the following formula: $$\:\text{B}\text{i}\text{n}\text{d}\text{i}\text{n}\text{g}\:\text{r}\text{a}\text{t}\text{e}\:\text{w}\text{i}\text{t}\text{h}\:\text{a}\text{C}\text{M}\text{s}\:\left(\%\right)=\frac{{W}_{I}-{W}_{U}}{{W}_{I}\:}\times\:100$$ where W I and W U is the weight of initially added CMB-NPs and the weight of unbound CMB-NPs after ultracentrifugation, respectively (unit: mg). Similarly, the binding rates of CMB-NPs with bCMs were determined using ultracentrifugation. For this, 200 µg FITC-BSA (FB, Solarbio, Beijing, China) was loaded into FB-CMB-NPs following the previously described protocol. Equal volumes of FB-CMB-NPs (2 mL) and bCMs were mixed and stirred for 1 hour. The mixture was then centrifuged twice. The free FB-CMB-NPs in the supernatant were lysed and quantified using a microplate reader (Biotek, USA). The binding rate of CMB-NPs with bCMs was calculated according to the following formulas: $$\:\text{B}\text{i}\text{n}\text{d}\text{i}\text{n}\text{g}\:\text{r}\text{a}\text{t}\text{e}\:\text{w}\text{i}\text{t}\text{h}\:\text{b}\text{C}\text{M}\text{s}\:\left(\%\right)=\frac{{W}_{T}-{W}_{F}}{{W}_{T}\:}\times\:100$$ where W T and W F is the weight of totally added FB-CMB-NPs and the weight of free FB-CMB-NPs in the supernatant after ultracentrifugation, respectively (unit: µg). The binding affinity of CMB-NPs to bCMs was further confirmed using a surface plasmon resonance (SPR) technique as previously described 33 . Briefly, a CM5 sensor chip was prepared by activating a cell with a mixture of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC, 200 µM) and N-hydroxysuccinimide (NHS, 50 µM), infused at a flow rate of 10 µL/min for 420 seconds. Subsequently, a solution containing 50 µL of KLO recombinant protein (1 mg/mL) mixed with 180 µL of 10 mM sodium acetate (pH 5.0) was immobilized on the cell surface at 10 µL/min for 420 seconds. This immobilization process was repeated twice. The cell was then blocked with 1 M ethanolamine infused at 10 µL/min for 10 minutes. A neighboring aisle, serving as a reference, underwent identical activation and blocking steps, but PBS (pH 5.0) was used for immobilization instead. Both aisles were equilibrated with PBS prior to testing. The molecule stock solution was diluted to various concentrations in PBS and flowed over the chip at 10 µL/min for 150 seconds per run. After each run, the cells were regenerated using a 10 mM glycine-HCl solution (pH 2.0) infused at 10 µL/min for 5 minutes. Data from the sample cell were recorded using Biacore T200 Control software (version 2.0, GE Healthcare) and subtracted from the reference cell data. Association and dissociation constants were derived through global fitting of the data to a 1:1 Langmuir binding model, facilitated by the software. The size and zeta potential of KLO-CMB-NPs were characterized using dynamic light scattering (DLS). The morphologies of aCMs, bCMs, KLO-ZWC-NPs, and KLO-CMB-NPs were observed using transmission electron microscopy (TEM, Hitachi, Japan). The storage stability of CMB-NPs at 4°C was assessed by monitoring changes in particle size and polydispersity index (PDI) over a period of 5 days. Encapsulation efficiency (EE) and loading capacity (LC) of KLO-CMB-NPs were calculated using a previously reported method 31 . The amount of unloaded KLO in the nanosuspension was quantified using a BCA protein assay kit (Thermo, USA). To examine the in vitro release of CMB-NPs, KLO was replaced by bovine serum albumin (BSA), and a dialysis method was employed 34 . Briefly, 1 mL of free BSA (5 mg/mL), BSA-ZWC-NPs, and BSA-CMB-NPs (containing 5 mg/mL BSA) were placed into dialysis bags with a molecular weight cutoff (MWCO) of 10 kDa. The dialysis bags were immersed in 45 mL of release medium at 37°C under gentle shaking. Initially, the bags were incubated in 0.1 M HCl (pH 1.2) for 2 hours, followed by PBS buffer (pH 6.8) for an additional 6 hours. At predetermined time points, 1 mL of the released sample was collected and replaced with an equal volume of fresh buffer. The cumulative release of BSA was measured using the BCA protein assay kit. Stability study using FRET FRET-CMB-NPs were prepared following a previously reported method with minor modifications 31 . FITC (0.1 mg, dissolved in deionized water) and RhoB (0.3 mg, dissolved in deionized water) were co-loaded into ZWC-NPs as described in the protocol. Unencapsulated FITC and RhoB were removed by ultrafiltration using a MWCO of 3 kDa. A lipid film was then prepared using the previously mentioned protocol and hydrated with the obtained FRET-ZWC-NPs. The resulting FRET-CMB-NPs were diluted and analyzed using a microplate reader, with excitation set at 450 nm and emission spectra measured from 480 to 700 nm. To assess stability, freshly prepared FRET-CMB-NPs (1 mL) were placed in a dialysis bag (MWCO 3 kDa) and submerged in a centrifuge tube containing 4 mL of 0.1 M HCl (pH 1.2) or phosphate buffer (pH 6.8). At predetermined time points, 100 µL of samples were withdrawn from the dialysis bag and analyzed using the microplate reader. Stability study by β-glucuronidase activity The stability of encapsulated KLO in KLO-CMB-NPs was further evaluated by assessing β-glucuronidase activity. Freshly prepared KLO-CMB-NPs (2 mL) were placed into a dialysis bag (MWCO 200 kDa) and incubated in simulated gastric fluid (100 mL) for 2 hours, followed by simulated intestinal fluid (100 mL) for 10 hours. As previously reported 23 , KLO specifically hydrolyzes 4-methylumbelliferyl β-D-glucuronide into 4-methylumbelliferone, which exhibits strong fluorescence. After incubation, the remaining KLO-CMB-NPs were washed three times with PBS and their β-glucuronidase activity was measured to determine the stability of the encapsulated KLO. Mucus layer penetration The mucus permeation of FITC-CMB-NPs in intestinal loops was visualized using a confocal laser scanning microscope (CLSM, ZEISS, LSM880, Germany) 35 . Briefly, a 2 cm segment of the jejunum was isolated from an overnight-fasted male Sprague Dawley rat. FITC-CMB-NPs were introduced into the isolated jejunum to prepare intestinal loops. The intestinal loops were maintained in 10 mL of oxygenated Krebs-Ringer buffer at 37°C with gentle shaking (100 rpm). After a 1-hour incubation, the intestinal loops were excised, stained with Alexa 555-WGA (Thermo Fisher Scientific, USA), and visualized under CLSM to assess mucus permeation of the FITC-CMB-NPs. Cellular uptake Caco-2 cells (1 × 10⁶ cells) were seeded in a confocal dish and allowed to adhere overnight. Upon reaching 90% confluence, the culture medium was replaced with medium containing FITC-ZWC (1.6 mg/mL), FITC-ZWC-NPs, or FITC-CMB-NPs (equivalent to FITC-ZWC at 1.6 mg/mL). After 1 hour of incubation, the treated cells were washed twice with cold PBS, fixed with 4% paraformaldehyde, stained with 4',6-diamidino-2-phenylindole (DAPI), and observed under a confocal laser scanning microscope (CLSM). For quantitative analysis, cellular uptake was measured using flow cytometry (BC-Cytoflex, Beckman, USA). Caco-2 cells (2 × 10⁶ cells/well) were seeded in a 24-well plate and cultured overnight. The cells were treated with FITC-ZWC, FITC-ZWC-NPs, or FITC-CMB-NPs (equivalent to FITC-ZWC at 1.6 mg/mL). After 2 hours of incubation, the fluorescence intensity in the collected cells was determined by the flow cytometer and analyzed using FlowJo software. To further investigate the endocytosis pathways of FITC-CMB-NPs in Caco-2 cells, cells were pretreated with specific endocytosis inhibitors for 2 hours. These inhibitors included chlorpromazine (50 µM), indomethacin (100 µM), colchicine (10 µM), and quercetin (10 µM). Cellular uptake in the presence of these inhibitors was also quantified using the flow cytometer to identify the predominant pathways involved Transcellular transport study Caco-2 cells were plated onto polycarbonate inserts (0.4 µm pore size, 4.67 cm² growth area, Corning) at a density of 5 × 10⁵ cells/well and cultured for 21 days prior to conducting transcellular transport studies, as previously described 36 . Only polarized cell monolayers with transepithelial electrical resistance (TEER) values exceeding 600 Ω were used in the subsequent experiments. FITC-ZWC (1.6 mg/mL), FITC-ZWC-NPs, and FITC-CMB-NPs (equivalent to FITC-ZWC at 1.6 mg/mL) were added to the apical side of the inserts, either in the absence or presence of Pluronic-L81 (2 mM) or oleic acid (2 mM). At 15, 30, 45, 60, 90, and 120 minutes, 0.5 mL of the sample from the basolateral chamber was collected and replaced with an equal volume of Hank's balanced salt solution (HBSS). The concentration of FITC-ZWC in the collected samples was determined using a microplate reader. The permeability coefficient (Papp) was calculated according to our reported method 37 . After 2 hours of incubation, the cell monolayers were washed three times with cold PBS, fixed with 4% paraformaldehyde, stained with DAPI, and visualized using a confocal laser scanning microscope (CLSM). The nanoparticle integrity of CMB-NPs was measured after the transcellular transport study using FRET. FRET-ZWC-NPs and FRET-CMB-NPs were loaded into the apical chamber of a polarized Caco-2 cell monolayer. At the indicated timepoints, transcellular nanoparticles were collected from the basolateral chamber, and then diluted and measured using the microplate reader. Lymphatic transport within Caco-2 cells Caco-2 cells were seeded in a confocal dish and allowed to adhere overnight. Following treatment with FITC-CMB-NPs (0.84 mg/mL) for 6 hours, ER-Tracker (1 µM, Beyotime Biotechnology, China) or Golgi-Tracker (333 µg/mL, Beyotime Biotechnology, China) was added to the dish and incubated at 4°C for 30 minutes. The cells were then fixed with 4% paraformaldehyde, stained with DAPI, and visualized using a confocal laser scanning microscope (CLSM). The fluorescence intensity and Pearson’s correlation coefficient were analyzed using ImageJ software to evaluate co-localization. Additionally, the co-localization of FITC-CMB-NPs with the ER and Golgi apparatus was further confirmed by transmission electron microscopy (TEM) as previously described 19 . Caco-2 cells were treated with FITC-CMB-NPs (0.84 mg/mL) for 6 hours, then fixed with 3% glutaraldehyde followed by 1% osmium tetroxide. The fixed cells were dehydrated through a graded ethanol series and embedded in epoxy resin. Ultra-thin sections were prepared from polymerized epoxy resin blocks and stained with 1% uranyl acetate and Reynold's lead citrate before being examined under TEM. CM-like lymphatic transport was assessed by analyzing L-FABP expression in Caco-2 cells, following a previously reported method 19 . Caco-2 cells were treated with CMB-NPs (0.84 mg/mL), TG (20 µL), or ZWC-NPs (0.3 mg/mL) for 24 hours. The cells were then harvested and lysed, and protein content was measured using a BCA protein assay kit (Thermo Fisher Scientific). Equal amounts of protein samples (30 µg) were separated on a 12% SDS-PAGE gel and electro-transferred onto PVDF membranes. The membranes were blocked with 5% BSA and incubated overnight at 4°C with primary antibodies against L-FABP (1:1,000, Abcam, ab222517) and GAPDH (1:2,000, Thermo Fisher Scientific). After incubation with secondary antibodies (1:2,000, Thermo Fisher Scientific), specific protein bands were visualized using a chemiluminescence detection kit (Thermo Fisher Scientific). Chemiluminescent signals were quantified using the ChemiDoc MP Gel Imaging System (Bio-Rad, USA). The presence of Apo-B48 (~ 250 kDa) anchored on the surface of CMB-NPs was verified in a Caco-2 cell monolayer. Following incubation with CMB-NPs (0.84 mg/mL), TG (20 µL), or ZWC-NPs (0.3 mg/mL) for 24 hours, the medium from the basolateral chamber was collected and total protein was precipitated. To isolate total protein, a mixture of medium (700 µL), methanol (175 µL), and chloroform (700 µL) was centrifuged at 21,000 × g for 10 minutes. The protein layer (middle phase) was collected and dissolved in a 2% SDS solution. Protein concentration was measured using the BCA protein assay kit. The samples were then separated on an 8% SDS-PAGE gel and transferred onto PVDF membranes. The membranes were blocked with a suitable blocking buffer and incubated overnight at 4°C with primary antibodies against Apo-B48 (1:500, Abcam, ab312318). After incubation with a secondary antibody, the protein bands were visualized using a chemiluminescence kit, and the signals were quantified. Additionally, the exocytosed CMB-NPs in the cell supernatant were visualized using TEM. After treating Caco-2 cell monolayers with CMB-NPs (0.84 mg/mL), TG (20 µL), or ZWC-NPs (0.3 mg/mL) for 24 hours, the medium from the basolateral chamber was collected and dialyzed in deionized water using a membrane with a MWCO of 500 Da for 12 hours. The dialyzed samples were then prepared for TEM observation by standard procedures, including deposition onto a grid, staining, and imaging to evaluate the morphology of exocytosed CMB-NPs. ILT study FITC-ZWC-NPs and FITC-CMB-NPs (equivalent to 2.4 mg/kg FITC-ZWC) were orally administered to overnight-fasted male Sprague Dawley rats (n = 3). Additionally, a group of rats was pretreated with cycloheximide (CHX, 3 mg/kg) via intraperitoneal injection 1 hour before FITC-CMB-NPs administration to inhibit lymphatic transport 38 , 39 . At 1-hour post-administration, the rats were sacrificed, and their gastrointestinal tracts were visualized using an IVIS spectrum imaging system (PerkinElmer, USA). The proportion of intestinal absorption via lymphatic transport (ILT) was determined by cannulation into the main mesenteric lymphatic duct of a Sprague Dawley rat 40 , 41 . Specifically, an overnight-fasted rat was administered olive oil (2 mL) to facilitate lymphatic duct cannulation. One hour later, the main mesenteric lymphatic duct became visible, and the rat was anesthetized for in situ single-pass intestinal perfusion. Warm KLO-CMB-NPs (containing 10 ng/mL KLO, diluted in PBS) were perfused at a flow rate of 0.20 mL/min for 1 hour using a syringe pump (Baoding Rongbai Precision Pump, China). Fresh blood was continuously supplied via the right jugular vein. At the designated time points, absorbed KLO-CMB-NPs were collected from the lymphatic duct and blood vessel using a cannulated catheter (external diameter 0.45 mm, internal diameter 0.22 mm). The KLO concentrations in lymph and blood samples were quantified using a commercial ELISA kit (Shanghai Tongwei, China). Pharmacokinetic study The pharmacokinetic study was conducted as previously reported 42 . KLO-CMB-NPs (equivalent to 20 µg/kg KLO) were administered orally to overnight-fasted Sprague Dawley (SD) rats (n = 6). A control group of rats was injected with free KLO (2 µg/kg) via the caudal vein. Blood samples were collected into heparinized tubes at specified time points, and KLO concentrations were measured using a commercial ELISA kit. The plasma concentration-time profiles were analyzed using DAS software (Shanghai, China) to evaluate pharmacokinetic parameters). Biodistribution study FITC-ZWC, FITC-ZWC-NPs, and FITC-CMB-NPs (equivalent to 2.4 mg/kg FITC-ZWC) were orally administered to overnight-fasted normal and unilateral ureteral obstruction (UUO) mice (n = 3 per group). At predetermined time points, three treated mice from each group were sacrificed by CO₂ inhalation. Tissue samples, including the heart, liver, spleen, lungs, kidneys, and gastrointestinal tract, were collected and visualized using the IVIS spectrum imaging system (PerkinElmer, USA) to assess the biodistribution of the nanoparticles. Therapeutic effects in UUO mice UUO-induced RF was generated in C57BL/6 mice as described previously 43 . Briefly, mice were anesthetized with 10% chloral hydrate (400 mg/kg, i.p.), and the right kidney was exposed through an incision in the right lateral dorsal surface. The ureter of the right kidney was ligated with 4–0 silk, and the incision was sutured. UUO mice were randomly divided into 5 groups (n = 8): (1) UUO (Model group); (2) Captopril (i.p., 10 mg/kg/day); (3) Free KLO (i.p., 10 µg/kg/day); (4) KLO-ZWC-NPs (p.o., equal to KLO 80 µg/kg/day) and (4) KLO-CMB-NPs (p.o., equal to KLO 80 µg/kg/day). Age-matched mice that underwent a similar surgery without ureter ligation served as the normal group. For the treatment groups, mice received daily intraperitoneal or intragastric administration starting 3 days before UUO induction and continuing until day 7 after surgery. Mice in the normal and model groups received an equal volume of distilled water. On day 8 post-surgery, all mice were sacrificed by CO₂ inhalation, and blood and kidney samples were collected. The right kidneys were decapsulated, washed, and dissected. Portions of each kidney were fixed in 10% formalin for histological analysis, while the remaining tissue was stored at − 80°C for western blot analysis. Serum creatinine levels were measured using a creatinine assay kit (Jiancheng Biotech. Co. Ltd., China), while serum blood urea nitrogen (BUN) levels were determined using a urea nitrogen content assay kit (Solarbio, China) 44 . Kidney samples were excised, weighed, formalin-fixed, and embedded in paraffin for sectioning. Histological examination was conducted under a microscope (Olympus, Japan) following hematoxylin and eosin (H&E) staining to assess general tissue morphology and Masson's trichrome staining to evaluate collagen deposition. Immunohistochemical staining was performed as previously reported 45 . Paraffin sections were quenched for endogenous peroxidase activity, pre-blocked with normal goat serum, and incubated with primary antibodies against collagen-I and fibronectin (1:100, Cell Signaling Technology, USA). The sections were visualized using DAB chromogen, counterstained with hematoxylin, and examined under a microscope. Kidney samples were prepared for western blot analysis as described previously 46 . Briefly, kidney tissues were homogenized, and total proteins were extracted using TRIzol reagent. The protein concentration was determined using a BCA protein assay kit (Thermo Fisher Scientific). Equal amounts of protein lysates were separated on an 8% SDS-PAGE gel and transferred onto PVDF membranes. The membranes were blocked with 5% BSA and incubated overnight at 4°C with primary antibodies against the following proteins: TGF-β1 (1:1000, Cell Signaling Technology, USA), α-SMA (1:1000, Proteintech, USA), E-cadherin (1:1000, Proteintech, USA), and GAPDH (1:1000, Abcam, USA). After incubation with secondary antibodies (1:1000, Cell Signaling Technology, USA), the specific protein bands were visualized using a chemiluminescence detection kit (Thermo Fisher Scientific). The chemiluminescent signals were quantified using the ChemiDoc MP Gel Imaging System (Bio-Rad, USA). Safety and biocompatibility evaluation Male C57BL/6 mice were administered daily doses of KLO-CMB-NPs (p.o., equivalent to KLO 80 µg/kg/day) for 7 consecutive days 47 . Mice in the control group received an equal volume of PBS daily. Throughout the study, mice were monitored for changes in body weight. On day 8, all mice were sacrificed by CO 2 inhalation, and their serum and organ samples were collected and stored at − 80°C. Various biochemical indicators, including liver enzyme activity and creatinine levels, were measured in the serum using assay kits (Jiancheng Biotech. Co. Ltd., China). The heart, liver, spleen, lung, and kidney tissues were fixed in formalin, sectioned, and stained with hematoxylin and eosin (H&E) for histopathological examination under a microscope. Declarations Data availability The data generated or analyzed in this study are included in the published article. These data will be made available upon request. Acknowledgements This study was supported by the Natural Science Foundation of China (Grant Nos. 82304411 and 52273299), the Guangdong Basic and Applied Basic Research Foundation (Grant No. 2022A1515010481), Guangdong Science and Technology Program (Grant No. 2023A1515011884) and the Shenzhen Science and Technology Program (Grant Nos. RCBS20221008093120049, 860000002111304, 827-00074220, GJHZ20210705141800002, JCYJ20220531102207016, D2403008 and KCXFZ20230731092802004). The authors thank the Instrumental Analysis Center of Shenzhen University for their technical support. Author contributions Yawen Yao : Writing – original draft, Methodology, Investigation. Cuihua Zhang : Writing – original draft, Methodology, Investigation. Jieying Zhou : Investigation. Shihao Xu : Investigation. Haiqiang Wu : Resources, Project administration, Supervision, Conceptualization. Hua Yu : Resources, Project administration, Formal analysis, Supervision, Funding acquisition, Conceptualization. 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Supplementary Files CMBSIXW20250324.docx Supporting Information: Chylomicron-biomimetic nanoparticles promote intestinal lymphatic transport by fast fusion with chylomicrons Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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Xiong","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA1UlEQVRIiWNgGAWjYFACxgaGBCDFD+Exk6BFsoF4LVBgcIBYLQbHm9skHlTcsdt8/vgzCYYK68QG9rMH8Gs5c7DZIOHMs+RtNxLSJBjOpCc28OQl4NVidiOx8UFi2+FksxsMxyQY2w4nNkjwGODXcv9hwwGQFuP+g20SjP+I0XKDEWyLnQFDMpsEYwMRWuzPJIL8cjhB4kYas0XCsXTjNp4c/Fok248/k/xRcdiev//4wxsfaqxl+9nP4NcCA4kNIDIBiNmIUg9yILEKR8EoGAWjYAQCAJJcSYCHSht5AAAAAElFTkSuQmCC","orcid":"","institution":"Shenzhen University","correspondingAuthor":true,"prefix":"","firstName":"Wei","middleName":"","lastName":"Xiong","suffix":""},{"id":447990077,"identity":"548ffd56-079c-4176-9f5c-3b49d706c047","order_by":1,"name":"Yawen Yao","email":"","orcid":"","institution":"University of Macau","correspondingAuthor":false,"prefix":"","firstName":"Yawen","middleName":"","lastName":"Yao","suffix":""},{"id":447990078,"identity":"bbf53e1d-eed0-4b5f-8d36-68ba8d88cf15","order_by":2,"name":"Cuihua Zhang","email":"","orcid":"","institution":"Shenzhen University","correspondingAuthor":false,"prefix":"","firstName":"Cuihua","middleName":"","lastName":"Zhang","suffix":""},{"id":447990079,"identity":"e2ab4de5-5eef-4ec3-b1b2-0d1cf0425421","order_by":3,"name":"Jieying Zhou","email":"","orcid":"https://orcid.org/0009-0001-1912-8894","institution":"Shenzhen University","correspondingAuthor":false,"prefix":"","firstName":"Jieying","middleName":"","lastName":"Zhou","suffix":""},{"id":447990080,"identity":"cc86ebe3-5912-4017-9e14-05cf7d97a900","order_by":4,"name":"Shihao Xu","email":"","orcid":"","institution":"Shenyang Pharmaceutical University","correspondingAuthor":false,"prefix":"","firstName":"Shihao","middleName":"","lastName":"Xu","suffix":""},{"id":447990081,"identity":"4099eddf-037a-45ea-ab06-7b2406395d91","order_by":5,"name":"Ying Li","email":"","orcid":"","institution":"Shenzhen University","correspondingAuthor":false,"prefix":"","firstName":"Ying","middleName":"","lastName":"Li","suffix":""},{"id":447990082,"identity":"db4377d2-f151-43cd-b43f-54bb5ec009d2","order_by":6,"name":"Haiqiang Wu","email":"","orcid":"","institution":"Shenzhen University","correspondingAuthor":false,"prefix":"","firstName":"Haiqiang","middleName":"","lastName":"Wu","suffix":""},{"id":447990083,"identity":"c43aca79-185e-40bb-adff-043939f85462","order_by":7,"name":"Hua Yu","email":"","orcid":"","institution":"University of Macau","correspondingAuthor":false,"prefix":"","firstName":"Hua","middleName":"","lastName":"Yu","suffix":""}],"badges":[],"createdAt":"2025-03-25 07:35:45","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6301196/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6301196/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":81498511,"identity":"0a15deb6-f286-4695-9888-0cd325f6a4b4","added_by":"auto","created_at":"2025-04-28 03:11:01","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":9249900,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSchematic illustration of the KLO-CMB-NPs promoting intestinal lymphatic transport (ILT) by fast fusion with chylomicrons (CMs).\u003c/strong\u003e \u003cstrong\u003ea\u003c/strong\u003e Lipid droplet (LD)formation serves as a rate-limiting step during ILT. \u003cstrong\u003eb\u003c/strong\u003e Development of KLO-CMB-NPs with high lipoprotein affinity. \u003cstrong\u003ec\u003c/strong\u003e KLO-CMB-NPs survive in the gastrointestinal tract. \u003cstrong\u003ed\u003c/strong\u003e KLO-CMB-NPs interact with CMs during the intracellular lymphatic pathway. \u003cstrong\u003ee\u003c/strong\u003e ILT bypasses first-pass hepatic metabolism, draininginto systemic circulation via the left subclavian vein.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-6301196/v1/463376f83e09dd5eabbf9ee8.png"},{"id":81499450,"identity":"4117bdd8-c09c-426f-afb5-1a69a4f320a7","added_by":"auto","created_at":"2025-04-28 03:27:01","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2131480,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe development of KLO-CMB-NPs with high binding affinity to chylomicrons (CMs).\u003c/strong\u003e Particle size and distribution of (\u003cstrong\u003ea\u003c/strong\u003e) natural chylomicrons (nCMs) and (\u003cstrong\u003eb\u003c/strong\u003e) artificial chylomicrons (aCMs). Screening of (\u003cstrong\u003ec\u003c/strong\u003e) triglyceride (TG) and (\u003cstrong\u003ed\u003c/strong\u003e) high-lipoprotein-affinity pharmaceutical excipients in the CMB-NPs formulation based on binding rates with aCMs. \u003cstrong\u003ee\u003c/strong\u003e Optimization of the CMB-NPs formulation confirmed by binding rates with nCMs. \u003cstrong\u003ef\u003c/strong\u003e Binding affinity of nCMs quantified using surface plasmon resonance (SPR). \u003cstrong\u003eg\u003c/strong\u003e Particle size stability of KLO-CMB-NPs at 4°C. \u003cstrong\u003eh\u003c/strong\u003e Particle sizes, zeta potentials, and TEM images of KLO-ZWC-NPs and KLO-CMB-NPs. \u003cstrong\u003ei \u003c/strong\u003eEncapsulation efficiency (EE) and loading capacity (LC). \u003cstrong\u003ej \u003c/strong\u003e\u003cem\u003eIn vitro\u003c/em\u003e release profiles of BSA-CMB-NPs in release media at pH 1.2 and 6.8. Data represent the means ± SD (n = 3), * \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, ** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, *** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, versus control group.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-6301196/v1/beb5d7114de60559d456aa6d.png"},{"id":81498529,"identity":"f470417a-3919-4c0b-a519-488a84939f4e","added_by":"auto","created_at":"2025-04-28 03:11:02","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":3816007,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eKLO-CMB-NPs maintained nanoparticle integrity in the gastrointestinal environment and enhanced permeability across the mucus layer.\u003c/strong\u003e \u003cstrong\u003ea \u003c/strong\u003eEmission spectra of FRET-CMB-NPs excited at 450 nm. \u003cstrong\u003eb\u003c/strong\u003e Emission spectra of FRET-CMB-NPs after 12-hour incubation in 0.1M HCl (pH 1.2) and PBS (pH 6.8). \u003cstrong\u003ec\u003c/strong\u003e β-glucuronidase activity of KLO-CMB-NPs following 12-hour incubation in simulated gastric and intestinal fluids. \u003cstrong\u003ed \u003c/strong\u003e3D visualization of FITC-CMB-NPs distribution within the mucus layer of the rat jejunum. \u003cstrong\u003ee\u003c/strong\u003e Fluorescence intensity profiles of FITC-ZWC at different longitudinal depths within the mucus layer. \u003cstrong\u003ef\u003c/strong\u003e Quantitative analysis of FITC-ZWC fluorescence intensity across the mucus layer. Data represent the means ± SD (n = 3), * \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, ** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, *** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, versus control group.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-6301196/v1/e1c091aeb64729d94e9b5a5a.png"},{"id":81498524,"identity":"4ff8b895-936e-4c4e-9110-0fc0b0790666","added_by":"auto","created_at":"2025-04-28 03:11:01","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":3640508,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eKLO-CMB-NPs enhanced transmembrane capability via lymphatic transport. \u003c/strong\u003eConfocal images (\u003cstrong\u003ea\u003c/strong\u003e) and quantitative analysis (\u003cstrong\u003eb\u003c/strong\u003e) of cellular uptake ofFITC-labeled ZWC (FITC-ZWC), FITC-ZWC-NPs, and FITC-CMB-NPs derived from FITC-ZWC in Caco-2 cells (scale bar: 10 μm). \u003cstrong\u003ec\u003c/strong\u003e Statistical analysis of fluorescence intensity during cellular uptake. \u003cstrong\u003ed\u003c/strong\u003e Effects of endocytosis inhibitors on the internalization of FITC-CMB-NPs in Caco-2 cells. \u003cstrong\u003ee\u003c/strong\u003e Apparent permeability of FITC-CMB-NPs in the presence of Pluronic-L81 or oleic acid in a Caco-2 cell monolayer.\u003cstrong\u003e f \u003c/strong\u003eVisualization of FITC-CMB-NPs crossing the Caco-2 cell monolayer. \u003cstrong\u003eg\u003c/strong\u003e Quantitative analysis ofFITC-CMB-NPs fluorescence intensity within the Caco-2 cell monolayer. Data represent the means ± SD (n = 3), * \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, ** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, *** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, versus control group.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-6301196/v1/a334a6beeb0b3315127b67bb.png"},{"id":81498749,"identity":"6434ac8d-75ec-4b99-a60d-b585b2b0a66e","added_by":"auto","created_at":"2025-04-28 03:19:02","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":13585758,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eKLO-CMB-NPs enhanced intestinal lymphatic transport (ILT) in a chylomicron (CM)-like manner. a \u003c/strong\u003eConfocal images showing colocalization of FITC-CMB-NPs with the endoplasmic reticulum (ER) and Golgi apparatus. Yellow signals in merged images indicate colocalization, with corresponding fluorescence intensity profiles across the cell along selected yellow lines. \u003cstrong\u003eb\u003c/strong\u003e TEM images illustrating colocalization of CMB-NPs with the ER or Golgi. CMB-NPs (black dots) are highlighted by yellow arrows, with ER and Golgi structures colored red and blue, respectively. \u003cstrong\u003ec \u003c/strong\u003eL-FABP expression in Caco-2 cells after 24-hour treatment with CMB-NPs. \u003cstrong\u003ed\u003c/strong\u003e Representative western blots and TEM images of ApoB-48 anchored to the surface of CMB-NPs in a Caco-2 cell monolayer after 24 hours (scale bar: 100 nm). \u003cstrong\u003ee\u003c/strong\u003e Proposed mechanism of CMB-NPs transcytosis across enterocytes via a CM-like pathway.\u003cstrong\u003e f \u003c/strong\u003eVisualization of ILT of FITC-CMB-NPs in rats.\u003cstrong\u003e g\u003c/strong\u003e Quantitative analysis of ILT of KLO-CMB-NPs viacannulation of the main mesenteric lymphatic duct. \u003cstrong\u003eh \u003c/strong\u003ePlasma concentration-time curves of KLO in rats following oral administration of KLO-CMB-NPs (1 μg/kg KLO) or intravenous injection of free KLO (0.1 μg/kg KLO) (n = 6).Data represent the means ± SD, * \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, ** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, *** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, versus control group.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-6301196/v1/f6d4572ce24420051d4db195.png"},{"id":81498513,"identity":"58450686-b20f-4241-8861-368107337e80","added_by":"auto","created_at":"2025-04-28 03:11:01","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":12575282,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eKLO-CMB-NPs improved kidney-targeted distribution and therapeutic efficacy in unilateral ureteral obstruction (UUO) mice. a\u003c/strong\u003e Biodistribution of FITC-CMB-NPs (equivalent to 2.4 mg/kg of FITC-ZWC) in normal and UUO mice following oral administration (n = 3). \u003cstrong\u003eb\u003c/strong\u003e Region-of-interest analysis of tissue accumulation. \u003cstrong\u003ec\u003c/strong\u003e Fluorescence images of obstructed kidney sections in UUO mice at 12 hours post-oral administration of FITC-CMB-NPs. \u003cstrong\u003ed\u003c/strong\u003e Schematic illustration of the treatment regimens. \u003cstrong\u003ee \u003c/strong\u003eRepresentative images of normal (left) and obstructed (right) kidneys from UUO mice. Serum levels of creatinine (\u003cstrong\u003ef\u003c/strong\u003e) and blood urea nitrogen (BUN) (\u003cstrong\u003eg\u003c/strong\u003e) in UUO mice (n = 8). \u003cstrong\u003eh\u003c/strong\u003e Expression of fibrosis-associated proteins in the obstructed kidneys of UUO mice. \u003cstrong\u003ei\u003c/strong\u003e Histological sections of obstructed kidneys in UUO mice stained with H\u0026amp;E, Masson, and antibodies against collagen-3 and fibronectin (scale bar: 200 μm). Data represent the means ± SD, * \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, ** \u003cem\u003ep\u003c/em\u003e\u0026lt; 0.01, *** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, versus control group.\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-6301196/v1/3b310c2823108b8b3b870d26.png"},{"id":82734241,"identity":"e199dde2-2c54-4dfe-bb75-a7ba5b12231f","added_by":"auto","created_at":"2025-05-14 15:28:48","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":42705189,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6301196/v1/cad5e603-de8a-49b4-a7ad-d0a72801b050.pdf"},{"id":81498523,"identity":"5ed06065-7bb4-415c-a629-c021a21f661a","added_by":"auto","created_at":"2025-04-28 03:11:01","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":4588738,"visible":true,"origin":"","legend":"Supporting Information: Chylomicron-biomimetic nanoparticles promote intestinal lymphatic transport by fast fusion with chylomicrons","description":"","filename":"CMBSIXW20250324.docx","url":"https://assets-eu.researchsquare.com/files/rs-6301196/v1/99acd5ab6a2495de7176ba21.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Chylomicron-biomimetic nanoparticles promote intestinal lymphatic transport by fast fusion with chylomicrons","fulltext":[{"header":"Introduction","content":"\u003cp\u003eOral administration of macromolecular drugs is the preferred option for patients requiring daily and long-term medication\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Most oral macromolecular drugs, however, fail to survive in the gastrointestinal tract\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e, penetrate the mucus layer\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e, cross the intestinal epithelial barrier\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e, and reach target sites at therapeutic concentration\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. While encapsulation\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e and PEGylation technologies\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e have successfully addressed the first two challenges, the latter two remain significant hurdles, resulting in low bioavailability and poor therapeutic efficacy for oral macromolecular drugs.\u003c/p\u003e \u003cp\u003eInspired by the endogenous transport of macromolecules from the intestinal epithelium into lymphatic vessels rather than the bloodstream\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e, rerouting drug absorption to intestinal lymph transport (ILT) has attracted widespread attention\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. ILT is the primary pathway for dietary lipid transport, such as triglycerides (TGs), following absorption by enterocytes\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. During this process, orally administered TGs are digested into fatty acids and 2-monoglycerides (MGs) in the intestinal lumen, absorbed by enterocytes, and re-esterified to TGs. These TG droplets then bind with lipoproteins, including L-FABP and ApoB-48, to form chylomicrons (CMs) in the endoplasmic reticulum (ER) and Golgi apparatus. CMs are subsequently secreted into mesenteric lymphatic vessels\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e, which drain directly into the systemic circulation via the left subclavian vein, bypassing first-pass hepatic metabolism\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. Recent studies have suggested that TG/MG-conjugated prodrugs\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e and nanoparticles\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e penetrated the intestinal epithelial barrier through ILT, enhancing oral bioavailability by 6.1-fold and 10.6-fold, respectively. The extent of ILT for these prodrugs and nanoparticles, however, remains unclear. Most studies have primarily assessed plasma drug levels with and without cycloheximide (an inhibitor of lipoprotein secretion), without directly quantifying lymphatic transport.\u003c/p\u003e \u003cp\u003eHere, we report the significant finding that chylomicron-biomimetic nanoparticles (CMB-NPs) with high lipoprotein affinity effectively cross the intestinal epithelial barrier intact via ILT. Basing on the components of CMs\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e, we constructed CMB-NPs with a TG-rich core surrounded by phospholipids, cholesterol, and Brij-O10. Since TG resynthesis and lipid droplet (LD) formation represent rate-limiting steps in ILT\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e, we screened 319 pharmaceutical excipients for lipoprotein affinity and identified Brij-O10 as a key component that enhances lymphatic transport by facilitating rapid fusion with CMs (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed). Moreover, we established a novel animal model by cannulating the main mesenteric lymphatic duct of a rat, enabling direct quantification of ILT.\u003c/p\u003e \u003cp\u003eTo explore the therapeutic applications, we encapsulated the anti-fibrotic macromolecular drug klotho (KLO) within zwitterionic chitosan nanoparticles (KLO-ZWC-NPs) to facilitate oral delivery for the treatment of renal fibrosis (RF). Due to the overexpression of the OCTN2 transporter in the kidney, ZWC-NPs demonstrated kidney-specific distribution. Accordingly, KLO-loaded ZWC-NPs (KLO-ZWC-NPs) were further encapsulated within CMB-NPs (KLO-CMB-NPs) using a modified film dispersion method (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). This dual-encapsulation strategy enabled the transport of KLO-CMB-NPs into systemic circulation via ILT, bypassing first-pass hepatic metabolism and achieving an absolute oral bioavailability of up to 2.7% (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec). In the bloodstream, KLO-CMB-NPs are hydrolyzed by lipoprotein lipase, releasing KLO-ZWC-NPs for kidney-targeted delivery (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee). Both \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e studies confirmed kidney-specific accumulation and demonstrated robust therapeutic efficacy against RF following oral administration of KLO-CMB-NPs.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eConstruction and characterization of KLO-CMB-NPs\u003c/h2\u003e \u003cp\u003eTo develop CMB-NPs with high lipoprotein affinity, we first obtain natural chylomicrons (nCMs) from the main mesenteric lymphatic duct of a rat by cannulation (Supplementary Fig.\u0026nbsp;1). From a single rat, only small amounts of nCMs (80\u0026ndash;100 \u0026micro;L) were collected, with an average particle size of approximately 93.6 nm (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). To address the limited availability of nCMs, we developed artificial chylomicrons (aCMs) using a film dispersion method\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. These aCMs were formulated with components mimicking nCMs, including olive oil, l-α-phosphatidylcholine, lysophosphatidylcholine, cholesteryl oleate, and cholesterol, in a molar ratio of 70:22.7:2.3:3.0:2.0\u003csup\u003e22\u003c/sup\u003e. The resulting aCMs formed an emulsion with an average particle size of approximately 160.3 nm (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003eAs TGs are the primary component of CMB-NPs, we initially screened different TG types for their binding affinity with aCMs. Interestingly, CMB-NPs prepared using triolein (TO) exhibited a significantly higher binding rate with aCMs (70.7%) compared to trimyristin (TM), tripalmitin (TP), and tristearin (TS) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). To further enhance the binding rate with aCMs, we evaluated 319 pharmaceutical excipients for their lipoprotein affinity using a cluster analysis method. We identified Brij-C10, Brij-S10, Brij-O10, TPGS, Span-40, Span-60, and Span-80 as candidates with potential to improve lipoprotein affinity (Supplementary Fig.\u0026nbsp;2).\u003c/p\u003e \u003cp\u003eThese excipients were subsequently incorporated into the aqueous phase during the preparation of CMB-NPs. Among them, Brij-O10 significantly increased the binding rate from 70.7\u0026ndash;84.8% (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed). The optimized CMB-NP formulation, comprising a TG-rich core surrounded by Brij-O10, demonstrated a binding rate comparable to that of nCMs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee). Binding affinity between CMB-NPs and nCMs was further quantified using surface plasmon resonance (SPR) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ef). Consistent with the binding rate results, CMB-NPs containing Brij-O10 exhibited a higher association rate constant (ka\u0026thinsp;=\u0026thinsp;7.8 \u0026times; 10⁶ a/Ms) and a lower dissociation constant (KD\u0026thinsp;=\u0026thinsp;1.4 \u0026times; 10⁻⁹ M) than CMB-NPs without Brij-O10. These findings highlight the role of Brij-O10 in enhancing the binding affinity of CMB-NPs with nCMs.\u003c/p\u003e \u003cp\u003eNext, we prepared KLO-ZWC-NPs and further encapsulated within CMB-NPs using the optimized formulation. Both KLO-ZWC-NPs and KLO-CMB-NPs exhibited similar particle sizes, measuring 172.1 nm and 176.3 nm, respectively, with a smooth spherical structure and neutral charge (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eh). The particle size of KLO-CMB-NPs remained stable after storage at 4\u0026deg;C for 5 days, indicating good storage stability (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eg). Furthermore, the encapsulation efficiency (EE) and loading capacity (LC) of KLO-CMB-NPs were 98.6% and 6.0%, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ei).\u003c/p\u003e \u003cp\u003eTo investigate the drug release behavior of CMB-NPs, KLO was replaced with bovine serum albumin (BSA) as a model protein and subjected to dialysis in release media at pH 1.2 and 6.8. BSA-CMB-NPs displayed similar particle size, zeta potential, and LC to those of KLO-CMB-NPs (Supplementary Table\u0026nbsp;1). Free BSA, as a water-soluble macromolecule, exhibited rapid release in the release media, with 65.7% released. By contrast, BSA-CMB-NPs released only 18.4% in the pH 1.2 medium and 27.5% in the pH 6.8 medium (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ej). The controlled release profile of CMB-NPs suggests that the encapsulated macromolecules are likely to survive in the harsh gastrointestinal environment.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eKLO-CMB-NPs survive in the gastrointestinal tract\u003c/h3\u003e\n\u003cp\u003eTo evaluate the protective effects of KLO-CMB-NPs, we used F\u0026ouml;rster resonance energy transfer (FRET) to validate nanoparticle integrity \u003cem\u003ein vitro\u003c/em\u003e. FRET-CMB-NPs were developed by co-loading fluorescein isothiocyanate (FITC, FRET donor) and rhodamine B (RhoB, FRET acceptor). A FRET signal was generated when FRET-CMB-NPs remained intact, indicating nanoparticle stability (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea).\u003c/p\u003e \u003cp\u003eTo assess the integrity of FRET-CMB-NPs in the gastrointestinal environment, they were incubated in 0.1M HCl (pH 1.2) and PBS (pH 6.8) for 12 hours. The FRET signal was observed at the indicated time points, demonstrating that FRET-CMB-NPs retained their structural integrity under harsh pH conditions for up to 12 hours (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). These findings confirm the stability of CMB-NPs in gastrointestinal pH environments.\u003c/p\u003e \u003cp\u003eKLO specifically hydrolyzes β-D-glucuronide into fluorescent 4-methylumbelliferone, enabling assessment of its enzymatic activity\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. To further confirm the protective effects of KLO-CMB-NPs, we therefore evaluated the β-glucuronidase activity of the encapsulated KLO. After incubation in simulated gastric fluid and simulated intestinal fluid for 12 hours, KLO-CMB-NPs retained 78.3% of their β-glucuronidase activity compared to freshly prepared KLO-CMB-NPs (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). These findings demonstrate that encapsulation within CMB-NPs effectively protects KLO, allowing it to survive in the gastrointestinal tract.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eKLO-CMB-NPs increase transport through mucus layer and epithelium\u003c/h3\u003e\n\u003cp\u003eDespite KLO-CMB-NPs survive in the gastrointestinal tract, they need to penetrate the mucus layers covering the apical side of the intestinal epithelium. To evaluate the mucus-penetrating capacity, we synthesized FITC-labelled ZWC (FITC-ZWC), which was used to produce FITC-ZWC-NPs and FITC-CMB-NPs for fluorescent tracing in the mucus layer. Compared to FITC-ZWC-NPs, FITC-CMB-NPs exhibited significantly stronger fluorescence intensity in the lower side of the jejunum of a rat within 1 hour of incubation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed), suggesting FITC-CMB-NPs penetrated the mucus layer more effectively than FITC-ZWC-NPs. Semiquantitative analysis of each slice further demonstrated that FITC-CMB-NPs increased the mucus permeability by 1.9-fold compared to FITC-ZWC-NPs (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee, f).\u003c/p\u003e \u003cp\u003eSubsequently, an \u003cem\u003ein vitro\u003c/em\u003e cellular uptake assay was performed using confocal laser scanning microscopy and flow cytometry. After 2 hours of incubation with Caco-2 cells, FITC-CMB-NPs demonstrated strong fluorescence intensity and increased cellular uptake by 11.2- and 1.7-fold compared to FITC-ZWC and FITC-ZWC-NPs, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea-c). To investigate the endocytosis pathways of FITC-CMB-NPs, cells were pretreated with specific endocytosis inhibitors. Cellular uptake was reduced to 38.6% and 44.4% of the original levels following treatment with chlorpromazine and indomethacin, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed). These results indicate that FITC-CMB-NPs are primarily internalized via clathrin- and caveolae-mediated pathways.\u003c/p\u003e \u003cp\u003eNext, we constructed a Caco-2 cell monolayer to evaluate the permeation capability of FITC-CMB-NPs. FITC-CMB-NPs again exhibited a strong permeability coefficient (3.81\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04 \u0026times; 10⁻⁶ cm/s), which was 1.7 times higher than that of FITC-ZWC-NPs (2.31\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04 \u0026times; 10⁻⁶ cm/s) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee). Three-dimensional visualization further confirmed that FITC-CMB-NPs successfully crossed the cell monolayer (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ef, g). The permeation capability of FITC-CMB-NPs was significantly reduced to 26.0% of the original level in the presence of Pluronic-L81, a known chylomicron (CM) secretion inhibitor (Supplementary Fig.\u0026nbsp;3). Conversely, the addition of oleic acid, a CM secretion stimulator\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e, enhanced the transmembrane transport of FITC-CMB-NPs by 69.8% of the original level. These findings indicate that the transmembrane transport of CMB-NPs is mediated by CMs within the cells.\u003c/p\u003e \u003cp\u003eIn addition, we evaluated the integrity of CMB-NPs after transmembrane transport through the Caco-2 cell monolayer using FRET. The transmembrane FRET-CMB-NPs demonstrated an increasing FRET signal over incubation time (Supplementary Fig.\u0026nbsp;4), indicating that CMB-NPs remained intact after penetrating the cell monolayer.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eCM-like lymphatic transport within enterocytes\u003c/h3\u003e\n\u003cp\u003eBuilding on the finding that the enhanced transmembrane transport of FITC-CMB-NPs was mediated by CMs in Caco-2 cells, we hypothesized that FITC-CMB-NPs traverse cells via the lymphatic transport pathway. To test this hypothesis, lymphatic transport-associated organelles within the cells were labelled, and their colocalization of FITC-CMB-NPs was examined. Merged confocal micrographs revealed substantial colocalization of FITC-CMB-NPs with the ER and Golgi apparatus (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea), with high colocalization coefficient of 0.745\u0026thinsp;\u0026plusmn;\u0026thinsp;0.048 and 0.668\u0026thinsp;\u0026plusmn;\u0026thinsp;0.041 respectively. By contrast, FITC-ZWC-NPs did not show obvious colocalization with these labelled organelles. Transmission electron microscopy (TEM) provided more precise evidence of FITC-CMB-NPs colocalization with the ER and Golgi apparatus. CMB-NPs were observed within the ER and Golgi apparatus (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). These confocal and TEM observations confirm that the transport of CMB-NPs within cells depend on lymphatic transport pathway.\u003c/p\u003e \u003cp\u003eTo further elucidate the CM-like lymphatic transport mechanism of CMB-NPs, we established an \u003cem\u003ein vitro\u003c/em\u003e CM-assembly model using Caco-2 cells. CMB-NPs, TGs (positive control), and ZWC-NPs (negative control) were incubated with Caco-2 cells for 24 hours. L-FABP and ApoB-48 are two key proteins involved in the CM assembly process\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. As anticipated, CMB-NPs and TGs significantly increased cellular L-FABP expression by 2.6-fold and 2.9-fold, respectively, compared to the control group, while treatment with ZWC-NPs did not show a statistically significant difference (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec). Additionally, ApoB-48 presence, which adheres to the surface of CM particles after exocytosis, was evaluated in exocytosed CMB-NPs collected and concentrated from the basolateral chamber after 24-hour incubation. Following separation and incubation with an ApoB-48 antibody, exocytosed CMB-NPs were found to be anchored with ApoB-48 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed). Furthermore, TEM analysis revealed that exocytosed CMB-NPs had a garland-like layer surrounding the particle (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed), with a size of 298 nm, which was slightly larger than ZWC-NPs (280 nm).\u003c/p\u003e \u003cp\u003eIn summary, western blotting and TEM results demonstrated that CMB-NPs enhance L-FABP expression and are covered with ApoB-48 during lymphatic transport within cells, mimicking the CM assembly process\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ee).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eHaving successfully verified the \u003cem\u003ein vitro\u003c/em\u003e lymphatic transport of CMB-NPs, we next demonstrated their \u003cem\u003ein vivo\u003c/em\u003e ILT. Compared to FITC-ZWC-NPs, FITC-CMB-NPs exhibited significantly higher fluorescence intensity in the rat mesentery, 1 hour after oral administration (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ef). This mesenteric absorption was significantly inhibited by cycloheximide (CHX), confirming the involvement of ILT in the oral absorption process.\u003c/p\u003e \u003cp\u003eTo further quantify ILT, we measured KLO concentrations in the lymphatic vessels and blood using a novel rat model. In this model, an overnight-fasted rat underwent \u003cem\u003ein situ\u003c/em\u003e single-pass intestinal perfusion, followed by collection of absorbed KLO-CMB-NPs from the lymphatic ducts and blood vessels via a cannulated catheter (Supplementary Fig.\u0026nbsp;5). Notably, KLO concentrations in the lymph were higher than in the serum, with the area under the curve (AUC) in the lymph being 1.2-fold greater than that in the serum (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eg). These findings indicate that lymphatic transport accounted for most of the intestinal absorption of KLO-CMB-NPs. Consistent with these results, KLO-CMB-NPs achieved an absolute oral bioavailability of 2.7% (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eh and Supplementary Table\u0026nbsp;2). These data confirm that KLO-CMB-NPs significantly enhance the oral bioavailability of KLO via ILT, providing evidence to support further studies on biodistribution and therapeutic efficacy.\u003c/p\u003e\n\u003ch3\u003eKLO-CMB-NPs enhance kidney-targeted distribution\u003c/h3\u003e\n\u003cp\u003eWe next performed \u003cem\u003eex vivo\u003c/em\u003e fluorescence imaging to visualize the biodistribution of FITC-CMB-NPs in normal and unilateral ureteral obstruction (UUO) mice. As expected, normal mice treated with FITC-CMB-NPs exhibited significant fluorescence intensity in the kidneys up to 8 hours post-oral administration, along with strong signals in the liver (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea). The biodistribution of FITC-CMB-NPs in UUO mice was markedly different compared to normal mice. Specifically, FITC-CMB-NPs displayed stronger fluorescence signals in the kidneys, particularly in the obstructed kidney of UUO mice, rather than the liver. At 12 hours post-oral administration, the average fluorescence intensity in the obstructed kidney was 2.2-fold higher than that in the normal kidney (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003eTo further investigate this phenomenon, we collected the obstructed kidneys of UUO mice were collected 12 hours post-oral administration for renal sectioning and immunofluorescent observation. Immunofluorescence analysis revealed colocalization of OCTN2 transporter and FITC-CMB-NPs in the renal tubules (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec), indicating OCTN2-mediated kidney accumulation. These findings demonstrate FITC-CMB-NPs enhance kidney-targeted distribution, which is essential for their potential therapeutic application in treating RF.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eKLO-CMB-NPs improve therapeutic effects against RF\u003c/h2\u003e \u003cp\u003eIn our final set of analyses, we used the UUO-induced RF mouse model to evaluate the anti-fibrotic efficacy of KLO-CMB-NPs. Three days before UUO surgery, mice were orally administered KLO-CMB-NPs (80 \u0026micro;g/kg/day) once daily. In control animals, free KLO (10 \u0026micro;g/kg/day) and captopril (10 mg/kg/day) were intraperitoneally injected once daily (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ed). On day 8 post-surgery, the kidneys from all groups were collected for observation. In the UUO mice, the obstructed (right) kidneys exhibited typical pathological signs of RF, including atrophy, paleness, and reduced elasticity, compared to the healthy (left) kidneys (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ee). However, the obstructed kidneys in the KLO-CMB-NPs-treated group appeared nearly identical to the healthy kidneys, indicating a strong anti-fibrotic effect. Furthermore, treatment with KLO-CMB-NPs significantly reduced serum creatinine and blood urea nitrogen levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ef, g), demonstrating effective restoration of renal function.\u003c/p\u003e \u003cp\u003eConsistent with the kidney function results, histopathological analysis confirmed that KLO-CMB-NPs significantly inhibited RF (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ei). Mice treated with KLO-CMB-NPs exhibited reduced tubulointerstitial injury in H\u0026amp;E-stained kidney sections, including less tubular epithelial cell apoptosis and inflammatory cell infiltration, compared to mice injected with free KLO and captopril groups. Masson's trichrome staining further demonstrated significantly less fibrous tissue in the kidneys of KLO-CMB-NPs-treated mice compared to UUO mice, indicating reduced collagen accumulation and deposition. Additionally, immunohistochemical staining of kidney sections revealed significantly fewer brown granules representing fibronectin and collagen-3 in KLO-CMB-NPs-treated mice compared to UUO mice (Supplementary Fig.\u0026nbsp;6). These findings suggest that KLO-CMB-NPs effectively alleviated extracellular matrix accumulation.\u003c/p\u003e \u003cp\u003eTo further elucidate the therapeutic effects of KLO-CMB-NPs against RF, we examined the protein expression of the TGF-β1 signaling pathway in obstructed kidneys. TGF-β1 activation was pronounced in the obstructed kidneys, particularly in the untreated UUO group (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eh and Supplementary Fig. S7). Elevated TGF-β1 levels triggered epithelial-mesenchymal transition (EMT), a critical event in RF development, characterized by increased α-smooth muscle actin (α-SMA) expression and reduced E-cadherin expression. However, treatment with KLO-CMB-NPs significantly suppressed TGF-β1 and α-SMA expression, while enhancing E-cadherin expression in the obstructed kidneys. Additionally, KLO expression was markedly increased in the obstructed kidneys of KLO-CMB-NPs-treated mice.\u003c/p\u003e \u003cp\u003eThese findings demonstrate the efficient kidney-targeted delivery of KLO by KLO-CMB-NPs and underscore their therapeutic potential in mitigating RF through modulation of the TGF-β1 signalling pathway and inhibition of EMT.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eILT is a promising pathway for the efficient oral delivery of macromolecular drugs. Recent studies have focused on TG/MG-conjugated prodrugs or nanoparticles\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e, achieving satisfactory oral bioavailability. However, since TG resynthesis and LD formation represent rate-limiting steps in ILT (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea), we directly developed CMB-NPs based on the natural components of CMs.\u003c/p\u003e \u003cp\u003eTo enhance the lipoprotein affinity of CMB-NPs, we conducted a comprehensive formulation study. While the binding affinity to aCMs reached 70.7% by selecting TO as the TG core of CMB-NPs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec), further screening of 319 pharmaceutical excipients identified Brij-O10 as a key additive. Incorporating Brij-O10 into the CMB-NP formulation significantly increased the binding rate to 84.8% with aCMs and 88.1% with nCMs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee). SPR analysis confirmed rapid fusion between CMB-NPs and nCMs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ef). After loading with the anti-fibrotic protein KLO\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e, the optimized KLO-CMB-NPs were shown to be an effective drug carrier for daily oral delivery of KLO in the treatment of RF.\u003c/p\u003e \u003cp\u003eTo overcome the intestinal epithelial barrier, KLO-CMB-NPs must remain stable in the harsh gastrointestinal environment and effectively penetrate the mucus layer. Using the FRET technique, we confirmed that CMB-NPs remained intact for 12 hours under gastrointestinal pH conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). Additionally, KLO-CMB-NPs retained 78.3% of their β-glucuronidase activity after incubation in simulated digestive fluids, demonstrating enzymatic stability (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). Furthermore, mucus penetration studies showed that FITC-CMB-NPs rapidly diffused through the mucus layer (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed-f), highlighting their ability to traverse this critical barrier efficiently.\u003c/p\u003e \u003cp\u003eNext, we demonstrated that KLO-CMB-NPs enhance intestinal permeability via lymphatic transport \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e and elucidated the underlying mechanism. To better quantify the transcytosis pathway of CMB-NPs, we used fluorescent FITC-CMB-NPs in confocal imaging experiments, with FITC-ZWC-NPs serving as a control. Compared to FITC-ZWC-NPs, FITC-CMB-NPs significantly increased cellular uptake and the apparent permeability coefficient in Caco-2 cells and cell monolayers (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec, g). Additionally, the enhanced transmembrane transport of CMB-NPs was mediated by CMs within the cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee). Notably, CMB-NPs exhibited CM-like lymphatic transport. Confocal and TEM imaging revealed colocalization of CMB-NPs with the ER and Golgi apparatus (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea, b), indicating that CMB-NPs were primarily absorbed via lymphatic transport within cells. Furthermore, two key proteins, L-FABP and ApoB-48, were successfully identified and coated the surface of CMB-NPs (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec, d), achieving the intended design of the nanoparticles. Taken together, these findings confirm that the constructed CMB-NPs traffic across enterocytes via lymphatic transport in a manner resembling CMs (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ee).\u003c/p\u003e \u003cp\u003eSubsequently, we observed that ILT was involved in the oral absorption of FITC-CMB-NPs (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ef). To further quantify this process, we performed cannulation into the main mesenteric lymphatic duct of a rat (Supplementary Fig.\u0026nbsp;5). The results indicated that lymphatic transport predominantly facilitated the intestinal absorption of KLO-CMB-NPs (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eg). Although the KLO ELISA measurement presented challenges in the pharmacokinetic study\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e, we successfully quantified the administered KLO using an ELISA kit from Tongwei Biotechnology. This allowed us to report the pharmacokinetic parameters of KLO for the first time (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eh and Supplementary Table\u0026nbsp;2). The findings demonstrated that KLO-CMB-NPs achieved an absolute oral bioavailability of up to 2.7%, confirming their potential as an effective oral delivery system for KLO.\u003c/p\u003e \u003cp\u003eIn addition to enhancing oral absorption, the absorbed FITC-CMB-NPs demonstrated kidney-targeted distribution, particularly in the obstructed kidneys of UUO mice (Fig.\u0026nbsp;7a, b). This kidney-specific accumulation was attributed to the overexpression of the OCTN2 transporter in renal tubules, with ZWC-NPs serving as a substrate for OCTN2. In the anti-fibrosis study, daily oral administration of KLO-CMB-NPs (80 \u0026micro;g/kg/day) produced superior therapeutic effects compared to intraperitoneal injection of free KLO (10 \u0026micro;g/kg/day) in UUO mice. Improvements were observed in kidney appearance, renal function, reduced extracellular matrix accumulation, and alleviated pathological changes (Fig.\u0026nbsp;7e, f, and i). Furthermore, KLO-CMB-NPs preserved KLO expression in the obstructed kidneys of UUO mice (Fig.\u0026nbsp;7h), which is critical for delaying the progression of RF.\u003c/p\u003e \u003cp\u003eOur comprehensive studies have demonstrated significant enhancements in the oral bioavailability and anti-fibrotic effects of KLO-CMB-NPs; however, certain limitations should be acknowledged. First, while KLO-CMB-NPs were shown to be safe and biocompatible during the 7-day observation period (Supplementary Fig.\u0026nbsp;8), long-term biosafety evaluations are necessary before considering potential clinical applications. Second, although the anti-fibrotic efficacy of KLO-CMB-NPs was well-validated in a 10-day UUO mouse model, further investigations are warranted in chronic kidney disease (CKD) models over extended treatment periods to assess their efficacy in more clinically relevant conditions. Third, this study identified OCTN2-mediated kidney-targeted accumulation of KLO-CMB-NPs; however, the underlying mechanisms for the overexpression of OCTN2 in the obstructed kidneys of UUO mice remain unclear and should be explored in future research.\u003c/p\u003e \u003cp\u003eIn summary, we developed KLO-CMB-NPs as an efficient oral delivery system utilizing ILT for the treatment of RF. Our findings demonstrated that KLO-CMB-NPs effectively protected encapsulated KLO in the gastrointestinal environment, facilitated penetration through the mucus layer, enhanced oral bioavailability via lymphatic transport, and ultimately exerted significant therapeutic effects on RF. Overall, this study provides an innovative design approach for oral drug carriers targeting ILT.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eChemicals, reagents and animals\u003c/h2\u003e \u003cp\u003eThe chemicals and reagents used in this study included phosphatidylcholine, lysophosphatidylcholine, cholesteryl oleate, and cholesterol (AVT, Shanghai, China); olive oil, sodium tripolyphosphate, pluronic-L81, oleic acid, chlorpromazine, indomethacin, colchicine, quercetin, dichloromethane, and triglycerides (TGs) including triolein, trimyristin, tripalmitin, and tristearin (Aladdin, Shanghai, China); Brij-C10, Brij-S10, Brij-O10, TPGS, and Span-40/60/80 (Sigma-Aldrich, St. Louis, MO, USA); and recombinant mouse Klotho (aa 35\u0026ndash;982, R\u0026amp;D Systems, Minneapolis, MN, USA). ZWC and FITC-ZWC were synthesized following previously reported methods\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. All reagents and chemicals were of analytical grade.\u003c/p\u003e \u003cp\u003eCaco-2 cells (human colon epithelial cell line, passage number 10\u0026ndash;15) were obtained from Procell Life Science \u0026amp; Technology Co., Ltd. (Wuhan, China) and cultured in DMEM (Gibco, Beijing, China) supplemented with 20% FBS (TransGen Biotech, China), 1% penicillin/streptomycin (Gibco, USA), and 1% non-essential amino acids (Gibco, USA) at 37\u0026deg;C in a humidified incubator with 5% CO₂ (Thermo, USA). Cells were collected upon reaching 80% confluence, with the medium changed every 48 hours.\u003c/p\u003e \u003cp\u003eMale C57BL/6 mice (8 weeks old, 25\u0026ndash;28 g) and Sprague Dawley rats (12 weeks old, 260\u0026ndash;280 g) were obtained from Guangdong Medical Laboratory Animal Center (Foshan, China). The animals were maintained under standard conditions at 22\u0026deg;C with 60\u0026thinsp;\u0026plusmn;\u0026thinsp;10% humidity and a 12-hour light/dark cycle, with free access to fresh water and rodent chow. All experiments were conducted after a 1-week acclimation period to the new environment. Experimental protocols adhered to the National Research Council's \u003cem\u003eGuide for the Care and Use of Laboratory Animals\u003c/em\u003e and were approved by the Animal Research Ethics Committee (IACUC-202300106) of Shenzhen University, Shenzhen, China.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eCollection of nCMs\u003c/h2\u003e \u003cp\u003eAn overnight-fasted Sprague Dawley rat was administered olive oil (2 mL) to stimulate nCM secretion. After 1 hour, the rat was anesthetized and opened via a midline incision, with a catheter inserted into the main mesenteric lymphatic duct. Additionally, fresh blood was supplied through the right jugular vein. The milky white nCMs (approximately 80\u0026ndash;100 \u0026micro;L) were collected as they flowed out into a centrifuge tube (Supplementary Fig.\u0026nbsp;1).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eDevelopment of aCMs\u003c/h2\u003e \u003cp\u003eAs the quantity of collected nCMs was insufficient to support the formulation optimization study of CMB-NPs, artificial chylomicrons (aCMs) were prepared using a thin-film dispersion method, as previously described\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. In brief, 0.77 mL olive oil, 227 mg phosphatidylcholine, 23 mg lysophosphatidylcholine, and 20 mg cholesterol were dissolved in 20 mL dichloromethane. The solvent was evaporated to form a lipid film using a rotary evaporator (Buchi, Switzerland) under gentle stirring (30 rpm) for 2 hours.The lipid film was further dehydrated under nitrogen and hydrated overnight at 4\u0026deg;C with 25 mL deionized water. The resulting suspension was vortex-mixed vigorously for 60 seconds and then sonicated using a probe ultrasound (Scientz, China) at 400 W for 15 minutes. The milky white emulsion obtained was sequentially extruded through 0.45 \u0026micro;m and 0.22 \u0026micro;m PVDF filters (Millipore) and characterized by dynamic light scattering (DLS, Malvern Instruments, UK).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003ePreparation of KLO-CMB-NPs\u003c/h2\u003e \u003cp\u003eKLO-ZWC-NPs were prepared using a cross-linking method as previously described\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. Specifically, ZWC was dissolved in 0.2% (v/v) acetic acid at a concentration of 3 mg/mL. After filtration through a 0.45 \u0026micro;m PVDF filter (Millipore), 8 mL of sodium tripolyphosphate (0.5 mg/mL, dissolved in deionized water) was added dropwise to 8 mL of ZWC solution containing 20 \u0026micro;g of KLO, with stirring at 300 rpm for 30 minutes. The mixture was then processed using probe ultrasound (200 W, 10 minutes) under an ice bath and filtered through a 0.22 \u0026micro;m filter to produce uniformly dispersed KLO-ZWC-NPs.\u003c/p\u003e \u003cp\u003eKLO-CMB-NPs were prepared using the thin-film dispersion method with minor modifications\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. Specifically, 70 mg of triglyceride (triolein, trimyristin, tripalmitin, or tristearin), 23 mg of phospholipids, 2 mg of lysophosphatidylcholine, and 3 mg of cholesterol were dissolved in 20 mL dichloromethane. The mixture was evaporated to form a lipid film using a rotary evaporator. Any residual dichloromethane in the lipid film was removed under vacuum in a desiccator for 2 hours. Subsequently, 2 mg of a surfactant (Brij-C10, Brij-S10, Brij-O10, TPGS, or Span-40/60/80) was added to freshly prepared KLO-ZWC-NPs (15 mL), which were used to hydrate the lipid film with vigorous vortex mixing and stirring at 70 rpm for 60 minutes. The resulting nanosuspension was sonicated using probe ultrasound and sequentially extruded through 0.45 \u0026micro;m and 0.22 \u0026micro;m filters. Additionally, unloaded nanoparticles (CMB-NPs) were prepared following the same protocol in the absence of KLO. FITC-CMB-NPs were developed similarly, with ZWC replaced by FITC-ZWC.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eCharacterization of KLO-CMB-NPs\u003c/h2\u003e \u003cp\u003eAs CMB-NPs undergo fusion and increase in size after binding with aCMs, unbound CMB-NPs can be quantified using ultracentrifugation. Briefly, 2 mL of CMB-NPs were mixed with an equal volume of aCMs and stirred for 1 hour. The mixture was then centrifuged at 17,000 \u0026times; g for 30 minutes. This process was repeated twice to ensure complete separation. The unbound CMB-NPs in the supernatant were quantified using a commercial triglyceride (TG) colorimetric assay kit (Nanjing Jiancheng, China). The binding rate of CMB-NPs with aCMs was calculated using the following formula:\u003cdiv id=\"Equa\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\n$$\\:\\text{B}\\text{i}\\text{n}\\text{d}\\text{i}\\text{n}\\text{g}\\:\\text{r}\\text{a}\\text{t}\\text{e}\\:\\text{w}\\text{i}\\text{t}\\text{h}\\:\\text{a}\\text{C}\\text{M}\\text{s}\\:\\left(\\%\\right)=\\frac{{W}_{I}-{W}_{U}}{{W}_{I}\\:}\\times\\:100$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003ewhere \u003cem\u003eW\u003c/em\u003e\u003csub\u003e\u003cem\u003eI\u003c/em\u003e\u003c/sub\u003e and \u003cem\u003eW\u003c/em\u003e\u003csub\u003e\u003cem\u003eU\u003c/em\u003e\u003c/sub\u003e is the weight of initially added CMB-NPs and the weight of unbound CMB-NPs after ultracentrifugation, respectively (unit: mg).\u003c/p\u003e \u003cp\u003eSimilarly, the binding rates of CMB-NPs with bCMs were determined using ultracentrifugation. For this, 200 \u0026micro;g FITC-BSA (FB, Solarbio, Beijing, China) was loaded into FB-CMB-NPs following the previously described protocol. Equal volumes of FB-CMB-NPs (2 mL) and bCMs were mixed and stirred for 1 hour. The mixture was then centrifuged twice. The free FB-CMB-NPs in the supernatant were lysed and quantified using a microplate reader (Biotek, USA). The binding rate of CMB-NPs with bCMs was calculated according to the following formulas:\u003cdiv id=\"Equb\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equb\" name=\"EquationSource\"\u003e\n$$\\:\\text{B}\\text{i}\\text{n}\\text{d}\\text{i}\\text{n}\\text{g}\\:\\text{r}\\text{a}\\text{t}\\text{e}\\:\\text{w}\\text{i}\\text{t}\\text{h}\\:\\text{b}\\text{C}\\text{M}\\text{s}\\:\\left(\\%\\right)=\\frac{{W}_{T}-{W}_{F}}{{W}_{T}\\:}\\times\\:100$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003ewhere \u003cem\u003eW\u003c/em\u003e\u003csub\u003e\u003cem\u003eT\u003c/em\u003e\u003c/sub\u003e and \u003cem\u003eW\u003c/em\u003e\u003csub\u003e\u003cem\u003eF\u003c/em\u003e\u003c/sub\u003e is the weight of totally added FB-CMB-NPs and the weight of free FB-CMB-NPs in the supernatant after ultracentrifugation, respectively (unit: \u0026micro;g).\u003c/p\u003e \u003cp\u003eThe binding affinity of CMB-NPs to bCMs was further confirmed using a surface plasmon resonance (SPR) technique as previously described\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. Briefly, a CM5 sensor chip was prepared by activating a cell with a mixture of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC, 200 \u0026micro;M) and N-hydroxysuccinimide (NHS, 50 \u0026micro;M), infused at a flow rate of 10 \u0026micro;L/min for 420 seconds. Subsequently, a solution containing 50 \u0026micro;L of KLO recombinant protein (1 mg/mL) mixed with 180 \u0026micro;L of 10 mM sodium acetate (pH 5.0) was immobilized on the cell surface at 10 \u0026micro;L/min for 420 seconds. This immobilization process was repeated twice. The cell was then blocked with 1 M ethanolamine infused at 10 \u0026micro;L/min for 10 minutes. A neighboring aisle, serving as a reference, underwent identical activation and blocking steps, but PBS (pH 5.0) was used for immobilization instead. Both aisles were equilibrated with PBS prior to testing. The molecule stock solution was diluted to various concentrations in PBS and flowed over the chip at 10 \u0026micro;L/min for 150 seconds per run. After each run, the cells were regenerated using a 10 mM glycine-HCl solution (pH 2.0) infused at 10 \u0026micro;L/min for 5 minutes. Data from the sample cell were recorded using Biacore T200 Control software (version 2.0, GE Healthcare) and subtracted from the reference cell data. Association and dissociation constants were derived through global fitting of the data to a 1:1 Langmuir binding model, facilitated by the software.\u003c/p\u003e \u003cp\u003eThe size and zeta potential of KLO-CMB-NPs were characterized using dynamic light scattering (DLS). The morphologies of aCMs, bCMs, KLO-ZWC-NPs, and KLO-CMB-NPs were observed using transmission electron microscopy (TEM, Hitachi, Japan). The storage stability of CMB-NPs at 4\u0026deg;C was assessed by monitoring changes in particle size and polydispersity index (PDI) over a period of 5 days. Encapsulation efficiency (EE) and loading capacity (LC) of KLO-CMB-NPs were calculated using a previously reported method\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. The amount of unloaded KLO in the nanosuspension was quantified using a BCA protein assay kit (Thermo, USA).\u003c/p\u003e \u003cp\u003eTo examine the \u003cem\u003ein vitro\u003c/em\u003e release of CMB-NPs, KLO was replaced by bovine serum albumin (BSA), and a dialysis method was employed\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. Briefly, 1 mL of free BSA (5 mg/mL), BSA-ZWC-NPs, and BSA-CMB-NPs (containing 5 mg/mL BSA) were placed into dialysis bags with a molecular weight cutoff (MWCO) of 10 kDa. The dialysis bags were immersed in 45 mL of release medium at 37\u0026deg;C under gentle shaking. Initially, the bags were incubated in 0.1 M HCl (pH 1.2) for 2 hours, followed by PBS buffer (pH 6.8) for an additional 6 hours. At predetermined time points, 1 mL of the released sample was collected and replaced with an equal volume of fresh buffer. The cumulative release of BSA was measured using the BCA protein assay kit.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eStability study using FRET\u003c/h2\u003e \u003cp\u003eFRET-CMB-NPs were prepared following a previously reported method with minor modifications\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. FITC (0.1 mg, dissolved in deionized water) and RhoB (0.3 mg, dissolved in deionized water) were co-loaded into ZWC-NPs as described in the protocol. Unencapsulated FITC and RhoB were removed by ultrafiltration using a MWCO of 3 kDa. A lipid film was then prepared using the previously mentioned protocol and hydrated with the obtained FRET-ZWC-NPs. The resulting FRET-CMB-NPs were diluted and analyzed using a microplate reader, with excitation set at 450 nm and emission spectra measured from 480 to 700 nm. To assess stability, freshly prepared FRET-CMB-NPs (1 mL) were placed in a dialysis bag (MWCO 3 kDa) and submerged in a centrifuge tube containing 4 mL of 0.1 M HCl (pH 1.2) or phosphate buffer (pH 6.8). At predetermined time points, 100 \u0026micro;L of samples were withdrawn from the dialysis bag and analyzed using the microplate reader.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eStability study by β-glucuronidase activity\u003c/h2\u003e \u003cp\u003eThe stability of encapsulated KLO in KLO-CMB-NPs was further evaluated by assessing β-glucuronidase activity. Freshly prepared KLO-CMB-NPs (2 mL) were placed into a dialysis bag (MWCO 200 kDa) and incubated in simulated gastric fluid (100 mL) for 2 hours, followed by simulated intestinal fluid (100 mL) for 10 hours. As previously reported\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e, KLO specifically hydrolyzes 4-methylumbelliferyl β-D-glucuronide into 4-methylumbelliferone, which exhibits strong fluorescence. After incubation, the remaining KLO-CMB-NPs were washed three times with PBS and their β-glucuronidase activity was measured to determine the stability of the encapsulated KLO.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eMucus layer penetration\u003c/h2\u003e \u003cp\u003eThe mucus permeation of FITC-CMB-NPs in intestinal loops was visualized using a confocal laser scanning microscope (CLSM, ZEISS, LSM880, Germany)\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. Briefly, a 2 cm segment of the jejunum was isolated from an overnight-fasted male Sprague Dawley rat. FITC-CMB-NPs were introduced into the isolated jejunum to prepare intestinal loops. The intestinal loops were maintained in 10 mL of oxygenated Krebs-Ringer buffer at 37\u0026deg;C with gentle shaking (100 rpm). After a 1-hour incubation, the intestinal loops were excised, stained with Alexa 555-WGA (Thermo Fisher Scientific, USA), and visualized under CLSM to assess mucus permeation of the FITC-CMB-NPs.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eCellular uptake\u003c/h2\u003e \u003cp\u003eCaco-2 cells (1 \u0026times; 10⁶ cells) were seeded in a confocal dish and allowed to adhere overnight. Upon reaching 90% confluence, the culture medium was replaced with medium containing FITC-ZWC (1.6 mg/mL), FITC-ZWC-NPs, or FITC-CMB-NPs (equivalent to FITC-ZWC at 1.6 mg/mL). After 1 hour of incubation, the treated cells were washed twice with cold PBS, fixed with 4% paraformaldehyde, stained with 4',6-diamidino-2-phenylindole (DAPI), and observed under a confocal laser scanning microscope (CLSM).\u003c/p\u003e \u003cp\u003eFor quantitative analysis, cellular uptake was measured using flow cytometry (BC-Cytoflex, Beckman, USA). Caco-2 cells (2 \u0026times; 10⁶ cells/well) were seeded in a 24-well plate and cultured overnight. The cells were treated with FITC-ZWC, FITC-ZWC-NPs, or FITC-CMB-NPs (equivalent to FITC-ZWC at 1.6 mg/mL). After 2 hours of incubation, the fluorescence intensity in the collected cells was determined by the flow cytometer and analyzed using FlowJo software.\u003c/p\u003e \u003cp\u003eTo further investigate the endocytosis pathways of FITC-CMB-NPs in Caco-2 cells, cells were pretreated with specific endocytosis inhibitors for 2 hours. These inhibitors included chlorpromazine (50 \u0026micro;M), indomethacin (100 \u0026micro;M), colchicine (10 \u0026micro;M), and quercetin (10 \u0026micro;M). Cellular uptake in the presence of these inhibitors was also quantified using the flow cytometer to identify the predominant pathways involved\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eTranscellular transport study\u003c/h2\u003e \u003cp\u003eCaco-2 cells were plated onto polycarbonate inserts (0.4 \u0026micro;m pore size, 4.67 cm\u0026sup2; growth area, Corning) at a density of 5 \u0026times; 10⁵ cells/well and cultured for 21 days prior to conducting transcellular transport studies, as previously described\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. Only polarized cell monolayers with transepithelial electrical resistance (TEER) values exceeding 600 Ω were used in the subsequent experiments. FITC-ZWC (1.6 mg/mL), FITC-ZWC-NPs, and FITC-CMB-NPs (equivalent to FITC-ZWC at 1.6 mg/mL) were added to the apical side of the inserts, either in the absence or presence of Pluronic-L81 (2 mM) or oleic acid (2 mM). At 15, 30, 45, 60, 90, and 120 minutes, 0.5 mL of the sample from the basolateral chamber was collected and replaced with an equal volume of Hank's balanced salt solution (HBSS). The concentration of FITC-ZWC in the collected samples was determined using a microplate reader. The permeability coefficient (Papp) was calculated according to our reported method\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. After 2 hours of incubation, the cell monolayers were washed three times with cold PBS, fixed with 4% paraformaldehyde, stained with DAPI, and visualized using a confocal laser scanning microscope (CLSM).\u003c/p\u003e \u003cp\u003eThe nanoparticle integrity of CMB-NPs was measured after the transcellular transport study using FRET. FRET-ZWC-NPs and FRET-CMB-NPs were loaded into the apical chamber of a polarized Caco-2 cell monolayer. At the indicated timepoints, transcellular nanoparticles were collected from the basolateral chamber, and then diluted and measured using the microplate reader.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eLymphatic transport within Caco-2 cells\u003c/h2\u003e \u003cp\u003eCaco-2 cells were seeded in a confocal dish and allowed to adhere overnight. Following treatment with FITC-CMB-NPs (0.84 mg/mL) for 6 hours, ER-Tracker (1 \u0026micro;M, Beyotime Biotechnology, China) or Golgi-Tracker (333 \u0026micro;g/mL, Beyotime Biotechnology, China) was added to the dish and incubated at 4\u0026deg;C for 30 minutes. The cells were then fixed with 4% paraformaldehyde, stained with DAPI, and visualized using a confocal laser scanning microscope (CLSM). The fluorescence intensity and Pearson\u0026rsquo;s correlation coefficient were analyzed using ImageJ software to evaluate co-localization.\u003c/p\u003e \u003cp\u003eAdditionally, the co-localization of FITC-CMB-NPs with the ER and Golgi apparatus was further confirmed by transmission electron microscopy (TEM) as previously described\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. Caco-2 cells were treated with FITC-CMB-NPs (0.84 mg/mL) for 6 hours, then fixed with 3% glutaraldehyde followed by 1% osmium tetroxide. The fixed cells were dehydrated through a graded ethanol series and embedded in epoxy resin. Ultra-thin sections were prepared from polymerized epoxy resin blocks and stained with 1% uranyl acetate and Reynold's lead citrate before being examined under TEM.\u003c/p\u003e \u003cp\u003eCM-like lymphatic transport was assessed by analyzing L-FABP expression in Caco-2 cells, following a previously reported method\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. Caco-2 cells were treated with CMB-NPs (0.84 mg/mL), TG (20 \u0026micro;L), or ZWC-NPs (0.3 mg/mL) for 24 hours. The cells were then harvested and lysed, and protein content was measured using a BCA protein assay kit (Thermo Fisher Scientific). Equal amounts of protein samples (30 \u0026micro;g) were separated on a 12% SDS-PAGE gel and electro-transferred onto PVDF membranes. The membranes were blocked with 5% BSA and incubated overnight at 4\u0026deg;C with primary antibodies against L-FABP (1:1,000, Abcam, ab222517) and GAPDH (1:2,000, Thermo Fisher Scientific). After incubation with secondary antibodies (1:2,000, Thermo Fisher Scientific), specific protein bands were visualized using a chemiluminescence detection kit (Thermo Fisher Scientific). Chemiluminescent signals were quantified using the ChemiDoc MP Gel Imaging System (Bio-Rad, USA).\u003c/p\u003e \u003cp\u003eThe presence of Apo-B48 (~\u0026thinsp;250 kDa) anchored on the surface of CMB-NPs was verified in a Caco-2 cell monolayer. Following incubation with CMB-NPs (0.84 mg/mL), TG (20 \u0026micro;L), or ZWC-NPs (0.3 mg/mL) for 24 hours, the medium from the basolateral chamber was collected and total protein was precipitated. To isolate total protein, a mixture of medium (700 \u0026micro;L), methanol (175 \u0026micro;L), and chloroform (700 \u0026micro;L) was centrifuged at 21,000 \u0026times; g for 10 minutes. The protein layer (middle phase) was collected and dissolved in a 2% SDS solution. Protein concentration was measured using the BCA protein assay kit. The samples were then separated on an 8% SDS-PAGE gel and transferred onto PVDF membranes. The membranes were blocked with a suitable blocking buffer and incubated overnight at 4\u0026deg;C with primary antibodies against Apo-B48 (1:500, Abcam, ab312318). After incubation with a secondary antibody, the protein bands were visualized using a chemiluminescence kit, and the signals were quantified.\u003c/p\u003e \u003cp\u003eAdditionally, the exocytosed CMB-NPs in the cell supernatant were visualized using TEM. After treating Caco-2 cell monolayers with CMB-NPs (0.84 mg/mL), TG (20 \u0026micro;L), or ZWC-NPs (0.3 mg/mL) for 24 hours, the medium from the basolateral chamber was collected and dialyzed in deionized water using a membrane with a MWCO of 500 Da for 12 hours. The dialyzed samples were then prepared for TEM observation by standard procedures, including deposition onto a grid, staining, and imaging to evaluate the morphology of exocytosed CMB-NPs.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eILT study\u003c/h2\u003e \u003cp\u003eFITC-ZWC-NPs and FITC-CMB-NPs (equivalent to 2.4 mg/kg FITC-ZWC) were orally administered to overnight-fasted male Sprague Dawley rats (n\u0026thinsp;=\u0026thinsp;3). Additionally, a group of rats was pretreated with cycloheximide (CHX, 3 mg/kg) via intraperitoneal injection 1 hour before FITC-CMB-NPs administration to inhibit lymphatic transport\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. At 1-hour post-administration, the rats were sacrificed, and their gastrointestinal tracts were visualized using an IVIS spectrum imaging system (PerkinElmer, USA).\u003c/p\u003e \u003cp\u003eThe proportion of intestinal absorption via lymphatic transport (ILT) was determined by cannulation into the main mesenteric lymphatic duct of a Sprague Dawley rat\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. Specifically, an overnight-fasted rat was administered olive oil (2 mL) to facilitate lymphatic duct cannulation. One hour later, the main mesenteric lymphatic duct became visible, and the rat was anesthetized for \u003cem\u003ein situ\u003c/em\u003e single-pass intestinal perfusion. Warm KLO-CMB-NPs (containing 10 ng/mL KLO, diluted in PBS) were perfused at a flow rate of 0.20 mL/min for 1 hour using a syringe pump (Baoding Rongbai Precision Pump, China). Fresh blood was continuously supplied via the right jugular vein. At the designated time points, absorbed KLO-CMB-NPs were collected from the lymphatic duct and blood vessel using a cannulated catheter (external diameter 0.45 mm, internal diameter 0.22 mm). The KLO concentrations in lymph and blood samples were quantified using a commercial ELISA kit (Shanghai Tongwei, China).\u003c/p\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003ePharmacokinetic study\u003c/h2\u003e \u003cp\u003eThe pharmacokinetic study was conducted as previously reported\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. KLO-CMB-NPs (equivalent to 20 \u0026micro;g/kg KLO) were administered orally to overnight-fasted Sprague Dawley (SD) rats (n\u0026thinsp;=\u0026thinsp;6). A control group of rats was injected with free KLO (2 \u0026micro;g/kg) via the caudal vein. Blood samples were collected into heparinized tubes at specified time points, and KLO concentrations were measured using a commercial ELISA kit. The plasma concentration-time profiles were analyzed using DAS software (Shanghai, China) to evaluate pharmacokinetic parameters).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003eBiodistribution study\u003c/h2\u003e \u003cp\u003eFITC-ZWC, FITC-ZWC-NPs, and FITC-CMB-NPs (equivalent to 2.4 mg/kg FITC-ZWC) were orally administered to overnight-fasted normal and unilateral ureteral obstruction (UUO) mice (n\u0026thinsp;=\u0026thinsp;3 per group). At predetermined time points, three treated mice from each group were sacrificed by CO₂ inhalation. Tissue samples, including the heart, liver, spleen, lungs, kidneys, and gastrointestinal tract, were collected and visualized using the IVIS spectrum imaging system (PerkinElmer, USA) to assess the biodistribution of the nanoparticles.\u003c/p\u003e \u003cdiv id=\"Sec25\" class=\"Section3\"\u003e \u003ch2\u003eTherapeutic effects in UUO mice\u003c/h2\u003e \u003cp\u003eUUO-induced RF was generated in C57BL/6 mice as described previously\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. Briefly, mice were anesthetized with 10% chloral hydrate (400 mg/kg, i.p.), and the right kidney was exposed through an incision in the right lateral dorsal surface. The ureter of the right kidney was ligated with 4\u0026ndash;0 silk, and the incision was sutured. UUO mice were randomly divided into 5 groups (n\u0026thinsp;=\u0026thinsp;8): (1) UUO (Model group); (2) Captopril (i.p., 10 mg/kg/day); (3) Free KLO (i.p., 10 \u0026micro;g/kg/day); (4) KLO-ZWC-NPs (p.o., equal to KLO 80 \u0026micro;g/kg/day) and (4) KLO-CMB-NPs (p.o., equal to KLO 80 \u0026micro;g/kg/day). Age-matched mice that underwent a similar surgery without ureter ligation served as the normal group. For the treatment groups, mice received daily intraperitoneal or intragastric administration starting 3 days before UUO induction and continuing until day 7 after surgery. Mice in the normal and model groups received an equal volume of distilled water. On day 8 post-surgery, all mice were sacrificed by CO₂ inhalation, and blood and kidney samples were collected. The right kidneys were decapsulated, washed, and dissected. Portions of each kidney were fixed in 10% formalin for histological analysis, while the remaining tissue was stored at \u0026minus;\u0026thinsp;80\u0026deg;C for western blot analysis.\u003c/p\u003e \u003cp\u003eSerum creatinine levels were measured using a creatinine assay kit (Jiancheng Biotech. Co. Ltd., China), while serum blood urea nitrogen (BUN) levels were determined using a urea nitrogen content assay kit (Solarbio, China)\u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. Kidney samples were excised, weighed, formalin-fixed, and embedded in paraffin for sectioning. Histological examination was conducted under a microscope (Olympus, Japan) following hematoxylin and eosin (H\u0026amp;E) staining to assess general tissue morphology and Masson's trichrome staining to evaluate collagen deposition. Immunohistochemical staining was performed as previously reported\u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e. Paraffin sections were quenched for endogenous peroxidase activity, pre-blocked with normal goat serum, and incubated with primary antibodies against collagen-I and fibronectin (1:100, Cell Signaling Technology, USA). The sections were visualized using DAB chromogen, counterstained with hematoxylin, and examined under a microscope.\u003c/p\u003e \u003cp\u003eKidney samples were prepared for western blot analysis as described previously\u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. Briefly, kidney tissues were homogenized, and total proteins were extracted using TRIzol reagent. The protein concentration was determined using a BCA protein assay kit (Thermo Fisher Scientific). Equal amounts of protein lysates were separated on an 8% SDS-PAGE gel and transferred onto PVDF membranes. The membranes were blocked with 5% BSA and incubated overnight at 4\u0026deg;C with primary antibodies against the following proteins: TGF-β1 (1:1000, Cell Signaling Technology, USA), α-SMA (1:1000, Proteintech, USA), E-cadherin (1:1000, Proteintech, USA), and GAPDH (1:1000, Abcam, USA). After incubation with secondary antibodies (1:1000, Cell Signaling Technology, USA), the specific protein bands were visualized using a chemiluminescence detection kit (Thermo Fisher Scientific). The chemiluminescent signals were quantified using the ChemiDoc MP Gel Imaging System (Bio-Rad, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section3\"\u003e \u003ch2\u003eSafety and biocompatibility evaluation\u003c/h2\u003e \u003cp\u003eMale C57BL/6 mice were administered daily doses of KLO-CMB-NPs (p.o., equivalent to KLO 80 \u0026micro;g/kg/day) for 7 consecutive days\u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e. Mice in the control group received an equal volume of PBS daily. Throughout the study, mice were monitored for changes in body weight. On day 8, all mice were sacrificed by CO\u003csub\u003e2\u003c/sub\u003e inhalation, and their serum and organ samples were collected and stored at \u0026minus;\u0026thinsp;80\u0026deg;C. Various biochemical indicators, including liver enzyme activity and creatinine levels, were measured in the serum using assay kits (Jiancheng Biotech. Co. Ltd., China). The heart, liver, spleen, lung, and kidney tissues were fixed in formalin, sectioned, and stained with hematoxylin and eosin (H\u0026amp;E) for histopathological examination under a microscope.\u003c/p\u003e \u003c/div\u003e "},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data generated or analyzed in this study are included in the published article. These data will be made available upon request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by the Natural Science Foundation of China (Grant Nos. 82304411 and 52273299), the Guangdong Basic and Applied Basic Research Foundation (Grant No. 2022A1515010481), Guangdong Science and Technology Program (Grant No. 2023A1515011884) and the Shenzhen Science and Technology Program (Grant Nos. RCBS20221008093120049, 860000002111304, 827-00074220, GJHZ20210705141800002, JCYJ20220531102207016, D2403008 and KCXFZ20230731092802004). The authors thank the Instrumental Analysis Center of Shenzhen University for their technical support.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eYawen Yao\u003c/strong\u003e: Writing \u0026ndash; original draft, Methodology, Investigation. \u003cstrong\u003eCuihua Zhang\u003c/strong\u003e: Writing \u0026ndash; original draft, Methodology, Investigation. \u003cstrong\u003eJieying Zhou\u003c/strong\u003e: Investigation.\u003cstrong\u003e\u0026nbsp;Shihao Xu\u003c/strong\u003e: Investigation. \u003cstrong\u003eHaiqiang Wu\u003c/strong\u003e: Resources, Project administration, Supervision, Conceptualization. \u003cstrong\u003eHua Yu\u003c/strong\u003e: Resources, Project administration, Formal analysis, Supervision, Funding acquisition, Conceptualization. \u003cstrong\u003eWei Xiong\u003c/strong\u003e: Writing \u0026ndash; review \u0026amp; editing, Writing \u0026ndash; original draft, Supervision, Resources, Project administration, Funding acquisition, Formal analysis, Data curation, Conceptualization.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData will be made available on reasonable request from the corresponding author.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eGriffin BT, Guo J, Presas E, Donovan MD, Alonso MJ, O'Driscoll CM (2016) Pharmacokinetic, pharmacodynamic and biodistribution following oral administration of nanocarriers containing peptide and protein drugs. 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Int J Pharm 659:124261\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"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":"Intestinal lymphatic transport, Chylomicron, Klotho, Oral, Renal fibrosis","lastPublishedDoi":"10.21203/rs.3.rs-6301196/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6301196/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eIntestinal lymphatic transport (ILT) represents a promising pathway for the oral absorption of macromolecular drugs, but the formation of lipid droplet is a rate-limiting step during ILT. In this study, we developed a chylomicron (CM)-biomimetic nanoparticle constructed from CM components, characterized by high lipoprotein affinity, to enable efficient oral delivery of the anti-fibrotic protein klotho via ILT. This approach demonstrated potent therapeutic efficacy in the treatment of renal fibrosis. The nanoparticle exhibited size stability and retained 78.3% enzyme activity after 12 hours of incubation in simulated digestive fluid, while facilitating rapid diffusion through the mucus layer. Within enterocytes, the nanoparticle underwent a CM-like transcytosis process and showed a preference for ILT, as evidenced by cannulation into the main mesenteric lymphatic duct in rats. Notably, this biocompatible oral nanoparticle achieved an absolute bioavailability of 2.7%, delivering superior anti-fibrotic activity in a mouse disease model compared to a 125-fold higher dose of intraperitoneally administered captopril, a first-line anti-fibrotic drug. Our innovative nanoparticle design based on high lipoprotein affinity enables enhanced oral absorption of macromolecular drugs via ILT.\u003c/p\u003e","manuscriptTitle":"Chylomicron-biomimetic nanoparticles promote intestinal lymphatic transport by fast fusion with chylomicrons","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-28 03:10:54","doi":"10.21203/rs.3.rs-6301196/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":"f49dab3c-f154-4452-af0b-02211ae5d94e","owner":[],"postedDate":"April 28th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":47683852,"name":"Health sciences/Nephrology/Kidney diseases/Renal fibrosis"},{"id":47683853,"name":"Biological sciences/Biotechnology/Protein delivery"},{"id":47683854,"name":"Physical sciences/Nanoscience and technology/Nanobiotechnology/Nanoparticles"}],"tags":[],"updatedAt":"2025-05-14T15:20:22+00:00","versionOfRecord":[],"versionCreatedAt":"2025-04-28 03:10:54","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6301196","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6301196","identity":"rs-6301196","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}