Isorhamnetin alleviates ferroptosis-mediated colitis by activating the NRF2/HO-1 pathway and chelating iron.

Ferroptosis of intestinal epithelial cells (IECs) had been identified as a key factor in the development of ulcerative colitis (UC). Therefore, targeted inhibition of ferroptosis may provide a new strategy for the treatment of UC. Isorhamnetin (ISO) was an O-methylated flavonol with therapeutic effects on a variety of diseases, such as cardiovascular disease, neurological disorders and tumors. However, the role and mechanism of ISO in ferroptosis and associated colitis were rarely investigated. In this study, we demonstrated that ISO could effectively alleviate intestinal inflammation by inhibiting ferroptosis of IECs in DSS-induced mice. Moreover, our results shown that ISO acted as a potent and common ferroptosis inhibitor in multiple human and murine cell lines. Mechanistically, ISO inhibited ferroptosis independent of its previously reported targets MEK1 and PI3K, but alleviated oxidative stress by targeting and activating NRF2. Furthermore, ISO could also directly chelate iron to hinder ferroptosis. In conclusion, our study indicated that ISO as a novel potential ferroptosis inhibitor, providing a promising therapeutic strategy for ferroptosis-related colitis.

Identification of a targeted ACSL4 inhibitor to treat ferroptosis-related diseases.

Ferroptosis is a form of iron-dependent, lipid peroxidation-driven regulatory cell death that has been implicated in the pathogenesis of multiple diseases, including organ injury, ischemia/reperfusion, and neurodegenerative diseases. However, inhibitors that directly and specifically target ferroptosis are not yet available. Here, we identify the compound AS-252424 (AS) as a potent ferroptosis inhibitor through kinase inhibitor library screening. Our results show that AS effectively inhibits lipid peroxidation and ferroptosis in both human and mouse cells. Mechanistically, AS directly binds to the glutamine 464 of ACSL4 to inhibit its enzymatic activity, resulting in the suppression of lipid peroxidation and ferroptosis. By using nanoparticle-based delivery systems, treatment with AS-loaded nanoparticles effectively alleviate ferroptosis-mediated organ injury in mouse models, including kidney ischemia/reperfusion injury and acute liver injury (ALI). Thus, our results identify that AS is a specific and targeted inhibitor of ACSL4 with remarkable antiferroptosis function, providing a potential therapeutic for ferroptosis-related diseases.

Tivantinib alleviates inflammatory diseases by directly targeting NLRP3.

NLRP3 inflammasome-mediated immune responses are involved in the pathogenesis of multiple inflammatory diseases, but few clinical drugs are identified that directly target the NLRP3 inflammasome to treat these diseases to date. Here, we show that the anticancer agent tivantinib is a selective inhibitor of NLRP3 and has a strong therapeutic effect on inflammasome-driven disease. Tivantinib specifically inhibits canonical and non-canonical NLRP3 inflammasome activation without affecting AIM2 and NLRC4 inflammasome activation. Mechanistically, Tivantinib inhibits NLRP3 inflammasome by directly blocking NLRP3 ATPase activity and subsequent inflammasome complex assembly. In vivo, Tivantinib reduces IL-1ß production in mouse models of lipopolysaccharide (LPS)-induced systemic inflammation, monosodium urate (MSU)-induced peritonitis and Con A-induced acute liver injury (ALI), and also has remarkable preventive and therapeutic effects on experimental autoimmune encephalomyelitis (EAE). In conclusion, our study identifies the anticancer drug tivantinib as a specific inhibitor of NLRP3 and provides a promising therapeutic agent for inflammasome-driven disease.

Optimized Cationic Lipid-assisted Nanoparticle for Delivering CpG Oligodeoxynucleotides to Treat Hepatitis B Virus Infection.

PURPOSE: Hepatitis B virus (HBV) infection is such a global health problem that hundreds of millions of people are HBV carriers. Current anti-viral agents can inhibit HBV replication, but can hardly eradicate HBV. Cytosine-phosphate-guanosine (CpG) oligodeoxynucleotides (ODNs) are an adjuvant that can activate plasmacytoid dendritic cells (pDCs) and conventional dendritic cells (cDCs) to induce therapeutic immunity for HBV eradication. However, efficient delivery of CpG ODNs into pDCs and cDCs remains a challenge. In this study, we constructed a series of cationic lipid-assisted nanoparticles (CLANs) using different cationic lipids to screen an optimal nanoparticle for delivering CpG ODNs into pDCs and cDCs. METHODS: We constructed different CLANCpG using six cationic lipids and analyzed the cellular uptake of different CLANCpG by pDCs and cDCs in vitro and in vivo, and further analyzed the efficiency of different CLANCpG for activating pDCs and cDCs in both wild type mice and HBV-carrier mice. RESULTS: We found that CLAN fabricated with 1,2-Dioleoyl-3-trimethylammonium propane (DOTAP) showed the highest efficiency for delivering CpG ODNs into pDCs and cDCs, resulting in strong therapeutic immunity in HBV-carrier mice. By using CLANCpG as an immune adjuvant in combination with the injection of recombinant hepatitis B surface antigen (rHBsAg), HBV was successfully eradicated and the chronic liver inflammation in HBV-carrier mice was reduced. CONCLUSION: We screened an optimized CLAN fabricated with DOTAP for efficient delivery of CpG ODNs to pDCs and cDCs, which can act as a therapeutic vaccine adjuvant for treating HBV infection.

Physiological and transcriptome analyses reveal the photosynthetic response to drought stress in drought-sensitive (Fengjiao) and drought-tolerant (Hanjiao) Zanthoxylum bungeanum cultivars.

As an important economical plant, Zanthoxylum bungeanum is widely cultivated in arid and semi-arid areas. The studies associated with photosynthesis under drought stress were widely carried out, but not yet in Z. bungeanum. Here, the photosynthesis of two Z. bungeanum cultivars (FJ, Z. bungeanum cv. "Fengjiao"; HJ, Z. bungeanum cv. "Hanjiao") was analyzed under drought stress using physiological indicators and transcriptome data. Drought decreased stomatal aperture and stomatal conductance (Gsw), reduced transpiration rate (E) and sub-stomatal CO2 concentration (Ci), and lowered chlorophyll and carotenoid content, which reduced the net photosynthetic rate (Pn) of Z. bungeanum. The higher photosynthetic rate in HJ stemmed from its higher chlorophyll content, larger stomatal aperture and Gsw, and higher Ci. Weighted gene co-expression network analysis (WGCNA) identified several ABA signal transduction genes (PYL4, PYL9, and PYR1), LCH-encoding genes (LHCB4.3), and chlorophyll metabolism genes (CRD1, PORA, and CHLH). Additionally, seven transcription factor genes were identified as important factors regulating photosynthesis under drought conditions. In general, a photosynthetic response model under drought stress was built firstly in Z. bungeanum, and the key genes involved in photosynthesis under drought stress were identified. Therefore, the results in our research provide important information for photosynthesis under drought and provided key clues for future molecular breeding in Z. bungeanum.

Widely targeted metabolomic profiling combined with transcriptome analysis provides new insights into amino acid biosynthesis in green and red pepper fruits.

The type and content of amino acids in pepper are important indicators to reflect its nutritional value, largely affecting the purchasing behavior of consumers. Understanding the biosynthesis of amino acids in pepper fruit is beneficial to the development of pepper functional food. Widely targeted metabolomics, transcriptome analysis, correlation analysis, weighted gene co-expression network analysis (WGCNA), and canonical correlation analysis (CCA) were used to evaluate the quality characteristics of green and red pepper amino acids. The results showed 78 kinds of amino acids and their derivatives in the fruit of pepper. The essential amino acids were comprehensive and abundant. Especially, the contents of lysine and tryptophan were high. However, significant differences were found in the ratio of essential amino acids to total amino acids in green and red pepper. The ratio of essential amino acids to total amino acids in red pepper was up to 28.88%, while that in green pepper was up to 17.69%. WGCNA and CCA analyses were performed in combination with amino acid metabolism profiling and transcriptome analysis to further identify the main contributors to amino acid synthesis in green and red pepper. The results showed PK and PFK were the genes in the backbone of the amino acid biosynthesis pathway, which had a direct impact on the synthesis of various amino acids, and were the main genes for amino acid synthesis in pepper fruit. In this study, the amino acid biosynthesis rules for two kinds of pepper were analyzed by amino acid metabolism profiling and transcriptome analysis, which provided the basis for the development of amino acid nutritional supplements and pepper functional food.

UPLC-MS/MS Profile Combined With RNA-Seq Reveals the Amino Acid Metabolism in Zanthoxylum bungeanum Leaves Under Drought Stress.

Zanthoxylum bungeanum leaves have a unique taste and incomparable nutritional value and hence are popular as a food item and traditional medicine in China. However, the studies on the metabolites in Z. bungeanum leaves are quite limited, especially for amino acids. Therefore, this study explored the amino acid component in Z. bungeanum leaves and also the accumulation mechanism under drought stress in two Z. bungeanum cultivars using the widely targeted metabolome combined with transcriptome analysis. A total of 56 amino acids and their derivatives were identified in Z. bungeanum leaves, including eight essential amino acids. The total amino acid content with most individual amino acids increased under progressive drought stress. More differentially accumulated amino acids (DAAs) and differentially expressed genes (DEGs) were found in FJ (Z. bungeanum cv. 'Fengjiao') than in HJ (Z. bungeanum cv. 'Hanjiao'). The orthogonal projections to latent structures discriminant analysis identified nine and seven indicator DAAs in FJ and HJ leaves, respectively. The weighted gene co-expression network analysis (WGCNA) showed that the green module was significantly correlated with most indicator DAAs and revealed the important role of FBA3, DELTA-OAT, PROC, and 15 transcription factor genes in regulating the amino acid synthesis. Furthermore, the correlation analysis and redundancy analysis (RDA) identified four candidate synthesis genes (ASNS, AK, ASPS, and PK) in amino acid biosynthesis pathway. This study provided useful information for the development of Z. bungeanum leaves in food and nutrition industry and also laid the foundations for future molecular breeding.

Genome-Wide Identification of the NAC Gene Family in Zanthoxylum bungeanum and Their Transcriptional Responses to Drought Stress.

NAC (NAM, ATAF1/2, and CUC2) transcription factors (TFs) are one of the largest plant-specific TF families and play a pivotal role in adaptation to abiotic stresses. The genome-wide analysis of NAC TFs is still absent in Zanthoxylum bungeanum. Here, 109 ZbNAC proteins were identified from the Z. bungeanum genome and were classified into four groups with Arabidopsis NAC proteins. The 109 ZbNAC genes were unevenly distributed on 46 chromosomes and included 4 tandem duplication events and 17 segmental duplication events. Synteny analysis of six species pairs revealed the closely phylogenetic relationship between Z. bungeanum and C. sinensis. Twenty-four types of cis-elements were identified in the ZbNAC promoters and were classified into three types: abiotic stress, plant growth and development, and response to phytohormones. Co-expression network analysis of the ZbNACs revealed 10 hub genes, and their expression levels were validated by real-time quantitative polymerase chain reaction (qRT-PCR). Finally, ZbNAC007, ZbNAC018, ZbNAC047, ZbNAC072, and ZbNAC079 were considered the pivotal NAC genes for drought tolerance in Z. bungeanum. This study represented the first genome-wide analysis of the NAC family in Z. bungeanum, improving our understanding of NAC proteins and providing useful information for molecular breeding of Z. bungeanum.

Bioorthogonal in situ assembly of nanomedicines as drug depots for extracellular drug delivery.

Developing precise nanomedicines to improve the transport of anticancer drugs into tumor tissue and to the final action site remains a critical challenge. Here, we present a bioorthogonal in situ assembly strategy for prolonged retention of nanomedicines within tumor areas to act as drug depots. After extravasating into the tumor site, the slightly acidic microenvironment induces the exposure of cysteine on the nanoparticle surface, which subsequently undergoes a bioorthogonal reaction with the 2-cyanobenzothiazole group of another neighboring nanoparticle, enabling the formation of micro-sized drug depots to enhance drug retention and enrichment. This in situ nanoparticle assembly strategy remarkably improves the antimetastatic efficacy of extracellular-targeted drug batimastat, and also leads to the simultaneous enhanced retention and sustained release of multiple agents for combined cocktail chemoimmunotherapy to finally elicit a potent antitumor immune response. Such in situ assembly of nanomedicines represents a generalizable strategy towards extracellular drug delivery and cocktail chemoimmunotherapy.

Integrated LC-MS/MS and Transcriptome Sequencing Analysis Reveals the Mechanism of Color Formation During Prickly Ash Fruit Ripening.

Prickly ash peel is one of the eight major condiments in China and is widely used in cooking because of its unique fragrance and numbing taste. The color of prickly ash fruit is the most intuitive quality that affects consumer choice. However, the main components and key biosynthetic genes responsible for prickly ash fruit color have not yet been determined. To better understand the biosynthetic mechanisms and accumulation of prickly ash fruit color components, we performed an integrated transcriptomic and metabolomic analysis of red and green prickly ash fruit at different growth periods. The transcriptome analysis identified 17,269 differentially expressed genes (DEGs) between fruit of red and green prickly ash: 7,236 upregulated in green fruit and 10,033 downregulated. Liquid chromatography tandem mass spectrometry (LC-MS/MS) identified 214 flavonoids of 10 types. Flavonoids and flavonols are the main flavonoids in prickly ash, and the total flavonoid content of red prickly ash is higher than that of green prickly ash. Comprehensive analysis showed that the main colored metabolites that differed between green and red prickly ash were cyanidin-3-O-galactoside and cyanidin-3-O-glucoside, and differences in the contents of these metabolites were due mainly to differences in the expression of ANS and UFGT. Our results provide insight into the mechanisms underlying color differences in red and green prickly ash and will be useful for improving the quality of prickly ash fruit.

Tumor-Microenvironment-Activatable Polymer Nano-Immunomodulator for Precision Cancer Photoimmunotherapy.

Cancer nanomedicine combined with immunotherapy has become a promising strategy for treating cancer in terms of safety and potency; however, precise regulation of the activation of antitumor immunity remains challenging. Herein, a smart semiconducting polymer nano-immunomodulator (SPNI), which responds to the acidic tumor microenvironment (TME), for precision photodynamic immunotherapy of cancer, is reported. The SPNI is self-assembled by a near-infrared (NIR)-absorbing semiconducting polymer and an amphipathic polymer conjugated with a Toll-like receptor 7 (TLR7) agonist via an acid-labile linker. Upon arrival at tumor site, SPNI undergoes hydrolysis and triggers an efficient liberation of TLR7 agonist in response to the acidic TME for dendritic cell activation. Moreover, SPNI exerts photodynamic effects for direct tumor eradication and immunogenic cancer cell death under NIR photoirradiation. The synergistic action of released immunogenic factors and acidic-TME-activated TLR7 agonist can serve as an in situ generated cancer vaccine to evoke strong antitumor activities. Notably, such localized immune activation boosts systemic antitumor immune responses, resulting in enhanced cytotoxic CD8+ T infiltration to inhibit tumor growth and metastasis. Thereby, this work presents a general strategy to devise prodrug of immunotherapeutics for precise regulation of cancer immunotherapy.

Rational designs of in vivo CRISPR-Cas delivery systems.

The CRISPR-Cas system initiated a revolution in genome editing when it was, for the first time, demonstrated success in the mammalian cells. Today, scientists are able to readily edit genomes, regulate gene transcription, engineer posttranscriptional events, and image nucleic acids using CRISPR-Cas-based tools. However, to efficiently transport CRISPR-Cas into target tissues/cells remains challenging due to many extra- and intra-cellular barriers, therefore largely limiting the applications of CRISPR-based therapeutics in vivo. In this review, we summarize the features of plasmid-, RNA- and ribonucleoprotein (RNP)-based CRISPR-Cas therapeutics. Then, we survey the current in vivo delivery systems. We specify the requirements for efficient in vivo delivery in clinical settings, and highlight both efficiency and safety for different CRISPR-Cas tools.

Integrated Analysis of Metabolome and Transcriptome Data for Uncovering Flavonoid Components of Zanthoxylum bungeanum Maxim. Leaves Under Drought Stress.

Zanthoxylum bungeanum Maxim. leaves (ZBLs) are rich in flavonoids and have become popular in nutrition, foods and medicine. However, the flavonoid components in ZBLs and the mechanism of flavonoid biosynthesis under drought stress have received little attention. Here, we performed an integrative analysis of the metabolome and transcriptome of ZBLs from HJ (Z. bungeanum cv. "Hanjiao") and FJ (Z. bungeanum cv. "Fengjiao") at four drought stages. A total of 231 individual flavonoids divided into nine classes were identified and flavones and flavonols were considered the most abundant flavonoid components in ZBLs. The total flavonoid content of ZBLs was higher in FJ; it increased in FJ under drought stress but decreased in HJ. Nine-quadrant analysis identified five and eight differentially abundant flavonoids in FJ and HJ leaves, respectively, under drought stress. Weighted gene correlation network analysis (WGCNA) identified nine structural genes and eight transcription factor genes involved in the regulation of flavonoid biosynthesis. Moreover, qRT-PCR results verified the accuracy of the transcriptome data and the reliability of the candidate genes. Taken together, our results reveal the flavonoid components of ZBLs and document changes in flavonoid metabolism under drought stress, providing valuable information for nutrition value and food utilization of ZBLs.

Photo-Enhanced CRISPR/Cas9 System Enables Robust PD-L1 Gene Disruption in Cancer Cells and Cancer Stem-Like Cells for Efficient Cancer Immunotherapy.

Blocking immune checkpoint pathways with an antibody or small interfering RNA (siRNA) has become a promising method to reactivate antitumor responses for cancer treatment. However, both blockade strategies achieve only temporary inhibition of these immune checkpoints. Herein, a photoswitched CRISPR/Cas9 system for genomic disruption of the PD-L1 gene is developed to achieve permanent blockade of the PD-1/PD-L1 pathway; this system is constructed by using a photoactivated self-degradable polyethyleneimine derivative and the plasmid pX330/sgPD-L1 (expression of the Cas9 protein and single-guide RNA targeting PD-L1). Under light irradiation, this photoswitched CRISPR/Cas9 system efficiently genetically disrupts the PD-L1 gene in not only bulk cancer cells but also cancer stem-like cells. As a result, the photoswitched CRISPR/Cas9 system significantly increases the infiltration of CD8+ T cells into tumor tissue, leading to effective activation of a T cell-mediated antitumor response against cancer cells and cancer stem-like cells. This study provides an alternative strategy to block the PD-1/PD-L1 pathway for efficacious immune checkpoint therapy.

An All-in-One Nanomedicine Consisting of CRISPR-Cas9 and an Autoantigen Peptide for Restoring Specific Immune Tolerance.

Nanotechnology has shown great promise in treating diverse diseases. However, developing nanomedicines that can cure autoimmune diseases without causing systemic immunosuppression is still quite challenging. Herein, we propose an all-in-one nanomedicine comprising an autoantigen peptide and CRISPR-Cas9 to restore specific immune tolerance by engineering dendritic cells (DCs) into a tolerogenic phenotype, which can expand autoantigen-specific regulatory T (Treg) cells. In brief, we utilized cationic lipid-assisted poly(ethylene glycol)-b-poly(lactide-co-glycolide) (PEG-PLGA) nanoparticles to simultaneously encapsulate an autoimmune diabetes-relevant peptide (2.5mi), a CRISPR-Cas9 plasmid (pCas9), and three guide RNAs (gRNAs) targeting costimulatory molecules (CD80, CD86, and CD40). We demonstrated that the all-in-one nanomedicine was able to effectively codeliver these components into DCs, followed by simultaneous disruption of the three costimulatory molecules and presentation of the 2.5mi peptide on the genome-edited DCs. The resulting tolerogenic DCs triggered the generation and expansion of autoantigen-specific Treg cells by presenting the 2.5mi peptide to CD4+ T cells in the absence of costimulatory signals. Using autoimmune type 1 diabetes (T1D) as a typical disease model, we demonstrated that our nanomedicine prevented autoimmunity to islet components and inhibited T1D development. Our all-in-one nanomedicine achieved codelivery of CRISPR-Cas9 and the peptide to DCs and could be easily applied to other autoimmune diseases by substitution of different autoantigen peptides.

Dually regulating the proliferation and the immune microenvironment of melanoma via nanoparticle-delivered siRNA targeting onco-immunologic CD155.

Studies have shown that the simultaneous regulation of tumor cell proliferation and the suppressive tumor immune microenvironment (TIME) could achieve better therapeutic effects. However, the targets of the proliferation and the TIME are different, which greatly limits the development of cancer therapy. A recent study found CD155, a highly expressed poliovirus receptor in melanoma cells and melanoma-infiltrating macrophages, functions as both an oncogene and immune checkpoint. Thus, it is supposed that targeting CD155 could bring dual therapeutic effects. Herein, we propose silencing the CD155 of melanoma cells and melanoma-infiltrating macrophages by a nanoparticle-delivered small interference RNA (siRNA) targeting CD155 (siCD155). We encapsulated siCD155 into cationic lipid-assisted nanoparticles (CLANsiCD155) and demonstrated that the intravenous injection of CLANsiCD155 could efficiently deliver siCD155 into melanoma cells and melanoma-infiltrating macrophages. The downregulation of CD155 in melanoma cells directly inhibited their proliferation, and meanwhile, the downregulation of CD155 in melanoma-infiltrating macrophages increased the activation of NK cells and T cells. Owing to this dual effect, CLANsiCD155 significantly inhibited the growth of B16-F10 melanoma. Our study suggests that nanoparticle-delivered siCD155 may be a simple but effective strategy for inhibiting tumor proliferation and reprogramming TIME.

Programmable Delivery of Immune Adjuvant to Tumor-Infiltrating Dendritic Cells for Cancer Immunotherapy.

Tumor-infiltrating dendritic cells (TIDCs) are mostly immature and immunosuppressive, usually mediating immune inhibition. The utilization of cytosine-guanine oligodeoxynucleotides (CpG ODNs) to stimulate the activation of TIDCs has been demonstrated to be effective for improving antitumor immunity. However, a series of biological barriers has limited the efficacy of previous nanocarriers for delivering CpG to TIDCs. Herein, we developed a dual-sensitive dendrimer cluster-based nanoadjuvant for delivering CpG ODNs into TIDCs. We show that the tumor acidity triggers the rapid release of CpG conjugated polyamidoamine (PAMAM) dendrimers from the nanoadjuvant, thus facilitating its perfusion deep into tumors and phagocytosis by TIDCs. Thereafter, the reductive condition of the endolysosomes led to the subsequent release of CpG, which promotes the DCs activation and enhances antitumor immunotherapies. Programmable delivery of immune adjuvant efficiently overcomes the barriers for targeted delivery to TIDCs and provides a promising strategy for improving cancer immunotherapy.

Linear Well-Defined Polyamines via Anionic Ring-Opening Polymerization of Activated Aziridines: From Mild Desulfonylation to Cell Transfection.

Linear polyethylenimine (L-PEI), a standard for nonviral gene delivery, is usually prepared by hydrolysis from poly(2-oxazoline)s. Lately, anionic polymerization of sulfonamide-activated aziridines had been reported as an alternative pathway toward well-defined L-PEI and linear polyamines. However, desulfonylation of the poly(sulfonyl aziridine)s typically relied on harsh conditions (acid, microwave) or used a toxic solvent (e.g., hexamethylphosphoramide). In addition, the drastic change of polarity requires solvents, which keep poly(sulfonyl aziridine)s as well as L-PEI in solution, and only a limited number of strategies were reported. Herein, we prepared 1-(4-cyanobenzenesulfonyl) 2-methyl-aziridine (1) as a monomer for the anionic ring-opening polymerization. It was polymerized to well-defined and linear poly(sulfonyl aziridine)s. The 4-cyanobenzenesulfonyl-activating groups were removed under mild conditions to linear polypropylenimine (L-PPI). Using dodecanethiol and diazabicyclo-undecene (DBU) allowed ≥98% desulfonylation and a reliable purification toward polyamines with high purity and avoiding main-chain scission. This method represents a fast approach in comparison to previous methods used for postpolymerization desulfonylation and produces linear well-defined polyamines. The high control over molecular weight and dispersities achieved by living anionic polymerization are the key advantages of our strategy, especially if used for biomedical applications, in which molecular weight might correlate with toxicity. The synthesized polypropylenimine was further tested as a cell-transfection agent and proved, with 16.1% transfection efficiency of the cationic nanoparticles, to be an alternative to L-PEI obtained from the 2-oxazoline route. This general strategy will allow the preparation of complex macromolecular architectures containing polyamine segments, which were not accessible before.

Intratumor Performance and Therapeutic Efficacy of PAMAM Dendrimers Carried by Clustered Nanoparticles.

In recent years, small nanoparticles (NPs) with a diameter of less than 10 nm have aroused considerable interest in biomedical applications. However, their intratumor performance, as well as the antitumor efficacy, has not been well understood due to their size-dependent pharmacokinetics, which presents a formidable challenge for delivering a comparable amount of different small NPs to tumor tissues. Utilizing the multistage delivery strategy, we construct G3-, G5-, and G7-iCluster delivery systems by using poly(amidoamine) (PAMAM) dendrimers of different generations (G3-, G5-, and G7-PAMAM) as building blocks. The iCluster nanoparticles showed comparable pharmacokinetics and similar initial tumor deposition due to their similarity in size and surface chemistry. After accumulating at a tumor site, individual small dendrimers were released, and thus, their intratumor performance was comparatively investigated. Our results indicated that a subtle change in generation markedly affects their intratumor activities. G5-iCluster outperformed G3-iCluster and G7-iCluster in the treatment efficacy in an orthotopic pancreatic tumor model. The mechanistic study revealed that G3-PAMAM showed reduced particle retention in tumor tissue due to its small size and weak cell internalization, while G7-PAMAM was much less penetrative because of its relatively large size and strong particle-cell interaction. In contrast, G5-PAMAM exhibited balanced tumor penetration, cell internalization, and tumor retention. Our finding highlights the huge influence of the subtle difference of small NPs in their intratumor performance.

Enhanced Primary Tumor Penetration Facilitates Nanoparticle Draining into Lymph Nodes after Systemic Injection for Tumor Metastasis Inhibition.

Lymph nodes (LNs) are normally the primary site of tumor metastasis, and effective delivery of chemotherapeutics into LNs through systemic administration is critical for metastatic cancer treatment. Here, we uncovered that improved perfusion in a primary tumor facilitates nanoparticle translocation to LNs for inhibiting tumor metastasis. On the basis of our finding that an iCluster platform, which undergoes size reduction from ∼100 nm to ∼5 nm at the tumor site, markedly improved particle perfusion in the interstitium of the primary tumor, we further revealed in the current study that such tumor-specific size transition promoted particle intravasation into tumor lymphatics and migration into LNs. Quantitative analysis indicated that the drug deposition in LNs after iCluster treatment was significantly higher in the presence of a primary tumor in comparison with that after primary tumor resection. Early intervention of metastatic 4T1 tumors with iCluster chemotherapy and subsequent surgical resection of the primary tumor resulted in significantly extending animal survival, with 4 out of the 10 mice remaining completely tumor-free for 110 days. Additionally, in the more clinical relevant late metastatic model, iCluster inhibited the metastatic colonies to the lungs and extended animal survival time. This finding provides insights into the design of more effective nanomedicines for treating metastatic cancer.