Dexamethasone-loaded peptide micelles for delivery of the heme oxygenase-1 gene to ischemic brain

The R3V6 peptides, which are composed of a 3-arginine block and a 6-valine block, formed self-assembled micelles in aqueous solution. Dye quenching assays showed that a hydrophobic fluorescent dye, 5-dodecanoylaminofluorescein (DAF), interacted with and was loaded into the hydrophobic core of the micelles. In this study, dexamethasone-loaded R3V6 peptide micelles (R3V6-Dexa) were evaluated as a gene carrier. R3V6-Dexa had higher gene delivery efficiency in human embryonic kidney 293 cells compared to those of the R3V6 peptides and poly-L-lysine (PLL). Dexamethasone might stabilize the micelle structure of the R3V6 peptides by forming strong hydrophobic cores and enhanced the transfection efficiency. Furthermore, R3V6-Dexa reduced the expression of an inflammatory cytokine, interleukin-6 (IL-6), more efficiently in lipopolysaccharide (LPS)-induced Raw264.7 cells than did dexamethasone, suggesting that R3V6-Dexa is also a useful carrier for dexamethasone delivery. A focal brain ischemia-reperfusion model was produced by middle cerebral artery occlusion (MCAO). A heme oxygenase-1 (HO-1) expression plasmid DNA, pSV-HO-1, was delivered into the brain using R3V6-Dexa as a carrier. The pSV-HO-1/R3V6-Dexa complex was injected into the brain 1hr prior to MCAO. Twenty-four hours later, the HO-1 expression of the pSV-HO-1/R3V6-Dexa injection group was higher than those of the MCAO control, pβ-Luc/R3V6-Dexa, and pSV-HO-1/PEI25k injection groups. In addition, the infarct size was reduced due to the delivery of pSV-HO-1/R3V6-Dexa complex. Therefore, R3V6-Dexa may be a useful carrier for HO-1 gene delivery and stroke gene therapy.     

Use of ultrasound to enhance nonviral lung gene transfer in vivo

We have assessed if high-frequency ultrasound (US) can enhance nonviral gene transfer to the mouse lung. Cationic lipid GL67/pDNA, polyethylenimine (PEI)/pDNA and naked plasmid DNA (pDNA) were delivered via intranasal instillation, mixed with Optison microbubbles, and the animals were then exposed to 1 MHz US. Addition of Optison alone significantly reduced the transfection efficiency of all three gene transfer agents. US exposure did not increase GL67/pDNA or PEI/pDNA gene transfer compared to Optison-treated animals. However, it increased naked pDNA transfection efficiency by approximately 15-fold compared to Optison-treated animals, suggesting that despite ultrasound being attenuated by air in the lung, sufficient energy penetrates the tissue to increase gene transfer. US-induced lung haemorrhage, assessed histologically, increased with prolonged US exposure. The left lung was more affected than the right and this was mirrored by a lesser increase in naked pDNA gene transfer, in the left lung. The positive effect of US was dependent on Optison, as in its absence US did not increase naked pDNA transfection efficiency. We have thus established proof of principle that US can increase nonviral gene transfer, in the air-filled murine lung.     

Characterization and evaluation of the efficacy of cationic complex mediated plasmid DNA delivery in human embryonic palatal mesenchyme cells

The purpose of this study was to develop and test a non‐viral gene delivery system that can be employed to deliver genes of interest into a pre‐osteoblastic cell line. Human embryonic palatal mesenchymal (HEPM 1486) cells were transfected with vector‐plasmid DNA (pDNA) complexes. We explored calcium phosphate and polyethylenimine (PEI) as non‐viral vectors and compared their respective in vitro transfection efficacies. Plasmid DNA encoding luciferase protein (LUC) was complexed with PEI (with differing N:P ratios) and calcium phosphate (with differing Ca:P ratios), using established protocols. The complexes prepared were then characterized for size and surface charge, using a Malvern Zetasizer Nano‐ZS. The transfection efficiency and cytotoxicity of the prepared complexes were evaluated in HEPM cells. The PEI–pDNA complexes over the whole range of N:P ratios were found to be < 160 nm in size, while the calcium phosphate–pDNA complexes were relatively bigger. The PEI–pDNA complexes prepared at a N:P ratio of 10 were found to have maximum transfection efficiency at 4 h of treatment, with minimal cytotoxicity. The highest transfection efficiency obtained with calcium phosphate–pDNA complexes (Ca:P 200) was nearly 12‐fold lower than that obtained with PEI–pDNA complexes (N:P 10). Following this, transgene expression in the HEPM cells treated with complexes prepared at a N:P ratio of 10 was further examined, using pDNA coding for enhanced green fluorescent protein (EGFP‐N1) or therapeutically relevant platelet‐derived growth factor B (PDGF‐B). In conclusion, PEI was a more effective vector for delivering genes of interest to pre‐osteoblasts than calcium phosphate. Copyright © 2014 John Wiley & Sons, Ltd.     

Development of lysine-histidine dendron modified chitosan for improving transfection efficiency in HEK293 cells

Chitosan has potential as a biocompatible gene carrier. However, its gene transfection efficiency is low because of its slow endosomal escape rate. Histidine has buffering capacity in the pH range of endosomes/lysosomes. The structure of dendron consists of a central core with several chains radiating from it and many histidines could be conjugated on the surface, increasing the efficiency of histidine modification. The purpose of this study is to increase the gene transfection efficiency of chitosan by promoting its endosomal escape property. We developed fourth-generation lysine-histidine (KH) dendrons that can provide 8 histidines in one dendron molecule. Chitosan-dendron (Chi13k-D) was synthesized using 2-iminothiolane to form the linkage; this was confirmed by NMR and the ninhydrin test. The buffering range, as measured by pH titration, was broader in the Chi13k-D group than in chitosan. Enhanced endosomal escape of Chi13k-D/pDNA complexes was confirmed using fluorescence-labeled endosomes and pDNA. The intralysosomal pH of Chi13k-D/pDNA was also higher than that of chitosan/pDNA. The gene transfection efficiency of Chi13k-D/pDNA was higher than that of chitosan/pDNA in HEK293 cells. These results suggest that KH dendron modification could provide high buffering capacity, which would increase the gene transfection efficiency of chitosan.     

Controlled release of cell-permeable gene complex from poly(L-lactide) scaffold for enhanced stem cell tissue engineering

The use of tissue engineering to deliver genes to stem cells has been impeded by low transfection efficiency of the inserted gene and poor retention at the target site. Herein, we describe the use of non-viral gene transfer by cell-permeable peptide (CPP) to increase the transfection efficiency. The combination of this technique with the use of a controlled release concept using a poly (l-lactide) scaffold allowed for prolonged uptake in stem cells. High transfection efficiency was obtained using a human-derived arginine-rich peptide denoted as Hph-1 (YARVRRRGPRR). The formation of complex between pDNA and Hph-1 was monitored using gel retardation tests to measure size and zeta potential. Complex formation was further assessed using a DNase I protection assay. A sustained gene delivery system was developed using a fibrous 3-D scaffold coated with pDNA/Hph-1 complexes. Transfection efficiency and the mean fluorescence intensity of human adipose-derived stem cells (hASCs) on the sustained delivery scaffold were compared to those of cells transfected via bolus delivery. Plasmid DNA completely bound Hph-1 at a negative-to-positive (N/P) charge ratio of 10. After complex formation, Hph-1 appeared to effectively protect pDNA against DNase I attack and exhibited cytotoxicity markedly lower than that of the pDNA/PEI complex. Plasmid DNA/Hph-1 complexes were released from the scaffolds over 14 days and were successfully transfected hASCs seeded on the scaffolds. Flow cytometry revealed that the transfection efficiency in hASCs treated with pDNA/Hph-1 complex was approximately 5-fold higher than that in cells transfected using Lipofectamine. The sustained delivery system showed a significantly higher transfection efficiency and remained able to transfect cells for a longer period of time than bolus delivery. These results suggest that cell-scaffold-based tissue regeneration can be further improved by transduction concept using CPP and controlled release using polymeric scaffold.     

Peptide dendrimers as efficient and biocompatible gene delivery vectors: Synthesis and in vitro characterization

We report the synthesis and characterization of different generations of dendritic poly(l-lysine) vectors, and their use for in vitro gene transfection. Gel retardation assay revealed that the dendrimers could form complexes with plasmid DNAs (pDNAs), evident from the inhibition of the migration of pDNA at the N/P ratios of 0.5, 1 and 2 by G3, G4 and G5 dendritic generations, respectively. DNase I assay revealed the protection of pDNA acquired from the complexation with dendrimers from nuclease-catalyzed degradation, with the protection capacity of G5 being even stronger than poly(ethyleneimine) (PEI). Atomic force microscopy (AFM) revealed that all 4 generations of dendrimer/DNA complexes studied were of similar particle sizes within 100-200 nm. Zeta potential measurements showed that as the N/P ratio increased from 1 to 25, all dendrimer/pDNA complexes gradually changed from negative to positive charges. The higher generations tended to produce the greater positive potentials, indicating a stronger potency of the complexes to interact with negatively charged cell membranes. In vitro and in vivo cytotoxicity evaluations showed good biocompatibility of the dendrimers and their complexes over the different N/P ratios studied. In vitro gene transfection revealed higher efficiency of G5 than other dendrimers and insensitive variation to the presence of serum. Given its similar transfection efficiency to PEI but lower toxicity to cultured cells, dendrimer G5 could be a better candidate for gene delivery.     

地塞米松对家兔急性肺损伤早期中性粒细胞相关功能的影响

目的研究地塞米松对家兔急性肺损伤(ALI)早期中性粒细胞(PMN)相关功能的影响,为临床治疗提供理论依据。方法雄性新西兰家兔32只,随机对照组(N)8只,急性肺损伤组(L)10只,早期干预组(E)7只,预处理组(P)7只,建立内毒素ALI模型。E、P组分别于建模前、后一次性给予地塞米松2mg。结果L组在内毒素注射后血压、心率、pH、PMN下降明显。PMN表面CD11b表达强度进行性升高。E、P两组血压、心率降幅减小;CD11b表达强度的升高及3h血PMN计数下降被明显抑制;肺病理改变明显减轻。E组血压及PMN下降幅度大于P组。结论地塞米松对于感染中毒性休克有一定的改善作用,可以抑制ALI的发生、发展。在血压和外周血PMN计数两项指标上,实验结果表明激素的预处理对于实验动物有更好的保护作用。     

Water-soluble lipopolymer as an efficient carrier for gene delivery to myocardium

Water-soluble lipopolymer (WSLP), which consisted of polyethylenimine (PEI, 1800 Da) and cholesterol, was characterized as a gene carrier to smooth muscle cells and myocardium. Acid-base titration showed that WSLP had a proton-buffering effect. The size of WSLP/plasmid DNA (pDNA) complex was around 70 nm. WSLP/pDNA complex was transfected to A7R5 cells, a smooth muscle cell line. WSLP showed the highest transfection at a 40/1 N/P ratio. WSLP has higher transfection efficiency than PEI (1800 and 25 000 Da), SuperFect, and lipofectamine. In addition, WSLP has less cytotoxicity than PEI (25 000 Da), SuperFect, and lipofectamine. Since WSLP has cholesterol moiety, it may utilize cellular cholesterol uptake pathway, in which low-density lipoprotein (LDL) is involved. An inhibition study with free cholesterol or low-density lipoprotein (LDL) showed that transfection was inhibited by cholesterol or LDL, suggesting that WSLP/pDNA complex is transfected to the cells through the cholesterol uptake pathway. To evaluate the transfection efficiency to myocardium, WSLP/pDNA complex was injected into the rabbit myocardium. WSLP showed higher transfection than PEI and naked pDNA. WSLP expressed the transgene for more than 2 weeks. In conclusion, WSLP is an efficient carrier for local gene transfection to myocardium, and useful in in vivo gene therapy.     

Using magnetic forces to enhance non-viral gene transfer to airway epithelium in vivo

We have assessed whether magnetic forces (magnetofection) can enhance non-viral gene transfer to the airways. TransMAG(PEI), a superparamagnetic particle was coupled to Lipofectamine 2000 or cationic lipid 67 (GL67)/plasmid DNA (pDNA) liposome complexes. In vitro transfection with these formulations resulted in approximately 300- and 30-fold increase in reporter gene expression, respectively, after exposure to a magnetic field, but only at suboptimal pDNA concentrations. Because GL67 has been formulated for in vivo use, we next assessed TransMAG(PEI) in the murine nasal epithelium in vivo, and compared this to naked pDNA. At the concentrations required for in vivo experiments, precipitation of magnetic complexes was seen. After extensive optimization, addition of non-precipitated magnetic particles resulted in approximately seven- and 90-fold decrease in gene expression for naked pDNA and GL67/pDNA liposome complexes, respectively, compared to non-magnetic particles. Thus, whereas exposure to a magnetic field improved in vitro transfection efficiency, translation to the in vivo setting remains difficult.     

Evaluation of proinflammatory cytokine production induced by linear and branched polyethylenimine/plasmid DNA complexes in mice

The purpose of this study was to evaluate the cytokine response induced by linear and branched polyethylenimine (PEI)/plasmid DNA (pDNA) complex (polyplex) in relation to the ratio of PEI nitrogen and DNA phosphate (N/P ratio) of the polyplex, dose of pDNA, and structure and molecular weight of PEI, which are important for transfection efficacy of PEI polyplex. As a control, a N-[1-(2, 3-dioleyloxy) propyl]-n,n,n-trimethylammonium chloride/cholesterol liposome/pDNA complex (lipoplex) was selected for its high transfection efficacy in vivo. The concentration of proinflammatory cytokines such as tumor necrosis factor (TNF)-alpha were much lower after the administration of polyplex than lipoplex irrespective of the N/P ratio, dose of pDNA, or structure and molecular weight of PEI, although these factors affected the transfection efficacy in vivo. We demonstrated that the amount of activated nuclear factor-kappaB, which contributes substantially to the production of cytokines, was comparable with the control (no treatment) level, and significantly less than that obtained with lipoplex. Although the production of proinflammatory cytokines (TNF-alpha, interferon-gamma, and interleukin-12) was reduced on the administration of the linear PEI polyplex, serum alanine aminotransferase levels were significantly enhanced by pDNA in a dose-dependent manner, suggesting that such hepatic damage is not induced by proinflammatory cytokines.     

己酮可可碱对大鼠内毒素性急性肺损伤iNOS和NO的影响

目的 :探讨己酮可可碱 (PTX)对大鼠内毒素性急性肺损伤 (ALI)iNOS和NO的影响。方法 :采用大鼠内毒素ALI模型。 2 4只SD大鼠随机分为生理盐水对照组 (CON)、内毒素组 (LPS)和PTX组 ,每组 8只。观察PTX对氧合指数 (PaO2 /FiO2 )、支气管肺泡灌洗液 (BAL)中蛋白含量、肺组织iNOS、NO3- /NO2 - 、髓过氧化物酶 (MPO)活性的影响 ,计算肺湿 /干比值(W /D)并行肺组织病理学检查。结果 :PTX组自 2h起各时间点PaO2 /FiO2 均高于LPS组 (P <0 0 5 ) ,W /D、BAL中蛋白含量、肺组织iNOS、NO含量和MPO活性均较LPS组显著降低 (P <0 0 5 )。病理学检查显示PTX组肺组织损伤程度较LPS组减轻。结论 :PTX对内毒素性ALI的保护作用可能与其抑制肺组织iNOS活性和减少NO生成有关。     

Optimizing aerosol gene delivery and expression in the ovine lung.

We have developed the sheep as a large animal model for optimizing cystic fibrosis gene therapy protocols. We administered aerosolized gene transfer agents (GTAs) to the ovine lung in order to test the delivery, efficacy, and safety of GTAs using a clinically relevant nebulizer. A preliminary study demonstrated GTA distribution and reporter gene expression throughout the lung after aerosol administration of plasmid DNA (pDNA):GL67 and pDNA:PEI complexes. A more comprehensive study examined the dose-response relationship for pDNA:PEI and assessed the influence of adjunct therapeutic agents. We found that the sheep model can differentiate between doses of GTA and that the anticholinergic, glycopyrrolate, enhanced transgene expression. Dose-related toxicity of GTA was reduced by aerosol administration compared to direct instillation. This large animal model will allow us to move toward clinical studies with greater confidence.     

NO和iNOS在内毒素诱导的大鼠急性肺损伤的作用及大黄对其影响

目的 主要探讨一氧化氮（ＮＯ）和诱导型一氧化氮合酶（ｉＮＯＳ）在内毒素（ＬＰＳ）诱导的大鼠急性肺损伤（ＡＬＩ）的作用机制及大黄对其影响。方法 在雄性Ｗｉｓｔａｒ大鼠利用舌下静脉注射ＬＰＳ复制ＡＬＩ动物模型,动物分为４组：ＬＰＳ组,对照组,大黄治疗组,地塞米松组。观察大体标本,组织病理以及生物学标志：肺湿／干重比,肺泡灌洗液中性粒细胞比,蛋白含量,肺血管通透性和肺泡通透性指数。同时测定血浆ＮＯ和肺组     

Effect of tyrosine kinase inhibitors,imatinib and nilotinib,in murine lipopolysaccharide-induced acute lung injury during neutropenia recovery

Introduction

Neutrophil recovery has been implicated in deterioration of oxygenation and exacerbation of preexisting acute lung injury (ALI). The aim of this study was to investigate whether imatinib or nilotinib was effective on lipopolysaccharide (LPS)-induced ALI during neutropenia recovery in mice.

Methods

Mice were rendered neutropenic with cyclophosphamide prior to the intratracheal instillation of LPS. Imatinib or nilotinib was administrated by oral gavage during neutropenia recovery. In order to study the effects of drugs, mice were killed on day 5 and blood, bronchoalveolar lavage (BAL) fluid and lung tissue samples were obtained. The lung wet/dry weight ratio and protein levels in the BAL fluid or lung tissue were determined.

Results

Treatment with imatinib or nilotinib significantly attenuated the LPS-induced pulmonary edema, and this result was supported by the histopathological examination. The concentrations of tumor necrosis factor-α, interleukin (IL)-1β, IL-6 and myeloperoxidase in BAL fluid were significantly inhibited by imatinib or nilotinib in mice of ALI during neutropenia recovery. The mRNA expressions of platelet-derived growth factor receptor-β and c-KIT in imatinib or nilotinib group were significantly lower than LPS group.

Conclusions

Our data indicated that imatinib or nilotinib effectively attenuated LPS-induced ALI during neutropenia recovery. These results provide evidence for the therapeutic potential of imatinib and nilotinib in ALI during neutropenia recovery.     

大黄对内毒素诱导致急性肺损伤的保护作用

目的：探讨内毒素在急性肺损伤（ＡＬＩ０中的作用机制及大黄,地塞米松对ＡＬＩ的保护作用。方法：在Ｗｉｓｔａｒ大鼠舌下静脉注射内毒素（ＬＰＳ）复制ＡＬＩ动物模型。动物分为４组：ＬＰＳ致伤组,对照组（生理盐水）,地塞米松治疗组,大黄治疗组。肉眼观察肺大体标本;普通光镜检查肺组织病理变化;电镜观察肺组织超微结构;测定ＡＬＩ的生物学指标;肺湿重与干重比,肺泡灌洗液中中性粒细胞比例和蛋白含量,肺泡通透指数和肺     

Effects of combined naloxone and methylprednisolone on nuclear factor-kappaB expression in the lung in acute lung injury in rats]

OBJECTIVE: To investigate the effects of a combination of naloxone and methylprednisolone on nuclear factor-kappaB (NF-kappaB) p65 expression in the lung tissue in lipopolysaccharide (LPS)-induced acute lung injury (ALI) in rats. METHODS: ALI models were reproduced by intratracheal instillation of LPS (3 mg/kg). Four hours after LPS instillation, rats were randomly divided into five groups: normal saline group, LPS group, methylprednisolone group, naloxone group (LPS+naloxone) and combined drug group (LPS+naloxone+methylprednisolone). The level of interleukin-8 (IL-8) in serum was measured by immunoassay. Meanwhile, the expression of NF-kappaB p65 in the lung tissue was determined with immunohistochemical staining. RESULTS: Naloxone and methylprednisolone significantly reduced the LPS-induced increase in IL-8 concentrations in serum in vivo, and suppressed the activation of NF-kappaB p65 in the lung tissue. CONCLUSION: NF-kappaB activation is involved in the LPS-induced ALI in rats. Combination of naloxone and methylprednisolone could suppress the increase of IL-8 content and NF-kappaB activation in the lung tissue of rat in vivo in our experiment.     

Delivery and expression of pDNA embedded in collagen matrices.

Collagen matrices can be used as non-viral biocompatible gene carriers for localized implantable gene therapy. Collagen matrices embedding pDNA with enhanced binding through condensing agent linkage to the matrix or to the pDNA have been formulated, and characterized in various systems. pDNA and condensed pDNA were released intact from the matrices within 1-2 days. In vitro transfection with collagen matrices containing pDNA (luciferase encoding), pDNA in liposome (LIP), and pDNA with polyethylenimine (PEI) resulted in significantly higher expression levels in comparison to naked pDNA. pDNA-LIP matrices exhibited a dose response transfection of NIH 3T3, 293, MDA-MB-231 and smooth muscle cells (SMCs) in cell cultures. Subdermal implantations of collagen-polylysine-pDNA matrices in rats resulted in significantly higher gene expression levels in comparison to non-condensed pDNA matrices. Perivascular treatment with pDNA matrix and of naked pDNA solution in balloon-injured rat carotid arteries resulted in significant expression. In conclusion, a facile method for embedding cationic formulations of pDNA in collagen matrices was developed. These bioactive matrices seem to be suitable for tissue engineering and local gene therapy strategies.     

Lactose-conjugated polyion complex micelles incorporating plasmid DNA as a targetable gene vector system: their preparation and gene transfecting efficiency against cultured HepG2 cells.

alpha-Lactosyl-poly(ethylene glycol)-poly(2-(dimethylamino)ethyl methacrylate) block copolymer (lactose-PEG-PAMA) was synthesized to construct a PIC micellar-type gene vector potentially useful for selective transfection of hepatic cells. Lactose-PEG-PAMA spontaneously formed a polyion complex (PIC) micelle with plasmid DNA (pDNA) encoding luciferase (pGL3-Luc) in aqueous solution without any precipitate formation. The lactosylated PIC micelle thus prepared achieved substantially higher transfection efficiency compared to the control PIC micelle without lactose moieties against HepG2 cells possessing asialoglycoprotein (ASGP) receptors recognizing the beta-d-galactose residue. This pronounced transfection efficacy of the lactosylated PIC micelle was inhibited by the addition of excess asialofetuin (ASF), a natural ligand against the ASGP receptor, indicating ASGP receptor-mediated endocytosis to be a major route of the cellular uptake of the lactosylated micelles. Notably, the lactosylated PIC micelle revealed enhanced transfection compared to the control PIC micelle at a lower dose of pDNA, demonstrating the feasibility of using the ligand-conjugated PIC micellar vector for gene delivery to targeted cells.