抗DNA抗体阳离子质脂体复合物用于质粒DNA的定位递送

Anti-DNA Antibody/Cationic Lipid Micelles for Plasmid DNA Gene Delivery1Song Cunxian 1, Wang Manyan1, Jin Xu1, Leng Xigang1, Tang Lina1, Sun Hongfan1, and RJ Levy2 (1 Institute of Biomedical Engineering, Peking Union Medical College, Chinese Academy of Medical Sciences, Tianjin, P. R. China 300192;2 The Division of Cardiology， Children’s Hospital of Philadelphia，PA 19104，USA)AbstractWe reported for the first time a new type of plasmid DNA gene delivery systemcharacterized by DNA-(anti-DNA) Antibody-Cationic lipid (DAC) micelles andpermitted the highly efficient entrapment and delivery of plasmid DNA in vitro. Themicelles were characterized with respect to size, charge, and DNA entrapment. DACtriplex system formed a stable suspension of nanospheres, with a mean particlediameter of around 360nm, and a zeta potential of –15mV. Fluorescent DNA studiesdemonstrated greater cell uptake in vitro with DAC compared to the various controlformulations. Confocal microscopy studies confirmed nuclear entry in A10 cells withDAC, while formulations without the antibody demonstrated no nuclear entry. Cellculture studies revealed that DAC gave 4-fold greater level of transfection than theformulation without the antibody. It is concluded that anti-DNA antibodies canmarkedly enhance plasmid DNA gene transfer in A10 cells in culture. This effectappears to be strongly associated with anti-DNA antibody nuclear entry properties.The DAC micelles reported in this study represent one of the most potent plasmidDNA vector delivery systems reported thus far, with efficiencies significantly greaterthan conventional approaches, such as Lipofectin.Keywords: M icelle, DNA-anti-DNA, Nuclear Entry, Gene Delivery1 IntroductionA major problem for current gene therapy strategies is an inability to achieve a high level of expression of the gene product in vivo. Since plasmid DNA vectors have major safety advantages compared to viral vectors, there have been numerous attempts to improve plasmid DNA transfection, and the most promising of these approaches involves using cationic liposome [1,2,3]. In the present studies we investigated anti-DNA antibody-cationic lipid complex as a gene delivery system. It was hypothesized that anti-DNA antibody micelles could enhance DNA delivery through mechanisms involving: Increased levels of DNA incorporation into micelles due to antibody binding, enhanced cell entry via cell membrane-micelle interactions, entry into the cytoplasm of intact DNA-antibody-cationic lipid micelles, associated resistance to nuclease attack, and increased nuclear entry of DNA. Special interest1 Support by the China Specialized Research Fund for the Doctoral Program of Higher Educationunder Grant No. 20030023004, the Key Project Foundation of Tianjin City under Grant No.043803011and the National Natural Science Foundation of China under Grant No. 50473059of this study is the potential possibility of binding a plasmid DNA on such carries as nanoparticle or coronary stent through the antibody bridge for introvascular site-specific gene delivery.2 Materials and Methods2.1 M a t e r ia lsA Mouse anti-Bovine DNA IgM recognizing double- and single- stranded DNA was obtained from US Biological (Swampscott, MA). A β-gal DNA was labeled with Rhodamine and Lipofectin was labeled with fluorescent (BioDIPY) by using the LablelIT™labeling Kits (Mirus-Pan Vera, Madison, WI). Lipofectin, DOPE and DOTMA were purchased from SIGMA Chemical.2.2 Micelle fo rmula t io nFor preparing DAC complex, GFP-DNA in phosphate buffer (PBS) was mixed with the anti DNA IgM and incubated at 37 °C for 1 hour followed by the addition of 5 µl of Lipofectin. Control formulations were prepared in parallel.2.3 M ic e lle c h a r a c t e r iza t io nDAC micelles were assayed with a ZetaPlus Particle Sizer (Brookhaven Instruments Corp., NY). The concentration of DNA entrapped in micelles was determined by spectroscopy after phenol/chloroform extraction of particles separable from suspension by 0.2µm membrane filtration.2.4 Cellula r upta k eA Rhodamine-labeled DNA was used with fluorescent (BioDIPY) Lipofectin to assess the cellular internalization and localization of the DAC complex by confocal microscopy.2.5 In vitro c e ll t r a n sf e c t io nSmooth muscle cells (A10 cells) were incubated at 37°C for 18 hours prior introducing DNA to cells. The DNA complex in serum-free medium was added to replace the medium in cell culture plate. After 24 hours the cell culture medium was replaced with fresh medium. Following 24-48 hours more incubation, the cells were fixed and cover slide mounted using Vectashield Mounting medium with DAPI. The cell was observed under fluorescent microscope using FITC filter for GFP transfection and DAPI filter for total cell numbers. The percent of cells transfected was calculated by flow cytometry and by cell counting.3 Results and Discussion3.1 F o r m u l a t i o n a n d c h a r a c t e r i za t i o n o f D A C m i c e l l e sDAC formed stable micelles by a self-assembly process, when formulated by first combining plasmid DNA and anti-DNA antibody, followed by vortexing with Lipofectin. It is of interest to note that when Lipofectin and plasmid DNA were combined first, and then mixed with the antibody, immune precipitates formed. DAC micelles had an initial mean particle diameter of around 360nm and zeta potential of around -15mV. By comparison, formulation in the same ratio of plasmid DNA/Lipofectin but without anti-DNA (DC formulation), also formed detectable particles with a diameter of 190nm, and had a less electronegative zeta potential (-8.89mV). Despite the addition of cationic lipid to the DAC formulation, there was only a slight reduction in the electro-negative charge (Table 1).Formulation of anti-DNA and Lipofectin without DNA had a markedly large size (>1000nm). It was hypothesized that congeries were formed here for some unknown sake. DAC micelles were investigated for their stability in terms of size and charge, with timed incubations under simulated physiologic conditions (pH 7.4, PBS solution, 37°C). The results revealed that DAC complex had little change in size and zeta potential over the one-week observation period (Figure 1). Using Rhodamine-labeled DNA we observed relatively uniform DNA nanospheres in the DAC preparations (Figure 2).Table 1: Particle size and Zeta potential of various micelle formulations FORMULATION PARTICLE SIZE (nm±se) ZETA POTENTIAL (mV±se) DNA-antiDNA - Lipofectin 360.0 ± 3.1 -15.27 ± 4.45DNA- antiDNA Not stable -17.46 ± 1.42DNA- Lipofectin 191.7.0 ± 2.6 -8.89 ± 5.53AntiDNA-Lipofectin 1363.8 ± 178.2 -6.71 ± 1.86Figure 1: Change of particle size and charge of DAC micelles shake in PBS at 37°CFigure 2: Fluorescence micrograph of the Rhodamine-DACExtraction of DNA from DAC micelles accounted for 28.5±1.5% of the total initial DNA entrapped in the micelles, in comparison with the much lower retaining of DNA in DC formulation (13.6±0.8%) (Figure 3).Figure 3: DNA entrapment in DAC and DC micelles3.2 Cellular uptake of the micellesThe uptake of DAC compared to control formulation (DC) was studied using Rhodamine labeled DNA in a series of A10 cell culture experiments. DAC uptake was observed to be extensive after only 8 hours, with a very prominent cytoplasm-oriented presence at 24 hours. Apparent nuclear entry of DAC was observed with 48 hours cultures. The control experiment revealed qualitatively less amounts of cellular uptake and nuclear entry.In confocal microscopy studies, it was demonstrated that DAC entered cells as an intact antibody-DNA-lipid complex with persistent association of cationic lipid, even in the nuclei of the A10 cells (Figure 4A). By comparison, DNA-cationic lipid without anti-DNA antibody demonstrated little cationic lipid entry in association with fluorescent DNA, and neither fluorescent DNA nor fluorescent lipid were observed in the nuclei (Figure 4B). Thus, DAC is structurally stable under cell culture conditions, and furthermore, our results show that the entire complex can remain intact throughout cellular processing including nuclear entry, indicating that the anti-DNA antibody investigated in our studies has nuclear entry capability.Figure 4: Confocal microscopy studies of A10 cell uptake of (A) DAC consisted of Rhodamine-labeled DNA (red) and BioDIPY labeled cationic lipid (green) and anti-DNA-antibody, colocalized as indicated by yellow in the cytoplasm and nucleus of A10 cells. (B) DC lipoplexes illustrating no nuclear entry3.3 In Vitro A10c e lls t r a n s f e c t io nDAC triplex micelles markedly increased the level of gene transferred to A10 cells, with more than a 4-fold increase in transfection compared to the DC formulation (Figure5A-5B). The DA formulations (GFP DNA with antibody) resulted in no transfection. Flow cytometry studies demonstrated the magnitude of the differences between DAC and other transfection formulations (Figure 5C). It showed that anti-DNA-antibody greatly enhanced DNA transfection compared with same amount of Lipofectinwithout anti-DNA-antibody. Coomassie blue staining of A10 cell cultures following DAC mediated transfection revealed a normal cellular morphology, with no apparent toxicity due to DAC.Since DOTMA is the cationic lipid and DOPE is the neutral lipid in Lipofectin formulation, the question is which one is the contributing ingredient in the DAC system? The two chemicals were further tested separately in A10 cell transfection. The results revealed that DOTMA alone induced almost the same transfection efficiency with Lipofectin. On the other hand, DOPE alone did not show any positive effect (Figure 6). The results suggest that the positive charge of cationic lipid in the triplex micelles played an important role in harmonizing with the anti-DNA antibody.Figure5: Transfection of A10 cells in vitro with GFP-DNA (green fluorescent) heteroplexes formulated with the same amount of DNA. (A) GFP-DAC showed intense fluorescent. (B) GFP-DC without anti-DNA-antibody. (C) Flow cytometry studies demonstrated A10 cells transfection using GFP-DAC was significantly efficient than other control formulationsFigure 6: Results of A10 cell transfection by GFP-DNA-Anti-DNA-antibody with different lipid formulations: (A) DOTMA/DOPE: 2/1, 5µl. (B) DOTMA/DOPE=1:1, 5µl. (C) DOTMA/DOPE=1/2, 5µl. (D) DOPE, 5µl. (E) DOTMA 5µl. (F) DOTMA 10µl. (G) DOTMA 20µl4 ImplicationsThis is the first time to involve antibody in gene vector formulation. Although there were many issues waiting for further exploring, preliminary results from this study revealed greatly potential possibility of using anti-DNA antibody to facilitate high-level gene transfer and expression when harmonized with cationic lipid. With purification of the DAC micelle constructs, and elimination of free antibody, the risk for anti-DNA antibody adverse effects should be hypothetically low. Further investigations with this gene delivery system will need to be mindful of the potential for anti-DNA antibody related diseaseactivity. Furthermore, DNA-antibody can be expediently coupled onto the surface of such medical devices as coronary stent, therefore to deliver therapeutic polynucleotides to an intravascular site achieving efficient gene therapy.A c k n o w l e d g m e n t sThese studies were financially supported by projects from Tianjin Committee of Sciences and Technology and the NSFC. Dr. Levy in Philadelphia, USA is the supervisor and co inventor. 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