Background/Aims: Liver transplantation (LT) following total hepatectomy is a life-saving treatment for hepatocellular carcinoma (HCC). The HCC recurrence after LT hinders the effectiveness of the procedure. The objective of this study is to develop a pre-operative risk stratification model based on a liquid biopsy. Methods: We conducted a comprehensive multi-omics study of 260 HCC patients from three centers, including clinical data, low-coverage whole-genome sequencing of cell-free DNA (cfDNA) from plasma, as well as whole-exome, single-nucleus RNA, and spatial transcriptomics from matched tumor and non-tumor tissues. Results: We identified cfDNA-derived copy number alteration (CNA) signatures associated with post-transplant recurrence. By integrating cfDNA-derived CNA profiles with single-cell transcriptomic data, we traced recurrence-associated cfDNA to a distinct subpopulation of malignant cells within the primary tumor. These cells were embedded in a pro-metastatic microenvironment of specialized endothelial subtypes and cancer-associated fibroblasts. Notably, most recurrence-associated lesions were detectable in cfDNA prior to liver transplantation (LT). Building on these insights, we developed the ZJU Criteria based on CNA fragments and tumor markers, a pre-LT risk prediction tool that integrates conventional clinical factors with cfDNA-derived CNA signatures, and validated it using internal and independent external cohorts. Conclusion Our findings suggest that post-transplant recurrence commonly originates from advanced subclones that emerge late during tumor evolution. The ZJU Criteria provides an accurate, non-invasive strategy that significantly improves pre-LT risk stratification and clinical decision-making for patients with HCC.

ObjectiveTo elucidate the effects of autonomic neuromodulators [calcium chloride (CaCl₂)-acetylcholine (ACh)] on atrial electrical remodeling and histopathological changes in rats, thereby providing evidence for further investigation into the pathological mechanisms by which autonomic imbalance induces atrial fibrillation (AF). MethodsTen 8-week-old male Sprague-Dawley (SD) rats were randomly divided into experimental and control groups, with 5 rats in each group. The experimental group received daily tail vein injections of a CaCl₂-ACh mixed solution for 28 days, while the control group received an equal volume of saline. Surface electrocardiograms were recorded before and after daily administration. On day 28 of administration, under isoflurane inhalation anesthesia, echocardiography was performed, and then the rats were euthanized by exsanguination under isoflurane anesthesia for tissue collection. Body weight and heart weight of rats were measured, and electrophysiological parameters including AF inducibility, conduction velocity, and conduction dispersion in isolated rat hearts were monitored using the MappingLab multichannel electrophysiological mapping system. HE staining was performed to evaluate atrial tissue architecture and inflammatory cell infiltration. The expression levels of matrix metalloproteinase 9 (MMP-9) and interleukin-1 beta (IL-1β) were analyzed by Western blotting. Masson staining was used to quantitatively analyze the proportional distribution of collagen fibers and myocardial fibers, and to calculate the area of collagen deposition, thereby evaluating the degree of myocardial fibrosis. Wheat germ agglutinin (WGA) staining was used to evaluate morphological changes of cardiomyocytes. Dihydroethidium (DHE) staining was used to detect the level of oxidative stress in atrial myocytes. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining was used to detect atrial cell apoptosis. ResultsCompared with the control group, the experimental group showed obvious arrhythmias on electrocardiograms. Echocardiography after 28 days of administration revealed significant structural remodeling in the experimental group, with a marked increase in left atrial anteroposterior diameter (P < 0.05), and also showed cardiac dysfunction, with significant decreases in left ventricular ejection fraction and left ventricular fractional shortening (P < 0.01). Compared with the control group, the experimental group showed a significantly increased heart weight-to-body weight ratio (P < 0.05), a markedly higher AF inducibility (P < 0.001), a significantly slowed atrial conduction velocity (P < 0.05), an uneven isochronal map with irregular directions, and a significantly increased conduction dispersion (P < 0.01). HE staining showed that atrial myocytes in the experimental group were disorganized, with slight inflammatory cell infiltration. Western blotting showed that the expression levels of MMP-9 and IL-1β in the atrial tissue of the experimental group were significantly upregulated (P < 0.05). Masson staining showed a significant increase in collagen deposition and a significant increase in fibrosis area in the atrial tissue of the experimental group (P < 0.001). WGA staining showed that cardiomyocytes in the experimental group were significantly hypertrophied compared with the control group (P < 0.05). DHE staining and TUNEL staining revealed that the level of oxidative stress in atrial myocytes (P < 0.001) and the apoptotic rate (P < 0.01) were both significantly increased. Conclusion Daily tail vein injections of the autonomic neuromodulator CaCl₂-ACh mixed solution for 28 days can successfully induce AF in rats, accompanied by significant atrial structural remodeling, electrical remodeling, and oxidative stress.

ObjectiveHepatocellular carcinoma (HCC) represents 90% of all primary liver cancers. The main risk factors associated with HCC include viral hepatitis (B and/or C), alcohol abuse, and metabolic dysfunction-associated steatotic liver disease (MASLD), which progressively advance to liver fibrosis, cirrhosis, and ultimately evolve into HCC. Surgical resection represents the most effective treatment for HCC, while recent advances in immunotherapy, including immune checkpoint inhibitors and adoptive cell therapies, have provided improved treatment prospects for patients with unresectable HCC. However, the complex metabolic heterogeneity of HCC limits the therapeutic efficacy. Metabolic intermediates acyl-CoA not only provide energy and substrates for numerous biochemical reactions but also serve as donors for protein lysine acylation, a major class of post-translational modification (PTM). Therefore, a deeper understanding of the molecular mechanisms underlying protein lysine acylation and hepatocarcinogenesis is urgently needed. MethodsThe levels of protein lysine acylation and silence information regulator 5 (SIRT5) expression levels in clinical HCC samples were analyzed by Western blot. Quantitative malonylome and succinylome of HCC samples were analyzed by antibody-based affinity enrichment coupled with tandem mass spectrometry. The proliferation of HCC cells was analyzed with Cell Counting Kit-8 (CCK-8) assays, the apoptosis was quantified by Annexin V-FITC/propidium iodide (PI) staining coupled with flow cytometry, and the ability of cells to migrate was assayed by Transwell assays. The enzymatic activity of glutathione S-transferase Mu 1 (GSTM1) was quantified. Transgenic mice with hepatic overexpression of SIRT5 were constructed using CRISPR-Cas9, and primary hepatocarcinogenesis was induced by administration of diethylnitrosamine. ResultsWestern blot analysis indicated that the expression level of SIRT5 was elevated in clinical samples from HCC patients, and the levels of lysine malonylation, glutarylation, and succinylation were significantly reduced in HCC tissues. Knockout of SIRT5 in MHCC-97H and MHCC-97L hepatoma cells suppressed cell proliferation, and increased the percentage of apoptotic cells significantly. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses of the differentially malonylome and succinylome of HCC samples revealed significant enrichment in two major classes of biological processes: core energy metabolism (e.g., glycolysis/gluconeogenesis, tricarboxylic acid metabolic process, fatty acid beta oxidation) and detoxification and oxidative stress response (e.g., response to toxic substance, chemical carcinogenesis, reactive oxygen species (ROS)). SIRT5 removes malonylation from lysine residues in GSTM1 and restores its detoxification activity, which is crucial for the survival of hepatocytes under stressed conditions. More importantly, in vivo experiment indicated that hepatic-specific overexpression of SIRT5 in mice accelerated diethylnitrosamine-induced liver fibrosis and hepatocarcinogenesis, indicating the critical role of SIRT5 in HCC progression. ConclusionThis study highlights the previously unrecognized SIRT5-GSTM1 axis as a key regulator in hepatocarcinogenesis, and suggests a potential target for the treatment of patients with HCC.

ObjectiveThe present study aimed to investigate alterations in white matter microstructure and spontaneous neural activity in male college smokers, and to further explore their associations with nicotine dependence. Given that adolescence and early adulthood represent critical periods for brain maturation, particularly for white matter development, understanding the neural correlates of smoking behavior during this stage is of substantial importance for both neuroscience and public health. MethodsA total of 115 male undergraduate students were initially recruited for this study. After quality control and exclusion procedures, 52 male college smokers and 42 demographically matched healthy non-smokers were included in the final analysis. All participants underwent multimodal magnetic resonance imaging (MRI), including diffusion tensor imaging (DTI) and resting-state functional MRI (rs-fMRI). White matter fiber tracts were reconstructed using the automated fiber quantification (AFQ) method, which enables precise identification and quantification of major fiber bundles. Eighteen major white matter tracts were segmented for each participant. Along the core trajectory of each tract, 100 equidistant nodes were sampled. Fractional anisotropy (FA) was calculated at each node to assess white matter microstructural integrity, while amplitude of low-frequency fluctuation (ALFF) was computed to evaluate spontaneous neural activity within white matter tracts. Between-group differences in FA and ALFF were assessed using two-sample t-tests, with appropriate corrections applied for multiple comparisons. Furthermore, Pearson correlation analyses were conducted to examine the relationships between imaging-derived metrics (FA and ALFF values in regions showing significant group differences) and nicotine dependence severity, as measured by the Fagerström test for nicotine dependence (FTND). ResultsCompared with healthy non-smokers, male college smokers exhibited significantly increased FA values in several white matter tracts, including the left thalamic radiation, right corticospinal tract, forceps major of the corpus callosum, left uncinate fasciculus, and right arcuate fasciculus. These findings suggest altered microstructural organization or increased directional coherence within these pathways. In addition, smokers demonstrated significantly elevated ALFF values in the forceps major, right uncinate fasciculus, and left arcuate fasciculus, indicating enhanced spontaneous neural activity in these white matter regions. Correlation analyses revealed that FA values in the left thalamic radiation and right corticospinal tract were negatively correlated with FTND scores, suggesting that higher levels of nicotine dependence were associated with reduced microstructural integrity or altered fiber organization in these regions. In contrast, ALFF values in the forceps major and right uncinate fasciculus were positively correlated with FTND scores, indicating that greater nicotine dependence was associated with increased spontaneous neural activity in specific white matter pathways. ConclusionThe present study provides evidence that male college smokers exhibit distinct alterations in both white matter microstructure and functional activity. These abnormalities are not uniformly distributed but rather localized to specific fiber tracts implicated in sensorimotor processing, interhemispheric communication, and higher-order cognitive and emotional regulation. Importantly, the observed associations between imaging metrics and nicotine dependence severity suggest that these structural and functional alterations may reflect neurobiological mechanisms underlying addiction. The combination of AFQ-based tract profiling and multimodal MRI offers a sensitive approach for detecting subtle changes along white matter pathways, highlighting its potential utility in identifying neuroimaging biomarkers of nicotine dependence. Overall, these findings indicate that smoking during early adulthood may disrupt ongoing white matter maturation, potentially leading to long-term consequences for brain function. This study provides novel insights into the neural basis of nicotine dependence and underscores the importance of early intervention and prevention strategies targeting young smokers.

ObjectiveStroke poses a heavy burden due to its high mortality and morbidity rates. Accurate and real-time detection of lesions is pivotal for prompt clinical intervention and favorable prognosis. Electrical impedance tomography (EIT) and microwave tomography (MWT) have emerged as compelling alternatives for stroke screening, owing to their non-ionizing, non-invasive and portable nature. EIT provides information on tissue conductivity, and MWT offers high sensitivity to changes in dielectric properties. However, single-modality imaging is inherently limited, EIT suffers from low sensitivity to deep-seated tissues and severe ill-posedness of inverse problems, whereas MWT is challenged by strong nonlinearity in inverse scattering and susceptibility to modeling errors. Consequently, the clinical utility of standalone EIT or MWT for stroke diagnosis remains constrained by poor spatial resolution and imaging artifacts. To improve the accuracy and robustness of stroke imaging, a dual-modality fusion conditional denoising diffusion probabilistic model (DM-DDPM) was proposed for high-precision dual-modality image reconstruction. MethodsA dual-encoder network with a symmetric architecture and independently trained parameters was constructed to extract heterogeneous features separately from EIT boundary voltage measurements and MWT scattered field signals. Attentional feature fusion (AFF) is employed to integrate complementary information from the two modalities adaptively, generating robust fused priors that suppress redundant noise while preserving key physical characteristics. Subsequently, the fused priors are embedded into a Transformer-based diffusion model via a cross attention mechanism to guide the reverse denoising process. This approach effectively reduces artifacts and enhances the stability of conductivity distribution reconstruction. Time step embedding is introduced to enable the network to perceive the diffusion stage and further improve the accuracy of noise prediction. ResultsSimulated experiments demonstrated that DM-DDPM significantly outperforms single-modality and multi-modality networks under various noise levels. A head model simulation dataset was constructed based on COMSOL Multiphysics, and tests were carried out under 50 dB, 40 dB and 30 dB signal-to-noise ratio levels. At 30 dB, the average relative error (RE) was below 0.20, while the structural similarity index measure (SSIM) and correlation coefficient (CC) remained above 0.90 and 0.89, respectively. Compared with single-modality and multi-modality networks, artifacts were significantly reduced, lesion edges were clearer, and localization was more accurate. The model maintains high reconstruction quality and strong robustness for single, double, and triple lesions simultaneously. Furthermore, physical experiments were conducted using a 16-electrode EIT system and a 16-antenna MWT system with asynchronous data acquisition. These experiments confirmed the feasibility of the method in real-world scenarios and demonstrated that it can robustly reconstruct simulated lesions despite environmental interference and measurement noise, validating its reliability for practical clinical applications. ConclusionThe proposed method effectively combines complementary dual-modality information with a conditional diffusion model. Low accuracy and poor noise resistance in single-modality imaging were effectively addressed, while the noise amplification issue caused by direct multimodal data fusion was avoided. The proposed algorithm exhibits strong anti-noise interference ability and high imaging stability in both simulation and physical experiments. Precise localization of stroke lesions with different quantities was achieved, providing a high-precision, and practical technical support for clinical stroke detection.

ObjectiveTo investigate the effects of modified Xiaoyaosan （JJXYS） on behavioral abnormalities and hippocampal mitochondrial quality control （MQC） in the rat model of post-myocardial infarction depression （PMD） and preliminarily explore its potential mechanism. MethodsA rat model of PMD was established by left anterior descending coronary artery ligation combined with chronic unpredictable mild stress （CUMS）. Rats were randomized into a control group， a model group， a fluoxetine （FLX， 10 mg·kg^-1） group， and low-， medium-， and high-dose JJXYS （JJXYS-L/M/H， 1.12， 2.24， 4.48 g·kg^-1， respectively） groups. Depressive-like behaviors were evaluated by body weight monitoring， sucrose preference test， open field test， and forced swimming test. Hematoxylin-eosin staining and Nissl staining were used to observe hippocampal histomorphology and neuronal changes. Enzyme-linked immunosorbent assay was conducted to determine the serum levels of 5-hydroxytryptamine （5-HT）， dopamine （DA）， interleukin-1 beta （IL-1β）， interleukin-6 （IL-6）， and tumor necrosis factor-alpha （TNF-α）. The mRNA levels of MQC-related genes including peroxisome proliferator-activated receptor-gamma coactivator-1 alpha （PGC-1α）， nuclear respiratory factor 1 （Nrf1）， and transcription factor A， mitochondrial （TFAM） in the hippocampal tissue were measured by real-time PCR. The expression of proteins related to the dynamin-related protein 1 （Drp1）/PTEN-induced putative kinase 1 （PINK1）/Parkin signaling pathway was determined by Western blot. ResultsCompared with the control group， the model group showed restricted body weight gain， aggravated depressive-like behaviors， declined serum 5-HT and DA levels， evident hippocampal neuronal damage and reduced Nissl bodies， as well as downregulated expression of MQC-related genes and proteins （P<0.05）. Compared with the model group， both FLX and JJXYS alleviated the above changes to varying degrees. Moreover， the JJXYS-M and JJXYS-H groups showed more pronounced effects， improving behavioral performance， restoring 5-HT and DA levels， alleviating hippocampal pathological injury， and upregulating the expression of PGC-1α/Nrf1/TFAM mRNA and Drp1/PINK1/Parkin signaling pathway-related proteins （P<0.05）. ConclusionJJXYS can significantly alleviate depressive-like behaviors and neurotransmitter imbalance in the rat model of PMD by regulating hippocampal MQC and upregulating the Drp1/PINK1/Parkin-related pathway. This study provides experimental evidence for the intervention of PMD with JJXYS.

BACKGROUND:During bone remodeling,bone formation and bone resorption are spatially and temporally coordinated,involving intricate interactions between osteoclasts and osteoblasts.Isoginkgetin,a flavonoid found in Ginkgo biloba,has a wide range of anticancer activity and anti-reactive oxygen species activity;however,the effect of isoginkgetin on osteoclast differentiation is unknown.OBJECTIVE:To study the effect and mechanism of action of isoginkgetin on osteoclastogenesis.METHODS:In vitro studies were performed on mouse bone marrow-derived macrophages,and cell counting kit-8 cytotoxicity assay was used to detect the effect of isoginkgetin on cell viability of bone marrow-derived macrophages.Macrophage colony-stimulating factor and receptor activator of nuclear factor kappa-B ligand were used to induce the differentiation of bone marrow-derived macrophages to osteoclasts.Network pharmacology and molecular docking and molecular dynamics simulations were used to predict the processes and targets of the effects of isoginkgetin on the differentiation of osteoclasts.Tartrate-resistant acid phosphatase staining and F-actin staining were used to detect the effects of isoginkgetin on the differentiation and function of osteoclasts.Western blot and RT-PCR were used to detect the effects of isoginkgetin on the expression of genes and proteins related to osteoclast differentiation,reactive oxygen species,and PI3K/AKT pathways.Fluorescent probes were used to detect cellular and mitochondrial reactive oxygen species levels.Flow cytometry technology was used to detect reactive oxygen species levels in cells.RESULTS AND CONCLUSION:(1)Network pharmacology results showed that isoginkgetin affected osteoporosis mainly through the PI3K-AKT pathway and cellular response to drugs and hypoxia,and GSK3β,ESR1,MCL1 and CCNA2 were the key targets.(2)Cell counting kit-8 and tartrate-resistant acid phosphatase staining results showed that isoginkgetin at 8 μmol/L had the most significant inhibitory effect on osteoclastogenesis in vitro,and F-actin results showed that isoginkgetin inhibited osteoclast cytoskeletal actin ring formation in a concentration-dependent manner.(3)Molecular dynamics simulations showed that isoginkgetin bound well to osteoclastogenesis marker proteins(NFATc1,c-Fos,CTSK,and MMP9).Western blot and RT-PCR results indicated that isoginkgetin inhibited the expression of osteoclastogenesis marker proteins and genes(NFATc1,c-Fos,CTSK,and MMP9).(4)Western blot results showed that isoginkgetin inhibited the phosphorylation level of PI3K/AKT/GSK3β and suppressed osteoclastogenesis by activating the PI3K-AKT-GSK3β pathway.(5)The results of reactive oxygen species assay showed that isoginkgetin significantly reduced receptor activator of nuclear factor kappa-B ligand-induced cellular and mitochondrial reactive oxygen species production,and inhibited the differentiation of bone marrow-derived macrophages to osteoclasts.

BACKGROUND:β-Caryophyllene has a variety of pharmacological activities such as antioxidant,anti-inflammatory and anti-apoptotic,which may have a better therapeutic effect on osteoarthritis.OBJECTIVE:To investigate the effect and mechanism of β-caryophyllene on mouse osteoarthritis.METHODS:Forty C57BL/6J mice were randomly divided into sham group,model group,low-dose β-caryophyllene group and high-dose β-caryophyllene group,with 10 mice in each group.Hulth method was used to construct an osteoarthritis model in the latter three groups.Four weeks after modeling,70 and 140 mg/kg/d β-caryophyllene was intragastrically given in the low-and high-dose β-caryophyllene groups,respectively,and normal saline was given by gavage in the sham group and the model group,once a day,for 4 weeks.After administration,knee joint morphological changes were observed by hematoxylin-eosin staining,serum levels of inflammatory factors(tumor necrosis factor-α,interleukin-1β,interleukin-6,and interleukin-10)were detected by ELISA,and oxidative stress indexes(glutathione peroxidase,superoxide dismutase,and malondialdehyde)were detected by chemiluminescence.The expression levels of key proteins in the Sonic hedgehog(Shh)/glioma associated oncogene homolog 1(Gli1)signaling pathway were detected by immunohistochemistry and western blot.RESULTS AND CONCLUSION:(1)Compared with the sham group,a large number of inflammatory cells infiltrated in the knee joint of mice in the model group,cartilage tissue was seriously damaged,serum levels of tumor necrosis factor-α,interleukin-1β,interleukin-6,interleukin-10 and malondialdehyde were significantly increased(P＜0.01),the activities of glutathione peroxidase and superoxide dismutase were significantly decreased(P＜0.01),and the relative expression levels of Shh and Gli1 in the knee joint were significantly increased(P＜0.01).(2)Compared with the model group,in the low-and high-dose β-caryophyllene groups,inflammatory cell infiltration in the mouse knee joint was decreased,cartilage tissue injury was alleviated,serum levels of tumor necrosis factor-α,interleukin-1 β,interleukin-6 and malondialdehyde were significantly decreased(P＜0.05),the activities of glutathione peroxidase and superoxide dismutase were significantly increased(P＜0.01),and the expression levels of Shh and Gli1 in the knee joint were significantly decreased(P＜0.01).The above-mentioned improvements were more significant in the high-dose β-caryophyllene group than the low-dose β-caryophyllene group.To conclude,β-caryophyllene can improve osteoarthritis,and its mechanism may be related to reducing inflammation and oxidative stress damage by regulating the Shh/Gli1 signaling pathway.

OBJECTIVE:Based on different algorithms of machine learning,the prediction model of lumbar disc herniation has become a trend and hot spot in the development of precision medicine.However,there is limited evidence on the reporting quality and methodological quality of prediction models of lumbar disc herniation outcomes using machine learning.This article is aimed to explore the performance of machine learning algorithms in predicting the prognosis of lumbar disc herniation by comprehensively analyzing the report quality and risk of bias of previous studies that developed and validated prognosis prediction models based on machine learning through a comprehensive literature search,in order to explore the performance of machine learning algorithms in predicting the prognosis of lumbar disc herniation.METHODS:The databases of CNKI,WanFang,VIP,SinOMED,PubMed,Web of Science,Embase,and The Cochrane Library were searched by computer.Studies on the use of machine learning to develop(and/or validate)prognostic prediction models for lumbar disc herniation were collected from the inception of the database to December 31,2023.Two researchers independently screened the literature,extracted data,and assessed the risk of bias of the included studies.The reporting quality and risk of bias of the included studies were assessed by the Multivariable Transparent Reporting of Predictive Models(TRIPOD)statement and the Predictive Model Risk of Bias Assessment Tool(PROBAST).The results of the evaluation were analyzed using descriptive statistics and visual charts.RESULTS:(1)A total of 23 articles were included,and the TRIPOD compliance of each study ranged from 11％to 87％,with a median compliance of 54％.The quality of reporting of titles,detailed descriptions of treatment measures,blinding of predictors,handling of missing data,details of risk stratification,specific procedures for enrollment,model interpretation,and model performance was mostly poor,with TRIPOD adherence rates ranging from 4％to 35％.(2)Of all included studies,61％had a high risk of bias and 39％had an unclear overall risk of bias.The area under the curve,accuracy,sensitivity and specificity were used to evaluate the performance of the model.The areas under the curve of 20 models were reported,ranging from 0.561 to 0.999.Three models reported the accuracy of the model,ranging from 82.07％to 89.65％.(3)Among all included studies,the statistical analysis domain was most often assessed as having a high risk of bias,mainly due to the small number of valid samples,the selection of predictors based on univariate analysis and the lack of calibration and discrimination assessment of the model in the study.CONCLUSION:These results indicate that machine learning can achieve good predictive ability in the development and validation of prognostic models for lumbar disc herniation.The commonly used algorithms include regression algorithm,support vector machine,decision tree,random forest,artificial neural network,naive Bayes and other algorithms.Reasonable algorithms combined with clinical practice can improve the accuracy of prognosis prediction of lumbar disc herniation.However,the reporting and methodological quality of prognosis prediction models based on machine learning are poor,the prediction performance of different models varies greatly,and the generalization and extrapolation of research models are unclear.There is an urgent need to improve the design,implementation and reporting of such studies.To promote the application of machine learning in the clinical practice of lumbar disc herniation prediction models,it is necessary to comprehensively consider various predictors related to the prognosis of the disease before modeling,and strictly follow the relevant standards of PROBAST tool during modeling.

ObjectivePancreatic ductal adenocarcinoma (PDAC) exhibits a limited response to current treatments due to its dense fibrotic stroma and highly immunosuppressive tumor microenvironment. In recent years, advancements in cellular immunotherapy, particularly chimeric antigen receptor macrophage (CAR-M) therapy, have offered new hope for pancreatic cancer treatment. Although CAR-M therapy demonstrates dual potential in directly killing tumor cells and remodeling the immune microenvironment, it still faces challenges such as complex in vitro preparation processes and low in vivo targeting and delivery efficiency. Therefore, developing strategies for efficient and targeted in vivo delivery of CAR genes has become crucial for overcoming current therapeutic limitations. This study aims to develop an orally administrable nano-gene delivery system for the targeted delivery of CAR genes to pancreatic tumor sites. MethodsCore nano-gene particles (PNP/pCAR) were constructed by loading plasmid DNA encoding CAR (pCAR) with cationic polypeptides (PNP). Subsequently, PNP/pCAR was surface-modified with β-glucan to prepare the targeted nanoparticles (βGlus-PNP/pCAR). The loading efficiency of PNP for pCAR was quantitatively assessed by gel retardation assay. The particle size, Zeta potential, morphology, and storage stability of PNP/pCAR were characterized using a Malvern particle size analyzer and transmission electron microscopy. At the cellular level, RAW 264.7 macrophages were selected. The cytotoxicity of PNP/pCAR was evaluated using the CCK-8 assay. The cellular uptake efficiency and lysosomal escape ability of the nanoparticles were assessed via flow cytometry and confocal microscopy. Transfection efficiency was quantitatively evaluated by detecting the expression of the reporter gene GFP using flow cytometry. At the in vivo level, an orthotopic pancreatic cancer mouse model was established. Cy7-labeled βGlus-PNP/pCAR nanoparticles were administered orally, and the fluorescence distribution in mice was dynamically monitored at 1, 2, 4, 8, and 16 h post-administration using a small animal in vivo imaging system. Forty-eight hours after oral gavage, the mice were euthanized, and pancreatic tumor tissues were collected for further analysis of intratumoral fluorescence signals using the imaging system. Additionally, βGlus-PNP/pCAR-GFP nanoparticles loaded with the reporter gene (GFP) were administered orally. Forty-eight hours post-administration, pancreatic tumor tissues were harvested to prepare frozen sections, and GFP expression was observed and analyzed under a fluorescence microscope. ResultsThe PNP carrier exhibited a high loading capacity for pCAR. The successfully prepared PNP/pCAR nanoparticles were regular spheres with a hydrodynamic diameter of approximately (120±10) nm and a Zeta potential of about +(6±1) mV. They maintained good structural stability after incubation in PBS buffer for 7 d. Cell experiments demonstrated that PNP/pCAR exhibited no significant cytotoxicity in RAW 264.7 cells while being efficiently internalized and effectively escaping lysosomal degradation. The transfection positive rate of PNP/pCAR-GFP in RAW 264.7 cells reached (25±3)%, surpassing that of Lipofectamine 2000-loaded pCAR-GFP (Lipo/pCAR-GFP), which was (20±1)%.In vivo experiments revealed that, compared to unmodified PNP/pCAR, βGlus-PNP/pCAR exhibited strongerin situ pancreatic tumor targeting ability after oral administration. Furthermore, oral administration of βGlus-PNP/pCAR-GFP resulted in significant GFP protein expression detectable within pancreatic tumor tissues. ConclusionThis study successfully constructed and validated an orally administrable, pancreatic cancer-targeting polypeptide-based nano-gene delivery system. It provides an important technological foundation in delivery systems and experimental basis for the subsequent development of in situ CAR-M-based therapeutic strategies for pancreatic cancer.