Rab26 alleviates sepsis-induced immunosuppression as a master regulator of macrophage ferroptosis and polarization shift.

Macrophage dysfunction is a significant contributor to more than 70 % of sepsis-related deaths, specifically secondary bacterial infections, during the immunosuppression stage of sepsis. Nevertheless, the role of Rab26 in this context remains unclear. In this study, we observed a substantial decrease in Rab26 expression in macrophages during the immunosuppressive phase of sepsis, which was also found to be suppressed by high extracellular levels of HMGB1. During the progression of sepsis, Rab26 deficiency promotes a polarization shift from the M1 to the M2-like phenotype in macrophages, rendering them susceptible to ferroptosis. Subsequent experimentation has revealed that Rab26 deficiency facilitates the degradation of GPX4, thereby aggravating macrophage ferroptosis through the upregulation of levels of lipid ROS, MDA, and ferrous iron induced by RSL3, a ferroptosis inducer. Additionally, Rab26-deficient mice in the immunosuppressed phase of sepsis exhibit heightened susceptibility to secondary infections, leading to exacerbated lung tissue damage and increased mortality rate. Overall, these findings indicate that Rab26 plays a crucial role in sepsis-induced macrophage immunosuppression by regulating macrophage ferroptosis and polarization. Hence, it represents a potential novel target for sepsis therapy.

Rab26 promotes macrophage phagocytosis through regulation of MFN2 trafficking to mitochondria.

Acute respiratory distress syndrome (ARDS) is an inflammatory disorder of the lungs caused by bacterial or viral infection. Timely phagocytosis and clearance of pathogens by macrophages are important in controlling inflammation and alleviating ARDS. However, the precise mechanism of macrophage phagocytosis remains to be explored. Here, we show that the expression of Rab26 is increased in Escherichia coli- or Pseudomonas aeruginosa-stimulated bone marrow-derived macrophages. Knocking out Rab26 reduced phagocytosis and bacterial clearance by macrophages. Rab26 interacts with mitochondrial fusion protein mitofusin-2 (MFN2) and affects mitochondrial reactive oxygen species generation by regulating MFN2 transport. The levels of MFN2 in mitochondria were reduced in Rab26-deficient bone marrow-derived macrophages, and the levels of mitochondrial reactive oxygen species and ATP were significantly decreased. Knocking down MFN2 using small interfering RNA resulted in decreased phagocytosis and killing ability of macrophages. Rab26 knockout reduced phagocytosis and bacterial clearance by macrophages in vivo, significantly increased inflammatory factors, aggravated lung tissue damage, and increased mortality in mice. Our results demonstrate that Rab26 regulates phagocytosis and clearance of bacteria by mediating the transport of MFN2 to mitochondria in macrophages, thus alleviating ARDS in mice and potentially in humans.

Attenuating endothelial leakiness with self-assembled DNA nanostructures for pulmonary arterial hypertension.

Vascular endothelium dysfunction plays an important role in oncological and pulmonary diseases. Endothelial barrier dysfunction is the initial step of pulmonary vascular remodeling (PVR) and pulmonary arterial hypertension. Upregulation of a pro-autophagy protein Atg101 in the endothelial cells triggered a cascade of intracellular events that leads to endothelial dysfunction through apoptosis. Herein, we proposed a strategy that used endothelial targeting DNA nanostructures to deliver Atg101 siRNA (siAtg101) as a safe, biocompatible "band-aid" to restore pulmonary arterial endothelial barrier integrity within the intricate milieu of pulmonary cells and the pulmonary vasculature. The siAtg101 and aptamer conjugated DNA nanostructures were found to attenuate hypoxia-induced pulmonary endothelial leakiness with surprisingly high selectivity and efficacy. Further in vivo study revealed that functionalized DNA nanostructures likewise attenuated the vascular remodeling in a monocrotaline-induced PVR mouse model. Mechanistically, functionalized DNA nanostructures suppressed PVR by knocking down Atg101, which in turn, downregulated Beclin-1 and subsequently upregulated VE-cadherin to restore endothelial cells' adherin junctions. This work opened a new window for future nanomaterial design that directly addresses the interfacial endothelial cell layer that often stands between the blood and many diseased sites of nanotherapeutic interest.

Framework Nucleic Acids Enabled Pulmonary Artery Endothelial Cell Growth Inhibition by Targeting microRNA-152.

Pulmonary artery vascular endothelial dysfunction plays a pivotal role in the occurrence and progression of pulmonary vascular remodeling (PVR). To address this, aberrantly expressed non-coding microRNAs (miRNAs) are excellent therapeutic targets in human pulmonary artery endothelial cells (HPAECs). Here, we discovered and validated the overexpression of miRNA-152 in HPAECs under hypoxia and its role in endothelial cell dysfunction. We constructed a framework nucleic acid nanostructure that harbors six protruding single-stranded DNA segments that can fully hybridize with miRNA-152 (DNT-152). DNT-152 was efficiently taken up by HPAECs with increasing time and concentration; it markedly induced apoptosis, and inhibited HPAEC growth under hypoxic conditions. Mechanistically, DNT-152 silenced miRNA-152 expression and upregulated its target gene Meox2, which subsequently inhibited the AKT/mTOR signaling pathway. These results indicate that miRNA-152 in HPAECs may be an excellent therapeutic target against PVR, and that framework nucleic acids with carefully designed sequences are promising nanomedicines for noncancerous cells and diseases.

The TLR7/8 agonist R848 optimizes host and tumor immunity to improve therapeutic efficacy in murine lung cancer.

Treatment with the Toll­like receptor 7 (TLR7) agonist, resiquimod (R848), is effective in various types of cancer, such as breast, pancreatic and colorectal cancer. The reported antitumor effect of R848 in lung cancer is considered to be achieved by targeting macrophages. In the present study, it was demonstrated that TLR7 expression on various immune cell types initially rises, then declines in the late stage of lung cancer. Intraperitoneal injection of R848 resulted in a reduction in tumor burden and prolonged survival in both subcutaneous and metastatic lung cancer models in C57BL/6 mice. Initial treatment with R848 at an early stage was found to be the optimal choice. Systemic injection of R848 promoted the activation of innate and adaptive immune responses. Systemic administration of R848 upregulated TLR7 expression in dendritic cells (DCs) and enhanced the activation of DCs and natural killer (NK) cells. Moreover, this treatment also resulted in increased production of T helper cell­associated cytokines in serum, including IFN­Î³, TNF­α and IL­2. In addition, continuous treatment with R848 increased the proportion of DCs, NK and CD8+ T cells, and reduced that of Foxp3+ regulatory T cells in the tumor microenvironment. These findings supported the use of R848 treatment for lung cancer via TLR7 targeting and provided insight into the underlying therapeutic mechanism.

Extracellular HMGB1 Impairs Macrophage-Mediated Efferocytosis by Suppressing the Rab43-Controlled Cell Surface Transport of CD91.

High-mobility group box 1 (HMGB1) protein can impair phagocyte function by suppressing the macrophage-mediated clearance of apoptotic cells (ACs), thereby delaying inflammation resolution in the lungs and allowing the progression of acute lung injury (ALI) and acute respiratory distress syndrome (ARDS). However, the precise mechanism underlying this HMGB1-mediated inhibition of efferocytosis remains unknown. The aim of this study was to determine the effect of HMGB1 on macrophage-mediated efferocytosis. We discovered that HMGB1 prevented efferocytosis by bone marrow-derived macrophages (BMDMs) and suppressed the expression of Ras-related GTP-binding protein 43 (Rab43), a member of the Ras-associated binding (Rab) family. The downregulation of Rab43 expression resulted in impaired clearance of apoptotic thymocytes by BMDMs. Subsequent analysis of HMGB1-treated and Rab43-deficient BMDMs revealed the inhibited transport of cluster of differentiation 91 (CD91), a phagocyte recognition receptor, from the cytoplasm to the cell surface. Notably, Rab43 directly interacted with CD91 to mediate its intercellular trafficking. Furthermore, Rab43 knockout delayed the inflammation resolution and aggravated the lung tissue damage in mice with ALI. Therefore, our results provide evidence that HMGB1 impairs macrophage-mediated efferocytosis and delays inflammation resolution by suppressing the Rab43-regulated anterograde transport of CD91, suggesting that the restoration of Rab43 levels is a promising strategy for attenuating ALI and ARDS in humans.

Extracellular CIRP-Impaired Rab26 Restrains EPOR-Mediated Macrophage Polarization in Acute Lung Injury.

Acute lung injury (ALI)/acute respiratory distress syndrome (ARDS) is a condition with an imbalanced inflammatory response and delayed resolution of inflammation. Macrophage polarization plays an important role in inflammation and resolution. However, the mechanism of macrophage polarization in ALI/ARDS is not fully understood. We found that mice with lipopolysaccharide administration developed lung injury with the accumulation of extracellular cold-inducible RNA-binding protein (eCIRP) in the lungs. eCIRP, as a damage-associated molecular pattern (DAMP), inhibited M2 macrophage polarization, thereby tipping the balance toward inflammation rather than resolution. Anti-CIRP antibodies reversed such phenotypes. The levels of macrophage erythropoietin (EPO) receptor (EPOR) were reduced after eCIRP treatment. Myeloid-specific EPOR-deficient mice displayed restrained M2 macrophage polarization and impaired inflammation resolution. Mechanistically, eCIRP impaired Rab26, a member of Ras superfamilies of small G proteins, and reduced the transportation of surface EPOR, which resulted in macrophage polarization toward the M1 phenotype. Moreover, EPO treatment hardly promotes M2 polarization in Rab26 knockout (KO) macrophages through EPOR. Collectively, macrophage EPOR signaling is impaired by eCIRP through Rab26 during ALI/ARDS, leading to the restrained M2 macrophage polarization and delayed inflammation resolution. These findings identify a mechanism of persistent inflammation and a potential therapy during ALI/ARDS.

Assembling Defined DNA Nanostructure with Nitrogen-Enriched Carbon Dots for Theranostic Cancer Applications.

DNA nanostructures as scaffolds for drug delivery, biosensing, and bioimaging are hindered by its vulnerability in physiological settings, less favorable of incorporating arbitrary guest molecules and other desirable functionalities. Noncanonical self-assembly of DNA nanostructures with small molecules in an alternative system is an attractive strategy to expand their applications in multidisciplinary fields and is rarely explored. This work reports a nitrogen-enriched carbon dots (NCDs)-mediated DNA nanostructure self-assembly strategy. Given the excellent photoluminescence and photodynamic properties of NCDs, the obtained DNA/NCDs nanocomplex holds great potential for bioimaging and anticancer therapy. NCDs can mediate DNA nanoprism (NPNCD ) self-assembly isothermally at a large temperature and pH range in a magnesium-free manner. To explore the suitability of NPNCD in potential biomedical applications, the cytotoxicity and cellular uptake efficiency of NPNCD are evaluated. NPNCD with KRAS siRNA (NPNCD K) is further conjugated for KRAS-mutated nonsmall cell lung cancer therapy. The NPNCD K shows excellent gene knockdown efficiency and anticancer effect in vitro. The current study suggests that conjugating NCDs with programmable DNA nanostructures is a powerful strategy to endow DNA nanostructures with new functionalities, and NPNCD may be a potential theranostic platform with further fine-tuned properties of CDs such as near-red fluorescence or photothermal activities.

Efficacy of Shenqi Pollen Capsules for High-Altitude Deacclimatization Syndrome via Suppression of the Reoxygenation Injury and Inflammatory Response.

High-altitude deacclimatization syndrome (HADAS) is involved in hypoxia-reoxygenation injury and inflammatory response, induced a series of symptoms, and has emerged as a severe public health issue. Here, we investigated the mechanism as well as potential means to prevent HADAS using Shenqi pollen capsules (SPCs) in subjects with HADAS in a multicenter, double-blinded, randomized, placebo-controlled study. All subjects were at the same high altitude (3650 m) for 4-8 months before returning to lower altitudes. Subjects (n = 288) in 20 clusters were diagnosed with mild or moderate HADAS on the third day of the study. We randomly allocated 20 clusters of subjects (1 : 1) to receive SPCs or a placebo for 7 weeks, and they were then followed up to the 14th week. The primary endpoints were subjects' HADAS scores recorded during the 14 weeks of follow-up. Compared with the placebo, SPC treatment significantly decreased the subjects' HADAS scores and reduced the incidence of symptom persistence. SPC therapy also reduced the serum levels of CK, CK-MB, LDH, IL-17A, TNF-α, and miR-155 and elevated IL-10 and miR-21 levels. We thus demonstrate that SPCs effectively ameliorated HADAS symptoms in these subjects via suppression of the hypoxia-reoxygenation injury and inflammatory response.

Mutated p53 Promotes the Symmetric Self-Renewal of Cisplatin-Resistant Lung Cancer Stem-Like Cells and Inhibits the Recruitment of Macrophages.

It has been proposed that mutant p53 is correlated with the recurrence of lung cancer. Recently, a small population of cells with asymmetric or symmetric self-renewal potential has been identified in lung cancer, which was termed as cancer stem-like cells (CSCs) and was speculated to be the reason for cancer recurrence after chemotherapy. In this study, we used lung cancer cell lines with different TP53 backgrounds to elucidate the potential role of mutant p53 in regulating lung CSC self-renewal and on lung cancer recurrence. Cisplatin-resistant lung cancer cells with different TP53 backgrounds were generated in vitro by exposing A549, H460, and H661 lung cancer cell lines repeatedly to cisplatin. CD44+/CD90+ stem-like cells were identified in above cisplatin-resistant lung cancers (termed as cisplatin-resistant lung cancer stem-like cells, (Cr-LCSCs)) and stained with PKH26 dye which was used to define the self-renewal pattern. The proportion of symmetric divisions was significantly higher in Cr-LCSCs with mutant (mt) p53 compared with Cr-LCSCs with wild-type (wt) p53, and forced expression of mt p53 promoted the symmetric division of Cr-LCSCs. Furthermore, fewer macrophages accumulated in subcutaneously implanted xenografts consisting of mt p53 Cr-LCSCs compared with wt p53 Cr-LCSCs. These results indicated that mt p53 might accelerate the recurrence of lung cancer by regulating the self-renewal kinetics of Cr-LCSCs as well as the recruitment of macrophages.

Targeted Delivery of Rab26 siRNA with Precisely Tailored DNA Prism for Lung Cancer Therapy.

Programmable DNA nanostructures are a new class of biocompatible, nontoxic nanomaterials. Nevertheless, their application in the field of biomedical research is still in its infancy, especially as drug delivery vehicles for gene therapy. In this study, a GTPase Rab26 was investigated as a new potential therapeutic target using a precisely tailored DNA nanoprism for targeted lung cancer therapy. Specifically, a DNA nanoprism platform with tunable targeting and siRNA loading capability is designed and synthesized. The as-prepared DNA prisms were decorated with two functional units: a Rab26 siRNA as the drug and MUC-1 aptamers as a targeting moiety for non-small cell lung cancer. The number and position of both siRNA and MUC-1 aptamers can be readily tuned by switching two short, single-stranded DNA. Native polyacrylamide gel electrophoresis (PAGE) and dynamic light scattering technique (DLS) demonstrate that all nanoprisms with different functionalities are self-assembled with high yield. It is also found that the cellular uptake of DNA prisms is proportional to the aptamer number on each nanoprism, and the as-prepared DNA nanoprism show excellent anti-cancer activities and targeting capability. This study suggests that by careful design, self-assembled DNA nanostructures are highly promising, customizable, multifunctional nanoplatforms for potential biomedical applications, such as personalized precision therapy.

Predictive Value of Combined LIPS and ANG-2 Level in Critically Ill Patients with ARDS Risk Factors.

To investigate the predictive value of the acute physiology and chronic health evaluation 2 (APACHE2) score and lung injury prediction score (LIPS) for acute respiratory distress syndrome (ARDS) when combined with biomarkers for this condition in patients with ARDS risk factors. In total, 158 Han Chinese patients with ARDS risk factors were recruited from the Respiratory and Emergency Intensive Care Units. The LIPS, APACHE2 score, primary diagnosis at admission, and ARDS risk factors were determined within 6 h of admission, and PaO2/FiO2 was determined on the day of admission. Blood was collected within 24 h of admission for the measurement of angiopoietin-2 (ANG-2), sE-selectin, interleukin-6 (IL-6), and interleukin-8 (IL-8) levels. ARDS was monitored for the next 7 days. Univariate and multivariate analyses and receiver operating characteristic (ROC) analyses were employed to construct a model for ARDS prediction. Forty-eight patients developed ARDS within 7 days of admission. Plasma ANG-2 level, sE-selectin level, LIPS, and APACHE2 score in ARDS patients were significantly higher than those in non-ARDS patients. ANG-2 level, LIPS, and APACHE2 score were correlated with ARDS (P < 0.001, P < 0.006, and P < 0.042, resp.). When the APACHE2 score was used in combination with the LIPS and ANG-2 level to predict ARDS, the area under the ROC curve (AUC) was not significantly increased. Compared to LIPS or ANG-2 alone, LIPS in combination with ANG-2 had significantly increased positive predictive value (PPV) and AUC for the prediction of ARDS. In conclusion, plasma ANG-2 level, LIPS, and APACHE2 score are correlated with ARDS. Combined LIPS and ANG-2 level displays favorable sensitivity, specificity, and AUC for the prediction of ARDS.

In vivo production of RNA nanostructures via programmed folding of single-stranded RNAs.

Programmed self-assembly of nucleic acids is a powerful approach for nano-constructions. The assembled nanostructures have been explored for various applications. However, nucleic acid assembly often requires chemical or in vitro enzymatical synthesis of DNA or RNA, which is not a cost-effective production method on a large scale. In addition, the difficulty of cellular delivery limits the in vivo applications. Herein we report a strategy that mimics protein production. Gene-encoded DNA duplexes are transcribed into single-stranded RNAs, which self-fold into well-defined RNA nanostructures in the same way as polypeptide chains fold into proteins. The resulting nanostructure contains only one component RNA molecule. This approach allows both in vitro and in vivo production of RNA nanostructures. In vivo synthesized RNA strands can fold into designed nanostructures inside cells. This work not only suggests a way to synthesize RNA nanostructures on a large scale and at a low cost but also facilitates the in vivo applications.

Isothermal Self-Assembly of Spermidine-DNA Nanostructure Complex as a Functional Platform for Cancer Therapy.

Programmable DNA nanostructure self-assembly offers great potentials in nanomedicine, drug delivery, biosensing, and bioimaging. However, due to the intrinsically negatively charged DNA backbones, the instability of DNA nanostructures in physiological settings poses serious challenges to their practical applications. To overcome this challenge, a strategy that combines the magnesium-free DNA self-assembly and functionalization is proposed in this study. We hypothesize that naturally abundant spermidine may not only mediate the self-assembly of DNA nanostructures, but also shield them from harsh physiological environments. As a proof of concept, a DNA nanoprism is designed and synthesized successfully through spermidine. It is found that spermidine can mediate the isothermal self-assembly of DNA nanoprisms. Compared to conventional Mg2+-assembled DNA nanostructures, the spermidine-DNA nanoprism complex shows higher thermal stability and better enzymatic resistance than Mg2+-assembled DNA nanoprisms, and more importantly, it has a much higher cellular uptake efficacy in multiple cancerous cell lines. The internalization mechanism is identified as clathrin-mediated endocytosis. To demonstrate the suitability of this new nanomaterial for biomedical applications, an mTOR siRNA, after being conjugated into the complex, is efficiently delivered into cancer cells and shows excellent gene knockdown efficacy and anticancer capability. These findings indicate that the spermidine-DNA complex nanomaterials might be a promising platform for biomedical applications in the future.

Maskless Hydrophilic Patterning of the Superhydrophobic Aluminum Surface by an Atmospheric Pressure Microplasma Jet for Water Adhesion Controlling.

Superhydrophobic surfaces with hydrophilic patterns have great application potential in various fields, such as microfluidic systems and water harvesting. However, many reported preparation methods involve complicated devices and/or masks, making fabrication of these patterned surfaces time-consuming and inefficient. Here, we propose a highly efficient, simple, and maskless microplasma jet (MPJ) treatment method to prepare hydrophilic patterns such as dots, lines, and curves on superhydrophobic aluminum substrates. Contact angles, sliding angles, adhesive forces, and droplet impact behavior of the created patterns are investigated and analyzed. The prepared "dot" patterns exhibit great water adhesion, whereas the "line" patterns show anisotropic adhesion. Additionally, the MPJ treatment does not obviously change the surface structures, which makes it possible to achieve repeatable patterning on one substrate. The adhesion behavior of these patterns could be adjusted using MPJs with different diameters. MPJs with larger diameters are efficient for the creation of patterns with high water adhesion, which can be potentially used for open-channel lab-on-chip systems (e.g., continuous water transportation), whereas MPJs with smaller diameters are preferable in preparing patterns with low water adhesion for diverse applications in biomedical fields (e.g., lossless liquid droplet mixing and cell screening).

Downregulation of serum miR-101 is associated with worse prognosis in non-small cell lung cancer.

Non-small cell lung cancer (NSCLC) is the leading cause of cancer-associated deaths, worldwide, and its prognosis is unfavorable. The aim of this study was to detect serum miR-101 levels in NSCLC patients and investigate its potential diagnostic and prognostic value. A total of 93 patients with NSCLC, 40 cases with various benign lung disease, and 55 healthy volunteers, were enrolled. Quantitative RT-PCR was performed to determine relative serum miR-101 levels in our participants. Decreased serum miR-101 expression was observed in patients with NSCLC and was closely associated with aggressive clinical characteristics. In addition, a significant increase in serum miR-101 levels was found in 36 NSCLC cases after tumor resection. Moreover, receiver-operating characteristic (ROC) curve analysis showed that serum miR-101 was an effective indicator for NSCLC diagnosis. Furthermore, Kaplan-Meier survival curve analysis revealed that low serum miR-101 expression predicted poor overall survival and disease-free survival. Finally, multivariate analysis confirmed serum miR-101 expression was an independent prognostic factor for NSCLC patients. In conclusion, serum miR-101 might serve as a potential biomarker for detection and prognosis evaluation of NSCLC.

Regulation on Toll-like Receptor 4 and Cell Barrier Function by Rab26 siRNA-loaded DNA Nanovector in Pulmonary Microvascular Endothelial Cells.

The small GTPase Rab26 is involved in multiple processes, such as vesicle-mediated secretion and autophagy. However, the mechanisms and functions of Rab26 in the human pulmonary microvascular endothelial cells (HPMVECs) are not clear. In this study, we thoroughly investigated the role and novel mechanism of Rab26 in permeability and apoptosis of HPMVECs using a self-assembled Rab26 siRNA loaded DNA Y-motif nanoparticle (siRab26-DYM) and Rab26 adenovirus. We found that siRab26-DYM could be efficiently transfected into HPMVECs in a time- and dose-dependent manner. Importantly, the siRab26-DYM nanovector markedly aggravated the LPS-induced apoptosis and hyper-permeability of HPMVECs by promoting the nuclear translocation of Foxo1, and subsequent activation of Toll-like receptor 4 (TLR4) signal pathway. Overexpression of Rab26 by Rab26 adenoviruses partially inactivated LPS-induced TLR4 signaling pathway, suppressed the cell apoptosis and attenuated the hyperpermeability of HPMVECs. These results suggest that the permeability and apoptosis of HPMVECs can be modulated by manipulating Rab26 derived TLR4 signaling pathway, and that Rab26 can be potential therapeutic target for the treatment of vascular diseases related to endothelial barrier functions.

Activation of PPARγ inhibits pro-inflammatory cytokines production by upregulation of miR-124 in vitro and in vivo.

Peroxisome proliferator-activated receptor gamma (PPARγ) and miR-124 have been reported to play important roles in regulation of inflammation. However, the underlying anti-inflammatory mechanisms remain not well understood. In the present study, we demonstrated that the expression level of PPARγ is positively correlated with that of miR-124 in patients with sepsis. Activation of PPARγ upregulates miR-124 and in turn inhibits miR-124 target gene. PPARγ bound directly to PPRE in the miR-124 promoter region, and enhanced the promoter transcriptional activity. PPARγ-induced miR-124 is involved in the suppression of pro-inflammatory cytokine in vitro and in vivo. These results suggest that PPARγ-induced miR-124 inhibits the production of pro-inflammatory cytokines is a novel PPARγ anti-inflammatory mechanism and also indicate that miR-124 may be a potential therapeutic target for the treatment of inflammatory diseases.

Self-assembled triangular DNA nanoparticles are an efficient system for gene delivery.

Developing an advanced nucleic acid drug delivery system is of great significance in order to achieve optimal gene delivery. Self-assembled nucleic acid nanoparticles are an excellent platform for the delivery of nucleic acids and other small molecular drugs. In this study, we developed the efficient, three-stranded, RNA/DNA hybrid triangular self-assembled nanoparticles, namely, mTOR single-stranded siRNA-loaded triangular DNA nanoparticles (ssRNA-TNP). The ssRNA-TNP is formed by the complementary association of the above mentioned three components and is more stable in complete medium than standard duplex siRNA. It could be efficiently transfected into NCI-H292 cells in a dose- and time-dependent manner, resulting in high transfection efficiency. Furthermore, ssRNA-TNP uptake is dependent on macropinocytosis and clathrin-mediated endocytosis pathways. Interestingly, ssRNA-TNP is more efficient to inhibit the expression of mTOR. This ssRNA-TNP has a simpler structure, better stability, and higher transfection efficiency; therefore it may become a novel nonviral nanosystem for gene delivery.