BODIPY-Based NIR Trackers with Acidity-Driven Amphiphilicity for STED Super-Resolution Imaging of Lysosomal Membranes.

Although super-resolution imaging provides a great opportunity to disclose the structures of living cells at the nanoscale level, resolving the structural details of organelles is highly dependent on the targeting accuracy and photophysical properties of fluorescence trackers. Herein, we report a series of ultrabright and photostable trackers of lysosomal membranes for super-resolution imaging using stimulated emission depletion microscopy (STED). These trackers are composed of lipophilic NIR BODIPY derivatives and ionizable tertiary amines. This structural feature enables accurate targeting of the lysosomal membrane through the formation of transient amphiphilicity driven by the acidity in the lysosome. As a representative, Lyso-700 is applied for STED-based super-resolution imaging of the lysosomal membrane of living macrophages. By use of Lyso-700, the interaction details between lysosomes of macrophages and fungi are visualized. Overall, these trackers display great potential as advanced lysosome trackers and merit further evaluation for lysosome-related studies.

Tetrazine-Based Ratiometric Nitric Oxide Sensor Identifies Endogenous Nitric Oxide in Atherosclerosis Plaques by Riding Macrophages as a Smart Vehicle.

Atherosclerosis (AS) is the formation of plaques in blood vessels, which leads to serious cardiovascular diseases. Current research has disclosed that the formation of AS plaques is highly related to the foaming of macrophages. However, there is a lack of detailed molecular biological mechanisms. We proposed a "live sensor" by grafting a tetrazine-based ratiometric NO probe within macrophages through metabolic and bio-orthogonal labeling. This "live sensor" was proved to target the AS plaques with a diameter of only tens of micrometers specifically and visualized endogenous NO at two lesion stages in the AS mouse model. The ratiometric signals from the probe confirmed the participation of NO during AS and indicated that the generation of endogenous NO increased significantly as the lesion progressed. Our proposal of this "live sensor" provided a native and smart strategy to target and deliver small molecular probes to the AS plaques at the in vivo level, which can be used as universal platforms for the detection of reactive molecules or microenvironmental factors in AS.

Adoptive macrophage directed photodynamic therapy of multidrug-resistant bacterial infection.

Multidrug-resistant (MDR) bacteria cause severe clinical infections and a high mortality rate of over 40% in patients with immunodeficiencies. Therefore, more effective, broad-spectrum, and accurate treatment for severe cases of infection is urgently needed. Here, we present an adoptive transfer of macrophages loaded with a near-infrared photosensitizer (Lyso700D) in lysosomes to boost innate immunity and capture and eliminate bacteria through a photodynamic effect. In this design, the macrophages can track and capture bacteria into the lysosomes through innate immunity, thereby delivering the photosensitizer to the bacteria within a single lysosome, maximizing the photodynamic effect and minimizing the side effects. Our results demonstrate that this therapeutic strategy eliminated MDR Staphylococcus aureus (MRSA) and Acinetobacter baumannii (AB) efficiently and cured infected mice in both two models with 100% survival compared to 10% in the control groups. Promisingly, in a rat model of central nervous system bacterial infection, we performed the therapy using bone marrow-divided macrophages and implanted glass fiber to conduct light irradiation through the lumbar cistern. 100% of infected rats survived while none of the control group survived. Our work proposes an efaficient and safe strategy to cure MDR bacterial infections, which may benefit the future clinical treatment of infection.

Analysis of the utilization value of different tissues of Taxus×Media based on metabolomics and antioxidant activity.

BACKGROUND: Taxaceae, is a class of dioecious and evergreen plant with substantial economic and ecology value. At present many phytochemical analyses have been performed in Taxus plants. And various biological constituents have been isolated from various Taxus species. However, the difference of compounds and antioxidant capacity of different tissues of T. media is not clear. RESULTS: In the present study, we investigated the metabolites and antioxidant activity of four tissues of T. media, including T. media bark (TB), T. media fresh leaves (TFL), T. media seeds (TS), T. media aril (TA). In total, 808 compounds, covering 11 subclasses, were identified by using UPLC-MS/MS. Paclitaxel, the most popular anticancer compound, was found to accumulate most in TS, followed by TB, TFL and TA in order. Further analysis found that 70 key differential metabolites with VIP > 1.0 and p < 0.05, covering 8 subclasses, were screened as the key differential metabolites in four tissues. The characteristic compounds of TFL mainly included flavonoids and tanninsis. Alkaloids and phenolic acids were major characteristic compounds of TS and TB respectively. Amino acids and derivatives, organic acids, saccharides and lipids were the major characteristic compounds of TA. Additionally, based on FRAP and ABTS method, TS and TFL exhibited higher antioxidant activity than TB and TA. CONCLUSION: There was significant difference in metabolite content among different tissues of T. media. TFL and TS had higher metabolites and antioxidant capacity than other tissues, indicating that TFL and TS were more suitable for the development and utilization of T. media in foods and drinks.

Evaluation of Lipid Droplet Size and Fusion in Bovine Hepatic Cells.

Lipid droplets (LDs) are organelles that play an important role in lipid metabolism and neutral lipid storage in cells. They are associated with a variety of metabolic diseases, such as obesity, fatty liver disease, and diabetes. In hepatic cells, the sizes and numbers of LDs are signs of fatty liver disease. Moreover, the oxidative stress reaction, cell autophagy, and apoptosis are often accompanied by changes in the sizes and numbers of LDs. As a result, the dimensions and quantity of LDs are the basis of the current research regarding the mechanism of LD biogenesis. Here, in fatty acid-induced bovine hepatic cells, we describe how to use oil red O to stain LDs and to investigate the sizes and numbers of LDs. The size distribution of LDs is statistically analyzed. The process of small LDs fusing into large LDs is also observed by a live cell imaging system. The current work provides a way to directly observe the size change trend of LDs under different physiological conditions.

Variations of main quality components of matcha from different regions in the Chinese market.

Matcha has a unique aroma of seaweed-like, which is popular with Chinese consumers. In order to effectively understand and use matcha for drinks and tea products, we roundly analyzed the variation of main quality components of 11 matcha samples from different regions in the Chinese market. Most of matcha samples had lower ratio of tea polyphenols to amino acids (RTA), and the RTA of 9 samples of matcha was less than 10, which is beneficial to the formation of fresh and mellow taste of matcha. The total volatile compounds concentrations by HS-SPME were 1563.59 ~ 2754.09 mg/L, among which terpenoids, esters and alcohols were the top three volatile components. The total volatile compounds concentrations by SAFE was 1009.21 ~ 1661.98 mg/L, among which terpenoids, heterocyclic compounds and esters ranked the top three. The 147 volatile components with high concentration (>1 mg/L) and no difference between samples are the common odorants to the 11 samples of matcha. The 108 distinct odorants had differences among the matcha samples, which were important substances leading to the different aroma characteristics. Hierarchical cluster analysis (HCA) and orthogonal partial least squares discriminant analysis (OPLS-DA) showed that 11 samples of matcha were well clustered according to different components. Japanese matcha (MT, MY, ML, MR, MJ) could be clustered into two categories. The aroma composition of Guizhou matcha (GM1, GM2) was similar to that of Japanese matcha, 45 volatile components (decanal, pyrazine, 3,5-diethyl-2-methyl-, 1-hexadecanol, etc. were its characteristic aroma components. The aroma characteristics of Shandong matcha and Japanese matcha (ML, MR, MJ) were similar, 15 volatile components (γ-terpinene, myrtenol, cis-3-hexenyl valerate, etc.) were its characteristic aroma components. While Jiangsu matcha and Zhejiang matcha have similar aroma characteristics due to 225 characteristic aroma components (coumarin, furan, 2-pentyl-, etc). In short, the difference of volatile components formed the regional flavor characteristics of matcha. This study clarified the compound basis of the flavor difference of matcha from different regions in the Chinese market, and provided a theoretical basis for the selection and application of matcha in drinks and tea products.

Transcriptional Analysis of Tea Plants (Camellia sinensis) in Response to Salicylic Acid Treatment.

Salicylic acid (SA) is an important plant hormone and signal required for establishing resistance to diverse pathogens and plant diseases. The abundant polyphenols in tea plants also defend plants from biotic and abiotic stresses. However, whether exogenous SA would increase the resistance of tea plants to adversity and the relationship between SA and polyphenols are still poorly understood. Here, we carried out SA treatment on tea seedlings and performed transcriptome sequencing. SA treatment inhibited the phenylpropanoid and flavonoid metabolic pathways but promoted the lignin metabolic pathways. The increased accumulation of lignin in tea leaves after treating with SA indicated that lignin might coordinate SA, enhance, and improve plant defense and disease resistance. Simultaneously, an SA-inducible flavonoid glucosyltransferase (CsUGT0554) specifically involved in 7-OH site glycosylation was characterized in vitro. These results provided valuable information about the effects of SA on tea seedlings and the molecular basis for SA-mediated immune responses.

Effect of thermal process on the key aroma components of green tea with chestnut-like aroma.

BACKGROUND: Chestnut-like aroma is one of the unique qualities of Chinese green tea and has become an important factor influencing consumer decisions. However, the chemical formation mechanism of chestnut-like aroma during green tea processing remains unclear. In this study, the dynamic changes of key components contributing to chestnut-like aroma and their precursors were analyzed in fresh leaves, fixation leaves, first baking tea leaves, and green tea. RESULTS: The thermal process had an important effect on volatile components in tea leaves, causing a significant decrease of alcohols and esters and a significant increase of ketones, acids, phenols, and sulfur compounds. Furthermore, 31 volatiles were identified as the key odorants responsible for chestnut-like aroma of green tea, including dimethyl sulfide, methyl isobutenyl ketone, 2-methylbutanal, 2,4-dimethylstyrene, d-limonene, methyl 2-methylvalerate, linalool, decanal, longifolene, phenylethyl alcohol, l-α-terpineol, jasmone, and so on. And the majority of these odorants were only formed in the drying stage. Additionally, isoleucine, theanine, methionine, and glucose were found to be involved in the formation of chestnut-like aroma of green tea. CONCLUSION: The drying process played a vital important role in the formation of chestnut-like aroma of green tea. © 2022 Society of Chemical Industry.

Cardiolipin-Targeted NIR-II Fluorophore Causes "Avalanche Effects" for Re-Engaging Cancer Apoptosis and Inhibiting Metastasis.

Restoring innate apoptosis and simultaneously inhibiting metastasis by a molecular drug is an effective cancer therapeutic approach. Herein, a large rigid and V-shaped NIR-II dye, DUT850, is rationally designed for potential cardiolipin (CL)-targeted chemo-phototheranostic application. DUT850 displays moderate NIR-II fluorescence, excellent photodynamic therapy (PDT) and photothermal therapy (PTT) performance, and ultra-high photostability. More importantly, the unique rigid V-shaped backbone, positive charge, and lipophilicity of DUT850 afford its specific recognition and efficient binding to CL; such an interaction of DUT850-CL induced a spectrum of physiological disruptions, including translocation of cytochrome c, Ca2+ overload, reactive oxygen species burst, and ATP depletion, which not only activated cancer cell apoptosis but also inhibited tumor metastasis both in vitro and in vivo. Furthermore, the tight binding of DUT850-CL improves the phototoxicity of DUT850 toward cancer cells (IC50 as low as 90 nM) under safe 808 nm laser irradiation (330 mW cm-2). Upon encapsulation into bovine serum albumin (BSA), DUT850@BSA exerted a synergetic chemo-PDT-PTT effect on the 4T1 tumor mouse model, eventually leading to solid tumor annihilation and metastasis inhibition, which could be followed in real time with the NIR-II fluorescence of DUT850. This work contributed a promising approach for simultaneously re-engaging cancer cell apoptotic networks and activating the anti-metastasis pathway by targeting a pivotal upstream effector, which will bring a medical boon for inhibition of tumor proliferation and metastasis.

Long-Wavelength Photoconvertible Dimeric BODIPYs for Super-Resolution Single-Molecule Localization Imaging in Near-Infrared Emission.

Sulfoxide-bridged dimeric BODIPYs were developed as a new class of long-wavelength photoconvertible fluorophores. Upon visible-light irradiation, a sulfoxide moiety was released to generate the corresponding α,α-directly linked dimeric BODIPYs. The extrusion of SO from sulfoxides was mainly through an intramolecular fashion involving reactive triplet states. By this photoconversion, not only were more than 100 nm red shifts of absorption and emission maxima (up to 648/714 nm) achieved but also stable products with bright fluorescence were produced with high efficiency. The combination of photoactivation and red-shifted excitation/emission offered optimal contrast and eliminated the interference from biological autofluorescence. More importantly, the in situ products of these visible-light-induced reactions demonstrated ideal single-molecule fluorescence properties in the near-infrared region. Therefore, this new photoconversion could be a powerful photoactivation method achieving super-resolution single-molecule localization imaging in a living cell without using UV illumination and cell-toxic additives.

Bio-orthogonal Toolbox for Monitoring Nitric Oxide in Targeted Organelles of Live Cells and Zebrafishes.

Nitric oxide (NO), playing crucial roles as a cellular messenger and as a toxic ROS, is highly related to the physiological and pathological states of living systems. The very wide but very uneven distribution of this radical gas in the inhomogeneous biological microenvironment imposes big challenges for specifically detecting its local level in certain subcellular areas, which calls for a long list of NO probes for each target. In order to simplify the syntheses and designs of these probes, herein it is proposed to construct a versatile NO-sensing toolbox based on a bio-orthogonal concept, i.e., inverse electron demand Diels-Alder click reaction between tetrazine and strained alkyne BCN. On the one hand, rhodamine-o-phenylenediamine as the NO-responsive scaffold is coupled with a tetrazine unit to generate a general probe TMR-Tz-NO, which, to our knowledge, is the first case of the tetrazine-coupled analyte-responsive probe. On the other hand, the BCN moiety is connected to different targeting groups, such as TPP, morpholine, and Ac4ManN, targeting to mitochondria, lysosomes, and membranes, respectively. It works well to use TMR-Tz-NO to match with any targetable BCN counterpart in this toolbox to achieve the imaging of NO in the corresponding subcellular area. For example, through metabolism, Ac4ManN-BCN is effectively taken and grows on the cell membranes. The bio-orthogonal reaction between TMR-Tz-NO and Ac4ManN-BCN makes the NO probe anchored to the membrane surface permanently. The zebrafish experiment revealed that this bio-orthogonal pair can track and image the NO produced during inflammation in vivo.

Outer Surface-Labeled Bacteria as Live Sensors Accurately Quantitating Interfacial pH: A Smart Technique for Antimicrobial Resistance.

The techniques to quantitatively monitor environmental factors surrounding the bacterial outer surface rather than the host's subcellular regions (e.g., lysosomes) should be the key to evaluate bacterial immune escape behavior. We report wild Staphylococcus aureus (SA) and methicillin-resistant Staphylococcus aureus (MRSA) labeled with a fluorescent resonance energy transfer probe, 4SR-L-BDP, on their outer surfaces as smart live sensors to quantify interfacial pH. The dual emission of 4SR-L-BDP affords high sensitivity to pH change in a ratiometric way in the pH range of 4-8 with high precision. Notably, 4SR-L-BDP possesses an anchoring group to fix on the bacterial surface for sensing the microenvironment encountered. Super-resolution imaging clearly demonstrates the specific labeling of bacterial membranes. These live sensors are applied in two-channel ratiometric imaging to dynamically visualize and quantify their interfacial pH changes during infection of macrophages. It is found that the interfacial pH of MRSA is lower by 0.2 units compared to that of SA. Such small but critical difference in pH reflects MRSA's ability to adapt to microenvironmental pH inside macrophages. These labeled bacteria as live sensors are also proven to be practically applicable in mice models with immune deficiency and immune activation.

Characterization of the Key Aroma Compounds of Shandong Matcha Using HS-SPME-GC/MS and SAFE-GC/MS.

Shandong matcha has the quality characteristics of bright green color, seaweed-like aroma and strong, fresh and brisk taste. In order to identify the characteristic aroma components and clarify the contribution of the grinding process to the aroma of Shandong matcha. Three grades of Shandong matcha and corresponding tencha material were firstly tested with sensory evaluation, and the volatile components were extracted with headspace solid-phase microextraction (HS-SPME) and solvent-assisted flavor evaporation (SAFE) and analyzed using GC-MS. The sensory evaluation results showed that high-grade matcha (M-GS) had prominent seaweed-like, fresh and roasted notes, whereas medium and low-grade matcha (M-G1, M-G2) were gradually coupled with grassy, fatty and high-fired aromas. GC-MS results showed that in the HS-SPME method, heterocyclic compounds (45.84-65.35%) were the highest in Shandong matcha, followed by terpenoids (7.44-16.92%) and esters (6.91-15.27%), while in the safe method, esters were the highest (12.96-24.99%), followed by terpenoids (10.76-25.09%) and heterocyclic compounds (12.12-17.07%). As a whole, the composition of volatile components between M-G1 and M-G2 is relatively close, and there are more differences in volatile components between them and M-GS. The volatile components unique to M-GS were screened using the odor activity value (OAV) evaluation method, with components such as 3-methyl-2-butene-1-thiol, 3-ethyl-Phenol, 2-thiophenemethanethiol, 2,4-undecadienal, (E,E)-2,6-nonadienal, (E,Z)- being evaluated. There were other differentially volatile components, that is, volatile components that coexist in the three grades of matcha, but with different concentrations and proportions. M-G1 and M-G2 contained more volatile substances with high-fired aroma, such as 2-ethyl-3-methyl-pyrazine, coumarin and 5,6,7,8-tetrahydroquinoxaline. The grinding process not only changes the appearance of tencha, but also increases the content of volatile components of matcha as a whole, enhancing the aroma and flavor characteristics of matcha. In this study, the contents of 24 volatile components in matcha were mainly increased, such as benzene, (2,2-dimethoxyethyl)-, cis-7-decen-1-al, safranal and fenchyl acetate. The dual factors of material tencha and matcha grinding technology are indispensable in forming the differences in aroma and flavor of Shandong matcha at different levels.

HS-SPME/GC-MS Reveals the Season Effects on Volatile Compounds of Green Tea in High-Latitude Region.

This study investigates the volatile compounds of green tea produced with different leaves from spring, summer, and autumn in high-latitude region. A total of 95 volatile compounds were identified by gas chromatography-mass spectrometry (GC-MS). Spring, summer and autumn green tea contained 68, 72 and 82 volatile compounds, respectively. Principal component analysis (PCA), partial least squares-discrimination analysis (PLS-DA), and hierarchical cluster analysis (HCA) classified the samples and showed the difference. And 32 key characteristic components were screened out based on variable importance in the projection (VIP) values higher than 1.0. The characteristic volatile compounds of spring green tea including 18 components, such as geranylacetone, phenethyl alcohol, geraniol, ß-ionone, jasmone, 1-octen-3-ol and longifolene. 13 components such as 2-methylfuran, indole, 1-octanol, D-limonene and ethanethiol were the key compounds in summer green tea. And 2,4,6-trimethylstyrene was the major differential volatile compounds in autumn green tea. The results increase our knowledge of green tea in different seasons and provide a theoretical basis for production control of green tea.

Polarity-Sensitive and Membrane-Specific Probe Quantitatively Monitoring Ferroptosis through Fluorescence Lifetime Imaging.

As a new form of regulated cell death, ferroptosis is closely related to various diseases. To interpret this biological behavior and monitor related pathological processes, it is necessary to develop appropriate detection strategies and tools. Considering that ferroptosis is featured with remarkable lipid peroxidation of various cell membranes, it is logical to detect membranes' structural and environmental changes for the direct assessment of ferroptosis. For this sake, we designed novel polarity-sensitive fluorescent probes Mem-C1C18 and Mem-C18C18, which have superior plasma membrane anchorage, high brightness, and sensitive responses to environmental polarity by changing their fluorescence lifetimes. Mem-C1C18 with much less tendency to aggregate than Mem-C18C18 outperformed the latter in high resolution fluorescence labeling of artificial vesicle membranes and plasma membranes of live cells. Thus, Mem-C1C18 was selected to monitor plasma membranes damaged along ferroptosis process for the first time, in combination with the technique of fluorescence lifetime imaging (FLIM). After treating HeLa cells with Erastin, a typical ferroptosis inducer, the mean fluorescence lifetime of Mem-C1C18 displayed a considerable increase from 3.00 to 4.93 ns, with a 64% increase (corresponding to the polarity parameter Δf increased from 0.213 to 0.232). Therefore, our idea to utilize a probe to quantitate the changes in polarity of plasma membranes proves to be an effective method in the evaluation of the ferroptosis process.

A Golgi-targeted viscosity rotor for monitoring early alcohol-induced liver injury.

We proposed to monitor the early stage of alcohol-induced liver injury through quantitatively detecting Golgi viscosity. Therefore, the first Golgi-targeted fluorescent rotor (GA-Vis) was developed. With the aid of GA-Vis, the changes in Golgi viscosity during alcohol-induced liver injury were quantitatively evaluated by fluorescence lifetime imaging in live cells and zebrafish. GA-Vis was qualified as a practical tool for future diagnoses of alcohol-induced liver injury.

Study on the Effect of Pyrolysis Conditions on the Combustion Behavior and Char Structure Evolution.

Pyrolysis polygeneration technology has been widely applied due to its wide adaptability to coal types and mild reaction conditions. In this paper, the in situ process test combining pyrolysis and combustion of Pingshuo coal was carried out using a thermogravimetric analyzer and a fluidized-bed reactor. The influence of pyrolysis conditions on the combustion behavior and char structure was studied. The reduction of the pyrolysis temperature and the increase in pyrolysis residence time resulted in a lower burnout temperature of char, thereby improving the combustion characteristics of char. Furthermore, the maximum CO2 release rate reached 2.8 mg/(g·s) during the combustion process. The decrease in the pyrolysis temperature was conducive to the increase of CO2 release mass, suggesting a more complete burning of char. The char combustion process was mainly controlled by the chemical reaction according to the reaction kinetics study. The apparent rate constant of the combustion process increased gradually with the decrease in the pyrolysis temperature of char, and the rate of the combustion reaction increased, which contributed to the enhancement of the combustion performance of the char within the range of 850-950 °C. Meanwhile, the crystal structure inside the char changed significantly after different pyrolysis conditions. However, it had little influence on the molar content of carbon structure, which suggested that the amount of CO2 released was mainly caused by the different resistances of oxygen diffusion to the reaction interface.

A new six-membered spiro-rhodamine probe for Cu2+ and its imaging in mitochondria and lysosomes of Hela cells.

Different from five-membered rhodamine spirolactam (Rh-OH), a new six-membered spiro-rhodamine probe (SRh-OH) bearing urea structure has been developed in this paper. Compared with five-membered Rh-OH, six-membered SRh-OH exhibited higher selectivity and sensitivity to Cu2+ in aqueous solution. Upon addition of 10 equiv. Cu2+, the molar extinction coefficient of SRh-OH at 563 nm can be up to 4.73 × 104 Lmol-1cm-1. In the range of Cu2+ from 0 to 28 µM, there was an excellent linear relationship between the absorption or emission intensity and Cu2+ concentration. The detection limit of SRh-OH was as low as 26 nM (S/N = 3). The reversible binding mode of SRh-OH with Cu2+ was further confirmed by S2- and EDTA addition. Bio-imaging showed SRh-OH can map the distribution of Cu2+ not only in mitochondria but also in lysosomes of Hela cells.

Adaptation by Type III CRISPR-Cas Systems: Breakthrough Findings and Open Questions.

CRISPR-Cas systems acquire heritable defense memory against invading nucleic acids through adaptation. Type III CRISPR-Cas systems have unique and intriguing features of defense and are important in method development for Genetics research. We started to understand the common and unique properties of type III CRISPR-Cas adaptation in recent years. This review summarizes our knowledge regarding CRISPR-Cas adaptation with the emphasis on type III systems and discusses open questions for type III adaptation studies.

Intratumoral delivery of IL-12 and IL-27 mRNA using lipid nanoparticles for cancer immunotherapy.

Cytokines are important immunotherapeutics with approved drugs for the treatment of human cancers. However, systemic administration of cytokines often fails to achieve adequate concentrations to immune cells in tumors due to dose-limiting toxicity. Thus, developing localized therapy that directly delivers immune-stimulatory cytokines to tumors may improve the therapeutic efficacy. In this study, we generated novel lipid nanoparticles (LNPs) encapsulated with mRNAs encoding cytokines including IL-12, IL-27 and GM-CSF, and tested their anti-tumor activity. We first synthesized ionizable lipid materials containing di-amino groups with various head groups (DALs). The novel DAL4-LNP effectively delivered different mRNAs in vitro to tumor cells and in vivo to tumors. Intratumoral injection of DAL4-LNP loaded with IL-12 mRNA was most potent in inhibiting B16F10 melanoma tumor growth compared to IL-27 or GM-CSF mRNAs in monotherapy. Furthermore, intratumoral injection of dual DAL4-LNP-IL-12 mRNA and IL-27 mRNA showed a synergistic effect in suppressing tumor growth without causing systematic toxicity. Most importantly, intratumoral delivery of IL-12 and IL-27 mRNAs induced robust infiltration of immune effector cells, including IFN-γ and TNF-α producing NK and CD8+ T cells into tumors. Thus, intratumoral administration of DAL-LNP loaded with IL-12 and IL-27 mRNA provides a new treatment strategy for cancer.