【精品论文】The Adsorption and Release of DNAMesoporous Silica.doc

精品论文The Adsorption and Release of DNA by Mesoporous SilicaMaterials with Different Pore Diameters.SUN Yan1, YAN Tingsheng1, LIU Xianbin25(1. Department of Life Science and School of Life Science and Technology, Harbin Institute ofTechnology, Harbin 150001;2. The School of Chemistry and Environmental Engineering, Harbin University of Science andTechnology, Harbin 150040)Abstract: Safe and efficient vector systems played a crucial role in the transgenic technology.10Mesoporous silica (MS) materials, which can be modified with functional groups, have the potential of being used as good vectors. In this study, the pore diameters of synthetic materials were from 2.3 nm to5.0 nm and the samples have a good dispersion. The MS meterials with different pore diameters were successfully amino-modified and studies about the adsorption and release properties of DNA werecarried out. The sample N-MCM-41-14 which had the smallest pore 2.3 nm could only adsorb DNA15fragment shorter than 750 bp. The sample N-SBA-15 with 5.0 nm pore could adsorb all DNA fragment shorter than 5000 bp. The pore size of MS materials had important implications on the DNA adsorption rate and the maximum adsorption capacity. However, pore size of the materials had little effect on the release rate of DNA. In sum, MS materials with different pore diameters had different adsorption properties of DNA, and could be as prefect gene delivery systems.20Keywords: mesoporous silica material; pore size; adsorption; release0IntroductionAs the development of genetic engineering technology, gene therapy shows great application potential in the future field of medicine. With the establishment of human genome database, more25and more genetic medicines were designed in the laboratory and then came to clinical application.The key factors of gene therapy were finding suitable gene vectors and exploring effective means to transfer these genetic materials into cellular environments. Currently viral vectors are widely used due to their good transmembrane characteristic and relatively high efficiency of gene transfer. But the human immune system has the ability to deal with viral invasion: viral vectors are easily30cause immune response. In addition, virus replicates itself into even more genetic variants; people are also deeply concerns about the safety of applications of viral vectors 1. Great strides have been made in the research of non-viral vectors during last few years, such as cytofection, ationic polymer, chitosan and its derivatives, and mesoporous silica, etc. Studies have shown that non-viral vectors have no immunogenicity as well as they are convenient to use, meanwhile they35can be produced in large amounts. On the basis of the above benefits, non-viral vectors are receiving increased attention lately 2.Mesoporous silica (MS) materials typically possess large surface areas, and large internal pore volumes and have narrow pore size distributions. Since the synthesis of mesoporous M41S materials in 19923, their applications to life science technologies and many other subjects are40attracting tremendous attention. Moreover, the special structure of the mesoporous wall makes it possible to tailor and modify whit functional groups, thus made these MS potential carrier vehicles for delivery and protection of target molecules 4-6.In recent years，many focused on the utilization of silica as nonviral vectors of DNA for geneFoundations: National Science Foundation of China (Grants 30900336), Doctoral Foundation of Ministry of Education of China (No. 20092302120070), Foundation of State Key Laboratory of Urban water Resource and Environment (QAK 200809)Brief author introduction:SUN Yan, Associate Professor, Research Area: Non-viral Drug/Gene DeliverySystem.Correspondance author: LIU Xianbin, Professor, Research Area: Porous Material. E-mail: liu_xb- 15 -delivery applications, if so, silica particles are required to be modified with functional substituent45such as amino groups for binding of DNA, because the negative DNA is generally liberated from the silica surface for the reason of electrostatic repulsion between them. Various of surface modifiers are available, however, surface modifications with shorter chain were more efficiency inthe enhancement of DNA fixed 7, 8. Radu et al. 9 reported a novel gene transfection system usinga polyamidoamine dendrimer-capped MCM-41-type mesoporous silica nanosphere, which were50efficient in protecting DNA against enzymatic cleavage and showed remarkable transfection enhancement into cells. The adsorption and release of DNA was strongly subject to the ion concentration of the circumstances, normally the fixed DNA was easily to get free in chaotropic salt. Other researches demonstrated the release of DNA had a great deal to do with the solution pH, which made it possible to accomplish controlled release of DNA. Sufficient evidence to show that55the DNA adsorption behaviors were highly dependent on the pore sizes of mesoporous silica, one stable adsorption manner in mesoporous silica was probably inclusion of DNA into the nanosize pores 10. Fei Gao, et al. 11 reported monodispersed mesoporous silica nanoparticles with very large pores could enhance the adsorption and release of DNA. Nevertheless, seldom articles have demonstrated the degree of possible impact of DNA lengths on the adsorption and release, nor yet60reported the relationship between pore sizes of mesoporous silica and its ability to accommodate different DNA. To clear these laws is of great significance in selecting proper mesoporous silica as vector of unique genes.In this work, mesoporous silica materials with different pore diameters were synthesize using various surfactants which had different length of carbon skeleton. Then modify the surface of MS65materials with 3-(2-aminoethyl aminopropyl) trimethoxysilane (ATMS) for binding of DNA.Particularly, we have focused on the influence of pore sizes on the adsorption treatment; meanwhile, we also noticed that the length of nucleic acid played an important role in the adsorption process. A series of experimental tests about the adsorption and release of DNA had been carried out to explain their characteristics.701Materials and methods1.1Synthesis of MCM-41We use hydrothermal method to synthesis MCM-41. 2.18 g of N-cetyltrimethy- lammonium bromide (CTAB) and 0.4 g NaOH was first added to 90 ml of pure water, the mixture was stirredand heated until completely dissolved，after cooling to room temperature, 10.4 g of TEOS was75slowly added with the stirring continued. After 1.5 hours, while the mixture gave rise to white precipitates, it was transfered to super high pressure polytetrafluoroethylene reaction pot to crystallize for 24 hours. The solid product was filtered, washed with deionized water andanhydrous ethyl alcohol, and dried at 80. In order to remove the surfactant template, theas-synthesized solid was calcined at 550 usingelectric meffle furnace for 5 h (heating ramp 580/min).MCM-41 samples with different pore diameters were prepared using various surfactants which had different length of carbon skeleton, besides CTAB, we also used octadecyl trimethyl ammonium chloride and tetradecyl trimethyl ammonium bromide as surfactants. The molar ratio of the synthesis mixture was fixed to TEOS / CTAB / NaOH / H2O: 1 / 0.12 / 0.2 / 100.851.2Synthesis of SBA-15The synthesis mixture contain 1.2g of P123, 2.4g of CTAB, 30g of H2O, 8 ml of anhydrous9095100105110115120125ethyl alcohol and 1.06g of 37% HCl, stirred until completely dissolved，the mixture was transferedto round bottom flask with three necks, 40 bath for 30 minutes, then 4g of TEOS was slowly added, stirred for one hour then increase the temperature to 80,stirred for another 8 hours. The solid product was filtered, washed with deionized water and anhydrous ethyl alcohol, and dried at80. In order to remove the surfactant template, the similar calcine process as MCM-41 was carried out.1.3Amino-functionalization of MSThe post-synthesis grafting method was adopt to modify the surface of MS. The calcined MS powder was dried at 80 to remove of excess water from the atmosphere, then 1g of dry solid was added into 100ml of toluene under stirring, and then 200 l of APMS was mixed with themixture. The amino-functionalization process was carried out at 80 for 12 hours. After the silanization reaction, the sample was filtered, washed with toluene and anhydrous ethyl alcohol,and dried at 80 to get N-MS.1.4Synthesis of N-MS-DNA ConjugatesIn a typical synthesis, 0.5 g of MS and 50mg DNA (Herring Sperm or pMD-18T plasmid) were suspended in 50 ml of water inside a triangular flask. The mixture was stirred for 8 h at room temperature with the aim of achieving maximum loading in the pores of the MS materials. The loaded solid was washed twice with deionized water and isolated by centrifuging treatment at10,000 rpm for 10 minutes, and dried at 40 for 12 hours.1.5Adsorption of DNATo investigate adsorption kinetics of DNA, 30 mg of N-MS and 0.6 ml of DNA (Herring Sperm, 5.0mg/ml) were suspended in 2.4 ml of deionized water at pH 7.0 inside a small glass bottle. The mixture was stirred at room temperature in order to facilitate the combination of DNA and MS. 100l of this suspension taken at regular intervals was centrifuged at 1 0000 rpm for 10 minutes, the precipitate was then washed twice with deionized water and centrifuged. Detect DNA content of all supernatant and cleaning solvent by ultra microspectrophotometer NanoDrop 2000c to calculate the amount of adsorbed DNA.To investigate adsorption isotherm of DNA, 1 mg of N-MS was put into centrifuge tubes foreach, then 100 l of DNA with different concentrations from 500 g/ml to 4000 g/ml was added , the mixture was shaked for 8 hours at room temperature with the aim of achieving maximum loading in the pores of the MS scaffolding. Similar centrifuging and washing treatments were carried out and calculated the amount of adsorbed DNA finally.Groups of 20bp, 8000bp and 15000bp DNA ladder solution were used to investigation the relationship between DNA size and pore size. 0.5 mg of N-MS was suspended in 50 l solution inside a centrifuge tube. After an absolutely adsorption for 8 hours, the mixture was centrifuged at1 0000 rpm for 10 minutes. The supernatant was taken for agarose gel electrophoresis studies.1.6Stability studies of N-MS-DNA conjugatesThe prepared N-MS-DNA (pMD-18T) was resuspended in deionized water at neutral pH, and then a certain amount complexes (containing 20 g of DNA)were digested with DNase(2 units)at room temperature overnight. After washed and centrifuged, the digested complex was put into NaCl 3 M aqueous solution agitating for 2 hours to release the loaded DNA. The mixture were centrifuged at 10 000 rpm for 15 min, then the supernatant was detected by agarose gel130135140145150155electrophoresis. In comparison, products extracted from N-MS-DNA conjugate with 3 M NaCl and the hydrolyzed free pMD-18T was also detected by agarose gel electrophoresis.1.7Materials CharacterizationLiquid nitrogen adsorption isotherms were measured in a Micromeritics ASAP 2000Flowsorb apparatus. Surface area calculations were carried out using the BET method, whereas the pore size distribution was calculated according to the BJH algorithm.Nanoparticle size measurements were conducted using a Zetasizer Nano ZS (MalvernInstruments Ltd.). Dried materials were resuspended in absolute ethanol at a concentration of5µg/ml. The mean hydrodynamic diameter was determined by cumulative analysis. The zeta-potential was measured using Zetasizer Nano ZS,the samples were suspended in deionized water yielding a suspension of about 0.1 mg/ml and sonicated for 1 min in a FinnSonicm08 ultrasound bath.Samples for transmission electron microscopy (TEM) were ultrasonically dispersed in ethanol and transferred to carbon coated copper grids. Scanning electron microscopy (SEM) micrographs were collected in a JEOL 6300 microscope operating at 20 kV.X-ray measurements were performed on a Rigaku 200EG.B1.C diffractometer using Cu K radiation, scanning 1.5 °10 ° with a rate of 2°/min.IR spectra were recorded on a Perkin Eimer Paragon 1000 instrument between 400 and 4000 cm1, diluting the solids in KBr pellets.2Results and discussion2.1X-rays diffractionX-rays diffraction analysis was effective in detecting the channel structures and pores array of mesoporous silica materials, typical X-ray diffraction pattern of mesoporous molecular sieves contains a series of (100), (110),(200) and (210) characteristic peaks standing for the regular hexagonal array of uniform channels.The X-ray powder diffraction data for the synthesized samples are shown in Fig. 1(A). The 3peaks observed respectively on the MCM-41 samples in the 2 range of 1.9-4.6° can be indexed on a hexagonal lattice. The SBA-15 sample, likewise, showed a good 2D hexagonal long-range order, as evidenced by the narrow reflection. The location of the first X-ray diffraction lines (100) of the MCM-41 materials altered when ammonium surfactants (CnH2n+l (CH3)3N+) with different alkyl chain lengths (n = 14, 16 and 18) were used: the longer alkyl chain of the surfactant, the smaller 2 degree (100) emerged, which can be indexed to the pore size of the calcined products.AB160Fig. 1 X-ray diffraction of MS materialsA: MCM-41 synthesized with different surfactants; B: 1.3 Amino-functionalization of MCM-41165170175180Compared to the MS samples, N-MS expressed a lower degree of long-range order, as theX-ray diffraction pattern of N-MCM-41-16 exhibited only one broad low-angle reflection (Fig.1(B). On the other hand, this phenomenon reflected the success of the amino-functionalization, too.2.2FTIR spectroscopyThe surface functional groups of MS materials were detected by FTIR spectroscopy, Fig. 2 displayed the infrared spectrograms of SBA-15 in different states. The results indicated that, by comparison with synthesized SBA-15 samples, calcination treatment can remove ammonium surfactants effectively, on account of the diminished or lost Infra-red adsorption bands around1400-1,500cm-1 and 3000cm-