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Fast, efficient, and inexpensive methods for delivering functional nucleic acids to primary human cell types are needed to advance regenerative medicine and cell therapies. Plasmid-based gene editing (such as with CRISPR-Cas9) can require the delivery of plasmids that are large (∼9.5-13 kbp) in comparison to common reporter plasmids (∼5-8 kbp). To develop more efficient plasmid delivery vehicles, we investigated the effect of plasmid size on the transfection of primary human dermal fibroblasts (HDFs) and induced pluripotent stem cells (iPSCs) using a heparin-treated trehalose-containing polycation (Tr4-heparin). Transfections with 4.7 kbp to 10 kbp plasmids exhibited high rates of polyplex internalization with both plasmid sizes. However, transfection with the large plasmid was nearly eliminated in HDFs and significantly reduced in iPSCs. Molecular additives were used to probe intracellular barriers to transfection. Chloroquine treatments were used to destabilize endosomes, and dexamethasone and thymidine were used to destabilize the nuclear envelope. Destabilizing the nuclear envelope resulted in significantly increased large-plasmid-transfection, indicating that nuclear localization may be more difficult for large plasmids. To demonstrate the potential clinical utility of this formulation, HDFs and iPSCs were treated with to dexamethasone-Tr4-heparin polyplexes encoding dCas9-VP64, synthetic transcription activator, targeted to collagen type VII. These transfections enhanced collagen expression in HDFs and iPSCs by 5- and 20-fold, respectively, compared to an untransfected control and were the more effective than the Lipofectamine 2000 control. Functional plasmid transfection efficiency can be significantly improved by nuclear destabilization, which could lead to improved development of nonviral vehicles for ex vivo CRISPR-Cas9 gene editing.

Hyperbranched bicyclo[3.3.1]nonane (BCN) polycations were synthesized by the reaction of the bivalent electrophile thiabicyclo[3.3.1]nonane dinitrate with a series of simple tris(pyridine) nucleophiles, including one alkyne-containing nucleophile to allow for postpolymerization functionalization. The hyperbranched polymers were found to be efficient binders of nucleic acid and exhibited higher efficiencies for oligo DNA and siRNA transfection than their linear counterparts, enabling knockdown at low siRNA concentrations to a superior extent than standard hyperbranched polyethylenimine and lipofectamine transfection agents. The use of an amide-containing linkage in the tris(pyridine) building block was found to be highly advantageous, but built-in fragmentability of the polycation structure, a unique potential feature of this new family of materials, did not give significantly better performance.

Various known and unknown viral diseases can threaten crustacean aquaculture. To develop prophylactic and therapeutic strategies against viruses, crustacean cell lines are urgently needed for immunology and virology studies. However, there are currently no permanent crustacean cell lines available. In this study, we developed a new method for preparing crayfish plasma (CP) and found that CP enhanced the proliferative capacity of haematopoietic tissue (hpt) cells from Cherax quadricarinatus by an EdU (5-ethynyl-2'-deoxyuridine) assay. The optimal CP concentration for hpt cell culture and the optimal subculture method are discussed. To achieve efficient expression of a foreign gene in hpt cells cultured in vitro, different transfection methods and vectors were analysed. We found that Lipofectamine 2000 could be used to efficiently transfect a foreign vector into hpt cells and exhibited a lower level of cytotoxicity than the other methods tested, and transfection of pEGFP-N1/w249 and pDHsp70-EGFP-FLAG resulted in high EGFP expression. By transmission electron microscopy (TEM) and virus copy number analysis, we found that white spot syndrome virus (WSSV) could infect hpt cells and multiply efficiently. Our results implied that the crayfish hpt cell culture system we improved could be used as a replacement for immortal crustacean cell lines in viral infection studies. Our findings provide a solid foundation for future immortalization and gene function studies in crustacean cells.

Mesenchymal stem cells (MSCs) are multipotent cells capable of differentiating into a wide range of cell types and provide a potential to transfer therapeutic protein in vivo, making them valuable candidates for gene therapy and cell therapy. However, using MSCs in in vivo is limited due to the low rate of transfection and transduction efficacy. Therefore, developing methods to efficiently transfer genes into MSCs would provide a number of opportunities for using them in the clinic. Here, we introduce a simple and robust method for efficient transduction of human adipose‐derived MSCs by modification under the culture condition of human embryonic kidney cells 293 (HEK293T) and MSCs. Moreover, as a transduction enhancer, polybrene was replaced with Lipofectamine, a cationic lipid. Therefore, we showed that transduction of primary cells can be increased efficiently by modifying the culture condition.

RNA interference shows a great strategy for biological studies; however, delivering of small interfering RNA (siRNA) remains challenging. Although several delivery vehicles, including cell-penetrating peptides, have been developed, their implementation is often restricted because of their endosomal entrapment. Herein, we report the formation of self-assembled nanostructures from rationally designed cyclic peptides and explore them for efficient delivery of functional biomacromolecules such as siRNA into mammalian cells. The newly obtained soft materials make stable complexes with siRNAs, thereby increasing their stability and deliver fluorescent labelled siRNA inside the cells as evident from confocal microscopy analysis. Flow cytometry analysis reveals that significant uptake of FAM-siRNA occurs in the presence of peptide nanostructures compared with siRNA alone. Peptide nanostructure-mediated delivery of very low concentration of siRNA causes significant knockdown of the target gene as observed at protein level by Western blot analysis, which is comparable to lipofectamine, commercially available transfection agent.

Mingxing Wang, Bo Wu, Sapana N Shah, Peijuan Lu, Qilong Lu McColl-Lockwood Laboratory for Muscular Dystrophy Research, Department of Neurology, Cannon Research Center, Carolinas Medical Center, Charlotte, NC 28203, USA Background: Antisense oligonucleotide (ASO)-mediated exon skipping has been feasible and promising approach for treating Duchenne muscular dystrophy (DMD) in preclinical and clinical trials, but its therapeutic applications remain challenges due to inefficient delivery. Methods: We investigated a few Saponins for their potential to improve delivery performance of an antisense 2′-Omethyl phosphorothioate RNA (2′-OMePS) in muscle cells and in dystrophic mdx mice. This study was carried out by evaluating these Saponins’ toxicity, cellular uptake, transduction efficiency in vitro, and local delivery in vivo for 2′-OMePS, as well as affinity study between Saponin and 2′-OMePS. Results: The results showed that these Saponins, especially Digitonin and Tomatine, enhance the delivery of 2′-OMePS with comparable efficiency to Lipofectamine 2k (LF-2k)-mediated delivery in vitro. Significant performance was further observed in mdx mice, up to 10-fold with the Digitonin as compared to 2′-OMePS alone. Cytotoxicity of the Digitonin and Glycyrrhizin was much lower than LF-2k in vitro and not clearly detected in vivo under the tested concentrations. Conclusion: This study potentiates Saponins as delivery vehicle for 2′-OMePS in vivo for treating DMD or other diseases. Keywords: antisense delivery, 2′-OMePS, exon skipping, saponin, muscular dystrophy

Background The blood-brain barrier (BBB) strongly restricts the entry of anti-glioma drugs into tumor tissues and thus decreases chemotherapy efficacy. Malignant gliomas are highly invasive tumours that use the perivascular space for invasion and co-opt existing vessels as satellite tumor form. Because regulation of the effect of noncoding RNA on BBB function is attracting growing attention, we investigated the effects of noncoding RNA on the permeability of glioma conditioned normal BBB and the mechanism involved using PIWI-associated RNA piR-DQ590027 as a starting point. Methods The mRNA levels of MIR17HG, miR-153, miR-377, ZO-1, occludin, and claudin-5 were determined using real-time PCR. Transient cell transfection was performed using Lipofectamine 3000 reagent. TEER and HRP flux were applied to measure the permeability of glioma conditioned normal BBB. Western blotting and immunofluorescence assays were used to measure ZO-1, occludin, and claudin-5 levels. Reporter vector construction and a luciferase reporter assay were performed to detect the binding sites of MIR17HG and piR-DQ590027, MIR17HG and miR-153 (miR-377), and FOXR2 and miR-153 (miR-377). RNA immunoprecipitation was used to test the interaction between miR-153 (miR-377) and its target proteins. Chromatin immunoprecipitation was performed to detect the interaction between the transcription factor FOXR2 and ZO-1, occludin, and claudin-5. Results piR-DQ590027 was expressed at low levels in glioma-conditioned ECs (GECs) of the in vitro glioma conditioned normal BBB model. Overexpression of piR-DQ590027 down-regulated the expressions of ZO-1, occludin, and claudin-5 and increased the permeability of glioma conditioned normal BBB. MIR17HG had high expression in GECs but miR-153 and miR-377 had low expression. piR-DQ590027 bound to and negatively regulated MIR17HG. FOXR2 was a downstream target of miR-153 and miR-377; MIR17HG bound separately to miR-153 and miR-377 and negatively regulated their ability to mediate FOXR2 expression. FOXR2 associated with the promoter regions of ZO-1, occludin, and claudin-5 in GECs to promote their transcription. Conclusion The piR-DQ590027/MIR17HG/miR-153 (miR-377)/FOXR2 pathway plays an important role in regulating glioma conditioned normal BBB permeability and provides a new target for the comprehensive treatment of glioma. Electronic supplementary material The online version of this article (10.1186/s13046-018-0886-0) contains supplementary material, which is available to authorized users.

RNA interference (RNAi) is a mechanism in which small interfering RNA (siRNA) silences a target gene. Herein, we describe a DNA hydrogel capable of producing siRNA and interfering with protein expression. This RNAi-exhibiting gel (termed I-gel for interfering gel) consists of a plasmid carrying the gene transcribing siRNA against the target mRNA as part of the gel scaffold. The RNAi efficiency of the I-gel has been confirmed by green fluorescent protein (GFP) expression assay and RNA production quantification. The plasmid stability in the I-gel results in an 8-times higher transcription efficiency than that of the free plasmid. We further applied the I-gel to live cells and confirmed its effect in interfering with the GFP expression. The I-gel shows higher RNAi effect than plasmids in free form or complexed with Lipofectamine. This nanoscale hydrogel, which is able to produce RNA in a cell, provides a platform technology for efficient RNAi system., Interfering RNA have a range of therapeutic and research based applications, issues with delivery have made systems that make siRNA in situ of interest. Here, the author report on the creation of a DNA hydrogel with improved stability and transcription efficiency over plasmid DNA.

Tri-block copolymers have exhibited great potentials in small interfering RNA (siRNA) therapeutics. To reveal structure-activity relationships , we here synthesized a series of tri-block copolymers with different hydrophobic segments, PEG-PAMA-P(C6A x-C7Ay-DPAz-DBAm) (EAAS) and PEG-PDAMAEMA-P(C6Ax-C7Ay-DPAz-DBAm ) (EDAS), termed from EAASa to EAASh and EDASa to EDASh, with pKa ranging from 5.2 to 7.0. Our data showed that the better gene silencing efficiency was located in pKa of 5.8–6.2, which was contributed from higher endosomal escape observed with confocal images and hemolysis assay. EAASc, the leader polymer, showed excellent gene knockdown at w/w ratio of 14.5 on HepG2 (89.94%), MDA-MB-231 (92.45%), 293A (83.06%), and Hela cells (80.27%), all better than lipofectamine 2000. Besides, EAASc mediated effective gene silencing in tumor when performed peritumoral injection. This work found out that polymers with pKa ranging from 5.8 to 6.2 were efficient in siRNA delivery, which provided an optimization strategy for siRNA delivery systems, especially for tri-block copolymers.

Three cationic lipids derived from [12]aneN3 modified with naphthalimide (1a), oleic acid (1b) and octadecylamine (1c) were designed and synthesized. In vitro transfection showed that all these liposomes can deliver plasmid DNA into the tested cell lines. Among these liposomes, 1a gave the best transfection efficiency (TE) in A549 cells, which was higher than that of lipofectamine 2000. More importantly, the TE of 1a was dramatically increased in the presence of 10% serum. These results suggested that 1a might be a promising non-viral gene vector, and also give further insight for developing novel high performance gene delivery agents.

IgE/FceRI signal pathway plays a crucial role in triggering allergic reactions, and there is no cross-recognition between IgE and FceRI in human and rats. In order to obtain the hFceRIα/ RBL-2H3 cell line, total RNA was extracted from U937 cells, and the human FceRIα gene was obtained by RT-PCR technology. Then the amplified product was digested and inserted into the pIRES2-EGFP vector. After the plasmid was transfected into the RBL-2H3 cells using lipofectamine, and the RBL-2H3 cell lines of stable expression were screened by G418. The transfection efficiency reached 60.45% with optimizing transfection parameters. The last the expression of hFceRIα was detected by RT-PCR, western blotting and fluorescent microscopy. The present results demonstrated that the pIRES2-EGFP-hFceRIα vector was constructed and a stable cell line of hFceRIα/ RBL-2H3 cells was established successfully. This cell line is promising tools for further research on the pathogenesis and drug development of allergic diseases.

Sorafenib is a first-line drug for hepatocellular carcinoma (HCC). Autophagy has been shown to facilitate sorafenib resistance. miR-375 has been shown to be an inhibitor of autophagy. In this study, miR-375 and sorafenib were co-loaded into calcium carbonate nanoparticles with lipid coating (miR-375/Sf-LCC NPs). The nanoparticles had high loading efficiency and were ∼50 nm in diameter. Besides, the NPs could increase the stability and residence time of both drugs. Moreover, we demonstrated that autophagy was activated in HCC cells by sorafenib but not by miR-375/Sf-LCC NPs. In vitro, miR-375/Sf-LCC NPs exhibited pH-dependent drug release and potent cytotoxicity. In vivo, miR-375/Sf-LCC NPs increased miR-375 and sorafenib uptake in tumor (2 folds compared with Lipofectamine 2000-miR-375 and 2–5 folds compared with free sorafenib). Furthermore, miR-375/Sf-LCC NPs showed greatly enhanced therapeutic efficacy in an HCC xenograft model. These findings suggest that miR-375/Sf-LCC NPs may be a promising agent for the HCC therapy. Statement of Significance Hepatocellular carcinoma (HCC) is the most common primary liver tumor and the third leading cause of cancer mortality globally. In this manuscript, miR-375 and sorafenib were co-loaded into calcium carbonate nanoparticles with lipid coating (miR-375/Sf-LCC NPs) to treat HCC. We demonstrated that miR-375/Sf-LCC NPs can deliver sorafenib and miR-375 into HCC cells and tumor tissues, increase drug retention time in tumor, significantly inhibit autophagy and produce enhanced anti-tumor effect.

Purpose The heavy subunit of the iron storage protein ferritin (FHC) is essential for the intracellular iron metabolism and, at the same time, it represents a central hub of iron-independent pathways, such as cell proliferation, angiogenesis, p53 regulation, chemokine signalling, stem cell expansion, miRNAs expression. In this work we have explored the ability of FHC to modulate gene expression in K562 cells, through the up-regulation of the lncRNA H19 and its cognate miR-675. Materials and methods Targeted silencing of FHC was performed by lentiviral-driven shRNA strategy. FHC reconstitution was obtained by full length FHC cDNA transfection with Lipofectamine 2000. ROS amounts were determined with the redox-sensitive probe H2DCFDA. H19, miR-675, miR-107, Twist1, ID3, EPHB6, GNS, ANK1 and SMAD6 mRNA amounts were quantified by Taqman assay and qPCR analysis. Results FHC silencing in K562 cells modulates gene expression through the up-regulation of the lncRNA H19 and its cognate miR-675. Experimental findings demonstrate that the molecular mechanism underlying this phenomenon is represented by an FHC knock-down-triggered increase in reactive oxygen species (ROS) production. Conclusions In this paper we uncover a so far not described function of the ferritin heavy subunit in the control of lncRNA pathways.

Background and Objectives: Nonviral carriers including those based on synthetic cationic lipids, offer several advantages over the viral counterparts. These carriers are able to form complexes with nucleic acids and deliver genes into the cells via the cellular endocytosis pathway, without significant toxicity. The level of transgenes expression depends on some experimental variables including cell type and density, Lipofectamine and DNA concentrations and Lipofectamine-DNA complexing time. The main objective of this study was to optimize transfection of SW480 colon cancer cells with Lipofectamine 2000. Methods: In this study, SW480 cells were transfected with plasmid containing green fluorescent protein reporter gene using Lipofectamine 2000. Green fluorescent protein expression was studied under a reverse fluorescence microscope and the results were analyzed with the ImageJ software. Effect of different quantities of plasmid DNA and different Lipofectamine 2000 volumes on cell transfection efficiency was evaluated. Results: The optimal volume of Lipofectamine and quantity of plasmid was 2 µl and 1µg, respectively, which showed 59% efficiency for the transfection of SW480 cells at 24 hours post-transfection. Conclusion: This study shows that Lipofectamine 2000 is an efficient reagent for the delivery of genes into SW480 cells. According to the results, the quantity of DNA per transfection and reagent concentrations are essential factors for a successful transfection. Keywords: Optimization; pEGFP-NI; Lipofectamine; SW480.

Carbon dots (CDs) focus great attention in a broad range of adhibitions because of their excellent optical properties and high biocompatibility and property adjustability. However, the developed CDs have rarely been used as effective gene vectors until now. In this work, we devised and synthesized novel fluorine-doped cationic CDs (FCDs) using tetrafluoroterephthalic acid as a fluorine source and using branched polyethylenimine to furnish positive charge sites. The FCDs achieve dramatic positive EGFP and luciferase gene transfection efficiency as well as low cytotoxicity in commonly used cell lines at a low weight ratio, even in primary and stem cells. It is worth pointing out that the FCDs possess superior efficiency and biocompatibility, compared to some widely used commercial reagents such as 25 kDa polyethylenimine and Lipofectamine 2000. In addition, the FCDs show excellent efficient transfection even at high serum concentration and low DNA dose, indicating potential practical applications.

Mast cells are important immune cells that have significant roles in mediating allergy and asthma. Therefore, studying the molecular mechanisms regulating these and other processes in mast cells is important to elucidate. Methods such as lipofection, transduction, and electroporation are often employed to dissect these mechanisms by disrupting gene expression in mast cell lines. However, as with other leukocytes, human mast cells (HMCs) are often refractory to the delivery of plasmids by lipofection. In this study, we investigated the utility of lipid nanoparticles (LNPs) containing the ionizable cationic lipids 1,2-dioleoyloxy-3-dimethylaminopropane, 1,2-dioleyloxy-3-dimethylaminopropane, or 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane for the delivery of plasmid DNA into HMC lines. Herein, we demonstrate for the first time the use of LNPs to achieve significant and reproducible levels of plasmid DNA transfection in HMC-1.2 and laboratory of allergic diseases 2 (LAD2) cells. These levels reached 53.2% and 16.0% in HMC-1.2 and LAD2 cells, respectively; and outperformed Lipofectamine 3000 in both cases. Moreover, cell viability in the transfected cells remained above 65% for all LNP conditions tested. Together, these observations illustrate the efficacy of this technique for mast cell researchers and further support the use of LNPs for nucleic acid delivery into leukocytes. Method based on DNA-lipid nanoparticles successfully and reproducibly transfects human mast cell lines while yielding levels of transgene expression and cell viability superior to traditional lipofection techniques.

In this study, transfection efficiency of human papillomavirus (HPV) E7 DNA and protein constructs into HEK-293T normal cell line, and A549 and TC-1 tumor cell lines was evaluated by four delivery systems including supercharge GFP, hPP10 cell penetrating peptide, TurboFect and Lipofectamine using fluorescence microscopy and flow cytometry. The results indicated that Lipofectamine 2000 and TurboFect produced more effective transfection for GFP and E7-GFP DNA constructs in HEK-293T cells compared to in A549 and TC-1 cells (p

The aim of the present study was to investigate the targeted interaction between microRNA (miR)-130b-5p and RAS protein activator like 1 (RASAL1) gene and elucidate the function of miR-130b-5p in cell proliferation, migration and invasion in gastric cancer. Expression of miR-130b-5p and RASAL1 in seven gastric cell lines was detected by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). MGC803 cells were selected for further study since they exhibited a marked increase in expression of miR-130b-5p accompanied by decreased expression of RASAL1. MGC803 cells were transfected with miR-130b-5p mimics and miR-130b-5p inhibitor using Lipofectamine 2000 for over- and underexpression, respectively, with cells transfected with negative control (NC) sequence as the control. In addition, a luciferase reporter gene assay was performed to evaluate the targeted interaction between miR-130b-5p and RASAL1. Then, alterations in RASAL1 expression were detected by RT-qPCR and western blot analysis following transfection with miR-130b-5p mimics and miR-130b-5p inhibitor. Cell proliferation, colony formation, and migration and invasion ability were detected by MTT, colony formation and Transwell assays, respectively. RASAL1 was demonstrated to be a target gene of miR-130b-5p by luciferase reporter gene assay. In addition, the expression of RASAL1 was significantly lower in MGC803 cells that were transfected with miR-130b-5p mimics and significantly higher in cells transfected with miR-130b-5p inhibitor in comparison with cells transfected with NC (P

One of the main problems in tumor therapy is immunosuppressive microenvironment of the tumor. To overcome this, we assessed targeted tumor cells with tumor necrosis factor-α (TNF-α) or CD40 Ligand (CD40L) to improve antigen presenting cells function and subsequently anti-tumor responses. 4T1 tumor cells were transfected with TNF-α and CD40L genes using lipofectamine and chitosan nanocomplexes. Engineered tumor cells were assessed in vitro and in vivo. Results showed that chitosan nanoparticles delivery of TNF-α or CD40L to 4T1 co-cultured with DCs induced expression of DCs co-stimulatory markers and enhanced the secretion of pro-inflammatory mediators IL-6, TNF-α and IL-12. The DCs were also able to enhance expansion of T cells with enhanced IFN-γ and decreased IL-4 production. TNF-α or CD40L engineered 4T1 cells resulted into delay in tumor growth. The study shows that nanoparticle manipulation of tumor cells by TNF-α or CD40L induce anti-tumor immune responses. Then, strategies that apply chitosan nanoparticles could provide a potent tool for tumor targeting and treatments.

Medication during pregnancy is often a necessity for women to treat their acute or chronic diseases. The goal of this study is to evaluate the potential of micelle-like nanoparticles (MNP) for providing safe drug usage in pregnancy and protect both foetus and mother from medication side effects. Clonazepam-loaded MNP were prepared from copolymers [polystyrene-poly(acrylic acid) (PS-PAA), poly(ethylene glycol)-b-poly(lactic acid) (PEG-PLA) and distearyl-sn-glycero-3-phosphoethanolamine-N-[methoxy-poly(ethylene glycol) (PEG-DSPE)] with varying monomer ratios and their drug-loading efficiency, drug release ratio, particle size, surface charge and morphology were characterised. The cellular transport and cytotoxicity experiments were conducted on clonazepam and MNP formulations using placenta-choriocarcinoma-BeWo and brain-endothelial-bEnd3 cells. Clonazepam-loaded PEG5000-PLA4500 MNP reduced the drug transport through BeWo cells demonstrating that MNP may lower foetal drug exposure, thus reduce the drug side effects. However, lipofectamine modified MNP improved the transport of clonazepam and found to be promising for brain and in-utero-specific drug treatment.

Objective Cervical squamous cell carcinoma seriously threats to patient's life and health. MiRNAs have role of regulating cell growth, proliferation, and death. MiRNAs can promote or inhibit cell growth and proliferation. This study discussed the role of miRNA373 in regulating cervical squamous cell carcinoma growth, proliferation, and apoptosis. Patients and methods MiRNA373 and scramble miRNA were synthetized and transfected to cervical squamous cell carcinoma SiHa cells by lipofectamine. IAPs plasmid and miRNA373 were sequentially transfected to SiHa cells. MTT assay, caspase-3 activity, and flow cytometry were applied to test miRNA373 and IAPs impacts on cell growth, proliferation, and apoptosis. Western blot was adopted to determine IAPs expression. Results MiRNA373 transfection obviously reduced SiHa cell growth, induced phosphatidylserine eversion and caspase-3 activation, and declined IAPs expression. IAPs interference significantly enhanced miRNA373 induced SiHa cell apoptosis. IAPs overexpression markedly restrained miRNA373 induced SiHa cell apoptosis. Conclusions MiRNA373 transfection suppressed SiHa cell growth and proliferation. MiRNA373 induced SiHa cell apoptosis possibly through downregulating IAPs, suggesting that IAPs might be a target for cervical squamous cell carcinoma treatment.

Background The ability to metastasize is one of the most important characteristics of neoplastic cells. An imbalance between the action of some matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs drives the invasion process. Some studies have suggested that MMP-2 is involved in metastasis, while other studies have reported that collagen production by cancer cells might also contribute to motility. However, decreased expression of microRNA-29b (miR-29b), which may control MMP-2 and collagen gene expression, has been shown in prostate cancer (PCa). The objectives of the present study were to clarify whether MMP-2 as well as collagens I and III (encoded by COL1A1 and COL3A1, respectively) are controlled by miR-29b and to determine whether metastasis is altered by this relationship. Methods PCa DU145 and PC-3 cells were transfected with 100 μL of OPTI-MEM I containing 100 nmol of miR-29b (or its inhibitor) along with 1.5 μL of lipofectamine. Positive and negative controls were prepared using the same protocol. MMP-2, COL1A1 and COL3A1 messenger RNA (mRNA) levels were evaluated via real-time polymerase chain reaction (qRT-PCR). For qRT-PCR, 6 × 104 cells were used. Invasion studies were conducted with Matrigel assays, which simulate invasion of the extracellular matrix by neoplastic cells. After transfection of 3 × 104 cells, invasion was allowed to proceed for 48 h. Invasive cells were counted under an optical microscope. Each experiment was performed in triplicate. Results MMP-2 mRNA was not expressed in DU145 cells after transfection with miR-29b. After transfection of cells with the miR-29b inhibitor, COL1A1 (p = 0.02) and COL3A1 (p = 0.06) mRNA expression was increased in DU145 cells, and a large number of transfected DU145 and PC3 cells invaded the Matrigel membrane. Conclusions In vitro studies showed that reducing the amount of miR-29b may lead to higher PCa cell invasion via a process that is independent of MMP-2. Collagen expression, controlled by miR-29b, may facilitate this motility process. Thus, the present study suggests that collagen production plays an active role in metastasis control and restoration of miR-29b levels may decrease metastasis. Altogether, these findings support further exploration of drug therapy targeting this aspect of the metastasis circuit.

Herein, five new α-tocopheryl cationic gemini lipids with hydroxyethyl bearing headgroups (THnS, n = 4, 5, 6, 8, 12) have been synthesized for efficient plasmid DNA (pDNA) delivery into cancer cells. Among these gemini lipid formulations, the lipid with an octamethylene [-(CH2)8] spacer (TH8S) showed the highest transfection efficiency (TE) that was comparable to that of the commercial standard lipofectamine 2000 (L2K) in terms of luciferase expression in HepG2 (liver hepatocellular carcinoma) cells. The addition of the helper lipid DOPE (1,2-dioleoyl phosphatidyl ethanolamine) with cationic lipids in mixed liposomes further enhanced the TE and the optimized molar ratio was 2 : 1 (DOPE : cationic lipid). The optimized co-liposomal formulation of TH8S (DOPE : TH8S = 2 : 1) showed a higher TE in HepG2, A549 (human lung carcinoma) and MCF7 (human breast adenocarcinoma) cells than other optimized co-liposomal formulations and was also significantly more potent than L2K. The comparison of the TE of DOPE–TH8S (2 : 1) with the gemini lipid T8T (the headgroup devoid of the hydroxyl group) further demonstrated the importance of the hydroxyethyl functionality at the level of the headgroup. Relatively good binding efficiency and easy release of pDNA (pGL3) were also observed with DOPE–TH8S (2 : 1) in the ethidium bromide (EB)-exclusion and re-intercalation assay, which may be the plausible reason for high TE. The lipoplexes were also characterized by atomic force microscopy (AFM), dynamic light scattering (DLS), zeta potential and small angle X-ray diffraction experiments. Greater cellular internalization of fluorescein tagged pDNA was also observed with DOPE–TH8S (2 : 1) lipoplexes compared to that with L2K. Retention of the TE of DOPE–TH8S (2 : 1) lipoplexes under high serum conditions was conferred by the presence of the tocopherol backbone and also the hydroxyethyl functionalities. The cellular internalization pathway of the lipoplexes was characterized by performing transfection experiment in the presence of inhibitors of different endocytic pathways and it was found to be caveolae mediated. An MTT based cell viability assay indicated that the lipoplex mediated gene delivery vectors exhibited low toxicity in all the three cancer cell lines studied.

Oral bone defects are difficult to treat. Recently, endogenous miR-34a was shown to be involved in bone anabolism. Clinical application of such microRNAs requires the inherent instability of microRNAs to be overcome by an efficient delivery system. In this study, we employed N-acetyl-L-leucine-modified polyethylenimine (N-Ac-L-Leu-PEI) as an miR-34a carrier and evaluated its delivery ability, transfection efficiency, cytotoxicity and whether it enhanced osteogenic differentiation and bone formation in vitro and in vivo. Stable N-Ac-L-Leu-PEI/miR-34a nanocomplexes were synthesized at a mass ratio of 4 and had a small size (190.34 nm), a low zeta potential (21.1 mV), a high transfection efficiency (69.39%) and no cytotoxicity in MG63 cells. N-Ac-L-Leu-PEI-mediated miR-34a delivery in vitro promoted ALP activity and expression of osteogenic differentiation markers, Runx2, SP7 and ColI to higher levels than those produced by Lipofectamine 2000-mediated delivery. N-Ac-L-Leu-PEI also achieved delivery of miR-34a in vivo to a local cranial bone defect area with miR-34a retaining the ability to initiate significant new bone formation 12 weeks post-implantation. This demonstrates the potential for N-Ac-L-Leu-PEI as a gene therapy vehicle for the regeneration of bone defects.

For actively targeted delivery of small interfering RNA (siRNA) to solid tumors, we fabricated functionalized selenium nanoparticles (SeNPs) decorated with the polypeptide RGDfC. Herein, RGDfC was used as tumor-targeted moiety and installed onto the surface of SeNPs to enhance the cellular uptake. RGDfC-SeNPs@siRNA were internalized into the HepG2 cell mainly through clathrin-mediated endocytosis. The active efficacy of the RGDfC-SeNPs@siRNA was confirmed via gene silencing assay, MTT assay and flow cytometry analysis. Owing to the tumor-targeting effect of RGDfC, RGDfC-SeNPs@siRNA achieved an obvious improvement in gene silencing ability, which led to significant growth inhibition of HepG2 cells. Furthermore, treatment with RGDfC-SeNPs@siRNA resulted in greater antitumor efficacy than lipofectamine 2000@siRNA in vitro and in vivo. In addition, the RGDfC-SeNPs@siRNA was almost non-toxic to the key organs of mice. In sum, these findings provide an alternative therapeutic route for targeted cancer treatments.

Background: Signal transducer and activator of transcription 3 (STAT3) was strongly expressed and activated in psoriatic keratinocytes (KCs) and correlated with the severity of psoriasis. The study aimed to investigate the effects of STAT3 small interfering RNA (siRNA) combined with ultrasonic irradiation and SonoVue microbubbles on the proliferation and apoptosis in KCs of psoriatic lesions and the relative mechanisms. Methods: Psoriatic KCs were transfected under four experimental conditions: (1) STAT3 siRNA carried by Lipofectamine 3000 combined with ultrasonic irradiation and SonoVue microbubbles (LUS group); (2) STAT3 siRNA only carried by Lipofectamine 3000 (L group); (3) the negative control of siRNA carried by Lipofectamine 3000 combined with ultrasonic irradiation and SonoVue microbubbles (siRNA-NC); (4) not treated as Blank. Cell Counting Kit-8 assay was used to evaluate the cell proliferation. Cell cycle analysis was detected with cycle test Plus DNA reagent kit associated with flow cytometer. FITC Annexin V apoptosis detection kit associated with flow cytometer was applied for apoptosis analysis. Fluo calcium indicator associated with flow cytometer was used to analyze intracellular free calcium concentration ([Ca2+]i). The expressions of cyclin D1 and Bcl-xL were detected both at the mRNA level by real-time reverse transcription-polymerase chain reaction (RT-PCR) and at the protein level by Western blotting. The obtained data were statistically evaluated by two-way analysis of variance. Results: STAT3 siRNA inhibited the growth of KCs in a time-dependent manner showing the highest proliferation inhibition in LUS group with proliferation ratio of 45.38% ± 5.85% at 72h (P < 0.05 vs. L group, siRNA-NC, or Blank). STAT3 siRNA induced an altered cell cycle distribution of KCs showing the highest increases in G2/M-phase population up to 18.06% ± 0.36% in LUS group (P < 0.05 vs. L group, siRNA-NC, or Blank). STAT3 siRNA induced late apoptosis of KCs with the highest late apoptosis percentage of 22.87% ± 1.28% in LUS group (P < 0.05 vs. L group, siRNA-NC, or Blank). STAT3 siRNA induced the elevation of [Ca2+]iof KCs with the highest calcium fluorescence intensity mean of 1213.67 ± 60.51 in LUS group (P < 0.05 vs. L group, siRNA-NC, or Blank). STAT3 siRNA induced the downregulation of cyclin D1 and Bcl-xL expressions of KCs at mRNA and protein levels with the lowest expressions in LUS group with cyclin D1 expression of 51.81% ± 9.58% and 70.17% ± 4.22% at mRNA level and at protein level, respectively, and with Bcl-xL expression of 37.58% ± 4.92% and 64.06% ± 7.78% at mRNA level and at protein level, respectively (P < 0.05 vs. L group, siRNA-NC, or Blank). Conclusions: STAT3 siRNA inhibited the growth and induced the apoptosis in psoriatic KCs likely partly through altering cell cycle distribution, elevating [Ca2+]i, and downregulating cyclin D1 and Bcl-xL expressions. Silencing the target gene STAT3 in psoriatic KCs with siRNA combined with ultrasonic irradiation and microbubbles would contribute to a significant innovation as a new clinical therapy for psoriasis.

Systemic, non-viral siRNA delivery for cancer treatment is mainly achieved via condensation by cationic materials (e.g., lipids and cationic polymers), which nevertheless, suffers from poor serum stability, non-specific tissue interaction, and unsatisfactory membrane activity against efficient in vivo gene knockdown. Here, we report the design of a metastable, cancer-targeting siRNA delivery system based on two functional polymers, PVBLG-8, a cationic, helical cell-penetrating polypeptide, and poly((L)-glutamic acid) (PLG), an anionic random-coiled polypeptide. PVBLG-8 with rigid, linear structure showed weak siRNA condensation capability, and PLG with flexible chains was incorporated as a stabilizer which provided sufficient molecular entanglement with PVBLG-8 to encapsulate the siRNA within the polymeric network. The obtained PVBLG-8/siRNA/PLG nanoparticles (PSP NPs) with positive charges were sequentially coated with additional amount of PLG, which reversed the surface charge from positive to negative to yield the metastable PVBLG-8/siRNA/PLG@PLG (PSPP) NPs. The PSPP NPs featured desired serum stability during circulation to enhance tumor accumulation via the enhanced permeability and retention (EPR) effect. Upon acidification in the tumor extracellular microenvironment and intracellular endosomes, the partial protonation of PLG on PSPP NPs surface would lead to dissociation of PLG coating from NPs, exposure of the highly membrane-active PVBLG-8, and surface charge reversal from negative to positive, which subsequently promoted tumor penetration, selective cancer cell internalization, and efficient endolysosomal escape. When siRNA against epidermal growth factor receptor (EGFR) was encapsulated, the PSPP NPs showed excellent tumor penetration capability, tumor cell uptake level, EGFR silencing efficiency, and tumor growth inhibition efficacy in U-87 MG glioblastoma tumor spheroids in vitro and in xenograft tumor-bearing mice in vivo, outperforming the PSP NPs and several commercial reagents such as Lipofectamine 2000 and poly((L)-lysine) (PLL). This study therefore demonstrates a facile and unique design approach of metastable and charge reversal NPs, which overcomes multiple biological barriers against systemic siRNA delivery toward anti-cancer treatment.

This study performed a biophysical characterization (electrochemistry, structure and morphology) and assessment of the biological activity and cell biocompatibility of GCL/DOPE-pDNA lipoplexes comprised of plasmid DNA and a mixed lipid formed by a DOPE zwitterionic lipid and a gemini cationic lipid N-N'-(1,3-phenylene bis (methylene)) bis (N,N-dimethyl-N-(1-dodecyl) ammonium dibromide (12PH12) containing an aromatic spacer or its monomeric counterpart surfactant, N-benzyl-N,N-dimethyl-N-(1-dodecyl) ammonium bromide (12PH). Electrochemical results reveal that i) the gemini cationic lipid (12PH12) and the plasmid pDNA yield effective charges less than their nominal charges (+2 and -2/bp, respectively) and that ii) both vectors (12PH12/DOPE and 12PH/DOPE) could compact pDNA and protect it from DNase I degradation. SAXS and cryo-TEM experiments indicate the presence of a lamellar lyotropic liquid crystal phase represented as alternating layers of mixed lipid and plasmid. Transfection efficiency (by FACS and luminometry) and cell viability assay in COS-7 cells, performed with two plasmid DNAs (pEGFP-C3 and pCMV-Luc VR1216), confirm the goodness of the proposed formulations (12PH12/DOPE and 12PH/DOPE) to transport genetic material, with efficiencies and biocompatibilities comparable to or better than those exhibited by the control Lipofectamine 2000*. In conclusion, although major attention has been paid to gemini cationic lipids in the literature, due to the large variety of modifications that their structures may support to improve the biological activity of the resulting lipoplexes, it is remarkable that the monomeric counterpart surfactant with an aromatic group analyzed in the present work also exhibits good biological activity. The in vitro results reported here indicate that the optimum formulations of the gene vectors studied in this work efficiently transfect plasmid DNA with very low toxicity levels and, thus, may be used in forthcoming in vivo experiments.

Development of efficient non-viral gene delivery vector has aroused great attention in the past few decades. In this study, we reported a new gene delivery vector, positively charged fluorescent conjugated polymer nanoparticles (CPNPs), for efficient gene transfection and in-situ intracellular fluorescence imaging. The microscopic and spectroscopic characterizations demonstrated that these CPNPs possess decent fluorescence performance (e.g. with fluorescence quantum yield of 70.7±0.3%) and small size dimension of ~3.6±0.3 nm (DLS result). Fast and efficient cellular translocation capability was observed according to the time-dependent living cell imaging experiments. Nearly all of the cells were loaded with CPNPs after co-incubation for 2 h regardless of the cell type. In comparison with the commonly used gene delivery vector, lipofectamine 2000 (with gene transfection efficiency of 55±5% for pEGFP), the gene expression efficiency with the positively charged CPNPs (70±3% for pEGFP) was improved significantly. Intracellular fluorescence imaging results demonstrated that the CPNPs could actively assemble close to the periphery of nuclei. Disassembly was not observed even 36 h later, which greatly facilitates releasing of pDNA close to the periphery of nuclei and thus promotes the gene transfection efficiency.

Acute lung injury (ALI) is a lung inflammatory disease for which pulmonary delivery of drug and gene could be a useful strategy. In this study, cholesterol-conjugated polyamidoamine (PAM-Chol) was synthesized and characterized as a carrier for combined delivery of anti-inflammatory gene and drug into the lungs by inhalation. The PAM-Chol formed self-assembled micelles in an aqueous solution with a critical micelle concentration of 0.22 mg ml-1. An in vitro transfection assay to L2 lung epithelial cells showed that the PAM-Chol micelle had higher transfection efficiency than lipofectamine and polyethylenimine (25 kDa, PEI25k). As the anti-inflammatory drug, resveratrol was loaded into the cores of the PAM-Chol micelles using the oil-in-water emulsion/solvent evaporation method. In lipopolysaccharide (LPS)-activated macrophage cells, resveratrol-loaded PAM-Chol (PAM-Chol/Res) reduced pro-inflammatory cytokines, confirming the anti-inflammatory effects of resveratrol. In in vitro transfection assays to L2 cells, the PAM-Chol/Res micelles had transfection efficiency similar to that of PAM-Chol. The delivery of resveratrol or the heme oxygenase-1 gene (pHO-1) by inhalation was evaluated in an ALI animal model. Resveratrol delivery using the PAM-Chol/Res micelles inhibited the nuclear translocation of nuclear factor-κB (NF-κB) and reduced pro-inflammatory cytokines in the lungs. pHO-1 delivery using PAM-Chol induced HO-1 expression and reduced pro-inflammatory cytokines. However, the highest anti-inflammatory effects were obtained with combined delivery of pHO-1 and resveratrol using the pHO-1/PAM-Chol/Res complex, as demonstrated in cytokine assays and immunohistochemical studies. Therefore, the PAM-Chol micelle is an efficient carrier of resveratrol and pHO-1 into the lungs and could be useful for the treatment of ALI by inhalation.

Preeclampsia (PE) is a severe pregnancy-specific disorder. Previous findings indicated that pigment epithelium-derived factor (PEDF) was upregulated in placentas of women with PE. Here, we investigated the role of PEDF in trophoblast function, especially under hypoxia. The effects of hypoxia on the morphology of extravillous trophoblast (EVT)-derived HTR-8Svneo cells were observed under inverted microscope. Transfections with Lipofectamine LTX were performed according to the manufacturer's protocol. The expression of PEDF protein and mRNA were confirmed by immunofluorescence (IF) and quantitative real-time PCR (qPCR). Apoptosis was detected by transferase-mediated dUTP nick end labeling (TUNEL) assay, and proliferation of trophoblast was detected by CCK-8 method. The invasion capacity of trophoblast was assessed by Transwell assay. PEDF was expressed in HTR-8/SVneo under both normoxia and hypoxic stress. However, cells of hypoxia groups had higher expression level of PEDF, increased apoptosis and decreased invasion capability, as compared with normoxia group. Moreover, after transfection with plasmid expressing PEDF gene, overexpression of PEDF modulated trophoblast activities. In addition, PEDF expression was negatively associated with invasion while positively correlated with apoptosis.Our data suggest that PEDF is an important factor to maintain the biological function of trophoblast cells, thus representing a rational therapeutic target in PE.

Compared to traditional clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) system, CRISPR/dead Cas9 (dCas9) system can precisely regulate endogenous gene expression without damaging the host gene, representing a greater potential for cancer therapy. Cancer/testis antigen 45 (CT45) is proved to enhance platinum-based chemosensitivity for individualized ovarian cancer therapy. However, the development of a single nanocarrier codelivering CRISPR/dCas9 system and chemotherapeutics for synergistic cancer therapy still faces challenges. Herein, a reduction-sensitive fluorinated-Pt(IV) universal transfection nanoplatform (PtUTP-F) is developed for the CT45-targeted CRISPR/dCas9 activation to achieve synergistic and individualized treatment of ovarian cancer. Overcoming multiple physiological barriers, PtUTP-F condensed gene can efficiently transfect into different cells including 293T cells, A2780, SKOV3, A549, and A2780/cisplatin (DDP) cancer cells, which is superior to Lipofectamine 6000. With the responsive release of gene and Pt(II) in the intracellular reducing microenvironment, PtUTP-F/dCas9-CT45 can generate CRISPR/dCas9 activation of CT45 expression for protein phosphatase 4C (PP4C) activity inhibition to hinder the DNA repair pathway and thus enhances the sensitivity to Pt(II) drugs for individualized A2780 tumor therapy. The PtUTP-F not only represents a powerful nanoplatform for CRISPR/dCas9 system delivery but also initiates a novel strategy for synergistic and individualized treatment of CRISPR/dCas9-based gene therapy with chemotherapy.

Bioactive glass nanoparticles (BGNs) have attracted much attention in drug delivery and bone tissue regeneration, due to the advantages including biodegradation, high bone-bonding bioactivity, and facile large-scale fabrication. However, the wide biomedical applications of BGNs such as efficient gene delivery are limited due to their poor pore structure and easy aggregation. Herein, for the first time, this study reports novel monodispersed bioactive glass nanoclusters (BGNCs) with ultralarge mesopores (10-30 nm) and excellent miRNA delivery for accelerating critical-sized bone regeneration. BGNCs with different size (100-500 nm) are fabricated by using a branched polyethylenimine as the structure director and catalyst. BGNCs show an excellent apatite-forming ability and high biocompatibility. Importantly, BGNCs demonstrate an almost 19 times higher miRNA loading than those of conventional BGNs. Additionally, BGNCs-miRNA nanocomplexes exhibit a significantly high antienzymolysis, enhance cellular uptake and miRNA transfection efficiency, overpassing BGNs and commercial Lipofectamine 3000. BGNCs-mediated miRNA delivery significantly improves the osteogenic differentiation of bone marrow stromal stem cells in vitro and efficiently enhances bone formation in vivo. BGNCs can be a highly efficient nonviral vector for various gene therapy applications. The study may provide a novel strategy to develop highly gene-activated bioactive nanomaterials for simultaneous tissue regeneration and disease therapy.

Chitosan/alginate (Chi/Alg) nanoparticles as a non-viral vector for the Smad4 encoding plasmid were optimized utilizing D-optimal design based on the nanoparticles/plasmid ratio, Chi/Alg MW, and preparation method type. Following the optimization and validation of the best formula, morphology studies and FTIR measurements were performed to evaluate the optimized Chi/Alg/S NPs. Toxicity (MTT assay) and transfection studies were performed for the best formula in comparison with Lipofectamine 2000, and Polyethyleneimine (PEI) and evaluated using Green Fluorescence Protein (GFP) assay, Flow cytometry, and RT-PCR. The model predicted a particle size of 111 nm, loading efficacy (LE) of 43%, cumulative release (CMR) of 39%, the ζ-potential of +50 mV, and PDI of 0.13. The predicted point condition was as follows: NP ratio = 13, Chi/Alg MW ratio = 2.35, and preparation method type = 1. Microscopic findings revealed that the shape of nanoparticles was spherical. The Chi/Alg/S nanoparticles showed no toxicity and transfection efficacy of 29.9% was observed in comparison with Lipofectamine (35.5%) and PEI (30.9%).

No currently available treatment is able to generate new contractile tissue or significantly improve cardiac function after myocardial infarction (MI), a leading cause of morbidity and mortality worldwide. Although gene transfer-enhanced endothelial progenitor cells (GTE-EPCs) show effectiveness in MI treatment in small animal models, no clinical trials using GTE-EPCs have been documented. Before the introduction of GTE-EPCs into human trials, gene-transfer-mediated augmentation of EPC function in animal models that reflect the human MI scenario should be tested. In this regard, a porcine model is the best choice since pigs have cardiac size, hemodynamics and coronary anatomy similar to that of humans. To examine GTE-EPC therapeutic efficacy in pig MI models, an efficient method for gene transfer into pig EPCs is required, which however, has been poorly documented. Pig bone marrow mononuclear cells were isolated and cultured in EGM-2 medium to obtain bone marrow-derived EPCs (BM-EPCs) that were characterized by immunostaining and the tube formation assay. Gene transfer was optimized in 6-well plates using a GFP and a VEGF plasmid, and scaled up in T75 flasks. Gene transfer efficiency was determined by fluorescence microscopy and flow cytometry. VEGF levels were measured by ELISA. Cell proliferation was assayed by the CCK-8 kit. (1) BM-EPCs expressed VEGFR2 and eNOS but not CD45 protein, and formed tube structures on Matrigel; (2) several chemical compounds were explored with the highest transfection efficiency of 41.4% ± 5.8% achieved using Lipofectamine 3000; (3) the VEGF level in culture medium after VEGF transfection was 378 ± 48 ng/106 cells; and (4) BM-EPCs overexpressing VEGF had significantly enhanced proliferation than GFP-transfected EPCs. A simple, easy and cheap method that can be applied to produce a large number of genetically-modified BM-EPCs was established, which will facilitate the study of GTE-EPC therapeutic efficacy in pig MI model.

Psoriasis is a common inflammatory skin disease. Infiltration of inflammatory cells and excessive proliferation of keratinocytes are the histopathological markers of psoriasis. The transcription factor Wilms Tumor 1 (WT1) is overexpressed in several tumor types, and plays an important part in the proliferation and apoptosis of cells. Studies have found that, compared with normal skin, WT1expression in the skin lesions of patients with psoriasis are increased significantly. Knockdown of WT1 inhibited the proliferation of a human epidermal keratinocyte cell line (HaCaT cells) and promoted their apoptosis, whereas WT1 overexpression exhibited the opposite effect. WT1 was overexpressed or inhibited in HaCaT cells by transfection with the WT1 plasmid or WT1 small interferring RNA (siRNA) using Lipofectamine 2000. Transcriptome sequencing and bioinformatics analysis revealed significant differences in IL-1β expression between the experimental group and control group. Real-time reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and enzyme-linked immunosorbent assays showed that expression of IL-1β and WT1 were consistent. Subsequently, IL-1β was demonstrated to be a target of WT1 by chromatin immunoprecipitation (ChIP)-sequencing and luciferase reporter assay. ChIP-qPCR showed that WT1 regulated IL-1β expression by altering acetylation. Expression of WT1 mRNA was positively correlated with expression of IL-1β mRNA in psoriatic skin lesions. Our study suggested that WT1 likely promotes psoriasis development by regulating its target gene IL-1β, which shows high expression in psoriatic lesions and is involved in psoriasis development. These findings provide a new target for psoriasis treatment.

The grail of gene delivery is the development of delivery vectors as effective and non-cytotoxic as possible. In this regard, there is an urgent need of new tools for the straightforward and quantitative assessment of transfection efficiency and cytotoxicity simultaneously. We herein reported the development and validation of an easy-to-use lab-on-chip platform to perform cell transfection assays for unbiased, high-throughput selection of more and more effective gene delivery vectors by using two commercially sourced lipids, Lipofectamine 2000® and FuGene® 6. A single PDMS-layer platform was endowed with: i) a chaotic serial dilution generator, designed for the automatic generation of a linear lipoplex dilution (from 100% to 0% with 25% steps) independently delivered to; and ii) the downstream culture and transfection module consisting in five units, each composed of 33 serially connected and fluidically connected culture chambers for trapping small populations of ≈10 cells/chamber. In the absence of any transfectant, cells spread and duplicated up to 2 days. Besides, cells were transfected with EGFP-encoding reporter gene. The very facile visual inspection of the microdevice by means of a microscope and a semi-automated analytical method allowed pinpointing the best transfection conditions in terms of efficiency, cytotoxicity, cell doubling rates, and morphological changes at once.

Nobutaka Hanagata,1,2 Xianglan Li,1 Min-Hua Chen,1 Jie Li,1 Shinya Hattori1 1Nanotechnology Innovation Station, National Institute for Materials Science, Tsukuba, 2Graduate School of Life Science, Hokkaido University, Sapporo, Japan Abstract: Conventional class B cytosine-guanine (CpG) (CpG-B) oligodeoxynucleotide (ODNs) consisting of a single-stranded (ss) phosphorothioate (PT) backbone (ss CpG-B-PT) is converted from a proinflammatory cytokine inducer to a type-I interferon (IFN) inducer when complexed with cationic materials. In this study, we designed ss CpG-B and double-stranded (ds) CpG-B ODNs with a phosphodiester (PD) backbone (ss CpG-B-PD and ds CpG-B-PD, respectively) that became type-I IFN inducers upon complexation with Lipofectamine 2000 (Lipo), a cationic liposome. The ds CpG-B-PD complex induced higher IFN-β expression in mouse macrophage-like RAW264 cells than ss CpG-B-PD and ss CpG-B-PT complexes. The fold induction of IFN-β increased with the number of CpG motifs in ds CpG-B-PD, and a complex of ds CpG-B-PD consisting of 72 base pairs with nine CpG motifs (ds CpG-B72-PD) and Lipo showed the highest capacity to induce IFN-β. The materials and method used for complexation influenced the degree of IFN-β induction: ds CpG-B72-PD entrapped by calcium phosphate (CaP) (ds CpG-B72-PD/CaP) showed a higher induction capacity than ds CpG-B72-PD adsorbed onto the CaP surface. Entrapment of ds CpG-B72-PD by CaP also enhanced the induction of the proinflammatory cytokine interleukin-12. Vaccinating mice with ds CpG-B72-PD/CaP in conjunction with ovalbumin (OVA) increased the ratios of OVA-specific CD8+ T cells to total CD8+ T cells in peripheral blood and of OVA-specific IgG2a associated with helper T (Th)1 cells to OVA-specific IgG1 associated with Th2 cells. These results indicate that ds CpG-B72-PD/CaP is an effective vaccine adjuvant that can activate both cellular and Th1-type humoral immune responses. Keywords: CpG oligodeoxynucleotide, calcium phosphate, vaccine adjuvant, cellular immunity, humoral immunity

The study aimed to explore the regulation of heat shock protein 47 (HSP47) on expressions of receptors associated with hepatic stellate cell (HSC) in liver fibrosis mouse models induced bySchistosoma japonicum(S. japonicum). Mouse fibroblasts (NIH/3T3) were transfected with HSP47 shRNA plasmid by lipofectamine transfection, and experimental fibrosis in HSCs was studied inS. japonicummouse models treated with HSP47 shRNAin vivo. HSP47 expression was assessed using Western blot and real-time PCR. Flow cytometry was adopted to determine the expression of cell membrane receptors. HSP47-shRNA could markedly down-regulate the expression of collagen (Col1a1 and Col3a1). The expressions of HSP47, endothelin receptor A (ETAR) and endothelin receptor B (ETBR) significantly increased in the liver tissue of infected mice. However, the expressions of ETAR and HSP47 and ETBR remarkably decreased after the administration of HSP47 shRNAin vitroandin vivo. ETAR and ETBR levels were found to be positively correlated with HSP47 expression. HSP47 might exert influence on liver fibrosis via the regulation of ETAR and ETBR.

Stem cell-based gene therapy has been considered in the treatment of many degenerative diseases. Gene-modified stem cells should maintain its reproductive activity without losing stem cell properties, including genetic phenotype and differentiation potential. In the study, a novel poly (amidoamine) with pendant aminobutyl group (PAA-BA) designed by our group was used in the transfection of bone marrow mesenchymal stromal cells (BMSCs) and the cellular properties post-transfection were evaluated, including DNA content, colony forming capacity, genetic phenotype, and multi-directional differentiation. Two classical non-viral gene delivery vectors, polyethylenimine (PEI) and Lipofectamine 2000 (LP2000) were also used. Compared to non-transfected group, PAA-BA showed minor decreased DNA content but maintained BMSCs' phenotype, reproductive activity and multi-differentiation potential (osteogenic, chondrogenic, adipogenic, and neurogenic differentiation). Both PAA-BA and PEI transfected BMSCs demonstrated improved osteogenic differentiation ability at late stage but suppressed adipogenic as well as mature neural differentiation in vitro. LP2000 and PEI transfected BMSCs displayed significantly lower DNA content and reproductive activity. These findings suggest that PAA-BA is one of safe gene delivery vectors in BMSCs transfection and plays a role in stem cell's osteogenic and neurogenic differentiation. This study proposes the potential application of PAA-BA in BMSCs based gene therapy, in particular bone and nerve relative diseases. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 686-697, 2018.

One of the most important properties of extracellular double-stranded DNA related to the treatment of various diseases is its ability to activate effector cells of the immune system (anti-tumor and vaccinal immunity) through dendritic cells (DCs). The stimulatory effect of DNA on DCs is mediated by the TLR9 signaling pathway and/or through a system of cytosolic sensors and is manifested by increased expression of MHC class II antigens and costimulatory molecules and by increased synthesis of immunoregulatory cytokines. In this work, the expression of cytokines, differentiation antigens and transcription factor genes has been investigated in DCs activated by double-stranded human DNA (i) without any additional factors, (ii) using a lipophilic agent, and (iii) by blocking TLR9 with chloroquine. Evaluation of the DNA effect was carried out after the 6- and 24-hour exposure. It was found that the preparation of double-stranded DNA transfected by Lipofectamine 2000 boosts DCs at the same level as Poly(dA : dT), a synthetic equivalent of double-stranded DNA. It was discovered that combined application of DNA and chloroquine enhances expression of the IFN - α , IFN - β , IFN - γ , IL­8 , МСР1 , VEGF , CD25 , and CD83 genes by hour 24 of incubation. It was for the first time shown that genomic “self” double-stranded DNA as a mono agent activates mRNA synthesis of cytokines IFN - α , IFN - β , IFN - γ , IL­8 , IL­10 , and VEGF in DCs at 6 hours of induction.

Cationic lipid-guided nucleic acid delivery holds great promise in gene therapy and genome-editing applications for treating genetic diseases. However, the major challenge lies in achieving therapeutically relevant efficiencies. Prior findings, including our own, demonstrated that asymmetry in the hydrophobic core of cationic lipids imparted superior transfection efficiencies. To this end, we have developed a lipid nanocarrier system with an asymmetric hydrophobic core (PS-Lips) derived from a mixture of fatty acids of food-grade palmstearin and compared its efficiency with symmetric palmitic acid-based nanocarrier system (P-Lip). PS-Lips exhibited superior transfection efficiencies with both plasmid DNA (pDNA) and mRNA in multiple cultured cells than the control P-Lip. More importantly, PS-Lips exhibited 2-fold superior transfections with linear nucleic acid, green fluorescent protein (GFP) mRNA in hematopoietic cells, when compared with the commercial control lipofectamine RNAiMAX. PS-Lips was also found to be effective in delivering genome-editing tools (CRISPR/Cas9, sgRNA encoded pDNA with a reporter GFP construct) than P-Lip in HEK-293 cells. In the present study, we report that cationic liposomes derivatized from natural food-grade fat palmstearin with a natural hydrophobic core asymmetry are efficient in delivering both linear and circular nucleic acids. In particular, PS-Lips is efficient in delivering mRNA to hematopoietic cells. These findings can be further exploited in the genome-editing approach for treating β-globinopathies.

Aim: Aim of the study was the development of ζ potential changing nanoparticles as gene delivery system for the cystic fibrosis transmembrane conductance regulator gene. Methods: Chitosan and carboxymethyl cellulose were modified with phosphotyrosine, a substrate for the brush border enzyme alkaline phosphatase. With these synthesized derivatives, different nanoparticle formulations, including the cystic fibrosis transmembrane conductance regulator gene were prepared by ionic gelation. Results: A change from negative to positive ζ potential after enzymatic cleavage could be observed. Transfection studies with HEK-293 and Caco-2 cells showed transfection rates comparable to Lipofectamine 2000. Transfection efficiencies were significantly decreased when phosphate cleavage and thus ζ potential change was inhibited by phosphatase inhibitor. Conclusion: The developed nanoparticles represent a promising gene delivery system.

Lipid-based nanoparticles as gene vectors have attracted considerable attention for their high gene transfection efficiency and low cytotoxicity. In our previous work, we synthesized gold nanoparticles/dimethyldioctadecylammonium bromide (DODAB)/dioleoylphosphatidylethanolamine (DOPE) (GDD) as anionic lipid- and pH-sensitive gene vectors. To further realize targeted gene transfection, a series of gold nanoparticles/DODAB/DOPE/DOPE-folic acid (DOPE-FA) with various ratios of DOPE-FA were prepared and termed as GFn (for which n=1.0, 2.5, 5.0, 7.5, or 10.0 %). The gene transfection efficiency mediated by GF2.5 can reach about 85 % for MCF-7 (FA-receptor-positive cells), higher than those of the negative control (GDD, 35 %) and positive control (Lipofectamine 2000, 65 %). However, GF2.5 does not further promote gene transfection into A549 (FA-receptor-negative cells). The higher gene transfection efficiency for MCF-7 cells can be attributed to enhanced cellular uptake efficiency mediated by the FA targeting ability. Furthermore, GF2.5 was also found to accumulate at the specific tumor site and showed enhanced in vivo gene delivery ability. In addition, no significant harm was observed for the main tissues of the mice after treatment with GF2.5. Therefore, GF2.5, with the targeting ability and improved transfection efficiency, shows promise for its utility in gene therapy for tumor cells that overexpress FA receptors. We believe the results of this study will find more broad applications in gene therapy.

Background: the miR-17-92 cluster is known as an oncogene (oncomiR-1) overexpressed in most cancers. However, recent studies have shown that these miRNAs were downregulated in some cancers. Thus, some or all members of miR-17-92 cluster may have dual activity: apoptosis induction or inhibition. Objectives: For a better understanding of miR-17-92 cluster activities, we focused on the function of mir-17 and mir-20a as two members of miR-17-92 cluster in Jurkat cells activated by phytohemagglutinin (PHA). Materials and Methods: Jurkat cells were stimulated by PHA for 24 h. P-lenti-mico-GFP plasmids containing miR-17 or miR-20a and an empty vector (blank) were transfected into activated Jurkat cells using Lipofectamine 2000 reagent. Cell viability was measured using XTT assay after transfection. Putative targets of these miRNAs were predicted by Targetscan and miRWalk algorithms in JAK/STAT and PI3K/AKT signaling pathways. Then the expression of several putative targets was evaluated by quantitative RT-PCR. Results: Transfection of mir-17 and mir-20a decreased the proliferation in activated Jurkat cells. In silico investigations revealed that mir-17 and mir-20a potentially target JAK1, AKT1 and AKT3. Q-RT-PCR analysis illustrated that these targets were downregulated in Jurkat cells after transfection with miR-17 and miR-20a. Conclusions: According to our findings, it seems that mir-17 and mir-20a expression, two members of miR-17-92 cluster, is dependent on cell condition and cell type; as a result, their ectopic expression in activated Jurkat cells may lead to cell death.