Dihydroartemisinin Regulated the MMP-Mediated Cellular Microenvironment to Alleviate Rheumatoid Arthritis.

Rheumatoid arthritis (RA) is an autoimmune disease with features of synovial inflammation, cartilage erosion, bone destruction, and pain and is currently lacking a satisfactory treatment strategy. Dihydroartemisinin (DHA), the active metabolite of artemisinin, has exhibited outstanding suppressive effects on RA without obvious side effects. However, the underlying mechanisms remain unclear, which limits its further clinical application. The purpose of this study is to reveal the pharmacodynamic mechanism of DHA against RA by means of a combination of single-cell RNA sequencing (RNA-seq), proteomics, as well as transcriptomics both in vivo and in vitro. In our results, DHA effectively reduced the degree of redness, swelling, and pain in RA rats and dramatically changed the synovial tissue microenvironment under the pathological state. Within this microenvironment, fibroblasts, macrophages, B cells, and endothelial cells were the major affected cell types, primarily through DHA targeting the extracellular matrix (ECM) structural constituent signaling pathway. In addition, we confirmed that DHA regulated the ECM by modulating matrix metalloproteinase 2 (MMP2) and MMP3 in the synovial tissue of RA rats. Moreover, DHA induced apoptosis in MH7A cells, further validating the bioinformatics data. In conclusion, DHA effectively reduced the inflammatory response and improved the immune microenvironment in synovial tissue by inhibiting MMP2 and MMP3. Our findings provide a basis for the application of DHA in the treatment of RA.

Chemoproteomics-based profiling reveals potential antimalarial mechanism of Celastrol by disrupting spermidine and protein synthesis.

BACKGROUND: Malaria remains a global health burden, and the emergence and increasing spread of drug resistance to current antimalarials poses a major challenge to malaria control. There is an urgent need to find new drugs or strategies to alleviate this predicament. Celastrol (Cel) is an extensively studied natural bioactive compound that has shown potentially promising antimalarial activity, but its antimalarial mechanism remains largely elusive. METHODS: We first established the Plasmodium berghei ANKA-infected C57BL/6 mouse model and systematically evaluated the antimalarial effects of Cel in conjunction with in vitro culture of Plasmodium falciparum. The potential antimalarial targets of Cel were then identified using a Cel activity probe based on the activity-based protein profiling (ABPP) technology. Subsequently, the antimalarial mechanism was analyzed by integrating with proteomics and transcriptomics. The binding of Cel to the identified key target proteins was verified by a series of biochemical experiments and functional assays. RESULTS: The results of the pharmacodynamic assay showed that Cel has favorable antimalarial activity both in vivo and in vitro. The ABPP-based target profiling showed that Cel can bind to a number of proteins in the parasite. Among the 31 identified potential target proteins of Cel, PfSpdsyn and PfEGF1-α were verified to be two critical target proteins, suggesting the role of Cel in interfering with the de novo synthesis of spermidine and proteins of the parasite, thus exerting its antimalarial effects. CONCLUSIONS: In conclusion, this study reports for the first time the potential antimalarial targets and mechanism of action of Cel using the ABPP strategy. Our work not only support the expansion of Cel as a potential antimalarial agent or adjuvant, but also establishes the necessary theoretical basis for the development of potential antimalarial drugs with pentacyclic triterpenoid structures, as represented by Cel. Video Abstract.

Glycyrrhetinic acid inhibits non-small cell lung cancer via promotion of Prdx6- and caspase-3-mediated mitochondrial apoptosis.

Glycyrrhetinic acid (GA) shows great efficiency against non-small cell lung cancer (NSCLC), but the detailed mechanism is unclear, which has limited its clinical application. Herein, we investigated the potential targets of GA against NSCLC by activity-based protein profiling (ABPP) technology and the combination of histopathology and proteomics validation. In vitro and in vivo results indicated GA significantly inhibited NSCLC via promotion of peroxiredoxin-6 (Prdx6) and caspase-3 (Casp3)-mediated mitochondrial apoptosis. This original finding will provide theoretical and data support to improve the treatment of NSCLC with the application of GA.

Metabolomics in drug research and development: The recent advances in technologies and applications.

Emerging evidence has demonstrated the vital role of metabolism in various diseases or disorders. Metabolomics provides a comprehensive understanding of metabolism in biological systems. With advanced analytical techniques, metabolomics exhibits unprecedented significant value in basic drug research, including understanding disease mechanisms, identifying drug targets, and elucidating the mode of action of drugs. More importantly, metabolomics greatly accelerates the drug development process by predicting pharmacokinetics, pharmacodynamics, and drug response. In addition, metabolomics facilitates the exploration of drug repurposing and drug-drug interactions, as well as the development of personalized treatment strategies. Here, we briefly review the recent advances in technologies in metabolomics and update our knowledge of the applications of metabolomics in drug research and development.

Advanced Strategies for Overcoming Endosomal/Lysosomal Barrier in Nanodrug Delivery.

Nanocarriers have therapeutic potential to facilitate drug delivery, including biological agents, small-molecule drugs, and nucleic acids. However, their efficiency is limited by several factors; among which, endosomal/lysosomal degradation after endocytosis is the most important. This review summarizes advanced strategies for overcoming endosomal/lysosomal barriers to efficient nanodrug delivery based on the perspective of cellular uptake and intracellular transport mechanisms. These strategies include promoting endosomal/lysosomal escape, using non-endocytic methods of delivery to directly cross the cell membrane to evade endosomes/lysosomes and making a detour pathway to evade endosomes/lysosomes. On the basis of the findings of this review, we proposed several promising strategies for overcoming endosomal/lysosomal barriers through the smarter and more efficient design of nanodrug delivery systems for future clinical applications.

Metabolomics Identifies a Panel of Diagnostic Biomarkers for Early Human Embryonic Development Arrest.

Early embryonic development arrest (EEDA) is a unique form of early spontaneous abortion in pregnant women, which is previously suggested to be associated with metabolic abnormalities. Noninvasive biomarkers would significantly improve its diagnosis and clinical outcome. Here, we performed a targeted metabolomics study in plasma from EEDA patients (n = 27) and normal pregnant women (NPW, n = 27) using liquid chromatography coupled with mass spectrometry (LC-MS) to identify potential diagnostic marker metabolites. Our results showed significantly different plasma metabolic profiles between EEDA patients and NPW. Particularly, EEDA patients showed significant alterations in amino acid, carbohydrate, and vitamin metabolism, which were characterized by 21 significantly increased metabolites and five decreased metabolites in plasma. Further receiver operating characteristic analysis showed that an optimal combination of S-methyl-5'-thioadenosine, kynurenine, leucine, and malate could be used as a panel of metabolites for EEDA diagnosis. The area under the curve of the metabolite panel was 0.941, suggesting a better performance than any single metabolite for the diagnosis of EEDA. In summary, our study identifies a panel of differential metabolites in plasma that could act as potential biomarkers for the diagnosis of EEDA in clinical settings.

Induction of mouse totipotent stem cells by a defined chemical cocktail.

In mice, only the zygotes and blastomeres from 2-cell embryos are authentic totipotent stem cells (TotiSCs) capable of producing all the differentiated cells in both embryonic and extraembryonic tissues and forming an entire organism1. However, it remains unknown whether and how totipotent stem cells can be established in vitro in the absence of germline cells. Here we demonstrate the induction and long-term maintenance of TotiSCs from mouse pluripotent stem cells using a combination of three small molecules: the retinoic acid analogue TTNPB, 1-azakenpaullone and the kinase blocker WS6. The resulting chemically induced totipotent stem cells (ciTotiSCs), resembled mouse totipotent 2-cell embryo cells at the transcriptome, epigenome and metabolome levels. In addition, ciTotiSCs exhibited bidirectional developmental potentials and were able to produce both embryonic and extraembryonic cells in vitro and in teratoma. Furthermore, following injection into 8-cell embryos, ciTotiSCs contributed to both embryonic and extraembryonic lineages with high efficiency. Our chemical approach to totipotent stem cell induction and maintenance provides a defined in vitro system for manipulating and developing understanding of the totipotent state and the development of multicellular organisms from non-germline cells.

Targeting acetylcholine signaling modulates persistent drug tolerance in EGFR-mutant lung cancer and impedes tumor relapse.

Although first-line epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) therapy is effective for treating EGFR-mutant non-small cell lung cancer (NSCLC), it is now understood that drug-tolerant persister (DTP) cells escaping from initial treatment eventually drives drug resistance. Here, through integration of metabolomics and transcriptomics, we found that the neurotransmitter acetylcholine (ACh) was specifically accumulated in DTP cells, and demonstrated that treatment with EGFR-TKI heightened the expression of the rate-limiting enzyme choline acetyltransferase (ChAT) in ACh biosynthesis via YAP mediation. Genetic and pharmacological manipulation of ACh biosynthesis or ACh signaling could predictably regulate the extent of DTP formation in vitro and in vivo. Strikingly, pharmacologically targeting ACh/M3R signaling with an FDA-approved drug, darifenacin, retarded tumor relapse in vivo. Mechanistically, upregulated ACh metabolism mediated drug tolerance in part through activating WNT signaling via ACh muscarinic receptor 3 (M3R). Importantly, we showed that aberrant ACh metabolism in patients with NSCLC played a potential role in predicting EGFR-TKI response rate and progression-free survival. Our study therefore defines a therapeutic strategy - targeting the ACh/M3R/WNT axis - for manipulating EGFR TKI drug tolerance in the treatment of NSCLC.

Ginseng total saponins and Fuzi total alkaloids exert antidepressant-like effects in ovariectomized mice through BDNF-mTORC1, autophagy and peripheral metabolic pathways.

BACKGROUND: Shenfu decoction (SFD) is a classic Chinese medicine prescription that has a strong cardiotonic effect. The combination of ginseng (the dried root of Panax ginseng C. A. Meyer) and Fuzi (processed product of sub-root of Aconitum carmichaeli Debx), the main constituents of SFD, has been reported to improve the pharmacological effect of each other. Moreover, research has shown that the main active components of SFD, ginseng total saponins (GTS) and Fuzi total alkaloids (FTA), have antidepressant activity. However, the effects of these ingredients on depressive-like behavior induced by ovariectomy, a model of menopausal depression, have not been studied. PURPOSE: Our research aims to elucidate the antidepressant-like effects of GTS and FTA compatibility (GF) in ovariectomized mice and the potential mechanisms. METHODS: To elucidate the antidepressant-like effects of GF in mice in ovariectomy condition, behavioral tests were performed after 7 days of intragastric administration of different doses of GF. Underlying molecular mechanisms of CREB-BDNF, BDNF-mTORC1 and autophagy signaling were detected by western blotting, serum metabolites were examined by UPLC-QE plus-MS and dendritic spine density was determined by Golgi-Cox staining. RESULTS: GF remarkably decreased the immobility time in the forced swim test. GF also increased levels of pCREB/CREB, BDNF, Akt, mTORC1 and p62 in the prefrontal cortex and hippocampus, as well as decreased LC3-II/LC3-I in the prefrontal cortex and hippocampus of ovariectomized mice. Furthermore, 15 serum differential metabolites (9 of which are lipids and lipid molecules) were identified by metabonomics. Next, the antidepressant-like effects of GF was blocked by rapamycin, an inhibitor of mTORC1. The antidepressant actions of GF on levels of pCREB, mTORC1, LC3-Ⅱ/LC3-Ⅰ and p62 in the prefrontal cortex and the levels of BDNF, Akt, mTORC1 and p62 in the hippocampus were inhibited by rapamycin, and the dendritic spines density was also regulated. CONCLUSION: GF has antidepressant effects in ovariectomized mice, and like other antidepressants, these effects involve activation of BDNF-mTORC1, autophagy regulation and consequent effects on hippocampal synaptic plasticity. Moreover, metabolomic results suggest that GF also has effects on peripheral lipid profiles that may provide potential biomarkers for these antidepressant-like effects. These results indicate that GF is worthy of further exploration as a promising pharmaceutical treatment for depression. This study provides a new direction for the development of new indications for traditional Chinese medicine compounds.

Cancer-associated fibroblasts employ NUFIP1-dependent autophagy to secrete nucleosides and support pancreatic tumor growth.

Cancer-associated fibroblasts (CAFs) are one of the most prominent and active components in the pancreatic tumor microenvironment. Our data show that CAFs are critical for survival from pancreatic ductal adenocarcinoma (PDAC) on glutamine deprivation. Specifically, we uncovered a role for nucleosides, which are secreted by CAFs through autophagy in a nuclear fragile X mental retardation-interacting protein 1 (NUFIP1)-dependent manner, increased glucose utilization and promoted growth of PDAC. Moreover, we demonstrate that CAF-derived nucleosides induced glucose consumption under glutamine-deprived conditions and displayed a dependence on MYC. Using an orthotopic mouse model of PDAC, we found that inhibiting nucleoside secretion by targeting NUFIP1 in the stroma reduced tumor weight. This finding highlights a previously unappreciated metabolic network within pancreatic tumors in which diverse nutrients are used to promote growth in an austere tumor microenvironment.

Treatment of SARS-CoV-2-induced pneumonia with NAD+ and NMN in two mouse models.

The global COVID-19 epidemic has spread rapidly around the world and caused the death of more than 5 million people. It is urgent to develop effective strategies to treat COVID-19 patients. Here, we revealed that SARS-CoV-2 infection resulted in the dysregulation of genes associated with NAD+ metabolism, immune response, and cell death in mice, similar to that in COVID-19 patients. We therefore investigated the effect of treatment with NAD+ and its intermediate (NMN) and found that the pneumonia phenotypes, including excessive inflammatory cell infiltration, hemolysis, and embolization in SARS-CoV-2-infected lungs were significantly rescued. Cell death was suppressed substantially by NAD+ and NMN supplementation. More strikingly, NMN supplementation can protect 30% of aged mice infected with the lethal mouse-adapted SARS-CoV-2 from death. Mechanically, we found that NAD+ or NMN supplementation partially rescued the disturbed gene expression and metabolism caused by SARS-CoV-2 infection. Thus, our in vivo mouse study supports trials for treating COVID-19 patients by targeting the NAD+ pathway.

Circulatory metabolites trigger ex vivo arterial endothelial cell dysfunction in population chronically exposed to diesel exhaust.

BACKGROUND: Chronic exposure to diesel exhaust has a causal link to cardiovascular diseases in various environmental and occupational settings. Arterial endothelial cell function plays an important role in ensuring proper maintenance of cardiovascular homeostasis and the endothelial cell dysfunction by circulatory inflammation is a hallmark in cardiovascular diseases. Acute exposure to diesel exhaust in controlled exposure studies leads to artery endothelial cells dysfunction in previous study, however the effect of chronic exposure remains unknown. RESULTS: We applied an ex vivo endothelial biosensor assay for serum samples from 133 diesel engine testers (DETs) and 126 non-DETs with the aim of identifying evidence of increased risk for cardiovascular diseases. Environmental monitoring suggested that DETs were exposed to high levels of diesel exhaust aerosol (282.3 µg/m3 PM2.5 and 135.2 µg/m3 elemental carbon). Surprisingly, chronic diesel exhaust exposure was associated with a pro-inflammatory phenotype in the ex vivo endothelial cell model, in a dose-dependent manner with CCL5 and VCAM as most affected genes. This dysfunction was not mediated by reduction in circulatory pro-inflammatory factors but significantly associated with a reduction in circulatory metabolites cGMP and an increase in primary DNA damage in leucocyte in a dose-dependent manner, which also explained a large magnitude of association between diesel exhaust exposure and ex vivo endothelial biosensor response. Exogenous cGMP addition experiment further confirmed the induction of ex vivo biosensor gene expressions in endothelial cells treated with physiologically relevant levels of metabolites cGMP. CONCLUSION: Serum-borne bioactivity caused the arterial endothelial cell dysfunction may attribute to the circulatory metabolites based on the ex vivo biosensor assay. The reduced cGMP and increased polycyclic aromatic hydrocarbons metabolites-induced cyto/geno-toxic play important role in the endothelial cell dysfunction of workers chronic exposure to diesel exhaust.

Metabolic characterization of hypertrophic cardiomyopathy in human heart.

Hypertrophic cardiomyopathy (HCM) is a common inherited cardiovascular disease with heterogeneous clinical presentations, governed by multiple molecular mechanisms. Metabolic perturbations underlie most cardiovascular diseases; however, the metabolic alterations and their function in HCM are unknown. Here, we describe the metabolome and lipidome of heart and plasma samples from individuals with and without HCM. Correlation analyses showed strong association between metabolic alterations and cardiac function and prognosis of patients with HCM. Using machine learning we identified metabolite panels as potential HCM diagnostic markers or predictors of survival. Clustering based on metabolome and lipidome of heart enabled stratification of patients with HCM into three subgroups with distinct cardiac function and survival. Integration of metabolomics and proteomics data identified metabolic pathways significantly altered in patients with HCM, with the pentose phosphate pathway and oxidative stress being particularly upregulated. Thus, targeting the pentose phosphate pathway and oxidative stress may serve as potential therapeutic strategies for HCM.

Aberrant NAD+ metabolism underlies Zika virus-induced microcephaly.

Zika virus (ZIKV) infection during pregnancy can cause microcephaly in newborns, yet the underlying mechanisms remain largely unexplored. Here, we reveal extensive and large-scale metabolic reprogramming events in ZIKV-infected mouse brains by performing a multi-omics study comprising transcriptomics, proteomics, phosphoproteomics and metabolomics approaches. Our proteomics and metabolomics analyses uncover dramatic alteration of nicotinamide adenine dinucleotide (NAD+)-related metabolic pathways, including oxidative phosphorylation, TCA cycle and tryptophan metabolism. Phosphoproteomics analysis indicates that MAPK and cyclic GMP-protein kinase G signaling may be associated with ZIKV-induced microcephaly. Notably, we demonstrate the utility of our rich multi-omics datasets with follow-up in vivo experiments, which confirm that boosting NAD+ by NAD+ or nicotinamide riboside supplementation alleviates cell death and increases cortex thickness in ZIKV-infected mouse brains. Nicotinamide riboside supplementation increases the brain and body weight as well as improves the survival in ZIKV-infected mice. Our study provides a comprehensive resource of biological data to support future investigations of ZIKV-induced microcephaly and demonstrates that metabolic alterations can be potentially exploited for developing therapeutic strategies.

Simultaneous 3-Nitrophenylhydrazine Derivatization Strategy of Carbonyl, Carboxyl and Phosphoryl Submetabolome for LC-MS/MS-Based Targeted Metabolomics with Improved Sensitivity and Coverage.

Metabolomics is a powerful and essential technology for profiling metabolic phenotypes and exploring metabolic reprogramming, which enables the identification of biomarkers and provides mechanistic insights into physiology and disease. However, its applications are still limited by the technical challenges particularly in its detection sensitivity for the analysis of biological samples with limited amount, necessitating the development of highly sensitive approaches. Here, we developed a highly sensitive liquid chromatography tandem mass spectrometry method based on a 3-nitrophenylhydrazine (3-NPH) derivatization strategy that simultaneously targets carbonyl, carboxyl, and phosphoryl groups for targeted metabolomic analysis (HSDccp-TM) in biological samples. By testing 130 endogenous metabolites including organic acids, amino acids, carbohydrates, nucleotides, carnitines, and vitamins, we showed that the derivatization strategy resulted in significantly improved detection sensitivity and chromatographic separation capability. Metabolic profiling of merely 60 oocytes and 5000 hematopoietic stem cells primarily isolated from mice demonstrated that this method enabled routine metabolomic analysis in trace amounts of biospecimens. Moreover, the derivatization strategy bypassed the tediousness of inferring the MS fragmentation patterns and simplified the complexity of monitoring ion pairs of metabolites, which greatly facilitated the metabolic flux analysis (MFA) for glycolysis, the tricarboxylic acid (TCA) cycle, and pentose phosphate pathway (PPP) in cell cultures. In summary, the novel 3-NPH derivatization-based method with high sensitivity, good chromatographic separation, and broad coverage showed great potential in promoting metabolomics and MFA, especially in trace amounts of biospecimens.

Integrated cytokine and metabolite analysis reveals immunometabolic reprogramming in COVID-19 patients with therapeutic implications.

Cytokine release syndrome (CRS) is a major cause of the multi-organ injury and fatal outcome induced by SARS-CoV-2 infection in severe COVID-19 patients. Metabolism can modulate the immune responses against infectious diseases, yet our understanding remains limited on how host metabolism correlates with inflammatory responses and affects cytokine release in COVID-19 patients. Here we perform both metabolomics and cytokine/chemokine profiling on serum samples from healthy controls, mild and severe COVID-19 patients, and delineate their global metabolic and immune response landscape. Correlation analyses show tight associations between metabolites and proinflammatory cytokines/chemokines, such as IL-6, M-CSF, IL-1α, IL-1ß, and imply a potential regulatory crosstalk between arginine, tryptophan, purine metabolism and hyperinflammation. Importantly, we also demonstrate that targeting metabolism markedly modulates the proinflammatory cytokines release by peripheral blood mononuclear cells isolated from SARS-CoV-2-infected rhesus macaques ex vivo, hinting that exploiting metabolic alterations may be a potential strategy for treating fatal CRS in COVID-19.

Illuminating a time-response mechanism in mice liver after PM2.5 exposure using metabolomics analysis.

PM2.5 is recognized as an atmospheric pollutant that seriously jeopardizes human health. Emerging evidence indicates that PM2.5 exposure is associated with metabolic disorders. Existing epidemiology and toxicology studies on the health effects of PM2.5 usually focused on its different components and doses, the effects on susceptible populations, or the effects of indoor and outdoor pollution. The underlying mechanisms of exposure time are poorly understood. Liver, as the central organ involved in various metabolisms, has special signaling pathways non-existed in lung and cardiovascular systems. Exacerbation in liver by the prolonged exposure of PM2.5 leads to hepatic function disorder. It is therefore essential to elucidate the mechanism underlying hepatotoxicity after PM2.5 exposure from the perspective of time-response relationship. In this study, targeted metabolomics was utilized to explore the hepatic injury in mice after PM2.5 exposure. Our results showed that prolonged exposure of PM2.5 would aggravate liver metabolic disorders. The metabolic process was divided into three phases. In phase I, it was found that PM2.5 exposure disturbed the hepatic urea synthesis. In phase II, oxidative damages and inflammations obviously occurred in liver, which would further cause neurobehavioral disorders and fat deposits. In phase III, the changes of metabolites and metabolic pathways indicated that the liver has been severely damaged, with the accelerated biosynthesis and fat metabolism. Finally, using ROC analysis coupled with their biological functions, 4 potential biomarkers were screened out, with which we established a method to classify and diagnose the progress of liver damage in mice after PM2.5 exposure. In this paper, we not only established the time-response relationship of PM2.5, but also provided new insights for the classification and prediction of the toxic injury stages in mice liver, which provides a ground work for the future drug intervention to prevent oxidative damage of PM2.5.

Emerging Applications of Metabolomics in Clinical Pharmacology.

Metabolic disturbances have been associated with many human diseases, including cancer, diabetes, and cardiovascular disease. Metabolomics, a rapidly growing member of the -omics family, investigates cellular metabolism by quantifying metabolites on a large scale and provides a link between metabolic pathways and the upstream genome that governs them. With the advances in analytical technologies, metabolomics is becoming a powerful tool for identifying diagnostic biomarkers of diseases, elucidating the pathological mechanisms, discovering novel drug targets, predicting drug responses, interpreting the mechanisms of drug action, as well as enabling precision treatment of patients. In this review, we highlight the recent advances of technologies and methodologies in metabolomics and their applications to the field of clinical pharmacology. Recent publications from 2013 to 2018 are covered in the review, and current challenges and potential future directions in the field are also discussed.

Targeting the Oncogene KRAS Mutant Pancreatic Cancer by Synergistic Blocking of Lysosomal Acidification and Rapid Drug Release.

Survival of KRAS mutant pancreatic cancer is critically dependent on reprogrammed metabolism including elevated macropinocytosis, autophagy, and lysosomal degradation of proteins. Lysosomal acidification is indispensable to protein catabolism, which makes it an exploitable metabolic target for KRAS mutant pancreatic cancer. Herein we investigated ultra-pH-sensitive micelles (UPSM) with pH-specific buffering of organelle pH and rapid drug release as a promising therapy against pancreatic cancer. UPSM undergo micelle-unimer phase transition at their apparent p Ka, with dramatically increased buffer capacity in a narrow pH range (<0.3 pH). Cell studies including amino acid profiling showed that UPSM inhibited lysosomal catabolism more efficiently than conventional lysosomotropic agents ( e. g., chloroquine) and induced cell apoptosis under starved condition. Moreover, pH-triggered rapid drug release from triptolide prodrug-loaded UPSM (T-UPSM) significantly enhanced cytotoxicity over non-pH-sensitive micelles (T-NPSM). Importantly, T-UPSM demonstrated superior safety and antitumor efficacy over triptolide and T-NPSM in KRAS mutant pancreatic cancer mouse models. Our findings suggest that the ultra-pH-sensitive nanoparticles are a promising therapeutic platform to treat KRAS mutant pancreatic cancer through simultaneous lysosomal pH buffering and rapid drug release.