Oral Spatiotemporal Nanoassembly for Sequential Genetic Reprogramming from Gut to Periphery Organ

Oral Spatiotemporal Nanoassembly for Sequential Genetic Reprogramming from Gut to Periphery Organ | 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 Oral Spatiotemporal Nanoassembly for Sequential Genetic Reprogramming from Gut to Periphery Organ Yong-Hee Kim, Juhyeong Hong, Seon-jeong Chang, Seung-Hwan Joo, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8523305/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 Oral gene therapy for metabolic diseases necessitates physiologically aligned strategies to sequentially regulate the gut-organ axis. Nevertheless, the gastrointestinal environment and unpredictable biodistribution limit the efficiency of conventional gene delivery. Here, we develop Layer-by-Layer Chylomicron-Mimicking Self-Assembly (LbL-CMSA), which enables sequential gene reprogramming from the gut to the peripheral organs. Utilizing dynamic layer-shedding kinetics, the outer bile-salt shell peels off in enterocytes to release siRNA for local intestinal reprogramming. Subsequently, the inner lipopeptoplex layer mimics chylomicrons, entering lymphatic trafficking to bypass hepatic first-pass metabolism and deliver genes to distal metabolic organs. In an obesity model, LbL-CMSA down-regulates obesogenic genes in gut–adipose tissues by sequentially blocking intestinal fat influx and adipocyte storage. By modulating genetic cargos, LbL-CMSA attenuates endotoxin translocation across the leaky gut to amplify anti-fibrogenic effects in a steatohepatitis model. Overall, LbL-CMSA shows its potential as a modular nanoplatform for combination gene therapy to overcome complex metabolic disorders efficiently. Biological sciences/Biotechnology/Biomaterials/Drug delivery Biological sciences/Biotechnology/Gene delivery Biological sciences/Biotechnology/Nucleic-acid therapeutics Biological sciences/Biotechnology/Nanobiotechnology/Nanoparticles Layer-by-layer self-assembly Chylomicron-mimetic nanoparticle Oral gene therapy Sequential genetic reprogramming Gut-organ axis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Main Cross-environmental functionality is a design criterion of biomaterials delivering oral nucleic-acid, because the efficacy hinges on seamless performance from the small intestine to downstream peripheral tissues 1-5 . Even its difficulties, oral gene therapy is an attractive approach for chronic metabolic diseases, offering patient-friendly dosing, molecular specificity, and the ability to modulate the gut environment via gut–liver and gut–adipose axes 2-4,6-9 . However, the gastrointestinal tract presents major delivery barriers such as enzymatic degradation, acidic pH, and mucus that limit nucleic-acid stability and absorption 10-13 . In parallel, hepatic first-pass metabolism or hepatic accumulation by protein corona markedly reduces the bioavailability of orally administered nucleic acids limiting efficacy 6,13-17 . To address these problems, several layer-by-layer constructs were challenged, but they only function primarily as multicoated barrier shields or single-target carriers, lacking post-epithelial function and multiple organ-specific drug release 4, 5, 6, 7 . However, since the metabolic disease represents coordinated dysfunction of major organs, multiple reprogramming across metabolic tissues is necessary to outperform single-site reprogramming 18 . More importantly, conventional trials treat the gut merely as a passive barrier to traverse, ignoring its biological role as the primary gatekeeper for systemic lipid flux and inflammatory signaling which are prerequisites for metabolic homeostasis 19-21 . The next frontier in the oral nucleic-acid therapeutics R&D lies in transitioning from simple nucleic acid delivery to engineer oral biomaterials coordinating modulation of multi-organ 22,23 . This requires dynamic layer-shedding kinetics capable of sequential cargo delivery across interconnected organ axes, a strategy that remains an uncharted territory in oral therapeutics. We hypothesized that a platform with programmed hierarchical disassembly in a different physiological environment could orchestrate the transition from luminal protection to transport competence, thereby guiding biodistribution toward both intestinal and distal metabolic niches. To implement this approach, the endogenous pathway of dietary lipid absorption was utilized as a foundational model. In physiology, bile salts micellize dietary lipids to facilitate uptake in enterocyte, and absorbed lipids are packaged into nascent chylomicrons that translocate via the ER/Golgi apparatus to enter the lymphatic network, avoiding portal vein draining 24 . Once intravascular, lipolytic processing by lipoprotein lipase (LPL) at the vascular endothelium remodels these particles, enabling substrate delivery to peripheral tissues 25 . Inspired by this design logic, we developed Layer-by-Layer Chylomicron-Mimicking Self-Assembly (LbL-CMSA), a spatiotemporally programmed platform that integrates oral robustness, enterocyte transcytosis, lymphatic routing for sequential genetic reprogramming (Fig. 1a). LbL-CMSA comprises (i) an outer deoxycholic acid (DCA) shell that stabilizes the assembly and facilitates ASBT-mediated enterocyte entry, (ii) an intermediate siRNA layer designed to dissociate early for local intestinal reprogramming, and (iii) an inner apolipoprotein-coated lipopeptoplex (aLPP) core engineered for lymphatic export (Fig. 1b). Structural validation confirmed that the DCA shell preserved dimensional integrity, effectively suppressing premature burst release under simulated gastrointestinal conditions 24,26-30 . Mechanistic studies identified ASBT-mediated uptake followed by caveola-mediated endocytosis as the predominant entry route, with FRET imaging and inhibition assay confirming ER/Golgi-coupled transcytosis and subsequent apolipoprotein-dependent lymphatic routing. By enriching the aLPP core with a ApoA-IV to bias cargo routing into the lacteal network and incorporating ApoC-II as a vascular cofactor to trigger lipoprotein lipase (LPL)-mediated uncoating, LbL-CMSA achieved a 5-fold increase in mesenteric lymph node accumulation while effectively bypassing ApoE-driven hepatic redirection 31-35 . Furthermore, LPL-mediated uncoating of peptoplex ensured therapeutic gene delivery to distal metabolic tissues via a receptor-targeting peptide fused to nona-arginine (9R). This programmed cascade enabled spatiotemporal separation of intestinal and peripheral programs, facilitated targeted silencing fatty acid binding proteins (FABPs) in gut-adipose tissue axis in obesity models. siFABP2 localized in the gut to attenuate lipid efflux, while the sh(FABP4/5) was delivered to adipocytes via 9R based targeting peptide (PBP9R, Prohibitin Binding Peptide 9R), triggering a brown-adipogenic shift 36-40 . This dual-silencing effects co-tune lipid metabolism and elicits robust metabolic benefits in body weight, insulin resistance, hyperlipidemia than single action components. Furthermore, the modularity of LbL-CMSA was demonstrated in fibrotic liver models by reconfiguring the payloads to interrupt the pathogenic gut–liver axis. By delivering siRNA against myosin light chain kinase 1 (MLCK1), a key regulator of paracellular permeability, intestinal tight junction recovery block endotoxin translocation to liver. Simultaneously, the delivery of a plasmid encoding heme oxygenase-1 (HO-1), a potent antioxidant and cytoprotective enzyme, dampened hepatic oxidative stress and TGF-β–mediated fibrogenic signaling synergistically. Collectively, these results establish LbL-CMSA as a programmable oral platform that coordinates gut-to-periphery genetic interventions by aligning nanoplatform kinetics with multi-compartment physiology. Design principles of LbL-CMSA We designed LbL-CMSA as a multi-stage vehicle that integrates gastrointestinal stability with the kinetic flexibility required for multi-compartment genetic interventions. Following the methods, LbL-CMSA was fabricated via stepwise self-assembly, yielding a double-layered liposomal morphology with a size of ~300 nm (Fig. 1c and Supplementary Fig. 1). Comparative studies with or without the DCA shell showed that colloidal stability and release behavior was markedly differed pH- and enzyme-dependently. DCA-coated LbL-CMSA retained dimensional integrity and a stable zeta potential, whereas LbL-CMSA (DCA−) exhibited pronounced size expansion and zeta-potential neutralization (Figs. 1d–f and Supplementary Figs. 1–2). Consistently, FT-IR analysis before and after FaSSGF/FaSSIF incubation confirmed preservation of multilayer signatures (C–H stretches ~2850–2920 cm⁻¹, amide I/II ~1650/1540 cm⁻¹, phosphate/C–O vibrations ~1200–1000 cm⁻¹), with only minor band shifts and modest intensity changes (Supplementary Fig.3). PCR-based release assays further showed that LbL-CMSA suppressed the initial burst and enabled sustained release of both pDNA and siRNA across an acidic-to-basic pH range, relative to LbL-CMSA (DCA−) (Fig. 1g and Supplementary Figs. 3–4). Similar stabilization was observed in enzyme-rich FaSSGF/FaSSIF solutions, where LbL-CMSA reduced early leakage and maintained sustained release for both cargos compared with the DCA− control (Fig. 1h and Supplementary Figs. 4–5). Endocytosis and transport mechanism investigation Beyond gastrointestinal protection, whether LbL-CMSA actively targets enterocytes via DCA-dependent way were measured using fluorescence-labelled pDNA and siRNA. After 4 h incubation under harsh conditions, cellular uptake was quantified in a differentiated Caco-2/HT29 mucus-forming epithelial layer (Fig. 2a). LbL-CMSA showed higher delivery efficacy of both genetic cargos in simulated gastrointestinal fluids, consistent with improved mucosal translocation, whereas DCA-deficient particles were trapped within the mucus barrier (Fig. 2a). To identify the dominant receptor involved in endocytosis, we assessed SR-B1, CD36 and ASBT which mediate dietary lipid and bile-acid absorption 41 . Receptor pre-blocking with antibody revealed ASBT as a key mediator of LbL-CMSA internalization, significantly decreasing the mean fluorescence intensity of pDNA and siRNA upon inhibition (Fig. 2b). Pharmacological inhibition of endocytic routes further indicated a primary role for caveola-mediated endocytosis, as β-cyclodextrin most effectively suppressed uptake (Fig. 2c). Confocal imaging confirmed colocalization of surface ASBT with LbL-CMSA, supporting an ASBT engagement–driven endocytosis (Fig. 2d). After internalization, both pDNA and siRNA exhibited reduced colocalization with late endosomes/lysosomes, indicating cytosolic release with lysosomal bypass (Fig. 2e and Supplementary Figs. 6–8). Together, these data define ASBT binding followed by caveola-mediated endocytosis as the predominant enterocyte entry mechanism for LbL-CMSA. The fluorescence imaging of the whole GI tracts after oral administration in C57BL/6 mice showed uniform distribution of pDNA and siRNA signals throughout the small intestine with LbL-CMSA, consistent with efficient enterocyte absorption (Fig. 2f and Supplementary Fig. 9). Fluorescence quantification of homogenized fecal samples collected over time revealed higher signal in the LbL-CMSA (DCA−) group, indicating increased loss of unabsorbed or degraded nanoparticles relative to LbL-CMSA (Fig. 2g). Fluorescence imaging of cryosection further confirmed retention and localization of LbL-CMSA signals within intestinal tissue (Fig. 2h). Collectively, the DCA shell provides a dual function of stabilizing the multilayer assembly during GI transit and enabling ASBT-driven epithelial uptake, thereby increasing intestinal delivery while reducing fecal loss compared with DCA-deficient formulations. Transcytosis mechanism after spatiotemporal disassembly The post-entry trafficking of LbL-CMSA within enterocytes were further investigated using a Caco-2/HT29 epithelium, in which paracellular transport is constrained by tight junctions under steady state (Transepithelial/transendothelial electrical resistance (TEER) ~8 Å) 42 . TEER remained unchanged for 24 h for both formulations, indicating that epithelial passage occurs via transcytosis (Fig. 3a). In a Transwell assay, LbL-CMSA produced a marked increase of pDNA–Cy5.5 in the basolateral compartment after 12 h while siRNA–FITC remained at basal levels, however, neither genetic cargo increased with LbL-CMSA (DCA−) (Supplementary Fig. 10). These data support a programmed sequence in which siRNA is released and retained within enterocytes, while pDNA loaded in aLPP is preferentially exported via epithelial transcytosis. To probe downstream transfer and sequential release behavior, indirect co-cultured transwell system were established with multiple cells including differentiated 3T3-L1 in the basolateral chamber as PBP9R target prohibitin on adipocytes (Fig. 3b and Supplementary Figs. 11–12). LbL-CMSA labeled siRNA-Cy5.5 and pDNA-FITC was administered after the pre-treatment with endocytosis/transcytosis inhibitors (Fig. 3c and Supplementary Figs. 13–16). After 24 h, pDNA signal decreased within the Caco-2/HT29 layer and appeared in the basal compartment, whereas siRNA signal remained confined to the epithelial monolayer. Inhibiting Golgi–ER trafficking with Brefeldin A (BFA) or Monensin (MON) significantly trapped the pDNA within the enterocyte layer and prevented exocytosis. This pharmacologically confirms that the aLPP core strictly follows the chylomicron-mimetic secretory pathway via the ER/Golgi apparatus, rather than non-specific transcytosis. Dual-label fluorescence resonance energy transfer (FRET) imaging (DiO labeled inner core and DiL labeled outer shell) further visualized time-dependent layer dissociation (Fig. 3d). Early internalized particles showed high FRET as evidence of intact assemblies, followed by reduced FRET with persistent donor signal in the cytoplasm, indicating outer-shell loss and core exposure (Figs. 3e,f). Intestinal sections after oral dosing mirrored this temporal FRET-to-donor transition, supporting sequential uncoating within enterocytes followed by delivery of the exposed core to downstream compartments (Figs. 3g). Consistently, pDNA colocalized with ER/Golgi markers, whereas siRNA remained cytosolic post-escape (Figs. 3h-i). To define whether apolipoprotein pre-coating governs ER/Golgi-mediated transcytosis and lymphatic routing, ApoL-deficient LbL-CMSA (ApoL−) were prepared and pDNA was substituted with luciferase (pLuci) for functional quantification in adipocytes (Fig. 3j). ApoL deficiency reduced luciferase expression in 3T3-L1 cells and its effect was amplified under transcytosis inhibition, indicating that the apolipoprotein layer promotes productive transcytosis of the aLPP core (Fig. 3f and Supplementary Fig. 17). This behavior was modulated by lipoprotein lipase (LPL), which facilitated selective lipolytic processing pf apolipoprotein and promoted inner peptoplex release (Supplementary Fig. 18) 25 . In vivo, apolipoprotein pre-coating markedly increased lymphatic delivery as LbL-CMSA showed up to 5-fold higher fluorescence in mesenteric lymph nodes and 4.6-fold higher fluorescence in inguinal nodes compared with LbL-CMSA (ApoL−) (Figs. 3g-i and Supplementary Figs. 19-20). Finally, serum challenge assays indicated that apolipoprotein pre-coating also mitigates deleterious protein corona remodeling (Supplementary Fig. 21). Apolipoprotein pre-coated aLPP retained colloidal properties (hydrodynamic size, polydispersity, and ζ-potential) after incubation in human serum, and ELISA results of particle lysates showed preserved ApoA-IV and ApoC-II with minimal acquisition of other apolipoproteins. In contrast, the absence of the pre-coated apolipoprotein led to the acquisition of a heterogeneous corona enriched in ApoE. This suggests that the apolipoprotein coating actively acts as a barrier against opsonization, preventing ApoE-driven hepatic clearance. Accordingly, LbL-CMSA showed prominent pDNA localization in mesenteric lymph nodes with negligible liver signal, whereas LbL-CMSA (ApoL−) preferentially accumulated in the liver (Supplementary Fig. 22). Together, these results support a chylomicron-mimicking, ER/Golgi-coupled transcytosis route that enables lymphatic absorption while resisting ApoE-driven hepatic redirection. Sequential gene delivery from gut to peripheral organ Fatty acid-binding proteins (FABPs) are central regulators of lipid trafficking and metabolic homeostasis, making them attractive targets for obesity and type 2 diabetes 43-45 . FABP4/5, enriched in adipocytes and macrophages, promotes lipid handling programs linked to inflammation, insulin resistance and obesity-associated complications 46-48 . In parallel, enterocyte FABP2 facilitates intracellular fatty-acid shuttling required for chylomicron assembly and intestinal lipid export 49,50 . We engineered LbL-CMSA to compartmentalize siFABP2 and sh(FABP4/5) into distinct layers, enabling organ-resolved but sequential silencing along the enterocyte–adipocyte axis (Fig. 4a). In an obese type 2 diabetes model, oral LbL-CMSA was designed to first attenuate intestinal lipid transport via enterocyte delivery of siFABP2, and subsequently reprogram adipose metabolism by directing sh(FABP4/5) to white adipocytes. LbL-CMSA downregulated FABP2 in enterocytes in the transwell model and concomitantly reduced lipid-handling pathways, including DGAT1, ATGL and MGL, resulting in decreased fatty-acid absorption and triglyceride accumulation in enterocytes (Figs. 4b,c). In mature adipocytes (3T3-L1), sh(FABP4/5) efficiently suppressed FABP4 and FABP5 and induced a brown-adipogenic transcriptional shift, as reflected by increased expression of PPARγ, PGC1α, PRDM16 and UCP1 (Figs. 4d,e). The upregulation of PPARγ, PGC1α, PRDM16, and UCP1 indicates a fundamental reprogramming toward mitochondrial biogenesis and oxidative metabolism, effectively converting energy-storing white adipocytes into energy-expending phenotypes. The dual silencing of FABP4/5 and FABP2 provided a beneficial strategy for orally treating obesity and obesity-related metabolic disorders by reducing fat absorption and triglyceride accumulation and by concomitantly enhancing energy expenditure without toxicity (Fig. 4f and Supplementary Fig. 23). Quantitative analysis of therapeutic genes across major organs after oral dosing showed compartmentalized biodistribution consistent with the programmed cascade. siFABP2 was largely confined to the GI tract, whereas sh(FABP4/5) selectively accumulated in epididymal WAT (epiWAT) and subcutaneous WAT (subWAT) following post-epithelial transport (Fig. 4g). epiWAT exhibited the highest sh(FABP4/5) abundance, consistent with prohibitin enrichment and PBP9R-mediated targeting of the peptoplex core. Ex vivo imaging of dissected GI and adipose tissues further confirmed spatial separation of the two genetic cargos, indicating dissociation of aLPP from the siFABP2 layer and subsequent accumulation of sh(FABP4/5) within epiWAT (Figs. 4h,i and Supplementary Figs. 24–25). Together, these data establish LbL-CMSA as an orally administered, sequential dual-gene modality that couples enterocyte lipid-uptake suppression with adipose reprogramming. To validate the proposed translocation cascade under physiological conditions, in vivo pathway perturbation using selective inhibitors was conducted (Fig. 4j). SC-435 reduced genetic payload in GI tracts and lowered distribution in mesenteric lymph nodes and adipose depots, consistent with diminished bile acid–mediated enterocyte uptake. Pluronic L-81 increased intestinal retention while reducing lymph-node and adipose accumulation, supporting blockade of chylomicron-dependent lymphatic export. Tyloxapol-mediated inhibition of lipoprotein lipase decreased peptoplex core release, reduced adipose accumulation and produced relative increases in hepatic signal. Finally, control adipose depots with naturally low PHB expressions (such as anterior and neck fat) showed negligible payload accumulation despite systemic circulation, confirming that the core delivery is strictly driven by PBP9R-mediated active targeting to PHB-rich adipocytes. Collectively, these pharmacologic perturbations provide in vivo evidence that ASBT-mediated uptake, chylomicron-dependent lymphatic partitioning, and LPL-dependent core processing are sequential, operative steps underpinning LbL-CMSA transport and multi-organ gene delivery. Sequential FABP silencing ameliorates metabolic dysfunction in DIO model Oral administration of multi-FABP–silencing LbL-CMSA in a diet-induced obesity (DIO) C57BL/6J model produced robust metabolic benefits over 10 weeks, outperforming single-gene silencing (Figs. 5a–c). The LbL-CMSA (siFABP2–shFABP4/5) group achieved a 40.59% body-weight loss at 10 weeks versus PBS, exceeding LbL-CMSA (siFABP2) by 16.19% and LbL-CMSA (shFABP4/5) by 11.19%. Notably, weight reduction was maintained during a subsequent 4-weeks of drug-free follow-up without rebound, in contrast to the rapid regain often observed after cessation of GLP-1 receptor agonist therapy. Functional suppression of intestinal lipid uptake was supported by weekly fecal fatty-acid profiling (Figs. 5d,e). siFABP2 treatment increased fecal free fatty acids by ~7 nmol/kg per week relative to LbL-CMSA (vehicle) and by ~4 nmol/kg per week relative to LbL-CMSA (sh(FABP4/5)), despite no differences in dietary fat intake. Together, these outcomes support a synergistic benefit from sequential oral silencing of FABP2 in enterocytes and FABP4/5 in adipocytes without side effects (Supplementary Fig. 27). Mechanistically, analyses of harvested small intestine and visceral adipose tissue confirmed compartment-specific metabolic remodeling. In the intestine, siFABP2-containing formulations downregulated lipid-handling genes, including DGAT1, ATGL and MGL, consistent with reduced fatty-acid processing in enterocytes (Figs. 5f,h). Orthogonal protein quantification by ELISA corroborated as NPC1L1 and CD36 were reduced, indicating diminished long-chain fatty-acid transport capacity, while DGAT1 and SREBP1c abundance decreased, consistent with attenuated triglyceride synthesis and lipogenic signaling (Supplementary Fig. 26). In adipose tissue, sh(FABP4/5) increased expression of brown-adipogenic markers (UCP1, PPARγ, PRDM16 and PGC1α), indicating reprogramming toward an energy-expending state (Figs. 5g,h). Consistent with the combined intestinal and adipose remodeing, the sequential dual-gene intervention improved systemic lipid homeostasis, lowering circulating triglycerides and free fatty acids, and alleviated hepatocellular injury as reflected by reduced ALT and AST (Figs. 5i,j). Multi-FABP silencing by oral LbL-CMSA also mitigated liver pathology, consistent with systemic rebalancing of lipid flux and inflammatory tone. Histology and biochemical analysis showed reduced hepatic steatosis and fibrosis, with fewer steatotic hepatocytes, lower collagen deposition, decreased hepatic hydroxyproline and suppressed TGF-β signaling (Figs. 6a,b). In parallel, FABP silencing attenuated inflammatory mediators (TNF-α, IL-1β, IL-6 and CCL2), supporting efficacy in inflammation-coupled metabolic disease (Figs. 6c–f). These outcomes are consistent with organ-resolved, sequential gene silencing in which siFABP2 remodels intestinal lipid handling while shFABP4/5 reprograms adipose storage and thermogenesis. While single-gene treatments also showed benefits, the dual-targeting strategy achieved a qualitatively more complete suppression of the metabolic–inflammatory milieu. In chronic disease contexts, such layered control over both intestinal and adipose pathways is expected to offer superior long-term outcomes and lower relapse risks than single-pathway interventions. For translation, formulation storability is essential for quality control and sustained clinical deployment. LbL-CMSA retained structural stability over 4 weeks, with preserved activity of encapsulated shRNA and siRNA (Fig. 6g). In addition, LbL-CMSA after long-term storage showed therapeutic efficacy in vivo comparable to freshly prepared material (Fig. 6i). By combining spatiotemporal control over lipid metabolism with robust stability, LbL-CMSA emerges as a scalable platform for oral gene therapy. Its unique ability to execute sequential, multi-target interventions positions it as a practical solution for complex metabolic disorders. Modular reconfiguration of LbL-CMSA for MASLD To demonstrate the modularity of platform, we reconfigured LbL-CMSA to interrupt the pathogenic gut–liver axis in metabolic dysfunction-associated steatotic liver disease (MASLD) by substituting payloads with an intestinal siMLCK1 layer and a systemic pHO-1 core (Supplementary Fig. 28). This dual-targeting strategy was designed to simultaneously repair leaky gut via MLCK1 inhibition and dampen hepatic fibrogenic signaling through HO-1-mediated cytoprotection (Extended Data Fig.1a and Supplementary Note.1). LbL-CMSA restored tight-junction integrity (ZO-1, occludin) in LPS-inflamed Caco-2/HT29 epithelial layer, significantly reducing endotoxin flux (Extended Data Figs. 1b,c). Simultaneously, pHO-1 delivery attenuated inflammatory and fibrotic programs in HepG2 cells and induced brown-adipogenic shifting in 3T3-L1 adipocytes (Extended Data Fig.1d and Supplementary Fig. 29). To further evaluate in vivo study, a fibrosis-augmented MASLD model was prepared with administration high fructose high fat diet with intraperitoneal CCl₄ injection. Because PHB is overexpressed in damaged liver in MASLD, LbL-CMSA accumulated in the liver and adipose tissues, leading to improved systemic indices and reduced body-weight gain (Extended Data Fig.1e and Supplementary Figs. 30–31). siMLCK1-mediated barrier reinforcement was confirmed by reduced FITC-dextran leakage and normalized levels of microbial translocation biomarkers including D-lactate, LBP, and sCD14 (Extended Data Figs.1f–h). This indicates that the reinforced barrier effectively blocked the influx of gut-derived endotoxins, which are key drivers of hepatic inflammation. In the liver, combination therapy yielded the most significant normalization of hepatic architecture and reduction in fibrotic staining compared to single-payload controls (Fig. 7i). Transcriptomic and biochemical analyses showed broad suppression of lipogenic (Srebp1c), inflammatory (Tnf, Il1b), and fibrogenic (Tgfb1, Col1a1) mediators, alongside reduced hydroxyproline and serum injury markers (ALT, AST) (Extended Data Figs.1j–l). Flow cytometry further confirmed a shift toward an anti-inflammatory macrophage phenotype and enhanced antioxidant capacity (Extended Data Figs. 1m,n). Finally, WAT exhibited reduced inflammation and increased thermogenic gene expression, normalizing adipokine profiles (Extended Data Figs.1q,r). Collectively, these results establish LbL-CMSA as a programmable oral platform to coordinate multi-axis correction of intestinal dysfunction and peripheral organ pathology. Conclusion LbL-CMSA establishes a conceptually new class of spatiotemporally programmed oral gene-delivery materials in which interface switching confers deterministic post-epithelial routing and site-specific gene modulation, thereby addressing a central bottleneck that has limited oral nucleic-acid therapeutics. By coupling DCA–guided epithelial entry with chylomicron-mimetic lymphatic export and niche-specific uncoating, the platform enables multi-tissue genetic modulation from a single dose while minimizing hepatic redirection, and its payload- and targeting-swappable design supports rapid reprogramming toward distinct gut–organ disease axes. By replacing cold-chain-dependent injectables with shelf-stable oral formulations, this platform addresses critical barriers in patient adherence and healthcare accessibility. Moving forward, clinical translation will require addressing scalable manufacturing and biological variability. To bridge the gap from bench to bedside, the adoption of continuous microfluidic assembly will be pivotal in maintaining the intricate layer-by-layer architecture while ensuring high-throughput quality control. With appropriate development of personalized biomarkers, LbL-CMSA could open new avenues for treating neurodegenerative or cardiovascular disorders linked to gut dysbiosis, beyond metabolic syndromes. Methods Materials A peptoplex of a therapeutic plasmid DNA and an oligopeptide (PBP9R, C-KGGRAKD-RRRRRRRRR-C) comprising nona-arginine (9R) and a prohibitin-binding peptide (PBP, KGGRAKD) was prepared; then, stepwise thin-film hydration was performed to construct a layer-by-layer chylomicron-mimicking self-assembly (LbL-CMSA) with an apolipoprotein-coated lipopeptoplex (aLPP) and siFABP2 in the middle layer. Negatively charged plasmid DNA was allowed to interact with an oligopeptide containing a positively charged guanidino group via nona-arginine (RRRRRRRRR) and assembled at a weight ratio of 1:3 (plasmid DNA: oligopeptide) with strong genetic material entrapment: an encapsulation efficiency of >95%, as shown in several previous studies. The peptoplex was 182.43 ± 15.16 nm with a zeta-potential of 23.456 ± 5.16 mV. These properties confirmed that it was a cationic oligopeptide complexed with a genetic material, and it mediated endosomal escape; consequently, the peptoplex could target specific cells to deliver its genetic materials. A chylomicron-mimicking strategy through an apolipoprotein coating was used to enhance the transcytosis of the genetic materials encapsulated in the peptoplex within enterocytes and avoid first-pass metabolism via portal vein drainage. Consequently, aLPP loaded with the peptoplex could effectively deliver genetic materials within the lymphatic circulation instead of releasing it within enterocytes. A thin-film lipid layer was prepared using DSPC:DPPC:DSPE:cholesterol:apolipoprotein (ApoA-IV): apolipoprotein (ApoC-II) at a molar ratio of 0.01:1:1:0.2:0.5:0.5. The lipid mixture was dissolved in chloroform:methanol (2:1, v/v) and evaporated under vacuum in a rotary evaporator at 50°C for 30 min. The resulting thin film was left to dry overnight in a fume hood to remove residual organic solvents. For rehydration, the lipid film was hydrated in distilled water containing the peptoplex at room temperature for 2 h and serially extruded through 0.4 and 0.2 µm pore PVDF membranes to ensure a uniform vesicle size. After thin-film hydration, the Z-average was increased to 223.67 ± 11.17 nm, which corresponded to an increase of approximately 40 nm from the peptoplex alone, through the serial extrusion of aLPP through 0.4 and 0.2 μm pore PVDF membrane filters. Furthermore, the zeta-potential of aLPP was −23.78 ± 7.64 mV because the lipid covered the positive charge of the peptoplex. Cryo-TEM images confirmed the unilamellar liposome morphology of aLPP. Bile salt-based lipid shells (DPPC:DSPC:DPPE:sodium deoxycholate = 1:1:0.2:1 molar ratio) were prepared by dispersing lipids in a chloroform:methanol (2:1, v/v) solution and evaporating it in a rotary evaporator at 50°C for 30 min to deliver aLPP into enterocytes despite the physical and chemical barriers in the GI environment. After the bile salt-based lipid shell layer was prepared, aLPP was initially lyophilized to prevent destabilization during siRNA incorporation and thus spatially separate siFABP2 from aLPP while maximizing the loading efficiency. The lyophilized aLPP was then resuspended in an aqueous solution containing 5 µg of siRNA and incubated at 25°C under gentle agitation for 2 h to facilitate siRNA adsorption onto the lipid bilayer without disrupting the aLPP integrity. The final formulation was extruded through a 0.8 µm PTFE membrane to ensure a uniform double-layer lipid nanoparticle population. Thus, the aLPP/siRNA dual-loaded layer-by-layer chylomicron-mimicking self-assembly (LbL-CMSA) could target enterocytes following the oral administration and release of siFABP2 into the cytosol to downregulate mRNA; then, aLPP was translocated into the lymph nodes, and the peptoplex was released into blood vessels via lipoprotein lipase. The zeta-average diameter of LbL-CMSA was slightly higher (365.15 ± 34.16 nm), and its zeta-potential was −26.47 ± 5.73 mV. The efficiencies of sh(FABP4/5) encapsulation at different preparation steps were as follows: 99.99% ± 1.99% for the peptoplex preparation, 92.27% ± 2.45% for the aLPP preparation, and 89.00% ± 3.41% for the final formulation. siFABP2 added to the LbL-CMSA formulation with aLPP was encapsulated at an efficiency of 84.34% ± 5.16%, indicating that the sequential loading of genetic materials was highly effective. Drug loading and encapsulation efficiency During each preparation step of LbL-CMSA containing 10 μg of sh(FABP4/5) and 10 μg of siFABP2, the peptoplex, aLPP, and LbL-CMSA were ultracentrifuged at 50,000 × g for 30 min, and the supernatants were collected to quantify the remnant DNA and siRNA. Plasmid DNA was quantified via Taqman-based real-time PCR with specific primer sequences and probes. siRNA was detected using a siRNA quantitation kit in accordance with the manufacturer’s instructions, and synthetic siRNA was quantified with deoxythymidine d(TT) as a 3′-overhang. Drug loading and encapsulation efficiency were calculated using the following equations: Drug loading = Encapsulation efficiency = Cumulative drug release in gastrointestinal-mimicking environments For the analysis of drug release kinetics, 10 mg of LbL-CMSA and LbL-CMSA (DCA-) was dispersed in 5 ml of PBS with various pH levels (pH 2, 4, 7, and 8) at 37°C. For the drug release analysis of LbL-CMSA in gastrointestinal-mimicking environments, fasted-state simulated intestinal fluid and fasted-state simulated gastric fluid were purchased from Biorelevant, and 10 mg of LbL-CMSA was incubated in the desired volume. At each time point, LbL-CMSA and LbL-CMSA were collected through ultracentrifugation at 14,000 rpm for 20 min. pDNA and siRNA were isolated using a blood and tissue DNeasy kit to evaluate the therapeutic genes remaining inside the NPs. The isolated pDNA and siRNA were quantified using the methods described for drug loading and encapsulation efficiency. FT-IR analysis of LbL-CMSA before and after biorelevant media exposure. To characterize layer integrity and chemical changes associated with DCA coating and biorelevant media challenge, attenuated-total-reflection FT-IR spectra were collected on lyophilized nanoparticle samples as follows. LbL-CMSA were incubated under simulated FaSSGF (pH ≈ 1.6) and FaSSIF (pH ≈ 6.5) for 2 h at 37 °C, with gentle orbital agitation (100 rpm) to mimic physiological shear. After incubation, particles were recovered by centrifugation (20,000 × g, 30 min), washed twice with deionized water to remove excess buffer, and lyophilized overnight. FT-IR spectra were acquired in ATR mode on a Fourier-transform infrared spectrometer. For each sample, 2 mg of lyophilized material was placed onto the ATR crystal and gently pressed to ensure uniform contact. Spectra were recorded from 4000 to 400 cm⁻¹ with a spectral resolution of 4 cm⁻¹, averaging 64 scans per sample. A background spectrum was recorded before each set of measurements and subtracted automatically. 3T3-L1 adipocyte differentiation 3T3-L1 cells were purchased from ATCC (Virginia, USA). High-glucose DMEM (WelGENE, Seoul, Korea) with 10% FBS and 1% penicillin–streptomycin (100 U ml -1 ) was used for cell culture. 3T3-L1 cells were incubated at 37°C in 5% CO 2 and passed every other day. For adipocyte differentiation, they were treated with a differentiation medium containing a complete medium, 10 µg/ml insulin, 1 µM dexamethasone, and 0.5 mM IBMX for 72 h. The differentiation medium was replaced with a complete medium containing 10 µg/ml insulin and changed every 2 days. In vitro model for intestinal permeability Caco-2 and HT29 cells were purchased from ATCC (Virginia, USA). High-glucose DMEM (WelGENE, Seoul, Korea) with 10% FBS and 1% penicillin–streptomycin (100 U ml -1 ) was used for the cell culture. Caco-2 and HT29 cells were seeded at a density of 2.5 × 10 5 cells onto Transwell® polycarbonate filter supports (0.4 μm pore size and 12 mm diameter) at an initial seeding ratio of Caco‐2:HT29 of 9:1. They were cultured for at least 21 days before analysis. The medium was replaced with fresh DMEM every 2 days. Before use, the TEER value (~400 Ω × cm 2 ) was measured to verify the constructed monolayer. The following inhibitors were used to address the endocytosis and transcytosis mechanisms of LbL-CMSA (Table 1). Inhibitors M.W. Functions Concentrations Preparation Chlorpromazine 318.86 Inhibition of clathrin-mediated endocytosis 25 μM 8 µg/mL (DW) β-cyclodextrin 1135 Inhibition of lipid/raft pathway. Inhibition of caveolae-mediated endocytosis 10 mM 113 mg/mL (DW) Amiloride 229.627 Inhibition of macropinocytosis pathway blocking the Na + /H + exchanger 2.5 mM 574 µg/mL (DW) Brefeldin A 280.36 Golgi apparatus/ER-related inhibitor 25 mg/mL 25 mg/mL (DMSO) Monensin 670.871 Golgi apparatus-related inhibitor 33 mg/mL 33 mg/mL (DMSO) Table 1. Endocytosis and transcytosis inhibitors used for mechanistic studies of LbL-CMSA transport. A panel of pharmacological inhibitors was employed to delineate the cellular pathways involved in LbL-CMSA uptake and transcytosis across Caco-2/HT29 intestinal epithelial monolayers. Dual-label preparation and characterization. Dual-label LbL-CMSA particles were prepared by incorporating the donor fluorophore DiO into the inner, apolipoprotein-mimetic core and by labeling the outer deoxycholic acid (DCA) shell with the acceptor fluorophore DiL. DiO (donor) was premixed with core lipid components during thin-film formation at a final dye-to-lipid molar ratio of 0.2 % and the core was formed as described in Methods. The outer DCA shell was labeled post-assembly by incubating particles with DiL (acceptor) in ethanol:water (1:9 v/v) followed by purification by centrifugation (20,000 × g, 20 min) and three washes in PBS to remove unbound dye. Fluorescence spectra were recorded to confirm donor and acceptor peaks and to verify FRET in assembled particles. High-fat diet-induced type 2 diabetes mouse model All experimental procedures were approved by the Institutional Animal Care and Use Committee at Hanyang University (2020-0036). Six-week-old male C57BL/6J mice (Orient Bio) were randomly assigned to different treatment groups ( n = 5 per group). They were fed with normal chow (Orient Bio) for the first 2 weeks and with diet mixed with 60% of calories from fat (high-fat diet, HFD, Central Lab Animal, Inc.) for the succeeding weeks to induce obesity and metabolic syndrome. The proportion of HFD in the total rodent diet gradually increased for 6 weeks; then, the mice were fed with only HFD for 8 more weeks. All mice were housed under specific pathogen-free conditions. They became obese and insulin resistant after the 16th week; their body weights and glucose levels were 45–55 g and >250 mg/dL, respectively. After an HFD-induced type 2 diabetes mouse model was prepared, 0.5 mg/kg of sh(FABP4/5) loaded with LbL-CMSA was administered through oral gavage thrice a week. After 6 weeks of treatment, the mice were observed for 4 more weeks to determine further therapeutic effects and then sacrificed on the 11th week for ex vivo analyses. Ex vivo biodistribution using an in vivo imaging system. LbL-CMSA loaded with sh(FABP4/5)-FITC and siRNA-Cy5.5 was orally administered to the mice that were then sacrificed at the desired time point. The liver, spleen, kidneys, lungs, heart, visceral adipose tissues, and subcutaneous adipose tissues were harvested. The ex vivo Cy5.5 fluorescence and FITC fluorescence in each organ were detected using a fluorescence in vivo imaging system; the mean fluorescence intensity per tissue area was measured. Ex vivo biodistribution via RT-qPCR LbL-CMSA loaded with sh(FABP4/5) and siFABP2 was orally administered to the mice that were then sacrificed at the desired time point. Then, the liver, spleen, kidneys, lungs, heart, visceral adipose tissues, and subcutaneous adipose tissues were harvested. pDNA and siRNA were isolated using a blood and tissue DNeasy kit. Plasmid DNA was quantified using Taqman-based real-time PCR with specific primer sequences and probes. siRNA was detected using a siRNA quantitation kit in accordance with the manufacturer’s instructions, and synthetic siRNA was quantified with deoxythymidine d(TT) as a 3′-overhang. Insulin tolerance test After 6 h of fasting, the initial blood glucose level was measured by using an Accu-Chek Active model GC kit (Roche Diagnostics GmbH, USA). Insulin (0.75 unit/kg) was injected intraperitoneally. Blood samples were collected 0, 30, 60, 90, and 120 min post-injection for the insulin tolerance test. Blood glucose levels were measured by placing a small drop of blood on a new test strip and recorded. Ex vivo mRNA analysis Samples from the small intestines and visceral white adipose tissues were mechanically minced into small fragments and total RNA was extracted from the cell pellets by using a commercial RNA extraction kit (RNeasy Mini Kit, Qiagen) in accordance with the manufacturer’s instructions. Equal amounts of purified RNA were reverse-transcribed into cDNA by using a cDNA synthesis kit (iScript™ cDNA Synthesis Kit, BIO-RAD). Relative gene expression levels were quantified via real-time PCR (Applied Biosystems 7500 Real-Time PCR System, USA) with gene-specific primers. GAPDH was used as the internal control, and gene expression was calculated using the ΔΔCt method. Immunofluorescence assay Adipose tissues and small intestines were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned at 8 µm thickness. The paraffin sections were deparaffinized and rehydrated through a graded alcohol series. After the antigen was retrieved, the sections were blocked with 5% bovine serum albumin (BSA) in PBS at room temperature for 1 h and incubated with primary antibodies against the target protein at 4°C overnight. On the next day, they were thoroughly washed with PBS and incubated with FITC-conjugated anti-rabbit secondary antibody (Abcam, USA) at room temperature in the dark for 2 h. After the final washes, they were mounted with a Dako fluorescence mounting medium (DAKO, Denmark) and sealed with coverslips. Immunofluorescence images were acquired using an AxioScan.Z1 slide scanner (Zeiss, Germany). Ex vivo analysis of cytokines and lipids At the end of the study, blood samples were collected via cardiac puncture. The plasma was separated by centrifugation at 2,000 × g and 4°C for 10 min and stored at −80°C until analysis. Liver and adipose tissues were surgically harvested, rinsed with cold PBS to remove blood contaminants, blotted dry, snap-frozen in liquid nitrogen, and stored at −80°C. Cholesterol, triglyceride, and free fatty acid (FFA) concentrations in the plasma and tissue homogenates were measured enzymatically by using commercially available kits (Abcam, USA) in accordance with the manufacturer’s instructions. For cytokine quantification, tissue samples were homogenized in lysis buffer containing protease inhibitors and centrifuged at 12,000 × g and 4°C for 15 min to obtain the supernatants. The total cytokine concentrations of interleukin-1β (IL-1β), interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), and monocyte chemoattractant protein-1 (MCP-1), transforming growth factor-beta (TGF-β), adiponectin, leptin, resistin, plasminogen activator inhibitor 1 (PAI-1) in the plasma or tissue supernatants were determined using enzyme-linked immunosorbent assay (ELISA) kits (InvivoGen, USA) in accordance with the manufacturer’s protocols. Liver histology and liver panel analysis Liver tissues were fixed in 4% neutral buffered formalin and embedded in paraffin. The paraffin sections were deparaffinized, rehydrated in gradual mixtures of ethanol/water, blocked with a 5% BSA blocking solution for 1 h, and incubated with anti-HO-1 antibody overnight. On the next day, they were washed and mounted with a mounting solution containing DAPI. In addition, 6 μm paraffin sections were stained with hematoxylin and eosin, Masson’s trichrome, and Sirius red for histological analyses. Metabolic dysfunction-associated steatotic liver disease (MASLD) model Six-week-old Female C57BL/6 mice (Orient Bio), aged 8 weeks were injected intraperitoneally with carbon tetrachloride (CCl 4 ) at a dose of 300 μl/kg body weight, dissolved in olive oil, three times a week (total volume of 100 μl per dose) for 6 weeks. To accelerate the induction of a metabolic dysfunction-associated liver fibrosis model, the mice were fed with a 60% Kcal HFD (Central Lab Animal, Inc., Seoul, South Korea). All mice were housed under specific pathogen-free conditions. After an MASLD mouse model was prepared, 0.5 mg/kg of pHO-1 loaded with LbL-CMSA was administered through oral gavage thrice a week. After 4 weeks of treatment, the mice were sacrificed for ex vivo analyses. Quantification of leaky gut derivates and hepatic antioxidant enzyme activities LBP, sCD14, D-lactate, SOD, and catalase were quantified using commercially available assay kits according to the manufacturers’ instructions. Circulating LBP and sCD14 were measured in serum by sandwich ELISA, and concentrations were calculated from standard curves with samples diluted as needed to fall within the linear range. D-lactate levels were determined using a D-lactate dehydrogenase–based enzymatic colorimetric assay, with appropriate sample preparation performed as recommended by the kit. For antioxidant enzyme activities, liver tissues were processed to obtain clarified homogenate supernatants, and SOD activity was assessed using a WST-1/xanthine oxidase–based inhibition assay, whereas catalase activity was measured using an H₂O₂ decomposition–based activity assay; activities were normalized to total protein content where applicable. All measurements were performed with technical replicates and included blanks and internal controls for quality assurance. Statistical analysis All in vitro experiments were performed with at least three independent biological replicates to ensure reproducibility. For in vivo studies, a sample size of n = 5 mice per group was used to ensure adequate statistical power and relevance. Data analyses were conducted using GraphPad Prism version 8.0 for Windows (GraphPad Software, California, USA). For group comparisons, one-way ANOVA followed by Tukey’s multiple comparison test was performed to identify statistically significant differences among groups. Statistical significance was defined as p < 0.05, with the following notations used to indicate significance levels: ns (not significant), *p < 0.033, **p < 0.01, and ***p < 0.001. The details of data processing procedures and the rationale for sample size determination for each specific analysis were provided in the corresponding figure legends. Declarations Data availability The main data supporting the results of this study are available within the paper and Supplementary Information. Source data are provided in this paper. Acknowledgments This research was partially supported by grants from the National Research Foundation of Korea (NRF‐2019R1A2C3008992, NRF-RS-2023-00208982), the Brain Korea 21 Fostering Outstanding Universities for Research (BK21 FOUR, 5199990514440), the Korean Health Technology R&D Project through the Ministry of Health Industry Development Institute (KHIDI) funded by the Ministry of Health & Welfare, Republic of Korea (HI19C0753), and the Korea Drug Development Fund funded by the Ministry of Science and ICT, Ministry of Trade, Industry, and Energy, and Ministry of Health and Welfare (HN21C0885, Republic of Korea). A uthor information Affiliations Department of Bioengineering, Institute for Bioengineering and Biopharmaceutical Research Hanyang University, 04763 Seoul, South Korea Juhyeong Hong, Seon-jeong Chang, Seung-Hwan joo, Han-Seok Lim, Kyungdong Kim, Sangjin Kim, Minjoo Jang, Yong-Hee Kim Education and Research Group for Biopharmaceutical Innovation Leader, Hanyang University, 04763 Seoul, South Korea Juhyeong Hong, Seon-jeong Chang, Seung-Hwan joo, Han-Seok Lim, Kyungdong Kim, Sangjin Kim, Minjoo Jang, Yong-Hee Kim Contributions Juhyeong Hong and Yong-Hee Kim designed experimental design and protocol. Juhyeong Hong performed the in vitro , in vivo , andex vivo therapeutic assays. Seon-jeong Chang, Seung-Hwan joo, Han-Seok Lim, Juhyun Lee, Kyungdong Kim, Sangjin Kim, and Minjoo Jang supported them in vitro , in vivo , andex vivo therapeutic assays. Juhyeong Hong and Yong-Hee Kim discussed the results and wrote the paper. 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The LbL-CMSA platform delivers siMLCK1 to strengthen tight junctions and pHO-1 to metabolic tissues, thereby reducing luminal translocation of inflammatory factors and providing anti-inflammatory protection at tissue level. b, Quantification of RNA expression in Transwell Caco-2/HT29 monolayers following apical application of LPS to induce tight junction loss. LPS were incubated for 24 hours and LbL-CMSA were treated for next 24 hours. c, Apical to basolateral passage of lipopolysaccharide (LPS) across Transwell monolayers. LPS in the basolateral medium was quantified by LAL assay. d, Quantification of RNA expression in LPS induced fibrotic HepG2 cells. e, Systemic metabolic outcomes including body weight, food uptake, and fasting blood glucose (n=5). f, Quantification of intestinal barrier permeability test after oral administration of FITC-dextran. g, gene expression for tight junction, barrier-associated markers, and inflammation in small intestine. h, PS binding protein (LBP) and soluble CD4 (sCD4) and D-lactate concentrations in blood levels to measure intestinal barrier function. i, Representative images of liver histological images demonstrating reduction of fibrosis after treatment. j, gene expression level covering steatosis, inflammation, antioxidants, and fibrosis pathways. k,fibrogenic hydroxyproline level in liver. l, hepatic injury markers (ALT, AST, and ASP) in serum. m, Quantification of populations in hepatic macrophages. n, anti-ROS enzymatic activity marker in liver. o, Quantification of inflammatory and fibrogenic cytokines in serum. p, Serum lipid levels in serum. q, gene expression level covering brown-adipogenesis and inflammation marker in WAT. r, Quantification of adipokines in WAT. Data are presented as means ± SD, and p-values were calculated by one-way ANOVA with Tukey's post-test. ns = not significant, * P < 0.0332, ** P < 0.0021, *** P < 0.0002, **** P < 0.0001. 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. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8523305","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":579515863,"identity":"5b6b1ef4-b97f-4471-b4ba-8c8fa6210fb2","order_by":0,"name":"Yong-Hee Kim","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABDUlEQVRIiWNgGAWjYJACZiA2YGNgPoYsmECMFrY0ErUwMPCYEafF4Hjv4dcFFTbGfGJnvj34ueOwvDn/AsYPPxjS8nFqOXMuzXrGmTQzNunc7Ya9Zw4b7pzxgFmyhyHHsgGXlhs5Zsa8bYdtgFq2SQAZjBtuHGCQZmCoMMBpC1jLv/9ALTnPJP+2HbYHamH+TUCL8WPehgNAh+WwSQNtSdxwvoENaEsOTi2SZ86YMfMcSzZmk04zk5ZtS0/ecIOxzbLHIA2nFr7jPcafeWrsDOfPTn4m+bbN2nbD+cOHb/yoSMapReEAA5sEEr+ZgUEisQEcT7iAfAMD8wckfh0DA/8B3MpHwSgYBaNgRAIAF0RYC5VxsCoAAAAASUVORK5CYII=","orcid":"","institution":"Hanyang University","correspondingAuthor":true,"prefix":"","firstName":"Yong-Hee","middleName":"","lastName":"Kim","suffix":""},{"id":579515864,"identity":"bc3d992c-0ce3-422b-ab0c-df890d031b18","order_by":1,"name":"Juhyeong Hong","email":"","orcid":"","institution":"Hanyang University","correspondingAuthor":false,"prefix":"","firstName":"Juhyeong","middleName":"","lastName":"Hong","suffix":""},{"id":579515865,"identity":"a9eaa605-6a09-445c-b25a-ad12ccdb827b","order_by":2,"name":"Seon-jeong Chang","email":"","orcid":"","institution":"Hanyang University","correspondingAuthor":false,"prefix":"","firstName":"Seon-jeong","middleName":"","lastName":"Chang","suffix":""},{"id":579515866,"identity":"f8a4adec-fa8e-4e79-ac30-6404dd45b344","order_by":3,"name":"Seung-Hwan Joo","email":"","orcid":"","institution":"Hanyang University","correspondingAuthor":false,"prefix":"","firstName":"Seung-Hwan","middleName":"","lastName":"Joo","suffix":""},{"id":579515867,"identity":"ebdc50e8-f4ac-414b-b114-37298c7bb888","order_by":4,"name":"Han-Seok Lim","email":"","orcid":"","institution":"Hanyang University","correspondingAuthor":false,"prefix":"","firstName":"Han-Seok","middleName":"","lastName":"Lim","suffix":""},{"id":579515868,"identity":"15b19375-8149-47f5-b62f-3c9ccd9bdd01","order_by":5,"name":"Juhyun Lee","email":"","orcid":"","institution":"Hanyang University","correspondingAuthor":false,"prefix":"","firstName":"Juhyun","middleName":"","lastName":"Lee","suffix":""},{"id":579515869,"identity":"3ac70632-86fa-4a7c-bb99-436a3f2a1bd5","order_by":6,"name":"Kyungdong Kim","email":"","orcid":"","institution":"Hanyang University","correspondingAuthor":false,"prefix":"","firstName":"Kyungdong","middleName":"","lastName":"Kim","suffix":""},{"id":579515870,"identity":"e09bb8e7-7fa5-49f8-8cc8-3b0ba22d5ad9","order_by":7,"name":"Sangjin Kim","email":"","orcid":"","institution":"Hanyang University","correspondingAuthor":false,"prefix":"","firstName":"Sangjin","middleName":"","lastName":"Kim","suffix":""},{"id":579515871,"identity":"ad9d3ec7-660d-42e5-8ae5-3f4a12c0f915","order_by":8,"name":"Minjoo Jang","email":"","orcid":"","institution":"Hanyang University","correspondingAuthor":false,"prefix":"","firstName":"Minjoo","middleName":"","lastName":"Jang","suffix":""}],"badges":[],"createdAt":"2026-01-05 16:11:44","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8523305/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8523305/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":101190181,"identity":"65567225-deb8-48ef-9030-8ba65bc0ffcb","added_by":"auto","created_at":"2026-01-27 06:57:43","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":3784064,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePreparation and characterization of LbL-CMSA, and gastrointestinal stability. a, \u003c/strong\u003eLayer-by-Layer tailoring methods for the preparation of LbL-CMSA with an pDNA inside core of aLPP and siRNA in the outer layer.\u003cstrong\u003e b, \u003c/strong\u003eSchematic of the therapeutic strategy of LbL-CMSA.\u003cstrong\u003e c,\u003c/strong\u003e Cryo-TEM images of aLPP (left) and LbL-CMSA (right). Scale bar = 100 μm. \u003cstrong\u003ed, \u003c/strong\u003eZ-average (d. nm) and zeta potential (mV) of LbL-CMSA in conditioned PBS buffer with various pH levels. \u003cstrong\u003ee, f, \u003c/strong\u003eZ-average (d. nm) of LbL-CMSA and LbL-CMSA (DCA-) during incubation in PBS for 4 h followed by incubation for 20 h in fasted-state simulated gastric fluid (FASSGF) (e) and fasted-state simulated intestinal fluid (FASSIF) (f). \u003cstrong\u003eg, \u003c/strong\u003eGene release profile of LbL-CMSA and LbL-CMSA (DCA-) at pH = 2 and 8. pDNA and siRNA in nanoparticles were quantified through qPCR with specific primers and standard curve (n=3). \u003cstrong\u003eh,\u003c/strong\u003eGene release profiles of LbL-CMSA and LbL-CMSA (DCA-) in FaSSIF. Data are presented as means ± SD, and p-values were calculated via one-way ANOVA with Tukey’s post-test. ns = not significant, * \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.0332, **\u003cem\u003e P\u003c/em\u003e\u0026lt; 0.0021, *** \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0002, ****\u003cem\u003e P\u003c/em\u003e \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8523305/v1/d478ec25856b7c10b84e06e6.jpg"},{"id":101190401,"identity":"216e84f1-36f8-4725-a671-d2004b40f3e3","added_by":"auto","created_at":"2026-01-27 06:58:22","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2386271,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDCA-mediated endocytosis in the intestinal epithelium. a, \u003c/strong\u003ein vitro cellular uptake of LbL-CMSA and LbL-CMSA (DCA-) in the Caco-2/HT29 cell layer, as quantified by the flow cytometry of fluorescence-tagged pDNA-Cy5.5 and siRNA-FITC (n=3). \u003cstrong\u003eb,\u003c/strong\u003e In vitro\u003cem\u003e \u003c/em\u003ecellular uptake of LbL-CMSA and LbL-CMSA (DCA-) in the Caco-2/HT29 cell layer pre-incubated with antibodies against various intestinal receptors (CD36, fatty acid translocase; NPC1L1, cholesterol transporter; SR-B1, scavenger receptor class B type 1; ASBT, apical sodium-dependent bile acid transporter) for 2 h\u003cem\u003e. \u003c/em\u003e\u003cstrong\u003ec, \u003c/strong\u003eIn vitro cellular uptake of LbL-CMSA in the Caco-2/HT29 cell layer pre-incubated with endocytosis inhibitors (amiloride [AM], macropinocytosis inhibitor; chlorpromazine [CPZ], clathrin-mediated endocytosis inhibitor; β-cyclodextrin [β-CD], caveola/lipid raft-mediated endocytosis inhibitor) for 4 h. Mean fluorescence intensity was analyzed via flow cytometry. \u003cstrong\u003ed,\u003c/strong\u003e Confocal laser microscopy images of Caco-2 cells stained with an anti-ASBT antibody (yellow) and plasma membrane (red). pDNA and siRNA were labeled with FITC (green), and their signals were colocalized with ASBT (yellow). Scale bar = 100 μm. \u003cstrong\u003ee, \u003c/strong\u003eConfocal laser microscopy images of Caco-2 cells. Lysosomes are stained in red. Scale bar = 100 μm. \u003cstrong\u003ef, \u003c/strong\u003eEx vivo\u003cstrong\u003e \u003c/strong\u003efluorescence imaging of the small intestines 4 h after the oral administration of LbL-CMSA and LbL-CMSA (DCA-). \u003cstrong\u003eg, \u003c/strong\u003eFluorescence intensity of pDNA and siRNA in mouse feces after the oral administration of LbL-CMSA and LbL-CMSA (DCA-). \u003cstrong\u003eh, \u003c/strong\u003eFluorescence imaging of the cryo-block of the small intestine 4 h after the oral administration of LbL-CMSA and LbL-CMSA (DCA-). Data are presented as means ± SD, and p-values were calculated through one-way ANOVA with Tukey’s post-test. ns = not significant, * \u003cem\u003eP \u0026lt; \u003c/em\u003e0.0332, **\u003cem\u003e P \u0026lt; \u003c/em\u003e0.0021, *** \u003cem\u003eP \u0026lt; \u003c/em\u003e0.0002, ****\u003cem\u003e P \u0026lt; \u003c/em\u003e0.0001\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8523305/v1/ae1bfc3bdb2bd026f5abab3f.jpg"},{"id":101190312,"identity":"e934872d-d70c-4232-b84d-0be1af337ddb","added_by":"auto","created_at":"2026-01-27 06:58:01","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":4384885,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eChylomicron-mimicking lymphatic absorption of LbL-CMSA after layer shedding. a,\u003c/strong\u003eTransepithelial/transendothelial electrical resistance (TEER) measurement after treating a Caco-2/HT29 cell layer with LbL-CMSA and LbL-CMSA (DCA-). \u003cstrong\u003eb, \u003c/strong\u003eExperimental design of a Transwell system with a Caco-2/HT29 cell layer in the upper chamber and 3T3-L1 in the bottom chamber. Lipoprotein lipase was added to the uncoated apolipoprotein shell of aLPP to release the pDNA/PBP9R peptoplex. \u003cstrong\u003ec, \u003c/strong\u003eFlow cytometry analysis of LbL-CMSA after the treatment with endocytosis and transcytosis inhibitors in a Transwell system. After pre-incubation with endocytosis inhibitors (amiloride [AM], chlorpromazine [CPZ], β-cyclodextrin [β-CD]) and transcytosis inhibitors (brefeldin A [BFA], monensin [MON]), LbL-CMSA (pDNA-FITC and siRNA-Cy5.5) was treated. \u003cstrong\u003ed\u003c/strong\u003e, Fluorescence intensity of DiO labeled-aLPP, DiL-labeled DCA shell, and dual-labeled LbL-CMSA. \u003cstrong\u003ee\u003c/strong\u003e, FRET intensity levels were analyzed by Flow cytometry after treatment of dual-labeled LbL-CMSA in enterocytes. Quantification of FRET efficiency in enterocytes from in vitro assays showing progressive decline in sensitized emission and reciprocal increase in donor only signal. \u003cstrong\u003ef\u003c/strong\u003e, CLSM images of caco-2 cells after treatment of and dual-labeled LbL-CMSA. FRET signals were diminished as time elapsed and donor signals were increased. \u003cstrong\u003eg, \u003c/strong\u003eRepresentative ex vivo confocal images of small intestinal mucosa at 1,2 and 4 hours after oral dosing showing decline in tissue level FRET signal. in vivo FRET results represent sequential uncoating and lymphatic transport of core material. \u003cstrong\u003eh, \u003c/strong\u003eConfocal laser microscopy images of Caco-2 cells stained with a Golgi-staining dye. pDNA or siRNA labeled with FITC (green) were used to track intracellular location. \u003cstrong\u003ei, \u003c/strong\u003eConfocal laser microscopy images of Caco-2 cells stained with an ER-staining dye. \u003cstrong\u003ej,\u003c/strong\u003e Luminescence of 3T3-L1 in the bottom chamber of a Transwell plate treated with LbL-CMSA and LbL-CMSA (ApoL-) encapsulated with luciferase-expressing plasmid DNA (pLuci).\u003cstrong\u003e k, l, \u003c/strong\u003eFluorescence imaging (k), and mean fluorescence intensity (l) of organs after oral administration of LbL-CMSA and LbL-CMSA (ApoL-) in mice. \u003cstrong\u003em,\u003c/strong\u003e Fluorescence imaging of small intestines and lymph nodes after oral administration of LbL-CMSA. Data are presented as means ± SD, and p-values were calculated by one-way ANOVA with Tukey's post-test. ns = not significant, * \u003cem\u003eP \u0026lt; \u003c/em\u003e0.0332, **\u003cem\u003e P \u0026lt; \u003c/em\u003e0.0021, *** \u003cem\u003eP \u0026lt; \u003c/em\u003e0.0002, ****\u003cem\u003e P \u0026lt; \u003c/em\u003e0.0001.\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8523305/v1/c883dc3f81010e98a23271ce.jpg"},{"id":101190315,"identity":"006d9429-2a48-4c3f-a1c7-40c751311c51","added_by":"auto","created_at":"2026-01-27 06:58:02","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":3625187,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCompartment-specific multi-gene delivery in obesity model. a, \u003c/strong\u003eFABP2 silencing mechanism of LbL-CMSA in enterocytes. \u003cstrong\u003eb, \u003c/strong\u003eRelative mRNA expression levels of FABP2 and lipid metabolism-related biomarkers (DGAT1, triglyceride synthesis marker; ATGL, triglyceride hydrolysis marker; MGL, lipolysis marker) in Caco-2/HT29 cells of a Transwell system with enterocytes in the upper chamber/adipocytes in the bottom chamber. \u003cstrong\u003ec, \u003c/strong\u003eReduction of palmitic acid uptake into enterocytes following 24 h pre-treatment with LbL-CMSA. \u003cstrong\u003ed, \u003c/strong\u003eFABP4/5 silencing mechanism of LbL-CMSA in adipocytes. \u003cstrong\u003ee,\u003c/strong\u003eRelative mRNA expression levels of FABP4, FABP5, and a brown adipogenesis marker (PPARγ, PGC1α, PRDM16, and UCP-1) in 3T3-L1 cells of a Transwell system with enterocytes in the upper chamber/adipocytes in the bottom chamber. \u003cstrong\u003ef, \u003c/strong\u003eReduction of intracellular TG and FFA levels in 3T3-L1 cells after 24 h of pre-treatment with LbL-CMSA. \u003cstrong\u003eg, \u003c/strong\u003eBiodistribution kinetics of sh(FABP4/5) and siFABP2 analyzed via RT-qPCR after the oral administration of LbL-CMSA. \u003cstrong\u003eh, i, \u003c/strong\u003eFluorescence images of the small intestines and epiWAT after the oral administration of LbL-CMSA (sh(FABP4/5)-Cy5.5 and siFABP2-FITC). \u003cstrong\u003ej,\u003c/strong\u003e in vivo pharmacologic perturbation of the proposed translocation pathway. Mice were pretreated with SC-435 (ASBT inhibitor), Pluronic L-81 (chylomicron secretion inhibitor), Tyloxapol (LPL inhibitor) prior to oral dosing of LbL-CMSA. Tissues were harvested at 8 hours for qPCR analysis. Organ level qPCR for delivered sequence demonstrating altered distribution patterns after pathway inhibition. Data are presented as means ± SD, and p-values were calculated by one-way ANOVA with Tukey’s post-test. ns = not significant, * \u003cem\u003eP \u0026lt; \u003c/em\u003e0.0332, **\u003cem\u003e P \u0026lt; \u003c/em\u003e0.0021, *** \u003cem\u003eP \u0026lt; \u003c/em\u003e0.0002, ****\u003cem\u003eP \u0026lt; \u003c/em\u003e0.0001.\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8523305/v1/4bb4730a46742d48867ff0d9.jpg"},{"id":101190178,"identity":"b1bfe88c-7061-4efb-90e5-444010af0452","added_by":"auto","created_at":"2026-01-27 06:57:42","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":3655511,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eReprogramming of lipid metabolism in gut-adipose tissue axis. a,\u003c/strong\u003e Experimental protocols for body weight and insulin resistance tests (n=5). \u003cstrong\u003eb, \u003c/strong\u003eBody weight was monitored for 10 weeks of the 6-week treatment and additional 4-week post-treatment follow-up periods. \u003cstrong\u003ec,\u003c/strong\u003e Insulin resistance tests were conducted following an intraperitoneal injection of insulin into the fasted mice treated for 6 weeks. \u003cstrong\u003ed,\u003c/strong\u003e Amounts of HFD were measured every other day and normalized with the sum of the body weights of each group.\u003cstrong\u003e e, \u003c/strong\u003eFeces from each group were collected daily and homogenized in PBS for analysis with a free fatty acid detection kit. Each point represents the average amount of FFA in the feces collected in a week. \u003cstrong\u003ef,\u003c/strong\u003e Relative mRNA expression levels of FABP2 and lipid metabolism-related genes (DGAT1, ATGL, and MGL) in the small intestine. The mRNA levels measured by qPCR were normalized to that of GAPDH mRNA. \u003cstrong\u003eg,\u003c/strong\u003e Relative mRNA expression levels of FABP4, FABP5, brown adipogenesis markers (PRDM16, PPARγ, and PGC1α), and a mitochondrial biogenesis marker (UCP1) in the visceral adipose tissue. The mRNA levels were normalized to that of GAPDH mRNA. \u003cstrong\u003eh,\u003c/strong\u003e Immunofluorescence staining of FABP2 and DGAT1 in the small intestine, and FABP4, FABP5, and UCP1 in the visceral adipose tissue embedded in paraffin. Section thickness = 6 μm. Scale bar = 200 μm. \u003cstrong\u003ei,\u003c/strong\u003e Levels of free fatty acids and triglyceride in the serum, epiWAT, liver, and small intestine. \u003cstrong\u003ej,\u003c/strong\u003eLevels of ALT and AST in serum were analyzed by ELISA. Data are presented as means ± SD and p-values were calculated by one-way ANOVA with Tukey’s post-test. ns = not significant, * \u003cem\u003eP \u0026lt; \u003c/em\u003e0.0332, **\u003cem\u003e P \u0026lt; \u003c/em\u003e0.0021, *** \u003cem\u003eP \u0026lt; \u003c/em\u003e0.0002, ****\u003cem\u003e P \u0026lt; \u003c/em\u003e0.0001.\u003c/p\u003e","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8523305/v1/e60f5ac9956a38a69b6e62cc.jpg"},{"id":101190394,"identity":"2bfdaeaf-ee20-4914-b56a-32e3878db262","added_by":"auto","created_at":"2026-01-27 06:58:13","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":3091714,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRecovery of hepatic steatosis and inflammation, and long-term storability. a,\u003c/strong\u003e Representative image of H\u0026amp;E staining and picrosirius staining of the liver embedded in paraffin. Section thickness = 6 μm. Scale bar = 200μm. \u003cstrong\u003eb, \u003c/strong\u003eHydroxyproline and TGF-β levels in the liver were quantified. \u003cstrong\u003ec, d, e, f, \u003c/strong\u003eLevels of inflammatory cytokines (TNF-α, IL-1β, IL-6, and CCL2) in the serum (a), liver (b), epiWAT (c), and small intestines (d) measured by ELISA. \u003cstrong\u003eg,\u003c/strong\u003e Z-average (d.nm) and zeta potential (mV) of LbL-CMSA after long-term storage for 4 weeks at 4°C.\u003cstrong\u003e h, \u003c/strong\u003eEncapsulation efficiency and drug loading of LbL-CMSA after long-term storage for 4 weeks at 4°C. \u003cstrong\u003ei, \u003c/strong\u003eComparison of freshly prepared LbL-CMSA and LbL-CMSA stored for 4 weeks in terms of the effectiveness of body weight decrease and recovery of insulin resistance (n=5). Data are presented as means ± SD, and p-values were calculated by one-way ANOVA with Tukey's post-test. ns = not significant, * \u003cem\u003eP \u0026lt; \u003c/em\u003e0.0332, **\u003cem\u003e P \u0026lt; \u003c/em\u003e0.0021, *** \u003cem\u003eP \u0026lt; \u003c/em\u003e0.0002, ****\u003cem\u003eP \u0026lt; \u003c/em\u003e0.0001.\u003c/p\u003e","description":"","filename":"Figure6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8523305/v1/774f5708cd7afe75026d1835.jpg"},{"id":105034728,"identity":"b75ae75d-93da-467e-9554-07475747c987","added_by":"auto","created_at":"2026-03-20 07:23:57","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":22320743,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8523305/v1/c7245d43-5518-431a-83c4-fb631efef923.pdf"},{"id":101190347,"identity":"7c57401b-ee14-4c68-a46a-30259a6a4f90","added_by":"auto","created_at":"2026-01-27 06:58:03","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":27459314,"visible":true,"origin":"","legend":"Supplementary Figures and Note","description":"","filename":"SupportingInformationNat.Mat.docx","url":"https://assets-eu.researchsquare.com/files/rs-8523305/v1/b9b618d192f59b83cbb410d4.docx"},{"id":101190230,"identity":"9e4163fc-cee9-464a-967a-c6ce81f233d9","added_by":"auto","created_at":"2026-01-27 06:57:54","extension":"jpg","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":4657646,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExtended Data Fig. 1:\u003c/strong\u003e \u003cstrong\u003eRestoration of leaky gut and fibrotic liver in a MASLD model.\u003c/strong\u003e \u003cstrong\u003ea,\u003c/strong\u003eSchematic illustration of LbL-CMSA in MASLD model. High fat and high fructose feeding increases intestinal permeability and promotes hepatic exposure to intestinal inflammatory mediators. The LbL-CMSA platform delivers siMLCK1 to strengthen tight junctions and pHO-1 to metabolic tissues, thereby reducing luminal translocation of inflammatory factors and providing anti-inflammatory protection at tissue level. \u003cstrong\u003eb,\u003c/strong\u003e Quantification of RNA expression in Transwell Caco-2/HT29 monolayers following apical application of LPS to induce tight junction loss. LPS were incubated for 24 hours and LbL-CMSA were treated for next 24 hours. \u003cstrong\u003ec\u003c/strong\u003e, Apical to basolateral passage of lipopolysaccharide (LPS) across Transwell monolayers. LPS in the basolateral medium was quantified by LAL assay. \u003cstrong\u003ed,\u003c/strong\u003e Quantification of RNA expression in LPS induced fibrotic HepG2 cells.\u003cstrong\u003e e,\u003c/strong\u003e Systemic metabolic outcomes including body weight, food uptake, and fasting blood glucose (n=5). \u003cstrong\u003ef,\u003c/strong\u003e Quantification of intestinal barrier permeability test after oral administration of FITC-dextran. \u003cstrong\u003eg,\u003c/strong\u003e gene expression for tight junction, barrier-associated markers, and inflammation in small intestine. \u003cstrong\u003eh,\u003c/strong\u003e PS binding protein (LBP) and soluble CD4 (sCD4) and D-lactate concentrations in blood levels to measure intestinal barrier function. \u003cstrong\u003ei,\u003c/strong\u003e Representative images of liver histological images demonstrating reduction of fibrosis after treatment. \u003cstrong\u003ej,\u003c/strong\u003e gene expression level covering steatosis, inflammation, antioxidants, and fibrosis pathways. \u003cstrong\u003ek,\u003c/strong\u003efibrogenic hydroxyproline level in liver. \u003cstrong\u003el,\u003c/strong\u003e hepatic injury markers (ALT, AST, and ASP) in serum. \u003cstrong\u003em,\u003c/strong\u003e Quantification of populations in hepatic macrophages. \u003cstrong\u003en,\u003c/strong\u003e anti-ROS enzymatic activity marker in liver. \u003cstrong\u003eo,\u003c/strong\u003e Quantification of inflammatory and fibrogenic cytokines in serum. \u003cstrong\u003ep,\u003c/strong\u003e Serum lipid levels in serum. \u003cstrong\u003eq,\u003c/strong\u003e gene expression level covering brown-adipogenesis and inflammation marker in WAT. \u003cstrong\u003er,\u003c/strong\u003e Quantification of adipokines in WAT. Data are presented as means ± SD, and p-values were calculated by one-way ANOVA with Tukey's post-test. ns = not significant, * P \u0026lt; 0.0332, ** P \u0026lt; 0.0021, *** P \u0026lt; 0.0002, **** P \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"ExtendedDataFigure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8523305/v1/a28f2458ba80b235971dd746.jpg"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Oral Spatiotemporal Nanoassembly for Sequential Genetic Reprogramming from Gut to Periphery Organ","fulltext":[{"header":"Main ","content":"\u003cp\u003eCross-environmental functionality is a design criterion of biomaterials delivering oral nucleic-acid, because the efficacy hinges on seamless performance from the small intestine to downstream peripheral tissues \u003csup\u003e1-5\u003c/sup\u003e. Even its difficulties, oral gene therapy is an attractive approach for chronic metabolic diseases, offering patient-friendly dosing, molecular specificity, and the ability to modulate the gut environment via gut\u0026ndash;liver and gut\u0026ndash;adipose axes\u003csup\u003e2-4,6-9\u003c/sup\u003e. However, the gastrointestinal tract presents major delivery barriers such as enzymatic degradation, acidic pH, and mucus that limit nucleic-acid stability and absorption \u003csup\u003e10-13\u003c/sup\u003e. In parallel, hepatic first-pass metabolism or hepatic accumulation by protein corona markedly reduces the bioavailability of orally administered nucleic acids limiting efficacy \u003csup\u003e6,13-17\u003c/sup\u003e. To address these problems, several layer-by-layer constructs were challenged, but they only function primarily as multicoated barrier shields or single-target carriers, lacking post-epithelial function and multiple organ-specific drug release \u003csup\u003e4, 5, 6, 7\u003c/sup\u003e. However, since the metabolic disease represents coordinated dysfunction of major organs, multiple reprogramming across metabolic tissues is necessary to outperform single-site reprogramming\u003csup\u003e18\u003c/sup\u003e. More importantly, conventional trials treat the gut merely as a passive barrier to traverse, ignoring its biological role as the primary gatekeeper for systemic lipid flux and inflammatory signaling which are prerequisites for metabolic homeostasis\u0026nbsp;\u003csup\u003e19-21\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe next frontier in the oral nucleic-acid therapeutics R\u0026amp;D lies in transitioning from simple nucleic acid delivery to engineer oral biomaterials coordinating modulation of multi-organ\u003csup\u003e22,23\u003c/sup\u003e.\u0026nbsp;This requires dynamic layer-shedding kinetics capable of sequential cargo delivery across interconnected organ axes, a strategy that remains\u0026nbsp;an uncharted territory in oral therapeutics. We hypothesized that a platform with programmed hierarchical disassembly in a different physiological environment could orchestrate the transition from luminal protection to transport competence, thereby guiding biodistribution toward both intestinal and distal metabolic niches. To implement this approach, the endogenous pathway of dietary lipid absorption was utilized as a foundational model. In physiology, bile salts micellize dietary lipids to facilitate uptake in enterocyte, and absorbed lipids are packaged into nascent chylomicrons that translocate via the ER/Golgi apparatus to enter the lymphatic network, avoiding portal vein draining\u0026nbsp;\u003csup\u003e24\u003c/sup\u003e. Once intravascular, lipolytic processing by lipoprotein lipase (LPL) at the vascular endothelium remodels these particles, enabling substrate delivery to peripheral tissues\u0026nbsp;\u003csup\u003e25\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eInspired by this design logic, we developed Layer-by-Layer Chylomicron-Mimicking Self-Assembly (LbL-CMSA), a spatiotemporally programmed platform that integrates oral robustness, enterocyte transcytosis, lymphatic routing for sequential genetic reprogramming (Fig. 1a). LbL-CMSA comprises (i) an outer deoxycholic acid (DCA) shell that stabilizes the assembly and facilitates ASBT-mediated enterocyte entry, (ii) an intermediate siRNA layer designed to dissociate early for local intestinal reprogramming, and (iii) an inner apolipoprotein-coated lipopeptoplex (aLPP) core engineered for lymphatic export (Fig. 1b). Structural validation confirmed that the DCA shell preserved dimensional integrity, effectively suppressing premature burst release under simulated gastrointestinal conditions\u003csup\u003e24,26-30\u003c/sup\u003e.\u0026nbsp;Mechanistic studies\u0026nbsp;identified ASBT-mediated uptake followed by caveola-mediated endocytosis as the predominant entry route, with FRET imaging and inhibition assay confirming ER/Golgi-coupled transcytosis and subsequent apolipoprotein-dependent lymphatic routing.\u0026nbsp;By enriching the aLPP core with a ApoA-IV to bias cargo routing into the lacteal network and incorporating ApoC-II as a vascular cofactor to trigger lipoprotein lipase (LPL)-mediated uncoating, LbL-CMSA achieved a 5-fold increase in mesenteric lymph node accumulation while effectively bypassing ApoE-driven hepatic redirection\u0026nbsp;\u003csup\u003e31-35\u003c/sup\u003e. Furthermore, LPL-mediated uncoating of peptoplex ensured therapeutic gene delivery to distal metabolic tissues via a receptor-targeting peptide fused to nona-arginine (9R).\u003c/p\u003e\n\u003cp\u003eThis programmed cascade enabled spatiotemporal separation of intestinal and peripheral programs, facilitated targeted silencing fatty acid binding proteins (FABPs) in gut-adipose tissue axis in obesity models. siFABP2 localized in the gut to attenuate lipid efflux, while the sh(FABP4/5) was delivered to adipocytes via 9R based targeting peptide (PBP9R, Prohibitin Binding Peptide 9R), triggering a brown-adipogenic shift \u003csup\u003e36-40\u003c/sup\u003e. This dual-silencing effects co-tune lipid metabolism and elicits robust metabolic benefits in body weight, insulin resistance, hyperlipidemia than single action components. Furthermore, the modularity of LbL-CMSA was demonstrated in fibrotic liver models by reconfiguring the payloads to interrupt the pathogenic gut\u0026ndash;liver axis. By delivering siRNA against myosin light chain kinase 1 (MLCK1), a key regulator of paracellular permeability, intestinal tight junction recovery block endotoxin translocation to liver. Simultaneously, the delivery of a plasmid encoding heme oxygenase-1 (HO-1), a potent antioxidant and cytoprotective enzyme, dampened hepatic oxidative stress and TGF-\u0026beta;\u0026ndash;mediated fibrogenic signaling synergistically. Collectively, these results establish LbL-CMSA as a programmable oral platform that coordinates gut-to-periphery genetic interventions by aligning nanoplatform kinetics with multi-compartment physiology.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cbr\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDesign principles of LbL-CMSA\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe designed LbL-CMSA as a multi-stage vehicle that integrates gastrointestinal stability with the kinetic flexibility required for multi-compartment genetic interventions. Following the methods, LbL-CMSA was fabricated via stepwise self-assembly, yielding a double-layered liposomal morphology with a size of ~300 nm (Fig. 1c and Supplementary Fig. 1). Comparative studies with or without the DCA shell showed that colloidal stability and release behavior was markedly differed pH- and enzyme-dependently. DCA-coated LbL-CMSA retained dimensional integrity and a stable zeta potential, whereas LbL-CMSA (DCA\u0026minus;) exhibited pronounced size expansion and zeta-potential neutralization (Figs. 1d\u0026ndash;f and Supplementary Figs. 1\u0026ndash;2). Consistently, FT-IR analysis before and after FaSSGF/FaSSIF incubation confirmed preservation of multilayer signatures (C\u0026ndash;H stretches ~2850\u0026ndash;2920 cm⁻\u0026sup1;, amide I/II ~1650/1540 cm⁻\u0026sup1;, phosphate/C\u0026ndash;O vibrations ~1200\u0026ndash;1000 cm⁻\u0026sup1;), with only minor band shifts and modest intensity changes (Supplementary Fig.3). PCR-based release assays further showed that LbL-CMSA suppressed the initial burst and enabled sustained release of both pDNA and siRNA across an acidic-to-basic pH range, relative to LbL-CMSA (DCA\u0026minus;) (Fig. 1g and Supplementary Figs. 3\u0026ndash;4). Similar stabilization was observed in enzyme-rich FaSSGF/FaSSIF solutions, where LbL-CMSA reduced early leakage and maintained sustained release for both cargos compared with the DCA\u0026minus; control (Fig. 1h and Supplementary Figs. 4\u0026ndash;5).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEndocytosis and transport mechanism investigation\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBeyond gastrointestinal protection, whether LbL-CMSA actively targets enterocytes via DCA-dependent way were measured using fluorescence-labelled pDNA and siRNA. After 4 h incubation under harsh conditions, cellular uptake was quantified in a differentiated Caco-2/HT29 mucus-forming epithelial layer (Fig. 2a). LbL-CMSA showed higher delivery efficacy of both genetic cargos in simulated gastrointestinal fluids, consistent with improved mucosal translocation, whereas DCA-deficient particles were trapped within the mucus barrier (Fig. 2a). To identify the dominant receptor involved in endocytosis, we assessed SR-B1, CD36 and ASBT which mediate dietary lipid and bile-acid absorption\u003csup\u003e41\u003c/sup\u003e.\u0026nbsp;Receptor pre-blocking with antibody revealed ASBT as a key mediator of LbL-CMSA internalization, significantly decreasing the mean fluorescence intensity of pDNA and siRNA upon inhibition (Fig. 2b). Pharmacological inhibition of endocytic routes further indicated a primary role for caveola-mediated endocytosis, as\u0026nbsp;\u0026beta;-cyclodextrin\u0026nbsp;most effectively suppressed uptake (Fig. 2c). Confocal imaging confirmed colocalization of surface ASBT with LbL-CMSA, supporting an ASBT engagement\u0026ndash;driven endocytosis (Fig. 2d). After internalization, both pDNA and siRNA exhibited reduced colocalization with late endosomes/lysosomes, indicating cytosolic release with lysosomal bypass (Fig. 2e and Supplementary Figs. 6\u0026ndash;8). Together, these data define ASBT binding followed by caveola-mediated endocytosis as the predominant enterocyte entry mechanism for LbL-CMSA.\u003c/p\u003e\n\u003cp\u003eThe fluorescence imaging of the whole GI tracts after oral administration in C57BL/6 mice showed uniform distribution of pDNA and siRNA signals throughout the small intestine with LbL-CMSA, consistent with efficient enterocyte absorption (Fig. 2f and Supplementary Fig. 9). Fluorescence quantification of homogenized fecal samples collected over time revealed higher signal in the LbL-CMSA (DCA\u0026minus;) group, indicating increased loss of unabsorbed or degraded nanoparticles relative to LbL-CMSA (Fig. 2g). Fluorescence imaging of cryosection further confirmed retention and localization of LbL-CMSA signals within intestinal tissue (Fig. 2h). Collectively, the DCA shell provides a dual function of stabilizing the multilayer assembly during GI transit and enabling ASBT-driven epithelial uptake, thereby increasing intestinal delivery while reducing fecal loss compared with DCA-deficient formulations.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTranscytosis mechanism after spatiotemporal disassembly\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe post-entry trafficking of LbL-CMSA within enterocytes were further investigated using a Caco-2/HT29 epithelium, in which paracellular transport is constrained by tight junctions under steady state (Transepithelial/transendothelial electrical resistance (TEER) ~8 \u0026Aring;) \u003csup\u003e42\u003c/sup\u003e. TEER remained unchanged for 24 h for both formulations, indicating that epithelial passage occurs via transcytosis (Fig. 3a). In a Transwell assay, LbL-CMSA produced a marked increase of pDNA\u0026ndash;Cy5.5 in the basolateral compartment after 12 h while siRNA\u0026ndash;FITC remained at basal levels, however, neither genetic cargo increased with LbL-CMSA (DCA\u0026minus;) (Supplementary Fig. 10). These data support a programmed sequence in which siRNA is released and retained within enterocytes, while pDNA loaded in aLPP is preferentially exported via epithelial transcytosis.\u003c/p\u003e\n\u003cp\u003eTo probe downstream transfer and sequential release behavior, indirect co-cultured transwell system were established with multiple cells including differentiated 3T3-L1 in the basolateral chamber as PBP9R target prohibitin on adipocytes (Fig. 3b and Supplementary Figs. 11\u0026ndash;12). LbL-CMSA labeled siRNA-Cy5.5 and pDNA-FITC was administered after the pre-treatment with endocytosis/transcytosis inhibitors (Fig. 3c and Supplementary Figs. 13\u0026ndash;16). After 24 h, pDNA signal decreased within the Caco-2/HT29 layer and appeared in the basal compartment, whereas siRNA signal remained confined to the epithelial monolayer. Inhibiting Golgi\u0026ndash;ER trafficking with Brefeldin A (BFA) or Monensin (MON) significantly trapped the pDNA within the enterocyte layer and prevented exocytosis. This pharmacologically confirms that the aLPP core strictly follows the chylomicron-mimetic secretory pathway via the ER/Golgi apparatus, rather than non-specific transcytosis. Dual-label fluorescence resonance energy transfer (FRET) imaging (DiO labeled inner core and DiL labeled outer shell) further visualized time-dependent layer dissociation (Fig. 3d). Early internalized particles showed high FRET as evidence of intact assemblies, followed by reduced FRET with persistent donor signal in the cytoplasm, indicating outer-shell loss and core exposure (Figs. 3e,f). Intestinal sections after oral dosing mirrored this temporal FRET-to-donor transition, supporting sequential uncoating within enterocytes followed by delivery of the exposed core to downstream compartments (Figs. 3g). Consistently, pDNA colocalized with ER/Golgi markers, whereas siRNA remained cytosolic post-escape (Figs. 3h-i).\u003c/p\u003e\n\u003cp\u003eTo define whether apolipoprotein pre-coating governs ER/Golgi-mediated transcytosis and lymphatic routing, ApoL-deficient LbL-CMSA (ApoL\u0026minus;) were prepared and pDNA was substituted with luciferase (pLuci) for functional quantification in adipocytes (Fig. 3j). ApoL deficiency reduced luciferase expression in 3T3-L1 cells and its effect was amplified under transcytosis inhibition, indicating that the apolipoprotein layer promotes productive transcytosis of the aLPP core (Fig. 3f and Supplementary Fig. 17). This behavior was modulated by lipoprotein lipase (LPL), which facilitated selective lipolytic processing pf apolipoprotein and promoted inner peptoplex release (Supplementary Fig. 18) \u003csup\u003e25\u003c/sup\u003e. In vivo, apolipoprotein pre-coating markedly increased lymphatic delivery as LbL-CMSA showed up to 5-fold higher fluorescence in mesenteric lymph nodes and 4.6-fold higher fluorescence in inguinal nodes compared with LbL-CMSA (ApoL\u0026minus;) (Figs. 3g-i and Supplementary Figs. 19-20).\u003c/p\u003e\n\u003cp\u003eFinally, serum challenge assays indicated that apolipoprotein pre-coating also mitigates deleterious protein corona remodeling (Supplementary Fig. 21). Apolipoprotein pre-coated aLPP retained colloidal properties (hydrodynamic size, polydispersity, and \u0026zeta;-potential) after incubation in human serum, and ELISA results of particle lysates showed preserved ApoA-IV and ApoC-II with minimal acquisition of other apolipoproteins. In contrast, the absence of the pre-coated apolipoprotein led to the acquisition of a heterogeneous corona enriched in ApoE. This suggests that the apolipoprotein coating actively acts as a barrier against opsonization, preventing ApoE-driven hepatic clearance. Accordingly, LbL-CMSA showed prominent pDNA localization in mesenteric lymph nodes with negligible liver signal, whereas LbL-CMSA (ApoL\u0026minus;) preferentially accumulated in the liver (Supplementary Fig. 22). Together, these results support a chylomicron-mimicking, ER/Golgi-coupled transcytosis route that enables lymphatic absorption while resisting ApoE-driven hepatic redirection.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSequential gene delivery from gut to peripheral organ\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFatty acid-binding proteins (FABPs) are central regulators of lipid trafficking and metabolic homeostasis, making them attractive targets for obesity and type 2 diabetes \u003csup\u003e43-45\u003c/sup\u003e. FABP4/5, enriched in adipocytes and macrophages, promotes lipid handling programs linked to inflammation, insulin resistance and obesity-associated complications\u003csup\u003e46-48\u003c/sup\u003e. In parallel, enterocyte FABP2 facilitates intracellular fatty-acid shuttling required for chylomicron assembly and intestinal lipid export \u003csup\u003e49,50\u003c/sup\u003e. We engineered LbL-CMSA to compartmentalize siFABP2 and sh(FABP4/5) into distinct layers, enabling organ-resolved but sequential silencing along the enterocyte\u0026ndash;adipocyte axis (Fig. 4a). In an obese type 2 diabetes model, oral LbL-CMSA was designed to first attenuate intestinal lipid transport via enterocyte delivery of siFABP2, and subsequently reprogram adipose metabolism by directing sh(FABP4/5) to white adipocytes.\u003c/p\u003e\n\u003cp\u003eLbL-CMSA downregulated FABP2 in enterocytes in the transwell model and concomitantly reduced lipid-handling pathways, including DGAT1, ATGL and MGL, resulting in decreased fatty-acid absorption and triglyceride accumulation in enterocytes (Figs. 4b,c). In mature adipocytes (3T3-L1), sh(FABP4/5) efficiently suppressed FABP4 and FABP5 and induced a brown-adipogenic transcriptional shift, as reflected by increased expression of PPAR\u0026gamma;, PGC1\u0026alpha;, PRDM16 and UCP1 (Figs. 4d,e). The upregulation of PPAR\u0026gamma;, PGC1\u0026alpha;, PRDM16, and UCP1 indicates a fundamental reprogramming toward mitochondrial biogenesis and oxidative metabolism, effectively converting energy-storing white adipocytes into energy-expending phenotypes. The dual silencing of FABP4/5 and FABP2 provided a beneficial strategy for orally treating obesity and obesity-related metabolic disorders by reducing fat absorption and triglyceride accumulation and by concomitantly enhancing energy expenditure without toxicity (Fig. 4f and Supplementary Fig. 23).\u003c/p\u003e\n\u003cp\u003eQuantitative analysis of therapeutic genes across major organs after oral dosing showed compartmentalized biodistribution consistent with the programmed cascade. siFABP2 was largely confined to the GI tract, whereas sh(FABP4/5) selectively accumulated in epididymal WAT (epiWAT) and subcutaneous WAT (subWAT) following post-epithelial transport (Fig. 4g). epiWAT exhibited the highest sh(FABP4/5) abundance, consistent with prohibitin enrichment and PBP9R-mediated targeting of the peptoplex core. Ex vivo imaging of dissected GI and adipose tissues further confirmed spatial separation of the two genetic cargos, indicating dissociation of aLPP from the siFABP2 layer and subsequent accumulation of sh(FABP4/5) within epiWAT (Figs. 4h,i and Supplementary Figs. 24\u0026ndash;25). Together, these data establish LbL-CMSA as an orally administered, sequential dual-gene modality that couples enterocyte lipid-uptake suppression with adipose reprogramming.\u003c/p\u003e\n\u003cp\u003eTo validate the proposed translocation cascade under physiological conditions, in vivo pathway perturbation using selective inhibitors was conducted (Fig. 4j). SC-435 reduced genetic payload in GI tracts and lowered distribution in mesenteric lymph nodes and adipose depots, consistent with diminished bile acid\u0026ndash;mediated enterocyte uptake. Pluronic L-81 increased intestinal retention while reducing lymph-node and adipose accumulation, supporting blockade of chylomicron-dependent lymphatic export. Tyloxapol-mediated inhibition of lipoprotein lipase decreased peptoplex core release, reduced adipose accumulation and produced relative increases in hepatic signal. Finally, control adipose depots with naturally low PHB expressions (such as anterior and neck fat) showed negligible payload accumulation despite systemic circulation, confirming that the core delivery is strictly driven by PBP9R-mediated active targeting to PHB-rich adipocytes. Collectively, these pharmacologic perturbations provide in vivo evidence that ASBT-mediated uptake, chylomicron-dependent lymphatic partitioning, and LPL-dependent core processing are sequential, operative steps underpinning LbL-CMSA transport and multi-organ gene delivery.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSequential FABP silencing ameliorates metabolic dysfunction in DIO model\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOral administration of multi-FABP\u0026ndash;silencing LbL-CMSA in a diet-induced obesity (DIO) C57BL/6J model produced robust metabolic benefits over 10 weeks, outperforming single-gene silencing (Figs. 5a\u0026ndash;c). The LbL-CMSA (siFABP2\u0026ndash;shFABP4/5) group achieved a 40.59% body-weight loss at 10 weeks versus PBS, exceeding LbL-CMSA (siFABP2) by 16.19% and LbL-CMSA (shFABP4/5) by 11.19%. Notably, weight reduction was maintained during a subsequent 4-weeks of drug-free follow-up without rebound, in contrast to the rapid regain often observed after cessation of GLP-1 receptor agonist therapy. Functional suppression of intestinal lipid uptake was supported by weekly fecal fatty-acid profiling (Figs. 5d,e). siFABP2 treatment increased fecal free fatty acids by ~7 nmol/kg per week relative to LbL-CMSA (vehicle) and by ~4 nmol/kg per week relative to LbL-CMSA (sh(FABP4/5)), despite no differences in dietary fat intake. Together, these outcomes support a synergistic benefit from sequential oral silencing of FABP2 in enterocytes and FABP4/5 in adipocytes without side effects (Supplementary Fig. 27).\u003c/p\u003e\n\u003cp\u003eMechanistically, analyses of harvested small intestine and visceral adipose tissue confirmed compartment-specific metabolic remodeling. In the intestine, siFABP2-containing formulations downregulated lipid-handling genes, including DGAT1, ATGL and MGL, consistent with reduced fatty-acid processing in enterocytes (Figs. 5f,h). Orthogonal protein quantification by ELISA corroborated as NPC1L1 and CD36 were reduced, indicating diminished long-chain fatty-acid transport capacity, while DGAT1 and SREBP1c abundance decreased, consistent with attenuated triglyceride synthesis and lipogenic signaling (Supplementary Fig. 26). In adipose tissue, sh(FABP4/5) increased expression of brown-adipogenic markers (UCP1, PPAR\u0026gamma;, PRDM16 and PGC1\u0026alpha;), indicating reprogramming toward an energy-expending state (Figs. 5g,h). Consistent with the combined intestinal and adipose remodeing, the sequential dual-gene intervention improved systemic lipid homeostasis, lowering circulating triglycerides and free fatty acids, and alleviated hepatocellular injury as reflected by reduced ALT and AST (Figs. 5i,j).\u003c/p\u003e\n\u003cp\u003eMulti-FABP silencing by oral LbL-CMSA also mitigated liver pathology, consistent with systemic rebalancing of lipid flux and inflammatory tone. Histology and biochemical analysis showed reduced hepatic steatosis and fibrosis, with fewer steatotic hepatocytes, lower collagen deposition, decreased hepatic hydroxyproline and suppressed TGF-\u0026beta; signaling (Figs. 6a,b). In parallel, FABP silencing attenuated inflammatory mediators (TNF-\u0026alpha;, IL-1\u0026beta;, IL-6 and CCL2), supporting efficacy in inflammation-coupled metabolic disease (Figs. 6c\u0026ndash;f). These outcomes are consistent with organ-resolved, sequential gene silencing in which siFABP2 remodels intestinal lipid handling while shFABP4/5 reprograms adipose storage and thermogenesis. While single-gene treatments also showed benefits, the dual-targeting strategy achieved a qualitatively more complete suppression of the metabolic\u0026ndash;inflammatory milieu. In chronic disease contexts, such layered control over both intestinal and adipose pathways is expected to offer superior long-term outcomes and lower relapse risks than single-pathway interventions. For translation, formulation storability is essential for quality control and sustained clinical deployment. LbL-CMSA retained structural stability over 4 weeks, with preserved activity of encapsulated shRNA and siRNA (Fig. 6g). In addition, LbL-CMSA after long-term storage showed therapeutic efficacy in vivo comparable to freshly prepared material (Fig. 6i). By combining spatiotemporal control over lipid metabolism with robust stability, LbL-CMSA emerges as a scalable platform for oral gene therapy. Its unique ability to execute sequential, multi-target interventions positions it as a practical solution for complex metabolic disorders.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eModular reconfiguration of LbL-CMSA for MASLD\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo demonstrate the modularity of platform, we reconfigured LbL-CMSA to interrupt the pathogenic gut\u0026ndash;liver axis in metabolic dysfunction-associated steatotic liver disease (MASLD) by substituting payloads with an intestinal siMLCK1 layer and a systemic pHO-1 core (Supplementary Fig. 28). This dual-targeting strategy was designed to simultaneously repair leaky gut via MLCK1 inhibition and dampen hepatic fibrogenic signaling through HO-1-mediated cytoprotection (Extended Data Fig.1a and Supplementary Note.1). LbL-CMSA restored tight-junction integrity (ZO-1, occludin) in LPS-inflamed Caco-2/HT29 epithelial layer, significantly reducing endotoxin flux (Extended Data Figs. 1b,c). Simultaneously, pHO-1 delivery attenuated inflammatory and fibrotic programs in HepG2 cells and induced brown-adipogenic shifting in 3T3-L1 adipocytes (Extended Data Fig.1d and Supplementary Fig. 29).\u003c/p\u003e\n\u003cp\u003eTo further evaluate in vivo study, a fibrosis-augmented MASLD model was prepared with administration high fructose high fat diet with intraperitoneal CCl₄ injection. Because PHB is overexpressed in damaged liver in MASLD, LbL-CMSA accumulated in the liver and adipose tissues, leading to improved systemic indices and reduced body-weight gain (Extended Data Fig.1e and Supplementary Figs. 30\u0026ndash;31).\u0026nbsp;siMLCK1-mediated barrier reinforcement was confirmed by reduced FITC-dextran leakage and normalized levels of microbial translocation biomarkers including D-lactate, LBP, and sCD14 (Extended Data Figs.1f\u0026ndash;h). This indicates that the reinforced barrier effectively blocked the influx of gut-derived endotoxins, which are key drivers of hepatic inflammation.\u003c/p\u003e\n\u003cp\u003eIn the liver, combination therapy yielded the most significant normalization of hepatic architecture and reduction in fibrotic staining compared to single-payload controls (Fig. 7i). Transcriptomic and biochemical analyses showed broad suppression of lipogenic (Srebp1c), inflammatory (Tnf, Il1b), and fibrogenic (Tgfb1, Col1a1) mediators, alongside reduced hydroxyproline and serum injury markers (ALT, AST) (Extended Data Figs.1j\u0026ndash;l). Flow cytometry further confirmed a shift toward an anti-inflammatory macrophage phenotype and enhanced antioxidant capacity (Extended Data Figs. 1m,n). Finally, WAT exhibited reduced inflammation and increased thermogenic gene expression, normalizing adipokine profiles (Extended Data Figs.1q,r). Collectively, these results establish LbL-CMSA as a programmable oral platform to coordinate multi-axis correction of intestinal dysfunction and peripheral organ pathology.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eLbL-CMSA establishes a conceptually new class of spatiotemporally programmed oral gene-delivery materials in which interface switching confers deterministic post-epithelial routing and site-specific gene modulation, thereby addressing a central bottleneck that has limited oral nucleic-acid therapeutics. By coupling DCA\u0026ndash;guided epithelial entry with chylomicron-mimetic lymphatic export and niche-specific uncoating, the platform enables multi-tissue genetic modulation from a single dose while minimizing hepatic redirection, and its payload- and targeting-swappable design supports rapid reprogramming toward distinct gut\u0026ndash;organ disease axes. By replacing cold-chain-dependent injectables with shelf-stable oral formulations, this platform addresses critical barriers in patient adherence and healthcare accessibility. Moving forward, clinical translation will require addressing scalable manufacturing and biological variability. To bridge the gap from bench to bedside, the adoption of continuous microfluidic assembly will be pivotal in maintaining the intricate layer-by-layer architecture while ensuring high-throughput quality control. With appropriate development of personalized biomarkers, LbL-CMSA could open new avenues for treating neurodegenerative or cardiovascular disorders linked to gut dysbiosis, beyond metabolic syndromes.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003eMaterials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA peptoplex of a therapeutic plasmid DNA and an oligopeptide (PBP9R, C-KGGRAKD-RRRRRRRRR-C) comprising nona-arginine (9R) and a prohibitin-binding peptide (PBP, KGGRAKD) was prepared; then, stepwise thin-film hydration was performed to construct a layer-by-layer chylomicron-mimicking self-assembly (LbL-CMSA) with an apolipoprotein-coated lipopeptoplex (aLPP) and siFABP2 in the middle layer. Negatively charged plasmid DNA was allowed to interact with an oligopeptide containing a positively charged guanidino group via nona-arginine (RRRRRRRRR) and assembled at a weight ratio of 1:3 (plasmid DNA: oligopeptide) with strong genetic material entrapment: an encapsulation efficiency of \u0026gt;95%, as shown in several previous studies. The peptoplex was 182.43 \u0026plusmn; 15.16 nm with a zeta-potential of 23.456 \u0026plusmn; 5.16 mV. These properties confirmed that it was a cationic oligopeptide complexed with a genetic material, and it mediated endosomal escape; consequently, the peptoplex could target specific cells to deliver its genetic materials.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eA chylomicron-mimicking strategy through an apolipoprotein coating was used to enhance the transcytosis of the genetic materials encapsulated in the peptoplex within enterocytes and avoid first-pass metabolism via portal vein drainage. Consequently, aLPP loaded with the peptoplex could effectively deliver genetic materials within the lymphatic circulation instead of releasing it within enterocytes. A thin-film lipid layer was prepared using DSPC:DPPC:DSPE:cholesterol:apolipoprotein (ApoA-IV): apolipoprotein (ApoC-II) at a molar ratio of 0.01:1:1:0.2:0.5:0.5. The lipid mixture was dissolved in chloroform:methanol (2:1, v/v) and evaporated under vacuum in a rotary evaporator at 50\u0026deg;C for 30 min. The resulting thin film was left to dry overnight in a fume hood to remove residual organic solvents. For rehydration, the lipid film was hydrated in distilled water containing the peptoplex at room temperature for 2 h and serially extruded through 0.4 and 0.2 \u0026micro;m pore PVDF membranes to ensure a uniform vesicle size. After thin-film hydration, the Z-average was increased to 223.67 \u0026plusmn; 11.17 nm, which corresponded to an increase of approximately 40 nm from the peptoplex alone, through the serial extrusion of aLPP through 0.4 and 0.2 \u0026mu;m pore PVDF membrane filters. Furthermore, the zeta-potential of aLPP was \u0026minus;23.78 \u0026plusmn; 7.64 mV because the lipid covered the positive charge of the peptoplex. Cryo-TEM images confirmed the unilamellar liposome morphology of aLPP.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eBile salt-based lipid shells (DPPC:DSPC:DPPE:sodium deoxycholate = 1:1:0.2:1 molar ratio) were prepared by dispersing lipids in a chloroform:methanol (2:1, v/v) solution and evaporating it in a rotary evaporator at 50\u0026deg;C for 30 min to deliver aLPP into enterocytes despite the physical and chemical barriers in the GI environment. After the bile salt-based lipid shell layer was prepared, aLPP was initially lyophilized to prevent destabilization during siRNA incorporation and thus spatially separate siFABP2 from aLPP while maximizing the loading efficiency. The lyophilized aLPP was then resuspended in an aqueous solution containing 5 \u0026micro;g of siRNA and incubated at 25\u0026deg;C under gentle agitation for 2 h to facilitate siRNA adsorption onto the lipid bilayer without disrupting the aLPP integrity. The final formulation was extruded through a 0.8 \u0026micro;m PTFE membrane to ensure a uniform double-layer lipid nanoparticle population.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThus, the aLPP/siRNA dual-loaded layer-by-layer chylomicron-mimicking self-assembly (LbL-CMSA) could target enterocytes following the oral administration and release of siFABP2 into the cytosol to downregulate mRNA; then, aLPP was translocated into the lymph nodes, and the peptoplex was released into blood vessels via lipoprotein lipase. The zeta-average diameter of LbL-CMSA was slightly higher (365.15 \u0026plusmn; 34.16 nm), and its zeta-potential was \u0026minus;26.47 \u0026plusmn; 5.73 mV. The efficiencies of sh(FABP4/5) encapsulation at different preparation steps were as follows: 99.99% \u0026plusmn; 1.99% for the peptoplex preparation, 92.27% \u0026plusmn; 2.45% for the aLPP preparation, and 89.00% \u0026plusmn; 3.41% for the final formulation. siFABP2 added to the LbL-CMSA formulation with aLPP was encapsulated at an efficiency of 84.34% \u0026plusmn; 5.16%, indicating that the sequential loading of genetic materials was highly effective.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDrug loading and encapsulation efficiency\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDuring each preparation step of LbL-CMSA containing 10 \u0026mu;g of sh(FABP4/5) and 10 \u0026mu;g of siFABP2, the peptoplex, aLPP, and LbL-CMSA were ultracentrifuged at 50,000 \u0026times; \u003cem\u003eg\u003c/em\u003e for 30 min, and the supernatants were collected to quantify the remnant DNA and siRNA. Plasmid DNA was quantified via Taqman-based real-time PCR with specific primer sequences and probes. siRNA was detected using a siRNA quantitation kit in accordance with the manufacturer\u0026rsquo;s instructions, and synthetic siRNA was quantified with deoxythymidine d(TT) as a 3\u0026prime;-overhang. Drug loading and encapsulation efficiency were calculated using the following equations:\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eDrug loading\u0026thinsp;=\u0026thinsp;\u003cimg width=\"257\" height=\"45\" 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\" alt=\"image\"\u003e\u0026thinsp;\u003c/p\u003e\n\u003cp\u003eEncapsulation efficiency\u0026thinsp;=\u003cimg width=\"340\" height=\"45\" 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\" alt=\"image\"\u003e\u0026thinsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCumulative drug release in gastrointestinal-mimicking environments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor the analysis of drug release kinetics, 10 mg of LbL-CMSA and LbL-CMSA (DCA-) was dispersed in 5 ml of PBS with various pH levels (pH 2, 4, 7, and 8) at 37\u0026deg;C. For the drug release analysis of LbL-CMSA in gastrointestinal-mimicking environments, fasted-state simulated intestinal fluid and fasted-state simulated gastric fluid were purchased from Biorelevant, and 10 mg of LbL-CMSA was incubated in the desired volume. At each time point, LbL-CMSA and LbL-CMSA were collected through ultracentrifugation at 14,000 rpm for 20 min. pDNA and siRNA were isolated using a blood and tissue DNeasy kit to evaluate the therapeutic genes remaining inside the NPs. The isolated pDNA and siRNA were quantified using the methods described for drug loading and encapsulation efficiency.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFT-IR analysis of LbL-CMSA before and after biorelevant media exposure.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo characterize layer integrity and chemical changes associated with DCA coating and biorelevant media challenge, attenuated-total-reflection FT-IR spectra were collected on lyophilized nanoparticle samples as follows. LbL-CMSA were incubated under simulated FaSSGF (pH \u0026asymp; 1.6) and FaSSIF (pH \u0026asymp; 6.5) for 2 h at 37 \u0026deg;C, with gentle orbital agitation (100 rpm) to mimic physiological shear. After incubation, particles were recovered by centrifugation (20,000 \u0026times; g, 30 min), washed twice with deionized water to remove excess buffer, and lyophilized overnight. FT-IR spectra were acquired in ATR mode on a Fourier-transform infrared spectrometer. For each sample, 2 mg of lyophilized material was placed onto the ATR crystal and gently pressed to ensure uniform contact. Spectra were recorded from 4000 to 400 cm⁻\u0026sup1; with a spectral resolution of 4 cm⁻\u0026sup1;, averaging 64 scans per sample. A background spectrum was recorded before each set of measurements and subtracted automatically.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3T3-L1 adipocyte differentiation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e3T3-L1 cells were purchased from ATCC (Virginia, USA). High-glucose DMEM (WelGENE, Seoul, Korea) with 10% FBS and 1% penicillin\u0026ndash;streptomycin (100 U ml\u003csup\u003e-1\u003c/sup\u003e) was used for cell culture. 3T3-L1 cells were incubated at 37\u0026deg;C in 5% CO\u003csub\u003e2\u003c/sub\u003e and passed every other day. For adipocyte differentiation, they were treated with a differentiation medium containing a complete medium, 10 \u0026micro;g/ml insulin, 1 \u0026micro;M dexamethasone, and 0.5 mM IBMX for 72 h. The differentiation medium was replaced with a complete medium containing 10 \u0026micro;g/ml insulin and changed every 2 days.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIn vitro\u0026nbsp;model for intestinal permeability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCaco-2 and HT29 cells were purchased from ATCC (Virginia, USA). High-glucose DMEM (WelGENE, Seoul, Korea) with 10% FBS and 1% penicillin\u0026ndash;streptomycin (100 U ml\u003csup\u003e-1\u003c/sup\u003e) was used for the cell culture. Caco-2 and HT29 cells were seeded at a density of 2.5 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells onto Transwell\u0026reg; polycarbonate filter supports (0.4 \u0026mu;m pore size and 12 mm diameter) at an initial seeding ratio of Caco‐2:HT29 of 9:1. They were cultured for at least 21 days before analysis. The medium was replaced with fresh DMEM every 2 days. Before use, the TEER\u0026nbsp;value (~400 \u0026Omega; \u0026times; cm\u003csup\u003e2\u003c/sup\u003e) was measured to verify the constructed monolayer. The following inhibitors were used to address the endocytosis and transcytosis mechanisms of LbL-CMSA (Table 1).\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eInhibitors\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eM.W.\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eFunctions\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eConcentrations\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003ePreparation\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eChlorpromazine\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e318.86\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eInhibition of clathrin-mediated endocytosis\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e25 \u0026mu;M\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e8 \u0026micro;g/mL (DW)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026beta;-cyclodextrin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1135\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eInhibition of lipid/raft pathway.\u003c/p\u003e\n \u003cp\u003eInhibition of caveolae-mediated endocytosis\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e10 mM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e113 mg/mL (DW)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eAmiloride\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e229.627\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eInhibition of macropinocytosis pathway blocking the Na\u003csup\u003e+\u003c/sup\u003e/H\u003csup\u003e+\u003c/sup\u003e exchanger\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e2.5 mM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e574 \u0026micro;g/mL (DW)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eBrefeldin A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e280.36\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eGolgi apparatus/ER-related inhibitor\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e25 mg/mL\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e25 mg/mL (DMSO)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eMonensin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e670.871\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eGolgi apparatus-related inhibitor\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e33 mg/mL\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e33 mg/mL (DMSO)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1. Endocytosis and transcytosis inhibitors used for mechanistic studies of LbL-CMSA transport.\u003c/strong\u003e A panel of pharmacological inhibitors was employed to delineate the cellular pathways involved in LbL-CMSA uptake and transcytosis across Caco-2/HT29 intestinal epithelial monolayers.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDual-label preparation and characterization.\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eDual-label LbL-CMSA particles were prepared by incorporating the donor fluorophore DiO into the inner, apolipoprotein-mimetic core and by labeling the outer deoxycholic acid (DCA) shell with the acceptor fluorophore DiL. DiO (donor) was premixed with core lipid components during thin-film formation at a final dye-to-lipid molar ratio of 0.2 % and the core was formed as described in Methods. The outer DCA shell was labeled post-assembly by incubating particles with DiL (acceptor) in ethanol:water (1:9 v/v) followed by purification by centrifugation (20,000 \u0026times; g, 20 min) and three washes in PBS to remove unbound dye. Fluorescence spectra were recorded to confirm donor and acceptor peaks and to verify FRET in assembled particles.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHigh-fat diet-induced type 2 diabetes mouse model\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll experimental procedures were approved by the Institutional Animal Care and Use Committee at Hanyang University (2020-0036). Six-week-old male C57BL/6J mice (Orient Bio) were randomly assigned to different treatment groups (\u003cem\u003en\u003c/em\u003e = 5 per group). They were fed with normal chow (Orient Bio) for the first 2 weeks and with diet mixed with 60% of calories from fat (high-fat diet, HFD, Central Lab Animal, Inc.) for the succeeding weeks to induce obesity and metabolic syndrome. The proportion of HFD in the total rodent diet gradually increased for 6 weeks; then, the mice were fed with only HFD for 8 more weeks. All mice were housed under specific pathogen-free conditions. They became obese and insulin resistant after the 16th week; their body weights and glucose levels were 45\u0026ndash;55 g and \u0026gt;250 mg/dL, respectively. After an HFD-induced type 2 diabetes mouse model was prepared, 0.5 mg/kg of sh(FABP4/5) loaded with LbL-CMSA was administered through oral gavage thrice a week. After 6 weeks of treatment, the mice were observed for 4 more weeks to determine further therapeutic effects and then sacrificed on the 11th week for ex vivo analyses.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEx vivo biodistribution using an in vivo imaging system.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLbL-CMSA loaded with sh(FABP4/5)-FITC and siRNA-Cy5.5 was orally administered to the mice that were then sacrificed at the desired time point. The liver, spleen, kidneys, lungs, heart, visceral adipose tissues, and subcutaneous adipose tissues were harvested. The ex vivo Cy5.5 fluorescence and FITC fluorescence in each organ were detected using a fluorescence in vivo imaging system; the mean fluorescence intensity per tissue area was measured.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEx vivo biodistribution via RT-qPCR\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLbL-CMSA loaded with sh(FABP4/5) and siFABP2 was orally administered to the mice that were then sacrificed at the desired time point. Then, the liver, spleen, kidneys, lungs, heart, visceral adipose tissues, and subcutaneous adipose tissues were harvested. pDNA and siRNA were isolated using a blood and tissue DNeasy kit. Plasmid DNA was quantified using Taqman-based real-time PCR with specific primer sequences and probes. siRNA was detected using a siRNA quantitation kit in accordance with the manufacturer\u0026rsquo;s instructions, and synthetic siRNA was quantified with deoxythymidine d(TT) as a 3\u0026prime;-overhang.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInsulin tolerance test\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter 6 h of fasting, the initial blood glucose level was measured by using an Accu-Chek Active model GC kit (Roche Diagnostics GmbH, USA). Insulin (0.75 unit/kg) was injected intraperitoneally. Blood samples were collected 0, 30, 60, 90, and 120 min post-injection for the insulin tolerance test. Blood glucose levels were measured by placing a small drop of blood on a new test strip and recorded.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEx vivo\u0026nbsp;mRNA analysis\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSamples from the small intestines and visceral white adipose tissues were mechanically minced into small fragments and total RNA was extracted from the cell pellets by using a commercial RNA extraction kit (RNeasy Mini Kit, Qiagen) in accordance with the manufacturer\u0026rsquo;s instructions. Equal amounts of purified RNA were reverse-transcribed into cDNA by using a cDNA synthesis kit (iScript\u0026trade; cDNA Synthesis Kit, BIO-RAD). Relative gene expression levels were quantified via real-time PCR (Applied Biosystems 7500 Real-Time PCR System, USA) with gene-specific primers. GAPDH was used as the internal control, and gene expression was calculated using the \u0026Delta;\u0026Delta;Ct method.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunofluorescence assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAdipose tissues and small intestines were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned at 8 \u0026micro;m thickness. The paraffin sections were deparaffinized and rehydrated through a graded alcohol series. After the antigen was retrieved, the sections were blocked with 5% bovine serum albumin (BSA) in PBS at room temperature for 1 h and incubated with primary antibodies against the target protein at 4\u0026deg;C overnight. On the next day, they were thoroughly washed with PBS and incubated with FITC-conjugated anti-rabbit secondary antibody (Abcam, USA) at room temperature in the dark for 2 h. After the final washes, they were mounted with a Dako fluorescence mounting medium (DAKO, Denmark) and sealed with coverslips. Immunofluorescence images were acquired using an AxioScan.Z1 slide scanner (Zeiss, Germany).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEx vivo analysis of cytokines and lipids\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAt the end of the study, blood samples were collected via cardiac puncture. The plasma was separated by centrifugation at 2,000 \u0026times; \u003cem\u003eg\u003c/em\u003e and 4\u0026deg;C for 10 min and stored at \u0026minus;80\u0026deg;C until analysis. Liver and adipose tissues were surgically harvested, rinsed with cold PBS to remove blood contaminants, blotted dry, snap-frozen in liquid nitrogen, and stored at \u0026minus;80\u0026deg;C. Cholesterol, triglyceride, and free fatty acid (FFA) concentrations in the plasma and tissue homogenates were measured enzymatically by using commercially available kits (Abcam, USA) in accordance with the manufacturer\u0026rsquo;s instructions. For cytokine quantification, tissue samples were homogenized in lysis buffer containing protease inhibitors and centrifuged at 12,000 \u0026times; \u003cem\u003eg\u003c/em\u003e and 4\u0026deg;C for 15 min to obtain the supernatants. The total cytokine concentrations of interleukin-1\u0026beta; (IL-1\u0026beta;), interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-\u0026alpha;), and monocyte chemoattractant protein-1 (MCP-1), transforming growth factor-beta (TGF-\u0026beta;), adiponectin, leptin,\u0026nbsp;resistin, plasminogen activator inhibitor 1 (PAI-1) in the plasma or tissue supernatants were determined using enzyme-linked immunosorbent assay (ELISA) kits (InvivoGen, USA) in accordance with the manufacturer\u0026rsquo;s protocols.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLiver histology and liver panel analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLiver tissues were fixed in 4% neutral buffered formalin and embedded in paraffin. The paraffin sections were deparaffinized, rehydrated in gradual mixtures of ethanol/water, blocked with a 5% BSA blocking solution for 1 h, and incubated with anti-HO-1 antibody overnight. On the next day, they were washed and mounted with a mounting solution containing DAPI. In addition, 6 \u0026mu;m paraffin sections were stained with hematoxylin and eosin, Masson\u0026rsquo;s trichrome, and Sirius red for histological analyses.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMetabolic dysfunction-associated steatotic liver disease (MASLD) model\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSix-week-old Female C57BL/6 mice (Orient Bio), aged 8 weeks were injected intraperitoneally with carbon tetrachloride (CCl\u003csub\u003e4\u003c/sub\u003e) at a dose of 300 \u0026mu;l/kg body weight, dissolved in olive oil, three times a week (total volume of 100 \u0026mu;l per dose) for 6 weeks. To accelerate the induction of a metabolic dysfunction-associated liver fibrosis model, the mice were fed with a 60% Kcal HFD (Central Lab Animal, Inc., Seoul, South Korea). All mice were housed under specific pathogen-free conditions.\u0026nbsp;After an MASLD mouse model was prepared, 0.5 mg/kg of pHO-1 loaded with LbL-CMSA was administered through oral gavage thrice a week. After 4 weeks of treatment, the mice were sacrificed for ex vivo analyses.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eQuantification of leaky gut derivates and hepatic antioxidant enzyme activities\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLBP, sCD14, D-lactate, SOD, and catalase were quantified using commercially available assay kits according to the manufacturers\u0026rsquo; instructions. Circulating LBP and sCD14 were measured in serum \u0026nbsp;by sandwich ELISA, and concentrations were calculated from standard curves with samples diluted as needed to fall within the linear range. D-lactate levels were determined using a D-lactate dehydrogenase\u0026ndash;based enzymatic colorimetric assay, with appropriate sample preparation performed as recommended by the kit. For antioxidant enzyme activities, liver tissues were processed to obtain clarified homogenate supernatants, and SOD activity was assessed using a WST-1/xanthine oxidase\u0026ndash;based inhibition assay, whereas catalase activity was measured using an H₂O₂ decomposition\u0026ndash;based activity assay; activities were normalized to total protein content where applicable. All measurements were performed with technical replicates and included blanks and internal controls for quality assurance.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll in vitro experiments were performed with at least three independent biological replicates to ensure reproducibility. For in vivo studies, a sample size of n = 5 mice per group was used to ensure adequate statistical power and relevance. Data analyses were conducted using GraphPad Prism version 8.0 for Windows (GraphPad Software, California, USA). For group comparisons, one-way ANOVA followed by Tukey\u0026rsquo;s multiple comparison test was performed to identify statistically significant differences among groups. Statistical significance was defined as p \u0026lt; 0.05, with the following notations used to indicate significance levels: ns (not significant), *p \u0026lt; 0.033, **p \u0026lt; 0.01, and ***p \u0026lt; 0.001. The details of data processing procedures and the rationale for sample size determination for each specific analysis were provided in the corresponding figure legends.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe main data supporting the results of this study are available within the paper and Supplementary Information. Source data are provided in this paper.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was partially supported by grants from the National Research Foundation of Korea (NRF‐2019R1A2C3008992, NRF-RS-2023-00208982), the Brain Korea 21 Fostering Outstanding Universities for Research (BK21 FOUR, 5199990514440), the Korean Health Technology R\u0026amp;D Project through the Ministry of Health Industry Development Institute (KHIDI) funded by the Ministry of Health \u0026amp; Welfare, Republic of Korea (HI19C0753), and the Korea Drug Development Fund funded by the Ministry of Science and ICT, Ministry of Trade, Industry, and Energy, and Ministry of Health and Welfare (HN21C0885, Republic of Korea).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003c/strong\u003e\u003cstrong\u003eA\u003c/strong\u003e\u003cstrong\u003euthor\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAffiliations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDepartment of Bioengineering, Institute for Bioengineering and Biopharmaceutical Research Hanyang University, 04763 Seoul, South Korea\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eJuhyeong Hong, Seon-jeong Chang, Seung-Hwan joo, Han-Seok Lim, Kyungdong Kim, Sangjin Kim, Minjoo Jang, Yong-Hee Kim\u003c/p\u003e\n\u003cp\u003eEducation and Research Group for Biopharmaceutical Innovation Leader, Hanyang University, 04763 Seoul, South Korea\u003c/p\u003e\n\u003cp\u003eJuhyeong Hong, Seon-jeong Chang, Seung-Hwan joo, Han-Seok Lim, Kyungdong Kim, Sangjin Kim, Minjoo Jang, Yong-Hee Kim\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eContributions\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eJuhyeong Hong and Yong-Hee Kim designed experimental design and protocol. Juhyeong Hong performed the in vitro\u003cem\u003e,\u0026nbsp;\u003c/em\u003ein vivo\u003cem\u003e,\u0026nbsp;\u003c/em\u003eandex vivo therapeutic assays. Seon-jeong Chang, Seung-Hwan joo, Han-Seok Lim, Juhyun Lee, Kyungdong Kim, Sangjin Kim, and Minjoo Jang supported them in vitro\u003cem\u003e,\u0026nbsp;\u003c/em\u003ein vivo\u003cem\u003e,\u0026nbsp;\u003c/em\u003eandex vivo therapeutic assays. Juhyeong Hong and Yong-Hee Kim discussed the results and wrote the paper.\u003c/p\u003e\n\u003cp\u003eCorresponding Author\u003c/p\u003e\n\u003cp\u003eCorrespondence to Yong-Hee Kim ([email protected])\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eLee, Y.\u003cem\u003e et al.\u003c/em\u003e Hyaluronic acid-bilirubin nanomedicine for targeted modulation of dysregulated intestinal barrier, microbiome and immune responses in colitis. \u003cem\u003eNat Mater\u003c/em\u003e \u003cstrong\u003e19\u003c/strong\u003e, 118-126, doi:10.1038/s41563-019-0462-9 (2020).\u003c/li\u003e\n\u003cli\u003eLamson, N. G., Berger, A., Fein, K. C. \u0026amp; Whitehead, K. A. Anionic nanoparticles enable the oral delivery of proteins by enhancing intestinal permeability. \u003cem\u003eNat Biomed Eng\u003c/em\u003e \u003cstrong\u003e4\u003c/strong\u003e, 84-96, doi:10.1038/s41551-019-0465-5 (2020).\u003c/li\u003e\n\u003cli\u003eHunt, N. 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Nevertheless, the gastrointestinal environment and unpredictable biodistribution limit the efficiency of conventional gene delivery. Here, we develop Layer-by-Layer Chylomicron-Mimicking Self-Assembly (LbL-CMSA), which enables sequential gene reprogramming from the gut to the peripheral organs. Utilizing dynamic layer-shedding kinetics, the outer bile-salt shell peels off in enterocytes to release siRNA for local intestinal reprogramming. Subsequently, the inner lipopeptoplex layer mimics chylomicrons, entering lymphatic trafficking to bypass hepatic first-pass metabolism and deliver genes to distal metabolic organs. In an obesity model, LbL-CMSA down-regulates obesogenic genes in gut–adipose tissues by sequentially blocking intestinal fat influx and adipocyte storage. By modulating genetic cargos, LbL-CMSA attenuates endotoxin translocation across the leaky gut to amplify anti-fibrogenic effects in a steatohepatitis model. Overall, LbL-CMSA shows its potential as a modular nanoplatform for combination gene therapy to overcome complex metabolic disorders efficiently.","manuscriptTitle":"Oral Spatiotemporal Nanoassembly for Sequential Genetic Reprogramming from Gut to Periphery Organ","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-27 06:56:26","doi":"10.21203/rs.3.rs-8523305/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":"5b09ef6e-39f7-4d93-a609-bf87ee20a501","owner":[],"postedDate":"January 27th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":61663308,"name":"Biological sciences/Biotechnology/Biomaterials/Drug delivery"},{"id":61663309,"name":"Biological sciences/Biotechnology/Gene delivery"},{"id":61663310,"name":"Biological sciences/Biotechnology/Nucleic-acid therapeutics"},{"id":61663311,"name":"Biological sciences/Biotechnology/Nanobiotechnology/Nanoparticles"}],"tags":[],"updatedAt":"2026-03-18T16:26:53+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-27 06:56:26","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8523305","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8523305","identity":"rs-8523305","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}