Metabolic reprogramming targeting the mevalonate pathway represents an emerging innate immune activation target. However, its regulatory mechanisms remain incompletely elucidated. Here, we target the mevalonate pathway and construct a nano-granulated zoledronate (Nano-ZD) modulator. Following subcutaneous injection, Nano-ZD preferentially accumulates in draining lymph nodes rather than in bone tissues, enabling targeted delivery to innate immune cells. Nano-ZD functions as an immune-metabolic adjuvant, sensitizing and amplifying immune responses. By integrating Nano-ZD with the TLR4 agonist monophosphoryl lipid A (MPLA), MPLA-loaded Nano-ZD (Nano-ZDM) elicits robust humoral and antitumor cellular immunity. Mechanistically, Nano-ZD not only inhibits the isoprenylation of RhoA GTPases but also reduces coenzyme Q (CoQ) biosynthesis. CoQ deficiency disrupts oxidative phosphorylation (OXPHOS) and pyrimidine metabolism, causes mitochondrial ROS accumulation, induces mitochondrial antiviral protein (MAVS) oligomerization, and activates the pyrin inflammasome. This mevalonate-CoQ-OXPHOS/pyrimidine metabolism axis serves as a promising target for screening additional immune-metabolic adjuvants, and nanofabrication offers a paradigm for the lymph-targeted in vivo delivery of such adjuvants.

Radiofrequency ablation (RFA) has emerged as a predominant minimally invasive approach for liver tumors, achieving favorable therapeutic outcomes and triggering a systemic immune response via the release of tumor-associated antigens. However, postablation tumor recurrence remains a critical challenge in clinics. Our previous studies revealed that ablation-induced immunosuppression, primarily characterized by the expansion and activation of myeloid-derived suppressor cells (MDSCs), critically compromised the host's antitumor immunity, impeding residual tumor eradication and fostering a permissive niche for recurrence. To decisively address this fundamental limitation and harness the potential of ablation to stimulate antitumor immunity, we proposed a nanotherapeutic immunomodulatory strategy that synchronously reversed postablation MDSC-driven immunosuppression ("release the immunosuppressive brakes") while activating the stimulator of interferon gene (STING) pathway-mediated antitumor immune priming ("apply the immunostimulatory accelerator") in the systemic immune landscape. Through rational design, we engineered the nanotherapeutic immunomodulators (termed cA@NPs, where NPs are nanoparticles) coencapsulating the STING agonist 2',3'-cyclic guanosine adenosine monophosphate (cGAMP) and the MDSC-differentiating agent all-trans retinoic acid (ATRA), effectively overcoming their disparate pharmacokinetic profiles while ensuring spatiotemporally coordinated delivery. The residual tumor models provided definitive evidence that reversing postablation MDSC-driven immunosuppression was essential for achieving more effective antitumor immunity. Comprehensive preclinical evaluations further demonstrated the cA@NPs' robust capacity to suppress micrometastasis, distant tumors, and orthotopic lesions via multidimensional remodeling of the postablation immune landscape. These findings established a mechanistic foundation for the clinical translation of ablation-synergized nanotherapy, effectively bridging localized thermal intervention with systemic immune potentiation to address tumor recurrence.

The oral mucosa is a potent yet challenging site for vaccination, where physiological barriers and a tolerogenic immune microenvironment often limit effective immune response. However, existing delivery systems struggle to overcome these obstacles. Herein, we developed an oral mucosal nano-vaccine (MnZ-NV) via Mn2+-zoledronate coordination to co-encapsulate the Toll-like receptor 4 (TLR4) agonist Kdo2-lipid A (KLA) and antigen ovalbumin (OVA) within sub-50 nm particles. Upon sublingual administration, MnZ-NV demonstrated augmented mucosal penetration, lymph node targeting and dendritic cell activation, stimulating robust local and systemic cellular/humoral immunity. Furthermore, it induced trained immunity through splenic monocyte activation and bone marrow progenitor reprogramming, eliciting significant therapeutic and prophylactic efficacy against in-situ oral carcinoma, lung-metastatic melanoma, and pathogenic oral dysbiosis. This work provides a promising strategy that synergistically integrates innate, adaptive, and trained immunity for advanced mucosal immunotherapy.

In the post-pandemic era, the growing demands for vaccines in the prevention of infectious diseases and the treatment of tumors have accelerated the research and development of immune adjuvants. Recent efforts have focused on multi-component adjuvant systems that achieve immune potentiation through synergistic activation of distinct pathways. However, the role of multi-component adjuvants in the lymphatic delivery of vaccines to lymph nodes is often overlooked. Here, we developed a dual-component nano-adjuvant system with enhanced lymphatic delivery functionality. Based on the regulatory role of the mevalonate metabolism in innate immunity, we selected zoledronate (ZOL), an inhibitor of mevalonate pathway, and combined it with various canonical Toll-like receptor agonists (TLR-A) to construct a series of nano-vaccine systems containing dual adjuvant components (ZOL/TLR-A@Ag). Compared with treatment with TLR agonists alone, the addition of ZOL significantly enhanced the maturation of dendritic cells (DCs) and the secretion of pro-inflammatory cytokines. Nanoparticulated ZOL/TLR-A@Ag exhibited greater innate immune activation effects both in vitro and in vivo. More importantly, we demonstrated that nanoparticulated ZOL/TLR-A@Ag expanded lymphatic vessels and promoted lymphatic drainage. Furthermore, ZOL facilitated the penetration of ZOL/TLR-A@Ag into the deep regions of lymph nodes and amplified adaptive immune responses through dual effects on the subcapsular sinus macrophage (SSM) barrier in lymph nodes via cytotoxicity and immune activation. These effects ultimately resulted in robust antigen-specific IgG and T cell responses, as well as significant tumor suppression. Collectively, the dual-component nano-adjuvant system based on nanoparticulated zoledronate establishes a synergistic adjuvant paradigm that remodels lymphatic transport and amplifies immune responses through dual activation of metabolic and innate immune signaling pathways.

Therapeutic monoclonal antibodies have revolutionized the treatment of cancers, infectious diseases and immune disorders; however, their efficacy is compromised by limitations associated with systemic administration, including systemic toxicity, poor patient compliance and inadequate drug concentration at pathological sites. Hydrogels are promising carriers for localized antibody delivery, but conventional formulations fail to simultaneously address the dual challenges of uncontrolled antibody release and insufficient hydrogel retention at the target site. Herein, leveraging a peptide co-assembly strategy, we engineered an injectable dual-affinity self-assembling peptide hydrogel for localized antibody delivery. This hydrogel integrates three components: peptide FEK as the hydrogel matrix, MEP-FEK incorporating 4-mercaptoethylpyridine (MEP) for non-covalent antibody binding, and C1BP-FEK functionalized with the collagen-binding peptide TKKTLRT for tumor extracellular matrix anchoring. The dual-affinity hydrogel enabled controlled antibody release, exhibiting only 40% cumulative release by Day 90 and an 8.8-fold reduction in initial diffusion rate. In proof-of-concept studies using SKOV3 tumor-bearing mice, the hydrogel carrying trastuzumab as a model therapeutic antibody enhanced local antibody retention, reduced. systemic exposure, exhibited negligible toxicity, and achieved a tumor inhibition rate of 68% This dual-affinity strategy overcomes limitations of conventional affinity-based hydrogels, providing a biocompatible and versatile platform for localized antibody therapy in oncology and other biomedical applications.

Pancreatic ductal adenocarcinoma (PDAC) is driven by KRAS mutations in over 90% of cases yet remains refractory to most therapies due to poor antigen delivery and a suppressive tumor microenvironment. To address these challenges, we developed an exosome‑augmented, epitope‑focused mRNA nanovaccine. First, we engineered a series-connected (SC) mRNA that condenses five KRAS-derived immunogenic epitope segments, including G12D, G12V, G12R and two additional KRAS-derived sequences into a single open reading frame. This design minimizes non‑productive sequence and enhances antigen presentation compared to a conventional parallel-connected (PC) mixture of full‑length transcripts. Second, we cloaked β‑sitosterol LNPs with mature dendritic‑cell-derived exosomes (LNP@exo), endowing the particles with lymph‑node tropism and intrinsic adjuvanticity. The resulting mKRAS SC-LNP@exo triggered potent dendritic cell activation and Th1 cytokine release in vitro, rapidly accumulated in lymph nodes, and drove superior CD8⁺ T cell infiltration in a Pan02 tumor model. Remarkably, one‑third of mKRAS SC-LNP@exo treated mice achieved complete tumor regression without off‑target toxicity. These findings demonstrate that combining epitope‑condensed mRNA with exosome‑cloaked LNP delivery can convert "cold" KRAS‑mutant PDAC into an immunologically responsive tumor and provide a broadly applicable strategy for next‑generation mRNA cancer vaccines.

The access to non-equilibrium supramolecular structures and the systematic investigation of their dynamic transition processes hold great promise for the fields of biomaterials and supramolecular chemistry. However, the facile acquisition of non-equilibrium systems of self-assembling monomers for biomedical applications remains a formidable challenge. Herein, we attempted to prepare non-equilibrium supramolecular systems of self- assembling peptides by antisolvent precipitation methods. Based on extensive preparation, theoretical calculation and validation, it is revealed that previously inaccessible non-equilibrium nanoassemblies were successfully achieved in the aqueous phase under supersaturation control and solvent shifting mode selection during the precipitation process. This approach is applicable to a vast majority of self-assembling peptides investigated in this study. These nanoassemblies exhibited significantly distinct morphologies and secondary structures compared to their equilibrium states. Mechanistically, they underwent a three-phase process to transform into thermodynamically stable nanofibers. This study provides novel insights into the construction of diverse non- equilibrium nanoassemblies and sheds light on the underlying mechanisms governing their formation.

Rationale: Radiofrequency ablation (RFA), as a minimally invasive surgery strategy based on local thermal-killing effect, is widely used in the clinical treatment of multiple solid tumors. Nevertheless, RFA cannot achieve the complete elimination of tumor lesions with larger burden or proximity to blood vessels. Incomplete RFA (iRFA) has even been validated to promote residual tumor growth due to the suppressive tumor immune microenvironment (TIME). Therefore, exploring strategies to remodel TIME is a key issue for the development of RFA therapy. Methods: The negative effect of iRFA on colorectal cancer therapy was firstly investigated. Then a zoledronate-mineralized nanoparticle loaded with IFNγ (Nano-IFNγ/Zole) was designed and its tumor suppressive efficacy was evaluated. Finally, the metabolic reprogramming mechanism of Nano-IFNγ/Zole on tumor-associated macrophages (TAMs) was studied in detail. Results: We found iRFA dynamically altered TIME and promoted TAM differentiation from M1 to M2. Nano-IFNγ/Zole was fabricated to metabolically remodel TAMs. IFNγ in Nano-IFNγ/Zole concentrated in the ablation site to play a long-term remodeling role. Acting on mevalonate pathway, Nano-IFNγ/Zole was discovered to reduce lysosomal acidification and activate transcription factor TFEB by inhibiting isoprene modification of the Rab protein family. These mechanisms, in conjunction with IFNγ-activated JAK/STAT1 signaling, accelerated the reprogramming of TAMs from M2 to M1, and suppressed tumor recurrence after iRFA. Conclusions: This study elaborates the synergistic mechanism of zoledronate and IFNγ in Nano-IFNγ/Zole to reshape suppressive TIME caused by iRFA by remodeling TAMs, and highlights the important value of metabolically induced cellular reprogramming. Since both zoledronate and IFNγ have already been approved in clinics, this integrative nano-drug delivery system establishes an effective strategy with great translational promise to overcome the poor prognosis after clinically incomplete RFA.

A simple and real-time method for the direct and precise identification of safety margins is needed in ultrasound (US)-guided radiofrequency ablation (RFA). Additionally, modulation of the post-ablation immunosuppressive microenvironment is required. The aim of this study was to develop an injectable, ultrasound-visible and immunoregulatory hydrogel for ultrasonic monitoring and adjuvant immune regulation in liver tumor ablation. The experiment was performed in 2 steps. Step 1-The US visibility and stability of the markers were evaluated in vitro in bovine livers, followed by in vivo animal test in beagles. Step 2-Evaluation of the antitumoral effect after RFA of the specific US marker F127-MB@MSA-2, which has immunomodulating properties, in murine in vivo animal tumor models. In step 1, 24 candidate materials were tested for ultrasound visualization in bovine liver. Among these, air, 5 mm metal stick, Pluronic F127 hydrogel microbubbles (F127-MB), polyglycolic acid (PGA) absorbable sutures, ultrasonic couplant, and calcium alginate demonstrated clear ultrasonic visibility and enduring stability in vitro and in vivo. In step 2, the antitumoral effect of F127-MB@MSA-2 after incomplete RFA was demonstrated in murine H22 and CT26 tumor models. Tumor Flow cytometry and immunofluorescence staining revealed that F127-MB@MSA-2 gel reduced the infiltration of tumor-promoting cells, while it increased the proportion of CD8+ T cells within the tumors after RFA. Using animal models, we demonstrated the feasibility of US-guided administration of the multifunctional gel (F127-MB@MSA-2) prior to RFA, which helped to accurately ablate tumors and increase the anti-tumor effect.

Inducing immunogenic cell death (ICD) of cancer cells is the key for cytotoxic drugs to improve the synergy with immunotherapy. However, due to the immunosuppressive microenvironment and the cytotoxicity differences of drugs, ICD effects are often insufficient and difficult to effectively and durably activate anti-tumor immune responses. Here, we performed a network meta-analysis (NMA) based on clinical data of gastrointestinal cancer and identified oxaliplatin (Oxp) as an effective ICD-inducing drug. Meanwhile, to overcome the negative effects of immunosuppressive microenvironment on ICD, we constructed a nanoscale innate immune multiplier (NIIM) composed of nano-granulated manganous zoledronate, and designed a systemic delivery strategy to achieve the superposition of innate immune responses from multiple sites and amplify the ICD effect of Oxp. NIIM was first targeted to the tumor tissue, where it concurrently engaged tumor cells and innate immunocytes to reverse the immunosuppressive microenvironment. Following intravenous administration, NIIM was also distributed to the spleen, where it activated Ly6C+ inflammatory monocytes, directly augmenting the phagocytosis and elimination of tumor cells, and inducing the tumor-killing efficacy by cytotoxic T cells. Additionally, NIIM modulated the composition of hematopoietic progenitor cells in the bone marrow, inducing a durable innate immune response through trained immunity. The superposition of these immune effects allowed NIIM to significantly enhance the efficacy of oxaliplatin in gastrointestinal cancers after only one single injection. In summary, this strategy of simultaneously activating intratumoral, systemic, and trained immunity based on intravenous NIIM provides an effective new approach to amplify ICD of cytotoxic drugs and achieve the efficient synergy of chemotherapy with immunotherapy.

Achieving increasingly finely targeted drug delivery to organs, tissues, cells, and even to intracellular biomacromolecules is one of the core goals of nanomedicines. As the delivery destination is refined to cellular and subcellular targets, it is essential to explore the delivery of nanomedicines at the molecular level. However, due to the lack of technical methods, the molecular mechanism of the intracellular delivery of nanomedicines remains unclear to date. Here, we develop an enzyme-induced proximity labeling technology in nanoparticles (nano-EPL) for the real-time monitoring of proteins that interact with intracellular nanomedicines. Poly(lactic-co-glycolic acid) nanoparticles coupled with horseradish peroxidase (HRP) were fabricated as a model (HRP(+)-PNPs) to evaluate the molecular mechanism of nano delivery in macrophages. By adding the labeling probe biotin-phenol and the catalytic substrate H2O2 at different time points in cellular delivery, nano-EPL technology was validated for the real-time in situ labeling of proteins interacting with nanoparticles. Nano-EPL achieves the dynamic molecular profiling of 740 proteins to map the intracellular delivery of HRP (+)-PNPs in macrophages over time. Based on dynamic clustering analysis of these proteins, we further discovered that different organelles, including endosomes, lysosomes, the endoplasmic reticulum, and the Golgi apparatus, are involved in delivery with distinct participation timelines. More importantly, the engagement of these organelles differentially affects the drug delivery efficiency, reflecting the spatial–temporal heterogeneity of nano delivery in cells. In summary, these findings highlight a significant methodological advance toward understanding the molecular mechanisms involved in the intracellular delivery of nanomedicines.

Extracellular vesicles (EVs) are an effective tool to elucidate the bioeffect of nanomedicines. To clarify the interaction between oral nanomedicines and intestinal epithelial cells, and their bioeffects on downstream cells, polystyrene nanoparticles (PS-NPs) with different sizes were used as the model nanomedicines for EVs induction. Caco-2 monolayers were selected as the model of the intestinal epithelium and DLD-1 cells as the colorectal cancer model proximal to the gastrointestinal tract. It is found that compared with small-sized (25, 50, 100 nm) PS-NPs, the large-sized (200 and 500 nm) exhibited higher co-localization with multivesicular bodies and lysosomes, and more significant reduction of lysosomal acidification in Caco-2 cells. Proteomic and westernblotting analysis showed that the EVs remodeled by large-sized PS-NPs exhibited a higher extent of protein expression changes. The in vitro and in vivo signaling pathway detection in DLD-1 cells and DLD-1 cell xenograft nude mice showed that the remodeled EVs by large-sized PS-NPs inhibited the activation of multiple signaling pathways including Notch3, EGF/EGFR, and PI3K/Akt pathways, which resulted in the inhibition of tumor cell migration. These results primarily clarify the regulation mechanisms of nanomedicines-EVs-receptor cells chain. It provides a new perspective for the rational design and bioeffect evaluation of oral drug nanomaterials and sets up the fundamental knowledge for novel tumor therapeutics in the future.

Redox-responsive self-assembled prodrug nanoparticles have received extensive attention for their high loading efficiency and environmentally responsive properties. However, the intracellular metabolism and transportation kinetics were poorly understood, which limited the rational design and development of this delivery system. Herein, tetraphenylporphyrin-paclitaxel (TxP) prodrugs with thioether, disulfide, and dicarbon linkers (TsP, TssP, and TccP) were synthesized and self-assembled as nanoparticles. The redox responsiveness was investigated both in the simulated medium and in tumor cells via mass spectrometry. TxP NP and PTX concentrations in 4T1 whole cells, endosomal systems, and cytoplasm over time were quantified by UPLC-MS/MS and modeled using the nonlinear mixed effect (NLME) approach. Cytotoxicity was studied in 4T1 and MCF-7 cell lines, and antitumor efficacy was analyzed in 4T1 tumor-bearing mice. Mass spectrometry identified both oxidative and reductive metabolites in redox simulants for TssP NPs and TsP NPs. In 4T1 cells, only reductive metabolites for TssP NPs were detected, while both oxidative and reductive metabolites for TsP NPs were detected. The developed subcellular pharmacokinetic model suggested that the estimated metabolism rates of TxP NPs in endosomal systems were 10 to 27 times of the rates in cytoplasm, indicating that endosomal systems were the dominant place for intracellular metabolism. These rates were numerically higher for TsP NPs than TssP NPs in endosomal systems (1.7-fold) and the cytoplasm (2.5-fold). The internalization of nanoparticles was identified to be slow (kmax,int, 0.015 h-1) and saturable. The transportation rate constant across the endosomal membranes was fast for PTX (27.1 h-1) and slow for TxP NPs (0.098 h-1). TsP and TssP NPs had comparable in vivo antitumor efficacy, which was higher than that of TccP NPs. This study quantified the organelle-level transportation and metabolism kinetics for three prodrug nanoparticles with redox-responsive or inert linkers using a combined experimental and modeling approach. These findings and the modeling framework might inform future studies for redox-responsive prodrug design and drug delivery systems.

Conventional chemotherapy based on cytotoxic drugs is facing tough challenges recently following the advances of monoclonal antibodies and molecularly targeted drugs. It is critical to inspire new potential to remodel the value of this classical therapeutic strategy. Here, we fabricate bisphosphonate coordination lipid nanogranules (BC-LNPs) and load paclitaxel (PTX) to boost the chemo- and immuno-therapeutic synergism of cytotoxic drugs. Alendronate in BC-LNPs@PTX, a bisphosphonate to block mevalonate metabolism, works as both the structure and drug constituent in nanogranules, where alendronate coordinated with calcium ions to form the particle core. The synergy of alendronate enhances the efficacy of paclitaxel, suppresses tumor metastasis, and alters the cytotoxic mechanism. Differing from the paclitaxel-induced apoptosis, the involvement of alendronate inhibits the mevalonate metabolism, changes the mitochondrial morphology, disturbs the redox homeostasis, and causes the accumulation of mitochondrial ROS and lethal lipid peroxides (LPO). These factors finally trigger the ferroptosis of tumor cells, an immunogenic cell death mode, which remodels the suppressive tumor immune microenvironment and synergizes with immunotherapy. Therefore, by switching paclitaxel-induced apoptosis to mevalonate metabolism-triggered ferroptosis, BC-LNPs@PTX provides new insight into the development of cytotoxic drugs and highlights the potential of metabolism regulation in cancer therapy.

Nanocrystals exhibit significant advantages in improving the oral bioavailability of poorly soluble drugs. However, the complicated absorption properties of nanocrystals and the differences in physiological characteristics between children and adults limit pediatric applications of nanocrystals. To elucidate the absorption differences and the underlying mechanisms between children and adults, the pharmacokinetics and tissue distribution of aprepitant crystals with different particle sizes (NC200, NC500, and MC2.5) in rats and mice at different ages were studied, and their absorption mechanisms were investigated in Caco-2 cells, mice, and rats. It was found that childhood animals demonstrated higher bioavailability compared with adolescent and adult animals, which was related to higher bile salt concentration and accelerated drug dissolution in the intestine of childhood animals. The majority of nanocrystals were dissolved and formed micelles under the influence of bile salts. Compared with intact nanocrystals, the bile salt micelle-associated aprepitant was absorbed through the chylomicron pathway, wherein Apo B assisted in the reassembling of the aprepitant micelles after endocytosis. Higher bile salt concentration and Apo B expression in the intestines of childhood animals are both responsible for the higher chylomicron transport pathways. Elucidation of the chylomicron pathway in the varied absorption of nanocrystals among children, adolescents, and adults provides strong theoretical guidance for promoting the rational and safe use of nanocrystals in pediatric populations.

Abstract PROteolysis TArgeting Chimeras (PROTACs) have been considered the next blockbuster therapies. However, due to their inherent limitations, the efficacy of PROTACs is frequently impaired by limited tissue penetration and particularly insufficient cellular internalization into their action sites. Herein, based on the ultra-pH-sensitive and enzyme-sensitive nanotechnology, a type of polymer PROTAC conjugated and pH/cathepsin B sequential responsive nanoparticles (PSRNs) are deliberately designed, following the construction of the PROTAC for Cyclin-dependent kinase 4 and 6 (CDK4/6). Colorectal cancer (CRC) which hardly responds to many treatments even immune checkpoint blockades was selected as the tumor model in this study. As a result, PSRNs were found to maintain nanostructure (40 nm) in circulation and efficiently accumulated in tumors via enhanced permeation and retention effect. Then, they were dissociated into unimers (<10 nm) in response to an acidic tumor microenvironment, facilitating tumor penetration and cellular internalization. Eventually, the CDK4/6 degrading PROTACs were released intracellularly following the cleavage of cathepsin B. Importantly, PSRNs led to the enhanced degradation of target protein in vitro and in vivo. The degradation of CDK4/6 also augmented the efficacy of immune checkpoint blockades, through the upregulation of programmed cell death-ligand 1 (PD-L1) expression in cancer cells and the suppression of regulatory T cells cell proliferation in tumor microenvironment. By combination with α-PD-1, an enhanced anti-tumor outcome is well achieved in CT26 tumor model. Overall, our study verifies the significance of precise intracellular delivery of PROTACs and introduces a promising therapeutic strategy for the targeted combination treatment of CRC.

Ex vivo or in vivo cell-hitchhiking has emerged as a potential means for efficient drug delivery and various disease therapies. However, many challenges remain, such as the complicated engineering process and dependence on ligand-receptor interaction. Here, we present a simple in vivo platelet-hitchhiking strategy based on self-assembling peptides without ligand modification. The engineered peptide nanofibers can hitchhike ultrafast (<5 s) and efficiently on both resting and activated platelets in a receptor-independent and species-independent manner. Mechanistic studies showed that unique secondary structure of nanofibers, which lead to surface exposure of hydrophobic and hydrogen bond-forming groups, might primarily contribute to the selective and efficient platelet-hitchhiking behavior. After intravenous injection, these peptide nanofibers hitchhiked in situ on circulating platelets and achieved almost 20-fold lung accumulation. Our study provides not only a different paradigm of in vivo platelet-hitchhiking beyond ligand-receptor recognition but also a potential strategy for lung-targeted drug delivery and pulmonary disease therapy.

KRAS gene is mutated in 40% of colorectal cancers (CRC), which induces malignant proliferation by regulating cellular nutrient metabolism and biosynthesis. It has been found that malignant proliferation of KRAS-mutant colorectal cancer relies on the upregulation of SLC25A22 protein expression, suggesting that inhibition the expression of both KRAS and SLC25A22 is a potential CRC therapeutics. Stably knocking down the oncogenic KRAS-G12V gene can achieve long-term gene therapy effects, while transient downregulation of SLC25A22, a normal functional gene most of the time, is preferred to kill tumor cells and minimize the side impact on normal cells. Here, two lipid nanoparticles (LNP) were designed to encapsulate KRAS-G12V CRISPR/Cas9 gene editing plasmids (pKRAS-LNPs) and SLC25A22 siRNA (siSLC-LNPs), respectively. Therapeutic effects of both nanoparticles alone and in combination on KRAS-G12V mutant colorectal cancer cells in vitro were first examined. The result showed that delivery of pKRAS-LNPs or siSLC-LNPs alone could effectively achieve KRAS-G12V gene editing or SLC25A22 gene silencing and inhibit tumor cell proliferation, while co-delivery of both LNPs could achieve stronger inhibition of tumor cell proliferation by inducing stronger apoptosis. Furthermore, we found that co-delivery of pKRAS-LNPs and siSLC-LNPs induced stronger apoptosis and cell proliferation inhibition compared to pKRAS&siSLC-LNPs that were constructed by pre-mixing pKRAS and siSLC and then encapsulating them. Finally, we validated that co-delivery of pKRAS-LNPs and siSLC-LNPs can achieve KRAS-G12V colorectal cancer treatment in vivo with a tumor inhibition rate of 61.15%. In summary, the delivery vectors constructed for nucleic acids targeting KRAS and SLC25A22 achieved therapeutic targeting of KRAS-G12V colorectal cancer in vitro and in vivo.

Compared with thermodynamically equilibrium supramolecular assemblies, non-equilibrium assemblies from the same building blocks have attracted increasing attentions because their diverse structures and dynamic natures may impart the assemblies with novel functionalities. However, facile access to non-equilibrium assemblies remains a formidable challenge. Herein, we endeavored to exploit various solvent-anti-solvent methods to achieve it using peptide amphiphile C16-VVAAEE-NH2 as a model. Through systematical utilization of dialysis, ultrasonic and stirring-dropping methods, as well as tuning of processing parameters, we demonstrated the successful formation of diverse non-equilibrium nanostructures with distinct morphologies and structures that significantly deviate from the thermodynamically favored twisted long ribbons. Additionally, these metastable nanostructures ultimately underwent spontaneous transformation into thermodynamically stable states. The transformation processes of three representative non-equilibrium assemblies were also demonstrated and characterized in detail using transmission electron microscopy, circular dichroism spectrum, and thioflavin T fluorescence spectrum. Furthermore, non-equilibrium assemblies exhibited various degrees of cytotoxic effects, which may stem from their spontaneous, dynamic transformation and interactions with cellular membranes. This study offers valuable approaches for direct access to diverse non-equilibrium supramolecular nanostructures from self-assembling peptide, and also has implications for the development of advanced materials with unprecedented biological functions.

Intracellular delivery crossing the endomembrane barrier is the "last mile to target" for nano delivery systems carrying biomacromolecules, including genetic medicines. Nevertheless, a mass of nanomedicines is currently restricted by their equivocal safety and delivery efficiency. Here, we establish a universal strategy independent of nanomaterials. Such a policy broadly facilitates the intracellular delivery of all kinds of tested nanomedicines, subtly by inducing ARF6 GTPases to their overactivated GTP-bound state. ARF6, one member of ARF subfamily in small GTPases, is verified to regulate intracellular vesicle transport and lipid metabolism through GTP/GDP conversion. ARF6 biased to GTP-bound state causes the increased endocytosis and reduced exocytosis of eleven types of nanoparticles. This universal effect is derived from the formation of a hybrid type of endosomes triggered by overactivated ARF6 via regulating cholesterol-associated vesicles and lipid raft/caveolae pathways. Due to the mild microenvironment in hybrid endosomes, the internalized protein and nanoparticles are steadily delivered to the cytoplasm, avoiding the intensive degradation in lysosomes. Based on these findings, we identify QS11, a safe small molecule inhibitor of ARF GTPase-activating proteins, significantly enhances the antitumor efficacy of siEGFR-loaded nanoparticles by inducing ARF6 overactivation. In sum, these findings reveal that the tactics of tuning ARF6 GTPases to GTP-bound form will widely benefit cellular nano delivery.