Abstract The clinical application of CRISPR-Cas9 remains limited by delivery challenges, particularly for oral administration. Lysine-specific demethylase 1 (Lsd1) plays a key role in colonic inflammation and tumorigenesis. Here, we developed an oral genome-editing platform (TPGS-RNP@LNP), where Lsd1-targeting ribonucleoproteins (RNPs) were encapsulated in mulberry leaf lipid nanoparticles (LNPs) and formulated with d-α-tocopherol polyethylene glycol succinate (TPGS). TPGS reinforced the lipid bilayer of LNPs, enhanced gastrointestinal stability, and facilitated colonic mucus penetration. Upon the galactose receptor–mediated endocytosis of TPGS-RNP@LNPs by macrophages, their fusion with the endosomal membrane and the presence of nuclear localization signals ensured the nuclear delivery of RNPs. TPGS-RNP@LNPs achieved 59.7% Lsd1 editing efficiency in macrophages, surpassing the commercial CRISPRMAX (43.0%). Oral TPGS-RNP@LNPs promoted H3K4 methylation to modulate epigenetic states, achieving inflammation mitigation, epithelial barrier restoration, and retardation of colitis and its associated tumorigenesis. As an LNP-based oral RNP delivery system, TPGS-RNP@LNPs provide a promising platform for precise treatment of colorectal diseases. __________________________________________________________________ Oral nanomedicines treat colonic diseases through epigenome editing. INTRODUCTION Ulcerative colitis (UC) is a chronic inflammatory disorder of the colon that substantially elevates the risk of colitis-associated colorectal cancer (CAC) ([54]1). Current treatments against colon diseases are largely palliative, and effective targeted therapies remain scarce. CRISPR-Cas9 genome editing offers a precise and versatile therapeutic strategy for addressing inflammatory diseases ([55]2), genetic disorders ([56]3), and various cancers ([57]4). However, its clinical application is hindered by major challenges in achieving efficient in vivo delivery. Among available formats, Cas9 ribonucleoprotein (RNP) complexes have distinct advantages over plasmid DNA– and mRNA-based approaches, including superior editing efficiency, reduced immunogenicity, and lower off-target effects ([58]5, [59]6). Nonetheless, their rapid degradation and poor cellular uptake highlight the requirement of an optimized delivery system. Recently, a variety of nonviral gene carriers have been exploited, with polymeric nanoparticles ([60]7), dendritic polymers ([61]8), and mesoporous silica nanoparticles ([62]9) being extensively used. Among these, lipid nanoparticles (LNPs) have demonstrated remarkable efficacy in facilitating the intracellular delivery of RNP complexes, enabling precise genome editing across diverse tissues, such as the skin, lung, and liver ([63]10–[64]12). However, existing LNP-based RNP delivery systems have been exclusively designed for intravenous injection, which is associated with multiple drawbacks, including patient noncompliance, infection risk, and rapid systemic drug distribution. On the contrary, oral administration represents a superior alternative for treating colonic diseases, providing a noninvasive, cost-effective, and localized approach for delivering therapeutics to the diseased mucosa in the gastrointestinal (GI) tract ([65]13). Despite these advantages, the application of oral LNPs for RNP delivery remains unexplored. Conventional LNP formulations struggle to withstand the harsh GI tract environment, where exposure to gastric acid, digestive enzymes, and gut microbiota leads to their rapid degradation ([66]14). In addition, efficient transport of the loaded drugs across the colonic mucus barrier remains a major hurdle, limiting the therapeutic efficacy of oral nanomedicines. Recent advances have identified edible plant–derived LNPs as a promising alternative because of their exceptional stability in the GI tract and the ability to encapsulate and transport a diverse range of therapeutic agents ([67]15). Building on this concept, our group has successfully used these naturally derived carriers for the delivery of small bioactive molecules ([68]16), carbon dots ([69]17), and magnetic particles ([70]18) via the oral route. To optimize CRISPR-Cas9 applications, it is essential to identify the critical regulatory factor in the development of UC and CAC. Lysine-specific demethylase 1 (Lsd1), the first identified lysine-specific histone demethylase ([71]19), can selectively remove methyl groups from mono- and dimethylated histone H3 at lysine-4 (H3K4) and histone H3 at lysine-9 (H3K9) ([72]20, [73]21), thereby playing a pivotal role in inflammation and oncogenesis ([74]22, [75]23). These properties position Lsd1 as a promising epigenetic regulation target for treating UC and CAC, particularly through CRISPR-Cas9 genome editing. In this study, we presented a laboratory-evolved platform wherein Cas9/single guide RNA (sgRNA) RNPs targeting Lsd1 knockout were encapsulated within LNPs composed of mulberry leaf lipids (MLLs) and the Food and Drug Administration–approved stabilizer [d-α-tocopherol polyethylene glycol succinate (TPGS)] ([76]Fig. 1A). The addition of TPGS significantly reinforced the lipid bilayer of LNPs, facilitating the efficient transport of RNPs through the harsh GI tract and enhancing their ability to penetrate the colonic mucus barrier. Subsequently, TPGS-RNP@LNPs accumulated in inflamed colonic tissues through the epithelial enhanced permeability and retention effect ([77]13, [78]14, [79]24) and specifically targeted macrophages via galactose receptors. Notably, the effective endosomal escape and nuclear delivery of RNPs are achieved via membrane fusion between TPGS@LNPs and endosomes, as well as nuclear localization signals (NLSs) in the Cas9. Eventually, oral TPGS-RNP@LNPs obtained robust genome-editing activity and favorable therapeutic effects against UC and CAC ([80]Fig. 1B). Fig. 1. Schematic illustration of efficient RNP delivery to enable cell-specific genome editing and precise treatment of colonic diseases mediated by TPGS@LNPs. [81]Fig. 1. [82]Open in a new tab (A) Fabrication procedure of TPGS-RNP@LNPs. Created in BioRender. Q. Gao (2025); [83]https://biorender.com/ehl3wlp. (B) Illustration of oral targeted delivery of TPGS-RNP@LNPs mediating specific genome editing in colonic epithelial cells and macrophages for treating UC and CAC. Upon oral administration, TPGS-RNP@LNPs can be efficiently internalized by colonic epithelial cells and preferably target macrophages via galactose receptor–mediated endocytosis following stable traversal of the GI tract and mucus barrier. Subsequently, the RNPs delivered by TPGS-RNP@LNPs are released and translocated into the nucleus through membrane fusion, enabling efficient genome editing. This process enhances H3K4 mono- and dimethylation, which modulates the transcription of multiple anticolitis genes and restores the expression of tight junction– and mucus-associated proteins. Consequently, the damaged intestinal epithelial barrier is repaired, the inflammatory response is mitigated, and inflammation-induced tumor growth is inhibited, eventually achieving the effective treatment of UC and CAC. Created in BioRender. Q. Gao (2025); [84]https://biorender.com/5rwjer6. RESULTS Construction and physicochemical characterization of TPGS-RNP@LNPs Although Lsd1 has been shown to play an important role in the progression of dextran sulfate sodium (DSS)–induced colitis in mice ([85]25), its expression patterns in human UC and CAC samples remain unreported. Accordingly, we benchmarked the expression profiles of LSD1 in the human colonic tissues using immunofluorescence staining ([86]Fig. 2A). In a confocal microscopy assay, the colon tissue sections from patients at different stages of UC and CAC showed overexpressed levels of LSD1 ([87]Fig. 2, B and C), suggesting the critical role of LSD1 in the pathogenesis of colonic diseases. Fig. 2. Expression profiles of LSD1 in the human colonic tissues and physicochemical characterization of various LNPs. [88]Fig. 2. [89]Open in a new tab (A) Schematic illustration of specimen collection, sectioning, immunostaining, and imaging of human colonic tissues. Created in BioRender. Q. Gao (2025); [90]https://biorender.com/zj6xl1h. (B) Fluorescence images and (C) the corresponding quantitative analysis of LSD1 protein in the human colonic tissue sections from patients at different stages of UC and CAC (n = 3). Scale bar, 200 μm. (D) Stabilities of LNPs, TPGS@LNPs, TPGS-RNP@LNPs, Lipo6000-RNP@LNPs, and CMAX-RNP@LNPs in the simulated gastric, small intestinal, and colonic fluids (n = 3). h, hours. (E) Optimization process of dynamics simulation of MLLs with or without the addition of TPGS in an aqueous solvent box. TPGS, MGDG, PG, FA, and OAHFA are represented in green, blue, purple, yellow, and blue-gray, respectively. (F) Root mean square deviation (RMSD; T[1]: equilibrium time for MLLs + TPGS; T[2]: equilibrium time for MLLs; ΔT = T[2] − T[1]), (G) radius of gyration (RG), and (H) surface area (SA) values of MLLs with or without the addition of TPGS. Particle size distribution profiles of (I) TPGS@LNPs and (J) TPGS-RNP@LNPs. (K) Transmission electron microscopy images of TPGS-RNP@LNPs. Scale bar, 100 nm. (L) Schematic diagram illustrating the substantial enhancement in the GI stability of mulberry leaf–derived LNPs after adding TPGS. Created in BioRender. Q. Gao (2025); [91]https://biorender.com/2g70ixm. Data are the means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. Epigenetic dynamics are crucial in identifying the target gene and dictating its knockout by precisely regulating specific transcriptional programs ([92]26). This study synthesized a candidate epigenetic sgRNA targeting the Lsd1 gene in mice, spanning ~100 bp (base pairs) (table S1 and fig. S1A), which was subsequently complexed with Cas9 protein. To preserve Cas9/sgRNA complex (RNP) integrity in the GI tract, we focused on LNPs because of their proven efficacy as RNA delivery carriers in preclinical and human studies ([93]27, [94]28). Our research group has demonstrated that plant-derived LNPs serve as a safe and effective platform for delivering a variety of drugs through the oral route ([95]16, [96]18, [97]29). It was found that mulberry leaf–derived LNPs showed turbulent size variations in the GI environment, indicating their inability to maintain structural stability ([98]Fig. 2D). This instability may be attributed to the failure of these LNPs to reestablish compact lipid structures during the “dissolution and reconstruction” process. Drawing inspiration from using straw to enhance the strength of mud walls ([99]30), TPGS was introduced to the LNPs as a stabilizer. As expected, the incorporation of TPGS significantly improved the GI stability of mulberry leaf–derived LNPs ([100]Fig. 2D). In particular, TPGS@LNPs with a mass ratio of 20:2 between LNPs and TPGS exhibited an optimal excipient ratio, revealing a desirable hydrodynamic particle size of 203.5 nm, a uniform size distribution, and a negative surface charge of −27.1 mV, as well as favorable stability in the GI tract ([101]Fig. 2I and fig. S1, B to E). Encouraged by the phenomenon that the inclusion of TPGS intriguingly improved the stability of LNPs in the GI fluids, we inspected its underlying mechanism by molecular dynamics simulations. It was noticed that TPGS rapidly and intimately interacted with the lipid components of LNPs, forming a stabilizing complex within 100 ns while reducing the equilibration time by 36 ns compared to the naked LNPs (without TPGS) ([102]Fig. 2, E and F). In addition, TPGS@LNPs exhibited significantly higher structural tightness than the naked LNPs ([103]Fig. 2, E, G, and H). These findings explain the sustained stability of TPGS@LNPs in the GI tract. After loading with RNPs, TPGS@LNPs demonstrated notable changes in the particle size (increasing to 253.7 nm) and zeta potential (reducing to −38.0 mV), along with a spherical morphology observed via transmission electron microscopy ([104]Fig. 2, J and K, and fig. S1D). Meanwhile, the addition of TPGS helps maintain the stability of RNPs in the GI tract, whereas the two commercial nucleic acid delivery reagents, Lipo6000 and CRISPRMAX (CMAX), fail to confer GI stability to RNPs ([105]Fig. 2D). In addition, TPGS-RNP@LNPs maintained excellent structural integrity and retained a consistent amount of encapsulated sgRNA after incubation at 37°C for 5 days (table S2 and fig. S1F). Collectively, these results imply the extraordinary potential of TPGS@LNPs as carriers for the oral delivery of RNPs ([106]Fig. 2L). Lsd1 knockout–mediated epigenetic alterations restore the damaged colonic epithelial barrier and mitigate inflammatory responses To examine whether TPGS@LNPs could deliver RNPs into the nucleus, we monitored the intracellular trafficking of their encapsulated enhanced green fluorescent protein (EGFP)–fused Cas9 protein by flow cytometry and confocal laser scanning microscopy (CLSM) analysis. Initially, the nontoxicity of TPGS-RNP@LNPs toward CT-26 cells (a murine colon cancer cell line) and RAW 264.7 macrophages (a murine leukemia monocyte-macrophage cell line) was guaranteed (fig. S2, A and B). Afterward, we observed that TPGS-RNP@LNPs were present in the cytoplasm of both cell lines with 1 hour, as green fluorescence was detectable ([107]Fig. 3A and fig. S2C). Notably, EGFP-fused RNPs were translocated into the nucleus gradually for 5 hours, which could account for the presence of NLSs on Cas9 ([108]10). The endocytosis percentages of TPGS-RNP@LNPs by CT-26 cells and RAW 264.7 macrophages were quantified as 85.9 and 96.1%, and their corresponding mean fluorescence intensities (MFIs) were determined to be 70617.0 and 100494.7, respectively ([109]Fig. 3, B to D, and fig. S2, D and E). The more notable phagocytosis of TPGS-RNP@LNPs by macrophages may be ascribed to the active interaction between galactose group–bearing LNPs and galactose receptors on the surface of macrophages. After pretreatment with free d-galactose for 4 hours, the uptake percentages of TPGS-RNP@LNPs by macrophages were reduced by 35.7, 56.4, and 45.6% at 1, 3, and 5 hours, respectively ([110]Fig. 3D), demonstrating the targeted delivery capability of TPGS-RNP@LNPs to macrophages. Fig. 3. In vitro cell internalization, Lsd1 knockout efficiency, and anti-inflammatory activity of various LNPs. [111]Fig. 3. [112]Open in a new tab (A) Intracellular distribution of TPGS-RNP@LNPs in macrophages after 1, 3, and 5 hours of incubation. Scale bars, 20 μm (small field) and 5 μm (large field). (B) Flow cytometric histograms and (C) MFIs of macrophages treated with TPGS-RNP@LNPs at corresponding time points (n = 3). (D) Cellular uptake of TPGS-RNP@LNPs by macrophages with or without d-galactose at 1, 3, and 5 hours (n = 3). (E) CLSM images of macrophages after incubation with TPGS-RNP@LNPs for 1, 4, and 8 hours and staining with LysoTracker Red. Scale bar, 5 μm. (F to H) Relative expression of Npc1, Lamp1, and Lamp2 of macrophages after 48 hours of treatment with TPGS@LNPs or TPGS-RNP@LNPs (n = 3). (I) T7E1 assay of Lsd1 locus in macrophages following 48 hours of treatment with CMAX and TPGS-RNP@LNPs. (J and K) Western blot and quantifications of H3K4Me1/Me2 in macrophages after 48 hours of incubation with TPGS@LNPs and TPGS-RNP@LNPs, respectively (n = 3). (L and M) TNF-α and IL-10 levels in supernatants of macrophages that received the treatment of various LNPs for 48 hours. Untreated and lipopolysaccharide-treated macrophages served as negative and positive controls, respectively. Both TPGS@LNP and TPGS-RNP@LNP groups were also treated with lipopolysaccharide (500 μl, 0.5 μg/ml) (n = 3). (N) Schematic diagram illustrating the mechanism by which TPGS-RNP@LNPs mediate Lsd1 gene editing via galactose receptor–mediated endocytosis and efficient endosomal escape, thereby enhancing H3K4 mono- and dimethylation levels and eventually achieving inflammation alleviation. Created in BioRender. Q. Gao (2025); [113]https://biorender.com/96n5xz9. Data are the means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001; ns, no significance. RNP escape from the endosome is crucial for achieving nucleus-based gene editing ([114]31). With the prolonged incubation period, the EGFP-fused RNPs gradually dissociated from endosomes in both cell lines, with a colocalization coefficient regression ([115]Fig. 3E and fig. S2F). To elucidate the molecular mechanism by which TPGS@LNPs facilitated the endosomal escape of RNPs, we performed a quantitative reverse transcription polymerase chain reaction analysis, and the corresponding primers are shown in table S3. NPC (Niemann-Pick C), encoded by intracellular cholesterol transporter 1 (Npc1), is a typical protein involved in membrane fusion and endosomal cargo regulation, playing a critical role in endosomal escape ([116]32–[117]34). Npc1 expression levels were significantly increased over 2.0-fold by the treatment of TPGS@LNPs and TPGS-RNP@LNPs ([118]Fig. 3F), whereas the expression levels of Lamp1 and Lamp2, encoding lysosome-associated membrane proteins 1 and 2, in the TPGS@LNP and TPGS-RNP@LNP groups were comparable to that of the control group, implying the integrity of endosomal structures, as there was no involvement in the breakdown and degradation of their membrane proteins ([119]Fig. 3, G and H). Together, these data strongly showcase that TPGS@LNPs can effectively achieve cell internalization, intracellular delivery, and cytosolic release of RNPs through membrane fusion with endosomes. Next, we explored the capacity of LNP-mediated RNP delivery for in vitro gene editing (fig. S2G and table S4). As shown in [120]Fig. 3I, the TPGS-RNP@LNPs achieved a remarkable indel frequency at the Lsd1 locus, demonstrating its highly efficient genome editing capability of up to 59.7% when the sgLsd1 dosage was 5 μg, which was also superior to the equivalent commercial RNP delivery reagent CMAX (43.0%). To investigate the impact of efficient Lsd1 knockout on inducing corresponding epigenetic modifications, we examined the mono- and dimethylation levels of H3K4 in macrophages following the treatment with TPGS-RNP@LNPs. Especially compared with TPGS@LNPs alone, RNP-encapsulated TPGS@LNPs significantly augmented the levels of H3K4 mono- and dimethylation by 2.0 and 1.6 times increases, respectively ([121]Fig. 3, J and K), supporting the hypothesis that the effective knockout of Lsd1 locus could induce epigenetic alterations. At the cellular level, we further focused on the impact of precise editing of the Lsd1 gene in repairing the colonic epithelial barrier and governing inflammatory responses. Our findings revealed that TPGS-RNP@LNPs effectively facilitated the migration of colonic epithelial cells (CT-26 cells) and accelerated the restoration of the damaged colonic epithelial barrier compared to the control group (fig. S2, H and I). These observations suggest that epigenetic changes following Lsd1 knockout have reparative potential for damaged colon mucosa. It is worth noting that the Lsd1-knockout macrophages by TPGS-RNP@LNPs performed a prominent anti-inflammatory feature, as evidenced by the down-regulated expression of the pro-inflammatory cytokine [tumor necrosis factor–α (TNF-α)] from 816.8 to 428.8 pg/ml ([122]Fig. 3L) and the up-regulated secretion of the anti-inflammatory factor [interleukin-10 (IL-10)] from 41.1 to 52.2 pg/ml ([123]Fig. 3M). The above results together demonstrate that RNPs, assisted by TPGS@LNPs, effectively edited the Lsd1 gene locus by efficient cellular uptake, endosomal escape, and nucleus delivery, thereby accelerating wound healing and alleviating inflammation through increased levels of mono- and dimethylation of H3K4 ([124]Fig. 3N). TPGS-RNP@LNPs penetrate the mucus layer and accumulate in the colitis-associated lesions Therapeutics must traverse the highly viscous intestinal mucus and undergo subsequent uptake by intestinal villi cells ([125]35, [126]36). We quantified the permeability of LNPs, TPGS@LNPs, and TPGS-RNP@LNPs across the artificial mucus by measuring their penetration depth. The naked LNPs obtained a moderate penetration distance. At the same time, the incorporation of TPGS significantly improved the mucus-penetrating performance of LNPs, with TPGS@LNPs and TPGS-RNP@LNPs showing 2.0- and 1.9-fold greater penetration depths than naked LNPs, respectively, while no significant difference was found between TPGS@LNPs and TPGS-RNP@LNPs ([127]Fig. 4, A to D). Notably, the participation of TPGS also significantly improved the motion performance of LNPs, as seen from higher mean square displacement (MSD) values and diffusion coefficients, as well as longer motion trajectories ([128]Fig. 4, E to H). After oral administration, the colonic accumulation of TPGS-RNP@LNPs in UC and CAC mice was assessed by tracking fluorescence signals from EGFP-fused RNPs. As shown in [129]Fig. 4 (I to K), the fluorescence signal in the colon increased rapidly, reaching a peak at 12 hours and remaining detectable for up to 24 hours. In contrast, only weak fluorescence was observed in the liver at 6 and 12 hours (fig. S3A). Over time, the fluorescence signal gradually decreased because of metabolic clearance, with no indication of redistribution to other organs. These findings further confirm the efficient colonic targeting and minimal systemic distribution of TPGS-RNP@LNPs in disease models. Furthermore, to exclude the possibility of premature uptake of TPGS-RNP@LNPs in the upper GI tract, gastric and small intestinal tissues from UC and CAC mice treated with TPGS-RNP@LNPs were collected at 6 hours for optimal cutting temperature (OCT) sectioning. It was found that no fluorescence signal from TPGS-RNP@LNPs was detected (fig. S3B). The specific accumulation of TPGS-RNP@LNPs in inflamed colonic tissues may be attributed to the epithelial enhanced permeability and retention effect, as well as their capacity to target macrophages through galactose receptor–mediated recognition. Fig. 4. Mucus penetration and in vivo biodistribution profiles of various LNPs. [130]Fig. 4. [131]Open in a new tab 3D mucus penetration images of (A) LNPs, (B) TPGS@LNPs, and (C) TPGS-RNP@LNPs. Scale bar, 200 μm. (D) Corresponding penetration depths of LNPs, TPGS@LNPs, and TPGS-RNP@LNPs (n = 3). (E) Mean square displacement (MSD) values and (F) diffusion coefficients of LNPs and TPGS@LNPs within the mucus-simulating hydrogel (n = 3). Motion trajectories of (G) naked LNPs and (H) TPGS@LNPs in the mucus-simulating hydrogel (n = 7). (I) Ex vivo fluorescence profiles of the GI tract from UC and CAC mice that received oral administration of TPGS-RNP@LNPs at different time points (6, 12, and 24 hours). Quantification of the relative MFIs of colon tissues from (J) UC mice and (K) CAC mice using ImageJ software in (I) (n = 3). (L) CLSM images of colonic tissue sections from the healthy, UC, and CAC mice after oral administration of TPGS-RNP@LNPs for 24 hours. Scale bar, 100 μm. (M) Quantifying the relative EGFP expression levels in the colonic tissues using ImageJ software in (L) (n = 3). (N) CLSM images of colonic tissue sections from the healthy, UC, and CAC mice after oral administration of TPGS-RNP@LNPs for 24 hours. Scale bar, 100 μm. Macrophages were visualized using an Alexa Fluor 647–labeled F4/80 antibody. (O) Schematic illustration of TPGS enhancing the mucus infiltration and lesion accumulation of TPGS-RNP@LNPs. Created in BioRender. Q. Gao (2025); [132]https://biorender.com/m5s4z5f. Data are the means ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001. We further estimated the homing capacity of TPGS-RNP@LNPs to the inflammatory lesions in vivo. After oral administration of TPGS-RNP@LNPs to the BALB/c mice with DSS-induced UC and azomethane (AOM)/DSS–induced CAC for 24 hours, the colon tissues were collected and sectioned for fluorescence microscope examination. The TPGS-RNP@LNPs primarily accumulated in the colon tissues of the UC and CAC mice rather than the healthy colon tissues ([133]Fig. 4L). The quantitative analysis revealed that the accumulation amounts of TPGS-RNP@LNPs in the colitis and colorectal tumor tissues were respectively 67.0- and 169.9-fold higher than those in healthy colon tissues ([134]Fig. 4M). After further labeling macrophages with an F4/80 antibody, it was observed that TPGS-RNP@LNPs were readily sequestered by the macrophages in the colon lesions of UC and CAC mice, as indicated by the colocalization of a large number of green fluorescent TPGS-RNP@LNPs and red fluorescent macrophages ([135]Fig. 4N). Consequently, these results demonstrate the exceptional mucosal penetration and excellent mobility of TPGS-RNP@LNPs while enabling effective macrophage targeting within the colitis and colorectal tumor tissues, underscoring the superb therapeutic potential of TPGS-RNP@LNPs against UC and CAC ([136]Fig. 4O). TPGS-RNP@LNPs targeting Lsd1 retard the progression of UC To further benchmark the impact of TPGS-RNP@LNPs on the progression of UC, oral administration of various formulas was carried out ([137]Fig. 5A). TPGS-RNP@LNP–mediated therapy effectively protected mice against DSS-induced body weight loss, colon shortening, and spleen coefficient increase. As depicted in [138]Fig. 5B, the TPGS-RNP@LNP group exhibited a significant alleviation of body weight loss, accounting for a decrease of 7.6% from their initial weight. In contrast, the clinically used 5-aminosalicylic acid (5-ASA) group experienced an exacerbation of body weight loss (11.1%), while DSS-induced mice exhibited an 11.0% decrease in body weight. Meanwhile, we also noticed a slightly reduced DSS effect in the TPGS@LNP group, which might be attributed to the presence of multiple bioactive lipids in MLLs that were well documented for their anti-inflammatory properties ([139]37), such as phosphatidylethanolamine ([140]38) and phosphatidylserine ([141]39). In addition, in comparison to the disability of 5-ASA and TPGS@LNPs, oral TPGS-RNP@LNPs also effectively restored colon length ([142]Fig. 5C and fig. S4C) and spleen coefficient ([143]Fig. 5D) in DSS-induced mice to 7.3 cm and 4.8 mg/g, respectively, approaching levels observed in healthy subjects (7.9-cm length and 4.1 mg/g spleen coefficient). Hence, we confirmed that the body weight, colon length, and spleen coefficient restoration in the TPGS-RNP@LNP group significantly outperformed those treated with 5-ASA and TPGS@LNPs. These typical colitis symptoms were alleviated in the TPGS-RNP@LNP group, likely due to the reduced infiltration of inflammatory cells and the damaged colonic mucosa restoration. Fig. 5. In vivo retardation effect of oral LNPs on UC progression. [144]Fig. 5. [145]Open in a new tab (A) Schematic representation of the treatment protocol. (B) Variations in mouse body weight across all groups throughout the entire study period (n = 6). (C) Colon lengths and (D) spleen coefficients of various groups at the end of experiments (n = 6). (E) H&E and PAS staining of the colonic tissues from mice in all experimental groups. Scale bar, 100 μm. Quantitative results of (F) H&E and (G) PAS staining of the colonic tissues (n = 3). (H) Representative Sanger sequencing results and (I) indel mutations of the Lsd1 locus of T-A cloning from the colonic tissues after co-incubation with TPGS-RNP@LNPs for 48 hours. (J) Relative expression levels of Lsd1 in the colonic tissues from different mouse groups (n = 3). (K) Principal components analysis from mice in the healthy control, DSS control, and TPGS-RNP@LNP groups (n = 4). (L) The heatmap illustrates the correlations between different groups (n = 4). (M) KEGG enrichment analysis between the TPGS-RNP@LNP group and the DSS control group. (N) The heatmap illustrates the expression levels of chemokines, inflammatory factors, colony-stimulating factors, and Ifi47 in different mouse groups (n = 4). (O) Molecular mechanism underlying the inhibitory effect of TPGS-RNP@LNPs on the progression of UC. Created in BioRender. Q. Gao (2025); [146]https://biorender.com/u8i27zv. Data are the means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. Strictly, hematoxylin and eosin (H&E) and periodic acid–Schiff (PAS) staining results in [147]Fig. 5E revealed that the colon tissues from the DSS control group presented the typical signs of inflammation, including infiltration of inflammatory cells, damage to the colonic mucosa, and apparent loss of crypts. In the context of conventional 5-ASA, this treatment failed to address these symptoms effectively. Conversely, oral TPGS-RNP@LNPs significantly alleviated the symptoms above, yielding histopathological scores and PAS staining that are highly comparable to those of the healthy colonic tissue ([148]Fig. 5, F and G). Thereafter, immunofluorescence staining analysis was performed to evaluate the reparative effect of various therapeutics on the damaged colonic epithelial barrier induced by DSS, with a focus on the expression of tight junction–associated protein [zona occludens 1 (ZO-1)] and mucus-correlative protein [mucin 2 (MUC2)]. The brightest fluorescence signals of ZO-1 and MUC2 were concurrently noticed in the healthy control mice and TPGS-RNP@LNP–treated mice. In contrast, relatively feeble fluorescence was observed in other mouse groups, including DSS control, TPGS@LNPs, and 5-ASA (fig. S4, A, D, and E). These results indicate that TPGS-RNP@LNP treatment can normalize the expression of the tight junction– and mucus-associated proteins and efficiently repair the intestinal epithelial barrier damaged by DSS-induced colitis. DSS treatment can inevitably trigger inflammatory cascade responses, leading to further exacerbation of UC progression ([149]40). To evaluate the effect of TPGS-RNP@LNPs on inflammatory regulation in mice with colitis, we detected the variations in the proportions of various immune cells in the colonic tissues, including CD4^+ and CD8^+ T lymphocytes, as well as Foxp3^+ regulatory T cells (T[reg] cells). According to the immunofluorescence staining results (fig. S4, B, F, and G), TPGS-RNP@LNPs evidently reduced the levels of CD4^+ and CD8^+ T lymphocytes, measuring reductions of 86.3 and 59.4% compared to the DSS control group, as well as 91.8 and 87.6% compared to the 5-ASA group, respectively. This outcome means that TPGS-RNP@LNPs can effectively extinguish the hyperactive inflammatory responses in DSS-induced UC mice. The observed appearance is likely attributed to an up-regulated level of Foxp3^+ T[reg] cells, which exert an immunosuppressive effect on activated inflammatory cells ([150]41). The TPGS-RNP@LNP group led to a substantial augmentation in the population of Foxp3^+ T[reg] cells by 9.5-, 4.3-, and 10.5-fold, respectively, compared to those detected in the DSS control, TPGS@LNP, and 5-ASA groups (fig. S4, B and H). These consequences strongly suggest that TPGS-RNP@LNPs can inactivate the inflammatory responses, thereby alleviating DSS-induced UC. Next, we found that the repeated oral administration of TPGS-RNP@LNPs did not yield discrepant indices or evident histopathological lesions in the five principal organs compared to healthy mice (fig. S4, I and K). Unlike 5-ASA that had lower hematocrit and platelet distribution width values, no abnormalities were detected in the blood routine parameters of mice following treatment with TPGS-RNP@LNPs (fig. S4J), demonstrating their excellent in vivo biosafety. To further elucidate the therapeutic mechanism of TPGS-RNP@LNPs in treating UC, we first confirmed their targeted genome editing at the Lsd1 locus using T-A cloning and Sanger sequencing ([151]Fig. 5, H and I). Compared to the DSS control mice, the relative expression level of Lsd1 in the colon tissues from the TPGS-RNP@LNP–treated mice was reduced by 70.8% ([152]Fig. 5J), suggesting that oral TPGS-RNP@LNPs achieved effective Lsd1 knockout. Avoiding unintended genome editing in nontarget organs remains a critical challenge in the development of CRISPR-Cas9–based therapeutic strategies. Considering the relatively weak fluorescence signal of TPGS-RNP@LNPs observed in the liver (fig. S3A), we designed specific primers targeting the top three off-target sites predicted by Cas-OFFinder ([153]www.rgenome.net/cas-offinder) to evaluate the potential off-target effects in this organ (tables S5 and S6). The analysis revealed no detectable mutations at the predicted off-target sites or genome variations surrounding the Lsd1 target locus (table S7). These findings further support the therapeutic safety of our therapeutic system. In addition, transcriptome analysis was conducted on colonic tissues after various treatments were completed. Primarily, the correlation between the TPGS-RNP@LNP group and the healthy control group was stronger, whereas it was weaker between the TPGS-RNP@LNP group and the DSS control group ([154]Fig. 5, K and L). Compared to the DSS control mice, there were 1400 up-regulated genes and 1104 down-regulated genes after implementing TPGS-RNP@LNPs (fig. S4L). On the contrary, only 137 genes showed differential expression profiles between the healthy control group and the TPGS-RNP@LNP group (fig. S4M). The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis revealed that the signaling pathways involving TNF, IL-17, inflammatory bowel disease, mitogen-activated protein kinase (MAPK), NOD (nucleotide oligomerization domain)–like receptor, and T helper 17 cell differentiation were activated in the DSS control mice compared to the TPGS-RNP@LNP–treated mice ([155]Fig. 5M). On the basis of the KEGG pathway function enrichment analysis, we further found that oral TPGS-RNP@LNPs significantly down-regulated the expression levels of the genes associated with chemokines (encoded by Ccl2, Cxcl1, Cxcl2, Cxcl3, and Cxcl5), pro-inflammatory factors (encoded by Il-6, Il-1b, Il-17, and Il-22), and colony-stimulating factors (encoded by G-CSF) ([156]Fig. 5N and fig. S5) in the UC mice, which were beneficial for relieving inflammation and maintaining immune tolerance. Synchronously, the antitumor regulatory factors, such as IL-21 (encoded by Il-21) and interferon-γ–inducible protein 47 (encoded by Ifi47), were significantly up-regulated in mice receiving oral treatment of TPGS-RNP@LNPs. These effects are primarily attributed to the knockdown of Lsd1 via oral TPGS-RNP@LNPs, which influenced the expression of IL-17 receptor B (encoded by Il-17rb) within the IL-17 signaling pathway. This increase in IL-17RB promoted the expression of TNFRSF1A-associated via death domain (encoded by Tradd). It facilitated the recruitment of tumor necrosis factor receptor–associated factor 2 (encoded by Traf2), ultimately activating the MAPK pathway. In the MAPK pathway, we detected significant up-regulation of mitogen-activated protein kinase 6 (encoded by Mkk6) and P38 expression. This up-regulation led to the enhanced expression of MYC-associated factor X (encoded by Max), known for its roles in regulating cell growth, differentiation, and apoptosis ([157]42), and retinoic acid receptor–related orphan receptor γt (encoded by Rorγt), essential for maintaining the immune homeostasis ([158]43). Concurrently, this pathway suppressed the expression of activator protein 1 (encoded by Ap-1), a key regulator of tumor proliferation and metastasis ([159]44, [160]45), and retinoic acid receptor–related orphan receptor α (encoded by Rorα), paramount for modulating inflammation, metabolism, and immune responses and supporting nervous system development (fig. S6) ([161]46–[162]48). Collectively, oral TPGS-RNP@LNPs, via Lsd1 genome editing, can facilitate cell differentiation, promote anti-inflammatory response, and prevent tumor development in the UC mice ([163]Fig. 5O), providing robust evidence for effective mitigation of inflammation and restoration of the intestinal barrier. TPGS-RNP@LNPs targeting Lsd1 exert a therapeutic effect against UC We further explored the therapeutic efficacy of TPGS-RNP@LNPs in ameliorating the manifestations of chronic UC. [164]Figure 6A illustrates the establishment of the UC mouse model and the treatment processes. It was observed that only TPGS-RNP@LNPs showed a therapeutic effect against UC, as evidenced by the prevention of body weight loss ([165]Fig. 6B) and colon length shortening ([166]Fig. 6C). According to the histopathological examination and epithelium-associated immunofluorescence staining, treatment with TPGS-RNP@LNPs also protected the colonic epithelial layer from pathological damage ([167]Fig. 6, D to I). Peculiarly, after treatment with TPGS-RNP@LNPs, the colonic crypt structure became more intact and orderly, the ulcer area was less, and the tight junction– and mucus-associated proteins were richer. Fig. 6. In vivo therapeutic outcomes of oral LNPs against UC. [168]Fig. 6. [169]Open in a new tab (A) Protocol for the establishment of UC mouse models and treatment procedures. (B) Mouse body weight variations during the entire investigation (n = 5). (C) Colon lengths of various treatment groups at the end of experiments (n = 5). (D) H&E and PAS staining of the colonic tissues. Scale bar, 100 μm. Quantitative analysis of (E) H&E and (F) PAS staining of the colonic tissues (n = 3). (G) Immunofluorescence staining of ZO-1 and MUC2 in the colonic tissue sections. Scale bar, 100 μm. Quantitative fluorescence analysis of (H) ZO-1 and (I) MUC2 in the colonic tissues (n = 3). (J) Immunofluorescence staining of CD4^+ T cells, CD8^+ T cells, and Foxp3^+ T[reg] cells in the colonic tissues. Scale bar, 100 μm. Corresponding fluorescence quantification of (K) CD4^+ T cells, (L) CD8^+ T cells, and (M) Foxp3^+ T[reg] cells in the colonic tissues (n = 3). Data are the means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. In addition, oral TPGS-RNP@LNPs effectively reduced the DSS-induced elevation of CD4^+ and CD8^+ T lymphocytes while significantly increasing the expression of Foxp3^+ T[reg] cells to suppress excessive immune responses in the intestine ([170]Fig. 6, J to M). Notably, the repeated administration of TPGS-RNP@LNPs resulted in negligible damage to the mice, as there was no evidence-based harmful indication in the five principal organs and no statistically significant differences in hematological parameters (fig. S7), verifying the formulation’s excellent biosafety. These results signify that TPGS@LNPs represent a desirable oral drug delivery platform, and TPGS-RNP@LNPs can safely and effectively remit colitis status. TPGS-RNP@LNPs attenuate the progression of CAC The persistent activation of inflammatory responses in the colon can increase the susceptibility to developing CAC ([171]49). Considering the potent anti-inflammatory capability of TPGS-RNP@LNPs, we sought to evaluate their role in suppressing the progression of inflammation-associated CAC ([172]Fig. 7A). It was found that the TPGS-RNP@LNP group developed significantly fewer tumor numbers and smaller tumors than the AOM/DSS control and TPGS@LNP groups ([173]Fig. 7, B to D). Additional confirmation of the therapeutic efficacy of TPGS-RNP@LNPs was provided by the histological analyses ([174]Fig. 7, E to H). Compared with other treatments, the colon slices from the TPGS-RNP@LNP group showed minimal inflammatory signs and the fewest tumor cells. Moreover, the TPGS-RNP@LNP–treated mice also exhibited the highest levels of ZO-1 expression compared to both the AOM/DSS control mice and TPGS@LNP-treated mice ([175]Fig. 7M and fig. S8I), testifying the integrity of the colonic epithelial barrier. Fig. 7. In vivo inhibitory effect of orally administered LNPs on CAC progression. [176]Fig. 7. [177]Open in a new tab (A) Schematic diagram of CAC model establishment and treatment protocol. ip, intraperitoneally. (B) Photographs of colon tumors. Scale bars, 0.5 cm. (C) Total tumor numbers and (D) tumor size distributions across various treatment groups at the end of experiments (n = 6). (E) H&E and PAS staining of colonic tissues. Scale bar, 100 μm. (F) Corresponding histopathological scores of colonic tissues from all treatment groups (n = 3). (G) Corresponding PAS staining area of colonic tissues from all treatment groups (n = 4). (H) Ki67 staining of colonic tissues. The scale bars for the small field and large field of the images are 100 and 50 μm, respectively. (I) Immunofluorescence staining of CD4^+ T cells, CD8^+ T cells, and Foxp3^+ T[reg] cells in colonic tissue samples. Scale bar, 100 μm. Quantitative fluorescence analysis of (J) CD4^+ T cells, (K) CD8^+ T cells, (L) Foxp3^+ T[reg] cells, and (M) ZO-1 expression in the colonic tissues (n = 3). Data are the means ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001. Suppressing exaggerated immune responses is crucial in restraining malignant development during colitis-associated neoplastic progression ([178]50). Consistently, the abundance of AOM/DSS–induced immune-activated CD4^+ and CD8^+ T lymphocytes was effectively reduced after oral administration of TPGS-RNP@LNPs ([179]Fig. 6, I to K), exhibiting reductions that were 12.5- and 4.0-fold lower than those in the AOM/DSS control group, respectively. Conversely, this treatment significantly increased the population of immunosuppressive Foxp3^+ T[reg] cells ([180]Fig. 7, I and L) by 181.0 and 535.5% compared to both the AOM/DSS control and TPGS@LNP groups, thereby controlling the inflammatory responses in the colon. It was noticed that these treatments did not affect the histopathological and hemal changes of mice (fig. S8, A to H), indicating that the potential detrimental effects on mouse health can be considered negligible. DISCUSSION The intricate UC and its evolvement to CAC pose essential challenges to traditional treatment modalities. The current clinical medications for UC and CAC are suboptimal, mainly owing to the insufficient understanding of the underlying pathophysiological mechanism. Here, we concentrated on clinical human samples and identified an extraordinary up-regulation of LSD1 in the colonic tissues of patients with UC and CAC ([181]Fig. 2, B and C). It is widely recognized that Lsd1 is a key gene for regulating diverse biological processes through the selective demethylation of mono- and dimethylated H3K4 or H3K9 ([182]51, [183]52). These observations may pave the way for developing potential therapeutic strategies against colonic diseases by commanding Lsd1 knockout. Encouragingly, we developed a reinforced plant-derived lipid nanoplatform that enabled RNP-based CRISPR-Cas9 epigenome editing via the oral route. On the basis of the results of molecular dynamics simulations, we found that incorporating Food and Drug Administration–approved TPGS excipients into LNP formulations could enhance the tightness of the LNP lipid bilayer ([184]Fig. 2, E to H) and create an ideal oral carrier for safeguarding RNPs in the GI tract ([185]Fig. 2, D and L). It was observed that TPGS@LNPs served as efficient transporters of RNPs, facilitating successful GI traversing, mucus infiltration, and targeted accumulation in the colitis and colorectal tumor tissues ([186]Fig. 4, A to O). Subsequently, the galactose groups on the surface of LNPs effectively guided RNPs toward activated macrophages by specifically binding to their galactose receptors, achieving selective cell internalization ([187]Fig. 3D). We must acknowledge that the endosomal escape of RNPs is obligatory for successful intranuclear genome editing. A large amount of prior research suggests that the endosomal escape mechanism for therapeutics predominantly involves pore formation, proton sponge effects, membrane destabilization, photochemical disruption, and the strategic use of endosomal escape agents ([188]53). Alternatively, we observed a new escape mechanism based on membrane fusion to realize the endosomal escape of RNPs, which fundamentally differed from previously identified mechanisms. During the escape process of TPGS@LNPs and TPGS-RNP@LNPs, there was no significant membrane disruption, as evidenced by unaltered levels of key components (Lamp1 and Lamp2) associated with normal endosomal membranes ([189]Fig. 3, G and H). However, Npc1, a critical factor in membrane fusion, exhibited notable up-regulation after the treatment of TPGS@LNPs and TPGS-RNP@LNPs ([190]Fig. 3F). The findings indicate that RNPs can escape from the endosomes using the fusion between TPGS@LNPs and endosomal membranes. The escaped RNPs preferentially translocate to the nucleus for Lsd1 gene editing. Intriguingly, the macrophage-targeting TPGS-RNP@LNPs showed a high editing efficiency of 59.7% at a dosage of 5 μg of RNPs, surpassing the performance of the equivalent commercial CMAX product ([191]Fig. 3I). The precise editing targeting the Lsd1 locus effectively attenuated colonic inflammation and promoted colonic epithelial repair, demonstrating conspicuous therapeutic potential in mouse models of UC and CAC by modulating H4K4Me1 and H4K4Me2. It should be noted that 5-ASA is clinically administered using colon-targeted formulations to avoid premature absorption in the upper GI tract ([192]54, [193]55). In this study, 5-ASA was used as a basic positive control without special formulation, which may limit its therapeutic performance. Despite this, TPGS-RNP@LNPs demonstrated remarkable anti-inflammatory effects in both UC and CAC models, further underscoring the potent therapeutic potential of our delivery platform. Recent advances in small interfering RNA (siRNA) therapeutics, particularly the development of chemical modifications that enhance stability and reduce the required dosing frequency ([194]56, [195]57), suggest that Lsd1-targeted siRNA may offer therapeutic potential for the treatment of UC and CAC. However, RNP delivery presents distinct advantages by acting directly at the DNA level to achieve permanent gene disruption through a single administration, rendering it especially suitable for the treatment of diseases that necessitate long-term gene modulation, like Lsd1-driven colonic disorders. In addition, the transient presence of RNPs minimizes prolonged off-target effects and immune responses that are associated with repeated siRNA administration. These features position RNP-based therapies as a promising strategy for the durable and efficient treatment of colonic inflammatory and neoplastic diseases. Future investigations may focus on the integrated application of both RNP and siRNA technologies to optimize therapeutic precision and flexibility ([196]58). We also note certain limitations that present opportunities for future research. While MLLs were shown to be safe, low-cost, and effective in constructing the oral delivery system (TPGS-RNP@LNPs), and our group has previously reported the therapeutic potential of other plant-derived carriers against inflammatory and cancerous diseases ([197]59, [198]60), there are still no clinical studies validating the feasibility of plant-derived carriers for oral drug delivery. This gap underscores the limited immediate translational applicability of our approach. Therefore, future research should focus on systematically evaluating the safety, reproducibility, and large-scale manufacturability of plant-derived lipid nanocarriers, which will be critical for advancing this platform toward clinical application. Moreover, the therapeutic efficacy of the oral CRISPR-Cas9 RNP formulation was validated only in murine models. While rodent systems provide valuable proof-of-concept evidence, they cannot fully recapitulate the complexity of human physiology, GI barriers, and immune responses. To bridge this gap, it will be necessary to perform detailed safety, pharmacokinetic, and therapeutic evaluations in larger animal models, such as pigs and nonhuman primates, to obtain more reliable insights into its translational potential. These studies are currently being planned in our ongoing research. In summary, the above findings robustly support the efficacy of the plant-derived LNP platform for the oral delivery of CRISPR-Cas9 RNPs in mediating genome editing at the Lsd1 locus. The obtained TPGS@LNPs offer a practical solution to overcome the GI barriers for RNP delivery and serve as a general methodology applicable to other drug systems, thereby establishing their translational potential in medical applications. The described modular RNP delivery strategy will provide valuable guidance for rationalizing cell-specific gene engineering in a broad spectrum of preclinical and clinical carriers associated with intestinal inflammation–related diseases. MATERIALS AND METHODS For additional detailed methods, see the Supplementary Materials. Immunofluorescence staining of human colon tissue sections All human colon tissues from patients with UC and CAC were obtained from the First Affiliated Hospital of Nanchang University with approval from its Medical Research Ethics Committee [ethics number: (2023) CDYFYYLK (02-029)], and informed consent was obtained. Briefly, the colon tissues were embedded in the OCT compound and sliced into 5-μm-thick sections using a freezing microtome (RWD, FS800, China). Afterward, the colon tissue sections were stained with an anti-LSD1 antibody (Servicebio, [199]GB112427, China) and incubated overnight at 4°C in a humid chamber. Next, the sections were incubated with Cy3-conjugated Goat Anti-Rabbit IgG (immunoglobulin G) (Servicebio, GB21303, China) for 50 min at room temperature in the dark, followed by 10 min of staining with 4′,6-diamidino-2-phenylindole (DAPI) under the same condition. Last, these sections were imaged by CLSM (Olympus, FV-3000, Japan). Molecular dynamics simulation The intermolecular interaction between TPGS and LNPs was analyzed by Shiyanjia Lab ([200]www.shiyanjia.com). Briefly, two mixed lipid systems were constructed using the Packmol program. Thereinto, the compositions of system-1 were monogalactosyldiacylglycerol (MGDG), phosphatidylglycerol (PG), fatty acid (FA), and (O-acyl)-ω-hydroxy FA (OAHFA), whose corresponding mass ratios were 15.1, 17.2, 26.9, and 40.9%, respectively. In addition, the compositions of system-2 were TPGS, MGDG, PG, FA, and OAHFA, with mass ratios of 9.1, 13.7, 15.6, 24.4, and 37.2%, respectively. The initial structures of these two mixed lipid systems were prepared using the Amber 20 program and immersed in an explicit water (TIP3P) environment. Subsequently, Na^+ or Cl^− was introduced to neutralize the overall charge of the systems, resulting in a zero net charge. Following this, molecular dynamics simulations lasting for 100 ns were performed under conditions of T = 298 K and P = 1 bar until equilibrium was achieved. Thereafter, the trajectory of the two mixed lipid systems was analyzed, including the root mean square deviation (RMSD), radius of gyration (RG), and surface area (SA). Their motion models were drawn using PyMol visualization software. Preparation of RNPs The sgRNAs targeting the Lsd1 gene were designed using CRISPRdirect (https://crispr.dbcls.jp/), and sgLsd1 transcripts were obtained by following the manufacturer’s recommended procedures with the TranscriptAid T7 High Yield Transcription Kit (Thermo Fisher Scientific). Afterward, aqueous solutions of Cas9 proteins and sgRNAs were mixed in equal mass. Following mixing, the RNPs were allowed to form through 10 min of incubation at room temperature to ensure the complete self-assembly of the Cas9/sgRNA complex. Fabrication of various LNPs Mulberry leaves were pulverized into a fine powder using a crusher, followed by cold soaking in cyclohexane and rotary evaporation to obtain the crude lipid extract. The obtained crude products were purified by decolorization, yielding the MLLs. Subsequently, the TPGS aqueous solution was rapidly added to the MLL ethanol solution at a volume ratio 3:1 with a mass ratio of 10:1 (lipid:TPGS, w/w) to prepare TPGS@LNPs. To fabricate TPGS-RNP@LNP complexes, the RNP/TPGS mixture was rapidly added dropwise into the ethanol-dispersed MLLs at a volume ratio of 3:1 (RNPs/TPGS:MLLs, v/v) to form TPGS-RNP@LNPs. Notably, TPGS@LNPs and TPGS-RNP@LNPs were subjected to dialysis (molecular weight cutoff, 14 kDa) against double-distilled H[2]O for 2 hours to remove ethanol before conducting in vitro assays and animal experiments. CMAX-RNP@LNPs were prepared following the manufacturer’s protocol using the Lipofectamine CRISPRMAX reagent (Thermo Fisher Scientific). Briefly, tube 1 contained 1 ml of Opti-MEM medium, 25 μg of Cas9 proteins, 5 μg of sgRNAs, and 50 μl of Cas9 Plus reagent. Tube 2 contained 1 ml of Opti-MEM medium and 60 μl of CMAX reagent. The contents of tube 1 were added to tube 2 within 3 min, mixed, and incubated at room temperature for 10 min. The resulting LNPs were stored at 4°C for further experiments. Similarly, Lipo6000-RNP@LNPs were gained following the manufacturer’s protocol (Beyotime Biotechnology Research Institute). Briefly, tube 1 contained 1 ml of Opti-MEM medium, 20 μg of Cas9 proteins, and 20 μg of sgRNAs, while tube 2 contained 1 ml of Opti-MEM medium and 40 μl of Lipo6000 reagent. After incubating both tubes at room temperature for 5 min, the contents of tube 1 were added to tube 2, mixed, and incubated for another 5 min. Then, the resulting LNPs were stored at 4°C for subsequent experiments. Endosomal escape capacity of TPGS-RNP@LNPs CT-26 cells and RAW 264.7 macrophages were inoculated into 35-mm dishes with a density of 4.0 × 10^5 cells per well and cultured overnight at 37°C. Thereafter, the medium in each well was replaced with 2 ml of serum-free medium containing TPGS-RNP@LNPs [RNPs: GenCrispr NLS-Cas9-EGFP Nuclease (0.5 μg/ml) and sgLsd1 (0.5 μg/ml)]. After co-incubation for 1, 4 and 8 hours, cells were rinsed with cold phosphate-buffered saline (PBS) and fixed in a formalin solution for 30 min. Subsequently, cells were stained with LysoTracker Red for endosomal labeling for 30 min, followed by nuclear staining with DAPI for an additional 30 min. The endosomal escape capacity of TPGS-RNP@LNPs was evaluated by CLSM (FV3000, Olympus, Japan). Galactose receptor–mediated endocytosis by macrophages To elucidate the uptake mechanism of TPGS-RNP@LNPs, RAW 264.7 macrophages were seeded at a density of 2.0 × 10^5 cells per well in 12-well plates and incubated overnight. Before administration, 500 μl of serum-free medium containing galactose (500 μg/ml) was added to each well and co-incubated with the cells for 4 hours. The medium in each well was substituted with 1 ml of serum-free medium containing TPGS-RNP@LNPs [RNPs: GenCrispr NLS-Cas9-EGFP Nuclease (1 μg/ml) and sgLsd1 (1 μg/ml)]. After the treatment of TPGS-RNP@LNPs for 1, 3, and 5 hours, the cells were rinsed thrice with PBS, harvested using trypsin, centrifuged at 1000g for 5 min, and resuspended in PBS for flow cytometry analysis (ACEA NovoCytet, US). Animal experiments All animal experiments adhered to the guidelines set by the Institutional Animal Care and Use Committee of Southwest University (IACUC-20230918-01). All animals were from Enswell Biotechnology Co., Ltd. (Chongqing, China), they were free to access water and food, and their living condition was observed daily. In vivo biodistribution of TPGS-RNP@LNPs after oral administration To investigate the in vivo biodistribution of TPGS-RNP@LNPs, the GI tissues (stomach, small intestine, cecum, and colon) and the five principal organs (heart, liver, spleen, lung, and kidney) were collected from UC and CAC mice that orally received EGFP-fused TPGS-RNP@LNPs (5 μg of EGFP-fused Cas9 proteins and 5 μg of sgLsd1) for 6, 12, and 24 hours for IVIS fluorescence observation (FX Pro90200, Carestream, US). Subsequently, we thoroughly investigated the accumulation ability of TPGS-RNP@LNPs in the lesion tissues of mice with UC and CAC. The mice, including healthy mice and those with UC or CAC, were euthanized, and their colon tissues were collected after treatment with EGFP-fused TPGS-RNP@LNPs (5 μg of EGFP-fused Cas9 proteins) for 12 hours. These tissues were fixed in a 4% (v/v) paraformaldehyde solution, embedded in an OCT compound, and sectioned into 5-μm-thick slices. After DAPI staining for 10 min, a coverslip was placed, and the slides were imaged by CLSM (Olympus, FV-3000, Japan). To further investigate the colocalization capability of TPGS-RNP@LNPs with macrophages, we performed immunostaining on these tissue sections using an anti–F4/80 antibody (Servicebio, [201]GB113373, China) and incubated overnight at 4°C in a humid chamber. The sections were incubated with Cy3-conjugated Goat Anti-Rabbit IgG (Servicebio, GB21303, China) for 50 min in the dark, followed by 10 min of incubation with DAPI under the same conditions for CLSM observation (Olympus, FV-3000, Japan). In vivo retardation effect of TPGS-RNP@LNPs on UC BALB/c female mice (6 to 8 weeks of age) were divided into five groups (n = 6), namely the healthy control group, DSS control group, TPGS@LNP group, TPGS-RNP@LNP group, and 5-ASA group. The UC mouse model was induced by replacing the drinking water with the DSS solution (3.5%, w/v) from day 0 to day 13. Apart from consuming the DSS solution, the TPGS@LNP-treated mice, TPGS-RNP@LNP–treated mice, and 5-ASA–treated mice were orally administered with 200 μl of TPGS@LNPs, TPGS-RNP@LNPs (RNPs: 5 μg of Cas9 proteins and 5 μg of sgLsd1), and 5-ASA (5 mg/kg per mouse) every other day from day 2 to day 12. In parallel, mouse body weights were recorded daily. On day 13, mice were euthanized, and their colon lengths were measured. Colon tissues were used to evaluate the therapeutic efficacies of various LNPs, including inflammation remission and mucosal repair, through histological staining techniques, such as H&E, PAS, and immunofluorescence staining (MUC2, ZO-1, CD4^+ T cells, CD8^+ T cells, and Foxp3^+ T[reg] cells). The five principal organs were obtained for the H&E histological analysis, and the blood samples were collected for the routine hematological analysis to assess the in vivo biosafety of TPGS-RNP@LNPs (BC-2800 VET, Mindray, Guangdong, China). In vivo therapeutic outcomes of TPGS-RNP@LNPs against UC BALB/c female mice (6 to 8 weeks of age) were divided into five groups (n = 6), namely the healthy control group, DSS control group, TPGS@LNP group, TPGS-RNP@LNP group, and 5-ASA group. The UC mouse model was established by administering a 3.5% (w/v) DSS solution instead of drinking water from day 0 to day 12. Afterward, the UC mice were orally administered with TPGS@LNPs, TPGS-RNP@LNPs (RNPs: 5 μg of Cas9 proteins and 5 μg of sgLsd1), and 5-ASA (5 mg/kg per mouse) daily from day 12 to day 15. During the entire experiment, mouse body weights were recorded every day. On day 16, mice were euthanized, and their colon tissues, five principal organs, and blood samples were harvested. The colon tissues were used for evaluating the therapeutic efficacy through length measurement and histological staining techniques, such as H&E, PAS, and immunofluorescence staining (MUC2, ZO-1, CD4^+ T cells, CD8^+ T cells, and Foxp3^+ T[reg] cells). The histological analysis of the five principal organs and the hematological analysis of blood samples were conducted to perform a biosafety assessment. In vivo preventive effect of TPGS-RNP@LNPs on CAC BALB/c male mice (7 to 8 weeks of age) were randomly divided into three groups (n = 6), namely the AOM/DSS control group, TPGS@LNP group, and TPGS-RNP@LNP group. Briefly, mice were intraperitoneally injected with AOM (10 mg/kg per mouse) and maintained with normal drinking water and diet for 7 days. Thereafter, mice were exposed to two cycles of DSS treatment (3.5%, w/v), with each cycle consisting of 7 days of DSS administration spaced 2 weeks apart. In the meantime, mice were orally administered with TPGS@LNPs and TPGS-RNP@LNPs (RNPs: 5 μg of Cas9 proteins and 5 μg of sgLsd1) every alternate day, while their body weights were monitored daily from day 7 to day 42. On day 42, mice were euthanized, and the colorectal tumors were measured and analyzed by H&E, PAS, and immunofluorescence staining (ZO-1, CD4^+ T cells, CD8^+ T cells, and Foxp3^+ T[reg] cells). The histological analysis of the five principal organs and the hematological analysis of blood samples were conducted to perform a biosafety assessment. Statistical analysis All values are presented as the means ± SEM. All statistical analyses were performed using GraphPad Prism version 8.0 software (GraphPad Software Inc.). Unless otherwise noted, statistical significance was determined using Student’s t test or a one-way analysis of variance. A P value <0.05 was considered statistically significant, denoted by *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. Acknowledgments