microRNA Delivery System Research Articles

Functional micro-RNA drugs acting as a fate manipulator in the regulation of osteoblastic death.

Bone loss is prevalent in clinical pathological phenomena such as osteoporosis, which is characterized by decreased osteoblast function and number, increased osteoclast activity, and imbalanced bone homeostasis. However, current treatment strategies for bone diseases are limited. Regulated cell death (RCD) is a programmed cell death pattern activated by the expression of specific genes in response to environmental changes. Various studies have shown that RCD is closely associated with bone diseases, and manipulating the death fate of osteoblasts could contribute to effective bone treatment. Recently, microRNA-targeting therapy drugs have emerged as a potential solution because of their precise targeting, powerful curative effect, and limited side effects. Nevertheless, their clinical application is limited by their inherent instability, easy enzymatic degradation, and poor membrane penetrability. To address this challenge, a self-assembling tetrahedral DNA nanostructure (TDN)-based microRNA (Tmi) delivery system has been proposed. TDN features excellent biocompatibility, cell membrane penetrability, serum stability, and modification versatility, making it an ideal nucleic acid carrier for miRNA protection and intracellular transport. Once inside cells, Tmi can dissociate and release miRNAs to manipulate key molecules in the RCD signaling pathway, thereby regulating bone homeostasis and curing diseases caused by abnormal RCD activation. In this paper, we discuss the impact of the miRNA network on the initiation and termination of four critical RCD programs in bone tissues: apoptosis, autophagy, pyroptosis, and ferroptosis. Furthermore, we present the Tmi delivery system as a miRNA drug vector. This provides insight into the clinical translation of miRNA nucleic acid drugs and the application of miRNA drugs in bone diseases.

Construction and application of star polycation nanocarrier-based microRNA delivery system in Arabidopsis and maize

BackgroundMicroRNA (miRNA) plays vital roles in the regulation of both plant architecture and stress resistance through cleavage or translation inhibition of the target messenger RNAs (mRNAs). However, miRNA-induced gene silencing remains a major challenge in vivo due to the low delivery efficiency and instability of miRNA, thus an efficient and simple method is urgently needed for miRNA transformation. Previous researches have constructed a star polycation (SPc)-mediated transdermal double-stranded RNA (dsRNA) delivery system, achieving efficient dsRNA delivery and gene silencing in insect pests.ResultsHere, we tested SPc-based platform for direct delivery of double-stranded precursor miRNA (ds-MIRNA) into protoplasts and plants. The results showed that SPc could assemble with ds-MIRNA through electrostatic interaction to form nano-sized ds-MIRNA/SPc complex. The complex could penetrate the root cortex and be systematically transported through the vascular tissue in seedlings of Arabidopsis and maize. Meanwhile, the complex could up-regulate the expression of endocytosis-related genes in both protoplasts and plants to promote the cellular uptake. Furthermore, the SPc-delivered ds-MIRNA could efficiently increase mature miRNA amount to suppress the target gene expression, and the similar phenotypes of Arabidopsis and maize were observed compared to the transgenic plants overexpressing miRNA.ConclusionTo our knowledge, we report the first construction and application of star polycation nanocarrier-based platform for miRNA delivery in plants, which explores a new enable approach of plant biotechnology with efficient transformation for agricultural application.Graphical

MicroRNA-375: potential cancer suppressor and therapeutic drug.

MiR-375 is a conserved noncoding RNA that is known to be involved in tumor cell proliferation, migration, and drug resistance. Previous studies have shown that miR-375 affects the epithelial–mesenchymal transition (EMT) of human tumor cells via some key transcription factors, such as Yes-associated protein 1 (YAP1), Specificity protein 1 (SP1) and signaling pathways (Wnt signaling pathway, nuclear factor κB (NF-κB) pathway and transforming growth factor β (TGF-β) signaling pathway) and is vital for the development of cancer. Additionally, recent studies have identified microRNA (miRNA) delivery system carriers for improved in vivo transportation of miR-375 to specific sites. Here, we discussed the role of miR-375 in different types of cancers, as well as molecular mechanisms, and analyzed the potential of miR-375 as a molecular biomarker and therapeutic target to improve the efficiency of clinical diagnosis of cancer.

Liposomal Delivery of MicroRNA-7 Targeting EGFR to Inhibit the Growth, Invasion, and Migration of Ovarian Cancer.

Ovarian cancer is highly aggressive and has high rates of recurrence and metastasis. Due to the limited effects of current treatments, it is necessary to conduct research and develop new treatment options. The application of gene therapy in tumor therapy is gradually increasing and has exciting prospects. MicroRNA-7 (miR-7) has been reported to inhibit the growth, invasion, and metastasis of a variety of solid tumors. Cationic liposomes are safe and effective gene delivery systems for transfection in vivo and in vitro. To realize the application of miR-7 in the treatment of ovarian cancer, cationic liposomes were prepared with 1,2-dioleoyl-3-trimethylammonium-propane, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, and cholesterol. The miR-7 liposomes had a suitable particle size, potential, and a high cellular uptake rate. MiR-7 encapsulated by liposomes could be effectively delivered to ovarian cancer cells and successfully targeted to the tumor site in a mouse xenograft model of ovarian cancer. In vitro and in vivo experiments revealed that the miR-7 liposomes had a significant ability to inhibit the growth, invasion, and migration of ovarian cancer, probably by inhibiting the expression of the epidermal growth factor receptor. Our studies of miR-7 liposomes demonstrated a safe and efficient microRNA delivery system for the gene therapy of ovarian cancer.

A Peptide-Based Small RNA Delivery System to Suppress Tumor Growth by Remodeling the Tumor Microenvironment.

MicroRNAs can regulate a variety of physiological and pathological processes and are increasingly recognized as being involved in regulating the malignant progression of cancer, which is an important direction for the study and treatment of cancer. In addition, the tumor microenvironment has gradually become an important direction of study for combating cancer. Researchers can inhibit tumor growth by remodeling and suppressing an immunosuppressive phenotype in the tumor microenvironment. Therefore, the combination of microRNA delivery and tumor microenvironment remodeling may be a potential research direction. In a previous study, we developed a novel cationic and hydrophilic antimicrobial peptide, DP7, by computer simulation. It was found that cholesterol-modified DP7 (DP7-C) has dual functions as a carrier and an immune adjuvant. In this experiment, we used DP7-C to deliver microRNAs or inhibitors intratumorally, where it played a dual role as a carrier and an immune adjuvant. As a delivery vector, DP7-C has more advantages in terms of transfection efficiency and cytotoxicity than Lipo2000 and PEI25K. Components of the DP7-C/RNA complex can effectively escape endosomes after uptake via caveolin- and clathrin-dependent pathways. As an immune adjuvant, DP7-C can activate dendritic cells and promote macrophage polarization. Moreover, it can transform the immunosuppressive tumor microenvironment into an immune-activated tumor microenvironment, indicating its potential as an anticancer therapy. In conclusion, this study identifies a novel microRNA and inhibitor delivery system that can remodel the tumor microenvironment and introduces an alternative scheme for antitumor treatment.

Nanodiamond-based microRNA delivery system promotes pluripotent stem cells toward myocardiogenic reprogramming.

Gene therapy is the advanced therapeutics for supplying or replacing the genetic material in patients with inherited disorders. Recent clinical studies have made some progress in a wide range of applications, including monogenic disorders, neurodegenerative diseases, malignant tumors, and congenital diseases. Heart diseases, especially myocardial ischemia, remain one of the leading causes of mortality worldwide and usually result in irreparable cardiomyocyte damage and severe heart failure. Most advances in induced pluripotent stem cell (iPSC) technologies for promoting regenerative medicine and stem cell research. However, the driver molecules of myocardial-lineage differentiation and the functional reconstruction capacity of iPSC-derived cardiomyocytes are still an open question. Nanomedicine-based gene delivery provided a crucial platform to carry on the biogenomic materials for equipping functionalities and engineering the living organ environment. Nanodiamond (ND), a carbon-based nanomaterial, has been discovered and shown the high biocompatible and less toxicity for transporting protein, drug, and genomic plasmids. Here, we applied ND as a gene delivery vehicle to carry microRNA (miR-181a), and then transfected into iPS to promote cardiomyocyte-lineage differentiation. Notably, miR-181a plays a key role in iPS-derived cardiomyocyte differentiation which directly targets Hox-A11, leading to elevated MyoD expression and enhanced cardiomyocyte differentiation. Our study demonstrated that miR-181a promotes iPSC differentiation into functional cardiomyocytes. Delivery of NANO-DIAMOND-miR-181a may host clinical potential to enhance the differentiation and recovery of the cardiogenic function in injured cardiomyocytes.

The Effects of Combination of Mimic miR-155-5p and Antagonist miR-324-5p Encapsulated Chitosan in Ovarian Cancer SKOV3.

Objective:Ovarian cancer is a malignant tumor that attacks reproductive organs of women. MicroRNA is known to have an involvement in the prognosis of ovarian cancer. One of them is miR-155-5p which is down regulated and miR-324-5p which is up regulated. Chitosan is used as microRNA delivery system. The aims of this study is to find out the effects of combination microRNA encapsulated chitosan in cell line SKOV3. Methods:Cell line SKOV3 obtained from Stem Cell and Cancer Institute (Kalbe). Mimic miR-155-5p and Antagonist miR-324-5p formulated with chitosan. Total RNA was extracted from nine samples (three as control and six as treatment), and prepared for cDNA synthesis. Expression of RNA and mRNA target was measured using q-PCR Biorad CFX96 C.100 and Gen Ex 7 software. Statistics analysis was measured using SPSS 16.0.Results:The administration of combination microRNA encapsulated with chitosan affect the expression of miR-155-5p and miR-324-5p endogen (P<0.05). The expression of mRNA target HIF1α and GLI1 was down regulated after treatment. The correlation between expression of microRNA and mRNA target was strongly (P<0.05). Conclusion:This study successfully presented effects of combination of mimic miR-155-5p and antagonist miR-324-5p encapsulated chitosan which be considered as a potential therapy targets for ovarium cancer.

Casting Light on Intracellular Tracking of a New Functional Graphene-Based MicroRNA Delivery System by FLIM and Raman Imaging.

The theranostic ability of a new fluorescently labeled cationic cyclodextrin-graphene nanoplatform (GCD@Ada-Rhod) was investigated by studying its intracellular trafficking and its ability to deliver plasmid DNA and microRNA. The nanoplatform was synthesized by both covalent and supramolecular approaches, and its chemical structure, morphology, and colloidal behavior were investigated by TGA, TEM, spectroscopic analysis such as UV-vis, fluorescence emission, DLS, and ζ-potential measurements. The cellular internalization of GCD@Ada-Rhod and its perinuclear localization were assessed by FLIM, Raman imaging, and fluorescence microscopy. Biological experiments with pCMS-EGFP and miRNA-15a evidenced the excellent capability of GCD@Ada-Rhod to deliver both pDNA and microRNA without significant cytotoxicity. The biological results evidenced an unforeseen caveolae-mediated endocytosis internalization pathway (generally expected for particles <200 nm), despite the fact that the GCD@Ada-Rhod size is about 400 nm (by DLS and TEM data). We supposed that the internalization pathway was driven by physical-chemical features of GCD@Ada-Rhod, and the caveolae-mediated uptake enhanced the transfection efficiency, avoiding the lysosomal acid degradation. The cellular effects of internalized miRNA-15a on the oncogene protein BCL-2 were investigated at two different concentrations (N/P = 10 and 5), and a reduction of the BCL-2 level was detected at a low concentration (i.e., N/P = 10). miRNA-15a is considered an ideal cancer therapy molecule due to its activity on multiple transcription factors, and the elucidation of the correlation between the concentration of delivered miRNA-15a and the down-/up-regulation of the BCL-2 level, documented for the first time in this work, could be an important contribution to guide its clinical application.

P023 Development of an anti-inflammatory star-polypeptide microRNA delivery system with therapeutic applications in cystic fibrosis

P023 Development of an anti-inflammatory star-polypeptide microRNA delivery system with therapeutic applications in cystic fibrosis

Efficient Self-Assembled MicroRNA Delivery System Consisting of Cholesterol-Conjugated MicroRNA and PEGylated Polycationic Polymer for Tumor Treatment.

MicroRNA (miR), a key molecule involved in endogenous RNA interference, is a promising therapeutic agent. In vivo delivery of miR, however, is a major factor limiting its application because its polyanionic nature and vulnerability to breakdown make delivery of miR to targeted lesions difficult. To overcome these challenges, we developed a self-assembled miR delivery system consisting of cholesterol-conjugated miR and polyethylene glycol-grafted polyethylene imine. Nanosized complexes of miR with polyethylene imine, which protected miR and its delivery into targeted lesions in vivo, were successfully synthesized by polyethylene glycol grafting. The hydrophobicity of cholesterol improved the structural stability of the complex, preventing the loss of miR. Here, we report the preparation of this self-assembled complex. We examined the delivery efficiency and validated the therapeutic efficacy of the complex. In conclusion, our miR delivery system shows considerable potential for effective in vivo delivery of miR.

Targeted Delivery of miRNA 155 to Tumor Associated Macrophages for Tumor Immunotherapy.

Tumor associated macrophages (TAMs) are important components residing in the tumor microenvironment. They are immunosuppressive and promote tumor progression. Targeting TAMs and reprogramming their phenotype may be a promising strategy that can restore antitumor immune responses. In this study, we developed a microRNA delivery system based on lipid-coated calcium phosphonate nanoparticles (CaP/miR@pMNPs) containing conjugated mannose and sterically shielded with a pH-responsive material. The nanocarrier could respond to the low pH in the tumor microenvironment and expose mannose to promote cellular internalization in TAMs. The carrier could reactivate TAMs and reprogram their functions, reverse the immunosuppressive tumor microenvironment, and inhibit tumor growth in a tumor-bearing mouse model. In summary, redirecting the polarization of TAMs is a potential therapeutic strategy for tumor immunotherapy.

A PEGylated megamer-based microRNA delivery system activatable by stepwise microenvironment stimulation.

We developed a biodegradable, oncosensitive, megamer-based delivery system for miRNA therapy. The miRNA nanotherapeutics, activatable by stepwise stimulation of acidity and reduction mimicking tumor microenvironment, efficiently improve liver-specific miR-122 expression, increasing the possibility of translational application of miR-122 therapy against liver cancer.

MicroRNA-145-loaded poly(lactic-co-glycolic acid) nanoparticles attenuate venous intimal hyperplasia in a rabbit model

BackgroundMicroRNA-145 (miR-145) reportedly alters the phenotype of vascular smooth muscle cells (VSMCs) from a proliferative to a contractile state. So far, viral or plasmid vectors have been experimentally used to transduce microRNAs into VSMCs. We hypothesized that a simple ex vivo microRNA delivery system using miR–145-loaded poly(lactic-co-glycolic acid) (PLGA) nanoparticles (PLGA NPs) could control the VSMC phenotype and prevent intimal hyperplasia. MethodsJugular vein grafts of male Japanese white rabbits were soaked in phosphate-buffered saline, control microRNA (cont-miR)-loaded PLGA NP solution or miR–145-loaded PLGA NP solution for 30 minutes (n = 8 for each). Vein grafts were implanted in the ipsilateral carotid artery and assessed 2 weeks after the implantation. ResultsQuantitative polymerase chain reaction analysis showed significantly higher miR-145 expression in the miR–145-treated group. The neointimal area was significantly smaller in the miR–145-treated group (phosphate-buffered saline-treated vs cont–miR-treated vs miR–145-treated group; 1.63 ± 0.52 mm2 vs 1.67 ± 0.49 mm2 vs 0.88 ± 0.34 mm2, respectively; P < .01 for the miR–145-treated vs the cont–miR-treated group). In the miR–145-treated group, Ki–67-positive cells were significantly fewer, indicating lower VSMC proliferation. An inflammation-related molecule, CD40 expression was significantly reduced by miR–145-loaded PLGA NP treatment. ConclusionsLocal and sustained release of miR-145 by PLGA NPs attenuated intimal hyperplasia in the rabbit model by maintaining VSMCs in a contractile state. This simple ex vivo miR-145 delivery system would be promising toward broader clinical application.

Cancer Therapy: Integrated Combination Treatment Using a “Smart” Chemotherapy and MicroRNA Delivery System Improves Outcomes in an Orthotopic Colorectal Cancer Model (Adv. Funct. Mater. 28/2018)

Cancer Therapy: Integrated Combination Treatment Using a “Smart” Chemotherapy and MicroRNA Delivery System Improves Outcomes in an Orthotopic Colorectal Cancer Model (Adv. Funct. Mater. 28/2018)

Integrated Combination Treatment Using a “Smart” Chemotherapy and MicroRNA Delivery System Improves Outcomes in an Orthotopic Colorectal Cancer Model

AbstractMesoporous silica nanoparticles (MSN) can load and deliver potentially synergistic anticancer agents such as small molecule cytotoxics (like doxorubicin, DOX) and nucleic acids (like microRNA, miRNA). However, these cargos have different underlying chemical properties so overcoming respective intracellular delivery barriers is a key consideration. Strategies to deliver DOX from MSN frequently employ pH‐driven mechanisms that are restricted to the acidic environment of lysosomes. Conversely, strategies to deliver miRNA make use of approaches that deliberately compromise lysosomal membrane integrity to enable cytosolic delivery of the payload. To reconcile these two needs (lysosomal delivery of DOX and intracellular delivery of miRNA), a new methodology by “weaving” polyethylenimine on the MSN surface through disulfide bonds to achieve superior delivery of chemotherapy (DOX) and miRNA therapy (using miRNA‐145) is developed. Furthermore, an active targeting strategy based on a peptide ligand with affinity to glucose‐regulated protein 78 (GRP78), a cell surface protein overexpressed in colorectal carcinoma, is developed. The active targeting approach results in enhanced synergistic antitumor effect both in vitro and in vivo in an orthotopic murine model of colorectal cancer. Taken together, this work demonstrates the capability and advantages of “smart” MSN delivery systems to deliver anticancer cargo appropriately to targeted cancer cells.

Establishment of MicroRNA delivery system by PP7 bacteriophage-like particles carrying cell-penetrating peptide

MicroRNAs have great therapeutic potential in cancer and other diseases. However, their instability and low invivo delivery efficiency limits their application. Recombinant PP7 bacteriophage-based virus-like particles (VLPs) could protect microRNAs against rapid degradation by RNase by packaging specific exogenous pre-microRNAs using the pac site. Insertion of a cell-penetrating peptide (CPP) into the AB-loop of VLPs could significantly improve the delivery efficiency of microRNAs into mammalian cells. Unlike other microRNA delivery methods (viral or non-viral vectors), recombinant PP7 VLPs carrying a CPP and microRNA could be efficiently expressed in Escherichia coli using the one-plasmid double expression system. Here we showed that PP7 VLPs carrying a CPP penetrated hepatoma SK-HEP-1cells and delivered the pre-microRNA-23b, which was processed into a mature product within 24h; a concentration of 10nM was sufficient for the inhibition of hepatoma cell migration via the downregulation of liver-intestine cadherin expression. Furthermore, PP7 VLPs carrying a CPP and a pre-microRNA were not infectious, replicative, or cytotoxic. Therefore, recombinant PP7 VLPs can be used for simultaneous and targeted delivery of both microRNAs and peptides because of their ability to package specific exogenous RNA using the pac site and to display peptides.

Novel miR-122 delivery system based on MS2 virus like particle surface displaying cell-penetrating peptide TAT for hepatocellular carcinoma.

Current treatments for hepatocellular carcinoma (HCC) have shown inadequate. MicroRNA-122 (miR-122) mediated RNA interference brings new prospects. A safe, efficient miRNA delivery system is an indispensable assurance. Previously, we developed an MS2 bacteriophage virus-like particle (VLP)-based microRNA delivery system crosslinked with the HIV TAT peptide, which served as an effective inhibitor in the treatments of systemic lupus erythematosus and osteoporosis. However, defects, such as low crosslinking efficiency, high cost, and potential toxicity of the crosslinking agent, needed to be confronted. Therefore, TAT peptide was designed to display on the surface of MS2 VLPs, instead of being chemically crosslinked, using the platform of phage surface display. The results reflected that MS2 VLPs displaying TAT could effectively penetrate the cytomembrane and deliver miR-122. Additionally, its inhibitory effects on HCC were significant in Hep3B, HepG2, and Huh7 cells and Hep3B related animal models. Thus, we have established a novel miR-122 delivery system based on MS2 VLPs surface displaying TAT peptide, which could effectively perform the function of penetrating cytomembrane and the inhibition of HCC.

Using a novel microRNA delivery system to inhibit osteoclastogenesis.

Previously, we developed a novel microRNA (miRNA) delivery system based on bacteriophage MS2 virus-like particles (MS2 VLPs). In this current study, we used this system to transport miR-146a into human peripheral blood mononuclear cells (PBMCs), and demonstrated the inhibition of osteoclastogenesis in precursors. Two cytokines, receptor activator of NF-κB ligand (RANKL), and macrophage-colony stimulating factor (M-CSF) were used to induce osteoclastogenesis. MS2 VLPs were transfected into PBMCs. qRT-PCR was applied to measure expression levels of miR-146a and osteoclast (OC)-specific genes. Western blot (WB) was conducted to evaluate miR-146a downstream target proteins: epidermal growth factor receptor (EGFR) and tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6). The formation and activity of OCs were assessed by cytochemical staining and bone resorption assay, respectively. In PBMCs treated with MS2-miR146a VLPs, qRT-PCR assays showed increased expression of miR-146a (p < 0.01) and decreased expression of all four OC-specific genes (p < 0.05). WB results indicated decreased expression of EGFR (p < 0.01) and TRAF6 (p < 0.05). The number of OCs decreased markedly and bone resorption assay demonstrated inhibited activity. This miR-146a delivery system could be applied to induce overexpression of miR-146a and to inhibit the differentiation and function of OCs.

MicroRNA delivery system inhibits proliferation of androgen-independent prostate cancer

Objective To prepare a microRNA(miRNA)delivery system using the branched polyethyleneimine(BPEI),linear polyethylenimine(LPEI)and polyamidoamine dendrimers(PAMAM)loaded with miRNA-15 aand miRNA-16-1,which can inhibit prostate cancer PC3 cell proliferation,and to examine the zeta potential,intracellular uptake,and the inhibition effect on PC3 cells of the three constructed nano-complexes.Methods Particle size analyzer was used to determine the size and potential of the three kinds of nano-complexes,and the miRNA affinity capability of them was determined by agarose gel electrophoresis retardation assay.The uptake efficiency of the nano-complexes by PC3 cells was examined by NC-miRNA labeled with FAM.CCK8 method was used to determine the inhibitory effect of the nano-complexes loaded with miRNA-15 a and miRNA-16-1against PC3 cells,and PCR was used to analyze their inhibitory effect on expression of Bcl-2,Cylin D1 and Wnt3agene in PC3 cells.Results BPEI,LPEI and PAMAM loaded with miRNA could form stable nano-complexes.When N/P=5,the intracellular uptake of BPEI/miRNA-FAM by PC3 cells was significantly higher than that of LPEI/miRNA-FAM and PAMAM/miRNA-FAM(P0.05).BPEI,LPEI and PAMAM could all carry miRNA-15 aand miRNA-16-1into PC3 cells and block Bcl-2,Cylin D1 and Wnt3aexpression in PC3 cells.Conclusion BPEI,LPEI and PAMAM loaded with miRNA-15 aand miRNA-16-1can suppress proliferation of prostate cancer PC3 cells and block Bcl-2,Cylin D1 and Wnt3aexpression.

Polycation liposomes as a vector for potential intracellular delivery of microRNA

We previously developed a microRNA (miRNA) delivery system by using dicetyl phosphate-tetraethylenepentamine-based polycation liposomes (TEPA-PCL), applied it to miR-92a delivery, and demonstrated its gene-silencing potential and effective anti-angiogenic effects. In the present study, we investigated the mechanism of intracellular delivery of cholesterol-grafted miR-92a (miR-92a-C) into cells. To investigate the intracellular distribution of miR-92a-C/TEPA-PCL complex, we used human umbilical vein endothelial cells and examined certain points after transfection: (i) the time-course of miR-92a-uptake into the cells; (ii) the endocytosis pathway induced by miR-92a-C/TEPA-PCL; (iii) the capability of miR-92a-C/TEPA-PCL to escape from the endosomes; and (iv) the release of miR-92a-C from TEPA-PCL in the cytoplasm. Our data indicated that miR-92a-C formulated in TEPA-PCL accumulated in and was spread throughout the cytoplasm in a time-dependent manner, and was taken up into the cells by macropinosome-mediated endocytosis. In addition, the surface charge of miR-92a-C/TEPA-PCL was neutral at pH 7.4 and was charged positively at around pH 5.5, which is the inner pH of endosomes. When the late endosomes/lysosomes were stained with Lysotracker, miR-92a-C/TEPA-PCL efficiently escaped from the endosomes into the cytoplasm, possibly through the proton-sponge effect. Furthermore, miR-92a-C spread throughout whole cytoplasm and did not co-localize completely with TEPA-PCL, indicating that some of the miR-92a-C was present in free form in the cytoplasm. The results of the present study suggest that TEPA-PCL-based lipoplexes have an excellent potential to deliver microRNAs into the cytoplasm of cells and to induce RNA silencing action mediated by microRNAs and other small RNAs.