Chitosan-DNA nanoparticles delivered by intrabiliary infusion enhance liver-targeted gene delivery

The goal of this study was to examine the efficacy of liver-targeted gene delivery by chitosan-DNA nanoparticles through retrograde intrabiliary infusion (RII). The transfection efficiency of chitosan-DNA nanoparticles, as compared with PEI-DNA nanoparticles or naked DNA, was evaluated in Wistar rats by infusion into the common bile duct, portal vein, or tail vein. Chitosan-DNA nanoparticles administrated through the portal vein or tail vein did not produce detectable luciferase expression. In contrast, rats that received chitosan-DNA nanoparticles showed more than 500 times higher luciferase expression in the liver 3 days after RII; and transgene expression levels decreased gradually over 14 days. Luciferase expression in the kidney, lung, spleen, and heart was negligible compared with that in the liver. RII of chitosan-DNA nanoparticles did not yield significant toxicity and damage to the liver and biliary tree as evidenced by liver function analysis and histopathological examination. Luciferase expression by RII of PEI-DNA nanoparticles was 17-fold lower than that of chitosan-DNA nanoparticles on day 3, but it increased slightly over time. These results suggest that RII is a promising routine to achieve liver-targeted gene delivery by non-viral nanoparticles; and both gene carrier characteristics and mode of administration significantly influence gene delivery efficiency.     

Chitosan nanoparticles for oral drug and gene delivery

Chitosan is a widely available, mucoadhesive polymer that is able to increase cellular permeability and improve the bioavailability of orally administered protein drugs. It can also be readily formed into nanoparticles able to entrap drugs or condense plasmid DNA. Studies on the formulation and oral delivery of such chitosan nanoparticles have demonstrated their efficacy in enhancing drug uptake and promoting gene expression. This review summarizes some of these findings and highlights the potential of chitosan as a component of oral delivery systems.     

Magnetic nanoparticles for gene and drug delivery

Investigations of magnetic micro- and nanoparticles for targeted drug delivery began over 30 years ago. Since that time, major progress has been made in particle design and synthesis techniques, however, very few clinical trials have taken place. Here we review advances in magnetic nanoparticle design, in vitro and animal experiments with magnetic nanoparticle-based drug and gene delivery, and clinical trials of drug targeting.     

层层自组装纳米粒作为非病毒基因载体

基因治疗的效果严重依赖于基因载体。与传统包封技术相比,在自组装技术基础上发展起来的以DNA为聚阴离子,与荷正电的高分子材料在溶液中形成纳米粒的方法,已成为目前最重要的非病毒基因载体制备手段,具有良好的应用前景。采用层层自组装(layer-by-layer assembly,LbL)技术可提高基因装载率,其优势还在于纳米粒表面性质的可控性:在温和的条件下实现多种材料在载体表面的固定,实现载体多功能化等。本文将对近年来国内外有关层层自组装纳米粒作为非病毒基因载体的研究进展以及本课题组在此方向的研究进行简要综述。     

Enhanced gene transfer into brain capillary endothelial cells using Antp-modified DNA-loaded nanoparticles

Brain capillary endothelial cells (BCECs) have been considered as one of the primary targets for cerebral gene therapy. However, the cells, well-known for their poor function of endocytosis, are difficult to be transfected by general non-viral vectors. The aim of this study was to enhance the efficiency of transfection and expression in BCECs of DNA/polymer nanoparticles with the modification of membrane-penetrating peptide, Antennapedia peptide (Antp) polyethylenimine (PEI) and polyamidoamine (PAMAM) were chosen to prepare Antp-modified DNA-loaded nanoparticles with a complex coacervation technique. After a 20-min transfection, the efficiency, in terms of transfection and expression, of DNA/PEI NP or DNA/PAMAM NP was enhanced significantly with the modification of Antp. After a 3-h transfection of DNA/Antp/PEI NP, there was no difference in cellular uptake but an enhancement in gene expression, compared to DNA/PEI NP alone. However, both the transfection and expression efficiency of DNA/PAMAM NP were enhanced using Antp. These observations suggest that Antp can increase the membrane-penetrating ability of DNA-loaded nanoparticles, which can be employed as novel non-viral gene vectors.     

Drug and gene delivery using gold nanoparticles

Monolayer-functionalized gold nanoparticles provide attractive vehicles for pharmaceutical delivery applications as a result of their size and the unique properties and release mechanisms imparted by their monolayer. This review provides examples of recent advances in the field of drug and gene delivery using gold nanoparticles.     

无机纳米粒子作为基因载体的研究进展

转染是将具生物功能的核酸转移、运送到细胞内,并使其在细胞内维持生物功能的过程。作为现代生物化学和分子生物学中的一种主要技术手段,转染对于基因治疗有重要的意义。无机纳米粒子作为基因载体受到人们日益广泛的关注,其具有易于制备,可进行多样化的表面修饰等多种优势。本文将概述无机纳米粒子作为基因载体的现状及其对基因表达的影响。     

Electro‐mediated gene transfer and expression are controlled by the life‐time of DNA/membrane complex formation

Background

Electroporation is a physical method used to transfer molecules into cells and tissues. Clinical applications have been developed for antitumor drug delivery. Clinical trials of gene electrotransfer are under investigation. However, knowledge about how DNA enters cells is not complete. By contrast to small molecules that have direct access to the cytoplasm, DNA forms a long lived complex with the plasma membrane and is transferred into the cytoplasm with a considerable delay.

Methods

To increase our understanding of the key step of DNA/membrane complex formation, we investigated the dependence of DNA/membrane interaction and gene expression on electric pulse polarity and repetition frequency.

Results

We observed that both are affected by reversing the polarity and by increasing the repetition frequency of pulses. The results obtained in the present study reveal the existence of two classes of DNA/membrane interaction: (i) a metastable DNA/membrane complex from which DNA can leave and return to external medium and (ii) a stable DNA/membrane complex, where DNA cannot be removed, even by applying electric pulses of reversed polarity. Only DNA belonging to the second class leads to effective gene expression.

Conclusions

The life‐time of DNA/membrane complex formation is of the order of 1 s and has to be taken into account to improve protocols of electro‐mediated gene delivery. Copyright © 2009 John Wiley & Sons, Ltd.     

An efficient gene transfer method mediated by ultrasound and microbubbles into the kidney

BACKGROUND: Safety issues are of paramount importance in clinical human gene therapy. From this point of view, it would be better to develop a novel non-viral efficient gene transfer method. Recently, it was reported that ultrasound exposure could induce cell membrane permeabilization and enhance gene expression. METHODS: In this study, we examined the potential of ultrasound for gene transfer into the kidney. First, we transfected rat left kidney with luciferase plasmid mixed with microbubbles, Optison, to optimize the conditions (duration of ultrasound and concentration of Optison). Then, 4, 7, 14 and 21 days after gene transfer, luciferase activity was measured. Next, localization of gene expression was assessed by measuring luciferase activity and green fluorescent protein (GFP) expression. Expression of GFP plasmid was examined under a fluorescence microscope at 4 and 14 days after gene transfer. Finally, to examine the side effects of this gene transfer method, biochemical assays for aspartate aminotransferase (AST), alanine aminotransferase (ALT), blood urea nitrogen (BUN) and creatinine (Cre) were performed. RESULTS: Optison and/or ultrasound significantly enhanced the efficiency of gene transfer and expression in the kidney. Especially, 70-80% of total glomeruli could be transfected. Also, a significant dose-dependent effect of Optison was observed as assessed by luciferase assay (Optison 25%: 12.5 x 10(5) relative light units (RLU)/g tissue; 50%: 31.3 x 10(5) RLU/g tissue; 100%: 57.9 x 10(5) RLU/g tissue). GFP expression could be observed in glomeruli, tubules and interstitial area. Results of blood tests did not change significantly after gene transfer. CONCLUSIONS: Overall, an ultrasound-mediated gene transfer method with Optison enhanced the efficiency of gene transfer and expression in the rat kidney. This novel non-viral method may be useful for gene therapy for renal disease.     

Zinc improves gene transfer mediated by DNA/cationic polymer complexes

Background

The weak efficiency of plasmid transfer into the cytosol remains one of the major limiting factors to achieve an efficient transfection with DNA/cationic polymer complexes. We found that divalent metal Zn²⁺ can improve the polyfection efficiency, especially with DNA/histidylated polylysine (His‐pLK) complexes.

Methods and results

The supplementation of the transfection medium with 250 µM ZnCl₂ increased the polyfection of human hepatocarcinoma (HepG2) cells with a plasmid encoding EGFP complexed with pLK, polyethyleneimine and His‐pLK. Zn²⁺ is more efficient on DNA/His‐pLK complexes: the number of EGFP‐positive cells increased from 1% to more than 40%. This phenomenon is selective to Zn²⁺ because no effect was obtained with other divalent cations. The effect of zinc varies from cell to cell. The binding of Zn²⁺ to histidyl residues might increase zinc endosomal concentration favoring membrane fusion. Flow cytometry and confocal microscopy studies clearly indicate that with His‐pLK, the plasmid is better delivered in the cytosol as well as in the cell nucleus in zinc‐treated cells. An investigation conducted with the histidine‐rich peptide H5WYG showed that zinc inhibits membrane permeabilization but promotes membrane fusion as evidenced by resonance energy transfer.

Conclusions

Data reported here imply that the addition of zinc ions in the transfection medium can trigger an increase of the fusion of endosomes containing polyplexes which is more effective in the presence of histidine‐rich molecules. Consequently, the amount of plasmid in the cytosol available to reach the nucleus is increased leading to an improvement of polyfection. Copyright © 2002 John Wiley & Sons, Ltd.

     

纳米金探针在基因检测中的应用

传统的核酸分析中常采用放射性元素、荧光色素以及酶标记等基因探针,这些探针都存在着一些不足之处。近年来,纳米金探针作为一种新型的基因探针,己引起了广泛的关注。该探针具有优良的光谱特征和光化学稳定性,对核酸的非特异吸附性小,与核酸等生物大分子结合后不改变生物分子的活性。将纳米金探针用于基因检测,具有操作简便、快速、安全、实验成本低等优点。本文就纳米金探针的发展过程、纳米金探针的制备、检测原理及其在基因分析中的应用等几个方面作了系统而全面地概述,同时介绍了纳米金探针的最新研究进展,并对其发展前景作了简要评述。     

Human-derived NLS enhance the gene transfer efficiency of chitosan

Nuclear import is considered as one of the major limitations for non-viral gene delivery systems and the incorporation of nuclear localization signals (NLS) that mediate nuclear intake can be used as a strategy to enhance internalization of exogenous DNA.In this work, human-derived endogenous NLS peptides based on insulin growth factor binding proteins (IGFBP), namely IGFBP-3 and IGFBP-5, were tested for their ability to improve nuclear translocation of genetic material by non-viral vectors. Several strategies were tested to determine their effect on chitosan mediated transfection efficiency: co-administration with polyplexes, co-complexation at the time of polyplex formation, and covalent ligation to chitosan. Our results show that co-complexation and covalent ligation of the NLS peptide derived from IGFBP-3 to chitosan polyplexes yields a 2-fold increase in transfection efficiency, which was not observed for NLS peptide derived from IGFBP-5.These results indicate that the integration of IGFBP-NLS-3 peptides into polyplexes has potential as a strategy to enhance the efficiency of non-viral vectors.     

Chitosan-g-PEG/DNA complexes deliver gene to the rat liver via intrabiliary and intraportal infusions

BACKGROUND: Chitosan has been shown to be a non-toxic and efficient vector for in vitro gene transfection and in vivo gene delivery through pulmonary and oral administrations. Recently, we have shown that chitosan/DNA nanoparticles could mediate high levels of gene expression following intrabiliary infusion 1. In this study, we have examined the possibility of using polyethylene glycol (PEG)-grafted chitosan/DNA complexes to deliver genes to the liver through bile duct and portal vein infusions. METHODS: PEG (Mw: 5 kDa) was grafted onto chitosan (Mw: 47 kDa, deacetylation degree: 94%) with grafting degrees of 3.6% and 9.6% (molar percentage of chitosan monosaccharide units grafted with PEG). The stability of chitosan-g-PEG/DNA complexes was studied by measuring the change in particle size and by agarose gel electrophoresis against bile or serum challenge. The influence of PEG grafting on gene transfection efficiency was evaluated in HepG2 cells using luciferase reporter gene. Chitosan and chitosan-g-PEG/DNA complexes were delivered to the liver through bile duct and portal vein infusions with a syringe pump. Gene expression in the liver and the distribution of gene expression in other organs were evaluated. The acute liver toxicity of chitosan and chitosan-g-PEG/DNA complexes was examined by measuring serum alanine aminotranferase (ALT) and aspartate aminotransferase (AST) activities as a function of time. RESULTS: Both chitosan and chitosan-g-PEG displayed comparable gene transfection efficiency in HepG2 cells. After challenge with serum and bile, chitosan-g-PEG/DNA complexes, especially those prepared with chitosan-g-PEG (GD = 9.6%), did not form large aggregates like chitosan/DNA complexes but remained stable for up to 30 min. In addition, chitosan-g-PEG prevented the degradation of DNA in the presence of serum and bile. On day 3 after bile duct infusion, chitosan-g-PEG (GD = 9.6%)/DNA complexes mediated three times higher gene expression in the liver than chitosan/DNA complexes and yielded background levels of gene expression in other organs. On day 1 following portal vein infusion, gene expression level induced by chitosan/DNA complexes was hardly detectable but chitosan-g-PEG (GD = 9.6%) mediated significant transgene expression. Interestingly, transgene expression by chitosan-g-PEG/DNA complexes in other organs after portal vein infusion increased with increasing grafting degree of PEG. The ALT and AST assays indicated that grafting of PEG to chitosan reduced the acute liver toxicity towards the complexes. CONCLUSION: This study demonstrated the potential of chitosan-g-PEG as a safe and more stable gene carrier to the liver.     

超声破裂载基因微泡增强心肌细胞报告基因的转染与表达

目的:通过超声破裂载基因微泡介导报告基因心肌细胞转染,探讨其能否增强心肌细胞外源基因转染与表达.方法:以β-galactosidase质粒为报告基因,将其与自制氟碳气体微泡粘附,制备载基因微泡.利用诊断性超声破裂微泡进行体外心肌细胞基因转染;以磷酸钙共沉淀转染为阳性对照并将其以不同方式与超声破裂微泡技术联合应用,以期进一步增强基因转染效果.分别采用原位染色及酶学定量检测β-galactosidase表达水平,同时进行细胞活性检测.结果:超声破裂载基因氟碳气体微泡(PESDA)转染组心肌细胞β-galactosidase表达水平可达单纯质粒转染组60倍(P＜0.01).磷酸钙共沉淀转染3.67倍(P＜0.01)超声强度、微泡浓度对超声破裂介导基因转染效果有明显影响.超声破裂微泡技术与磷酸钙共沉淀联合应用可进一步提高报告基因的表达(P＜0.05),即使在磷酸钙转染后6 h,超声破裂微泡仍能明显增强报告的基因的表达(P＜0 05).结论:超声破裂微泡技术是一种高效基因转染方法,其不但能增加DNA转染,而且增强入胞后基因的表达.超声破裂微泡与其它基因转染技术联合应用能进一步增加基因转染效率.     

Surface modification of lipid nanoparticles for gene therapy

Gene therapies have the potential to target and effectively treat a variety of diseases including cancer as well as genetic, neurological, and autoimmune disorders. Although we have made significant advances in identifying non-viral strategies to deliver genetic cargo, certain limitations remain. In general, gene delivery is challenging for several reasons including the instabilities of nucleic acids to enzymatic and chemical degradation and the presence of restrictive biological barriers such as cell, endosomal and nuclear membranes. The emergence of lipid nanoparticles (LNPs) helped overcome many of these challenges. Despite its success, further optimization is required for LNPs to yield efficient gene delivery to extrahepatic tissues, as LNPs favor accumulation in the liver after systemic administration. In this mini-review, we provide an overview of current preclinical approaches in that LNP surface modification was leveraged for cell and tissue targeting by conjugating aptamers, antibodies, and peptides among others. In addition to their cell uptake and efficiency-enhancing effects, we outline the (dis-)advantages of the different targeting moieties and commonly used conjugation strategies.