Research Article www.acsami.org
Efficient Transfection by Using PDMAEMA-Modified SiNWAs as a Platform for Ca2+-Dependent Gene Delivery Jingjing Pan, Yuqi Yuan, Hongwei Wang,* Feng Liu, Xinhong Xiong, Hong Chen, and Lin Yuan* College of Chemistry, Chemical Engineering, and Materials Science, Soochow University, Suzhou 215123, People’s Republic of China S Supporting Information *
ABSTRACT: The major bottleneck for gene delivery lies in the lack of safe and efficient gene vectors and delivery systems. In order to develop a much safer and efficient transfection system, a novel strategy of combining traditional Ca2+dependent transfection with cationic polymer poly(N,Ndimethylamino)ethyl methacrylate (PDMAEMA) modified silicon nanowire arrays (SiNWAs) was proposed in this work. Detailed studies were carried out on the effects of the PDMAEMA polymerization time, the Ca2+ concentration, and the incubation time of Ca2+@DNA complex with PDMAEMAmodified SiNWAs (SN-PDM) on the gene transfection in the cells. The results demonstrated that the transfection efficiency of SN-PDM assisted traditional Ca2+-dependent transfection was significantly enhanced compared to those without any surface assistance, and SN-PDM with polymerization time 24 h exhibited the highest efficiency. Moreover, the optimal transfection efficiency was found at the system of a complex containing Ca2+ (100 mM) and plasmid DNA (pDNA) incubated on SN-PDM for 20 min. Compared with unmodified SiNWAs, SN-PDM has little cytotoxicity and can improve cell attachment. All of these results demonstrated that SN-PDM could significantly enhance Ca2+-dependent transfection; this process depends on the amino groups’ density of PDMAEMA on the surface, the Ca2+ concentration, and the available Ca2+@DNA complex. Our study provides a potential novel and excellent means of gene delivery for therapeutic applications. KEYWORDS: DNA, gene delivery, transfection efficiency, Ca2+-dependent transfection, PDMAEMA-modified SiNWAs, biocompatibility teeth.15,16 Thus, no traces of synthetic chemical components remain within the cells or the medium after the gene transfection. Apart from its biocompatibility, the superiority of this method also lies in the fact that calcium ions could help to achieve excellent gene transfection in many ways. For instance, calcium ions could be functional in successful delivery of DNA directly to the nucleus of a cell as a result of Coulombic interactions.17 It could enhance the nuclear uptake of DNA through the nuclear pore complex due to the involvement of calcium in intracellular signaling pathways.18 Furthermore, the addition of Ca2+ could cause the cytosolic calcium oscillations and thus strongly promote the efficiency of gene transfection and expression.19 Based on the above advantages, the Ca2+-dependent transfection techniques have been applied widely in many fields, especially in biomedical research, biotechnological applications, and gene therapy.20−22 However, this method is hampered by its insufficient efficiency and poor reproducibility compared with other nonviral carriers.21,23−25 One of the primary reasons is the partial therapeutical protection of the plasmid DNA by the encapsulating material as well as the limited accumulation of
1. INTRODUCTION Introducing exogenous DNA into target cells is one of the most important molecular techniques to manipulate cells, identify the function of specific genes, and analyze protein expression in vitro. Progress in the development of gene delivery has great therapeutic potential in the prevention and treatment of a variety of deadly diseases.1 Unfortunately, the nucleic acid itself cannot pass through a cell membrane barrier.2,3 Therefore, specific carriers and protectors are needed for the safe and efficient delivery of DNA inside the cell. Among various gene transfer techniques, the transfection dependent on calcium ions (Ca2+) and related substances remains a commonly well used nonviral vector in a wide variety of mammalian cells and is used as a routine laboratory procedure.4−6 This method relies heavily on the fact that Ca2+, as the divalent metal cation, can form ionic complexes with the helical phosphates of DNA (Ca2+@DNA).7 This method is simple, cost-effective, and generally suitable for a wide range of cell lines.8 What makes it even more fascinating is that it offers an advantage of low cell toxicity for its excellent biocompatibility and admirable biodegradability.9−12 Calcium is the fifth most abundant element in the human body and Earth’s crust, omnipresent in the living organism and culture medium. Ionic calcium plays a vital role in many cellular and biological processes13,14 and is also a substantial component of bones and © 2016 American Chemical Society
Received: April 19, 2016 Accepted: June 1, 2016 Published: June 1, 2016 15138
DOI: 10.1021/acsami.6b04689 ACS Appl. Mater. Interfaces 2016, 8, 15138−15144
Research Article
ACS Applied Materials & Interfaces the plasmid on a cell surface.23 Such partial protection makes the DNA highly susceptible to aggressive DNase attack and large amounts of DNA degradation in the intracellular acidic environment.26,27 In addition, the poor chemical stability of the unprotected Ca2+@DNA particles under physiological conditions21,28,29 and the very small proportion of DNA entering the nucleus after being delivered into cells30 are also responsible for the low transfection efficiency. These problems existed in Ca2+-dependent transfection has significantly limited its applications. Recently, great efforts have been devoted to obtain higher transfection efficiency based on Ca2+-dependent transfection technique. First of all, the formation of appropriate size of Ca2+@DNA particles is one of the critical factors for high transfection efficiency.31 Ca2+@DNA particles are usually carried across the cell membrane via endocytosis,10,32 in which, the size range of the particle is approximately 20−200 nm in diameter.33,34 The particle size in nanoscale would also help the protection of DNA from degrading enzymes.24 There have been many attempts in various laboratories to prepare particles on the nanoscale size smaller than 200 nm and have been proved to be efficient for better transfection.24,25,29,32−37 However, Ca2+@DNA nanoparticles prepared by these schemes are usually relatively large, and the actual size cannot be easily and truly controlled due to significant agglomeration.25,38 An alternative approach is combining positive effects of calcium with synthetic transfection reagents, especially cationic polymers and liposomes. This cooperation has been shown to stimulate the stability of the transfection complexes and the release of DNA into the cytoplasm.39,40 However, this strategy still cannot escape from the dependences of cellular uptake on the endocytosis of the transfection complexes and could not promise the maximal possibility of DNA to be transported into the nucleus. Therefore, apart from paying more attention to well-marked reduced toxicity, improved transfection efficiency, better stability of the complexes in vivo, there exists much space for developing novel and ideal gene transfection systems which exhibit excellent ability to pass through the barriers to cells or nuclei more easily and effectively.41,42 Recently, nanostructured silicon substrates have been widely used as biomaterials with many potential advantages for medical monitoring and biological research, including biocompatibility, being easily modified, and special topographic structure for cell attachment and spreading.43,44 Several studies have found that silicon nanowire arrays (SiNWAs) can be utilized as a new nonviral physical gene transfer substrate which could penetrate cell membranes and even nuclear membranes45,46 while maintaining the normal function of the cells.47 However, the transfection efficiency of SiNWAs alone is below 1%, which may mainly, due to the oxide layer on the surface of the unmodified SiNWAs, severely restrict the gene loading capacity.46,47 We have proposed to modify SiNWAs with PEI and found that it could increase the transfection efficiency.48 However, there exist several problems to be solved. First, PEI, especially high-molecular-weight PEI (HMW PEI; 25 kDa), may still have potential disadvantages and be toxic for therapeutic applications.49,50 Second, the capability and amount of DNA encapsulated by HMW PEI is still not high enough, and additional low-molecular-weight PEI is needed to increase the effective DNA amounts. In order to resolve the problems existing in the above two transfection systems and achieve high transfection efficiency, in
the present work, we proposed an integrated strategy combining Ca2+-dependent transfection and cationic polymers modified SiNWAs transfection platforms for best use of the advantages and for bypassing the disadvantages (Scheme 1). Scheme 1. Formation of the SN-PDM Assisted Ca2+Dependent Transfection System (SN-PDM with Ca2+@ DNA)
This strategy is also in keeping with the main stream in novel gene delivery.1,51 To avoid the cytotoxicity of PEI, poly(N,Ndimethylamino)ethyl methacrylate (PDMAEMA), which is a cationic polymer with better biocampability and has been successfully used in gene delivery,52,53 was modified on SiNWAs via surface-initiated ATRP.54 We studied the effect of the PDMAEMA polymerization time, the Ca2+ concentration, and the incubation time of the Ca2+@DNA complex with PDMAEMA-modified SiNWAs (SN-PDM) on the gene transfection of pRL-CMV plasmid into HeLa cells and found that the transfection efficiency of the SN-PDM assisted Ca2+dependent transfection system was significantly enhanced by about 590% compared to the traditional Ca2+-dependent transfection system. In this SN-PDM assisted Ca2+-dependent transfection system, SiNWAs can penetrate cell membranes, which ensures direct access to the cells’ interiors and enables more DNA to enter into the nucleus, to increase the gene transfection efficiency to a large extent. Ca2+ can promote the stability of the transfection complex outside the cell and the intercellular release of pDNA. Our study attempts to inspire new strategies and provides a novel and excellent means of gene delivery for a variety of therapeutic applications.
2. MATERIALS AND METHODS Materials. Silicon wafers [n-doped, (100)-orientation, 0.56 mm thickness, and a diameter of 100 mm (Guangzhou Semiconductor Materials, Guangzhou, China)] were cut into square chips of approximately 0.5 cm × 0.5 cm. 3-Aminopropyltriethoxysilane (APTES), 2-bromoisobutyryl bromide (BIBB), 2,2′-bipyridine (Bpy), and (N,N-dimethylamino)ethyl methacrylate (DMAEMA) were obtained from Aldrich. CH3OH and all the other chemical and biological reagents were purchased from the Sinopharm Chemical Reagent Co., Ltd. (Beijing, China) and purified before use. Preparation of PDMAEMA-Modified SiNWAs (SN-PDM). SiNWAs used in this study were prepared by chemical etching of silicon wafer as described before.48 Briefly, the silicon wafers were etched in the solution containing HF (5 M) and AgNO3 (15 mM) at 50 °C for 10 min followed by being immersed in 20% HNO3 for 1 min and then washed by deionized water. The silicon nanowires on the surface of SiNWAs have a width of substantially 52 nm and 10 μm in length. The modification of PDMAEMA was achieved through surfaceinitiated atom transfer radical polymerization (SI-ATRP).54 The general process for SN-PDM was carried out according to the steps described in our previous work55 and illustrated in Supporting Information Figure S1a. Surface Characterization. Transmission electron microscopy (TEM; G-200, Hitachi, Tokyo, Japan) was used to detect the surface properties of SN-PDM. It was performed at 200 kV. In order to study the thickness of the polymer layer on SiNWAs, single silicon 15139
DOI: 10.1021/acsami.6b04689 ACS Appl. Mater. Interfaces 2016, 8, 15138−15144
Research Article
ACS Applied Materials & Interfaces nanowires were obtained by proper sonication of SiNWAs modified with PDMAEMA. After sonication, nanowires could be broken off from the arrays and be suspended or dispersed evenly in an aqueous solution for better displaying the TEM images of the polymer layer on the material surface (Figure S5a). Surface wettability of the hydroxylated SiNWAs and SN-PDM was evaluated with an SL200C optical contact angle meter (USA Kino Industry Co., Ltd.) at room temperature. The sessile drop method was applied to measure their static water contact angles. Cell Culture. HeLa and L929 cells were cultured in DMEM medium (Hyclone) and RPMI-1640 medium (Hyclone), respectively. All media were supplemented with 10% fetal bovine serum (FBS, Gibco), 100 U/mL penicillin (Genview), and 100 μg/mL streptomycin (Solarbio). The cells were incubated at 37 °C with 5% CO2 in the humidified chamber. In Vitro Gene Transfection and Luciferase Assay. Transfection studies were performed in HeLa cells using the plasmid pRL-CMV as a reporter. In brief, Ca2+@DNA nanoparticles (containing 1.5 μg of pDNA) were prepared by the dropwise addition of Ca2+ solution into the DNA solution, followed by thorough mixing and incubation for 10 min at room temperature. The size and surface ζ potential of Ca2+@ DNA nanoparticles were examined by dynamic light scattering (DLS) using a Zetasizer Nano-ZS90 ζ potential analyzer (Malvern Instruments, Malvern, U.K.). When the concentration of Ca2+ was 100 mM, the diameter of Ca2+@DNA nanoparticles was about 70 nm and the surface ζ potential of Ca2+@DNA nanoparticles was negative (Figure S6). Sterile SiNWAs and SN-PDM were placed into a 48 well plate, and then Ca2+@DNA complexes were added into each well and further incubated for a period (Scheme 1). At the time of transfection, HeLa cells (5 × 104 cells/well) were seeded in each well and further incubated in media containing 10% FBS for 24 h at 37 °C with 5% CO2 in the humidified chamber. The luciferase assay and protein concentration measurement were conducted according to our recent work.48 The transfection efficiency was calculated as relative light units per milligram of cell protein lysate (RLU/(mg of protein)). Cell Viability Assay. For the evaluation of cell viability, L929 cells at a density of 1 × 104 cells/well in culture media were seeded and cultured on various samples placed for 24−72 h in a 48 well plate (Nunc). After the cells attached to the sample surfaces, the surfaces were washed twice with sterilized phosphate buffered saline (PBS, pH 7.4) and stained with diamidino-2-phenylindole (DAPI, Invitrogen). Cells were observed and imaged using an Olympus IX71 fluorescence microscope. Observation of Cell Morphology. The cell morphology was assessed by scanning electron microscope (SEM). SN-PDM with L929 cells was washed twice with sterilized PBS (pH 7.4), and then fixed in 4% glutaraldehyde solution (in PBS) for 30 min at room temperature. The fixed samples were then dehydrated by ethanol solution with deionized water (10, 30, 50, 70, 80, 90, 100% (v/v)) for 10 min each step. After drying at room temperature, all samples were coated with a thin layer of gold and imaged using SEM (Leica S440i, Cambridge, U.K.).
Figure 1. Surface properties of SN-PDM: (a) Water contact angles of the unmodified and modified SiNWAs surfaces. (SN-PDM 1−4 represent PDMAEMA-modified SiNWAs with a polymerization time of 12, 18, 24, and 36 h, respectively); (b) TEM image of a single silicon nanowire (about 52 nm in width and 10 μm in length) modified with PDMAEMA for 24 h; (c) TEM image of a single nanowire modified with PDMAEMA at high magnification, with the arrow showing a layer of PDMAEMA.
oxidation of silicon on the surface; after being modified with PDMAEMA, the material showed high hydrophobicity significantly with the water contact angle at about 108° when the polymerization time was 12 h (for SN-PDM 1). Further extending the polymerization time from 18 to 36 h (for SNPDM 2, SN-PDM 3, and SN-PDM 4) could not affect the hydrophobicity of the surface. This might be due to the significant and large change in surface wettability by grafting of polyelectrolyte on the surface of SiNWAs. At this point, even if there are differences in the polymer layer thickness in the four different SN-PDM surfaces with extending polymerization time, the values of the water contact angle may not exhibit significant changes. The results in Figure 1a mainly illustrated the successful grafting of polymer on the material surface. Even though there is not much difference in the hydrophobicity of the four SN-PDM surfaces, the values of water contact angle were all significantly higher than that of unmodified SiNWAs. However, the amino group density of SN-PDM 2, SN-PDM 3, and SN-PDM 4 increased after long-time polymerization compared with SN-PDM 1 (Figure S2). The XPS data affirmed the amino group density results and showed that the N element on the surface increased from 3.89% in SN-PDM 1 to 4.22% SN-PDM 2. Since the amino groups can carry positive charges under physiological environment, they have the property to bind negatively charged groups, such as the phosphate groups of DNA. In order to know the surface charge of SN-PDM, fluorescein sodium salt, a negatively charged dye, was applied on the materials. And the results showed that SN-PDM 3 had the most positive charges (Figure S3). These results above suggest that the biocompatible cationic polymer, PDMAEMA, had been successfully grafted to the surface of SiNWAs. Furthermore, the presence of PDMAEMA as a layer surrounding the single silicon nanowire could also be directly proved by TEM observation (Figure 1b,c), which significantly improved the surface properties to bind with DNA and cells. Gene Transfection of the SN-PDM Assisted Ca2+Dependent Transfection System. On the basis of the improvement in material surface, the gene transfection efficiency in the immobilized HeLa cells was investigated on SN-PDM using pRL-CMV encoding Renilla luciferase as a reporter gene in this work. In order to develop a possibly safer and more effective strategy based on the cooperative effect of nanostructure and Ca2+-dependent transfection, we first examined the effect of unmodified SiNWAs on the traditional Ca2+-dependent transfection; it could be seen that after introducing SiNWAs, the transfection efficiency could be increased by about 20% compared to the system without the help of SiNWAs. The results demonstrated that SiNWAs can
3. RESULTS AND DISCUSSION Surface Property of SN-PDM. The surface property of the material could determine the interaction of SN-PDM with DNA and cells. Since the modification of PDMAEMA was achieved through SI-ATRP grafting of PDMAEMA,54 the surface wettability, chemical composition, and physical structure would have been changed compared to unmodified SiNWAs. Physicochemical surface properties of SN-PDM were investigated by contact angle measurements, surface charge measurement, X-ray photoelectron spectroscopy (XPS), amino group density measurements, and TEM. Generally, compared to the superhydrophilic surface, cells prefer to attach to the hydrophobic surface.43,55 As shown in Figure 1a, SiNWAs before modification showed surperhydrophilicity with a water contact angle of about 1° due to the 15140
DOI: 10.1021/acsami.6b04689 ACS Appl. Mater. Interfaces 2016, 8, 15138−15144
Research Article
ACS Applied Materials & Interfaces
possible that excess of PDMAEMA might induce some cytotoxic effects and the cytotoxicity of SN-PDM may also increase with the polymerization time, leading to weakened efficiency. As shown in Figure 2b, the cooperation of Ca2+ and SN-PDM 3 could achieve a desired transfection efficiency as high as approximately 6.2 × 106 RLU/(mg of protein), six times greater than that of the traditional method only dependent on Ca2+. Therefore, it could be concluded that the property of PDMAEMA conjugated on the SiNWAs was a key factor in achieving high gene transfection efficiency. Factors Influencing the SN-PDM Assisted Ca2+Dependent Transfection System. The mechanism of the SN-PDM assisted Ca2+-dependent system promoting transfection efficiency might be not only because of the nanowires of SiNWAs penetrating into cells and cationic groups of PDMAEMA adsorbing DNA but also due to the package of DNA with Ca2+ and the compact of Ca2+@DNA on the SNPDM surface. Therefore, the concentration of calcium ions, the incubation time of Ca2+@DNA on SN-PDM, and even the loading amount of DNA (Figure S7) would influence the positive charge of the transfection system, the binding and compacting of Ca2+@DNA complex with PDMAEMA, and the stability of compacted nanoparticles outside and inside cells. The effects of the above factors on the transfection efficiency were studied on SN-PDM 3. The results showed that the transfection efficiency increased with increasing Ca2+ concentration at the range of 10−100 mM and reached its highest efficiency at 100 mM (Figure 3a). At 10−100 mM Ca2+, the
be utilized as a pretty potential substrate for gene transfer owing to its merits of penetrating cell membranes and even nuclear membranes while maintaining the normal function of the cells.45−47 However, in our previous study, we found that the transfection efficiency of SiNWAs alone is very low, and PEI modification could help the SiNWAs to achieve a much better performance in gene transfection.48 Herein we applied this cationic polymer modified SiNWAs into the traditional Ca2+dependent transfection system in order to explore a novel transfection method which is safer and more effective. In consideration of the potential cytotoxicity of PEI, PDMAEMA, a more biocompatible cationic polymer, was utilized to take the place of PEI. It was found that SN-PDM exhibits better transfection efficiency than PEI-modified SiNWAs (SN-PEI) without calcium addition (Figure S4), which might be due to the higher cell viability. After incubation of Ca2+@DNA complex with SN-PDM, it could be seen from Figure 2a that
Figure 2. Effect of SN-PDM on gene transfection: (a) In vitro transfection efficiency of the traditional way of using Ca2+ without and with the surface assistance of SN-PDM [(Control) transfection using SN-PDM alone (without Ca2+); (A) traditional transfection using Ca2+ without surface assistance of SN-PDM; (B) transfection using Ca2+ with surface assistance of SN-PDM]; (b) transfection efficiency after adsorbing Ca2+@DNA on SN-PDM with different PDMAEMA polymerization time (SN-PDM 1−4 represent PDMAEMA-modified SiNWAs with a polymerization time of 12, 18, 24, and 36 h, respectively). Data are presented as the mean ± SE (n = 3).
the transfection efficiency is greatly increased compared to that of traditional transfection using Ca2+ without the surface assistance of SN-PDM. The XPS results also showed that, after incubation of Ca2+@DNA complex with SN-PDM, the calcium and phosphorus elements were 0.18% and 0.26% on the surface, indicating that surface modification of SiNWAs with PDMAEMA can effectively increase the loading amount of Ca2+@DNA. A detailed study of the transfection efficiencies is presented for four kinds of SN-PDM with different polymerization time (12, 18, 24, or 36 h) in Figure 2b. The results showed that when the polymerization time was prolonged from 0 to 24 h, the transfection efficiency increased with the increase of the polymerization time and reached the maximum at a grafting time of 24 h; the transfection efficiency began to decline when the polymerization time exceeded 24 h but was still higher than that of the unmodified SiNWAs. The reason for the low transfection efficiency of SN-PDM 1 (polymerization time, 12 h) might be that the amount and density of PDMAEMA conjugated is relatively inadequate, or less than fully effective to provide enough positive charge on the surface and thus the sufficient loading of pDNA (Figures S2 and S3). However, too long a polymerization time (36 h) led to weakened transfection efficiency, caused by the thick and overcovered layer consisting of long polymer chains, which hindered SiNWAs to exert its full potential as a nanostructure and thus attenuate the pDNA loading capacity. Meanwhile, it is
Figure 3. Effect of Ca2+ concentration and incubation time on gene transfection efficiency: (a) Transfection efficiency after absorbing the mixture of Ca2+ of different concentrations and pDNA on SN-PDM; (b) transfection efficiency after absorbing the mixture of Ca2+ (100 mM) and pDNA on SN-PDM for different incubation times. Data are presented as the mean ± SE (n = 3).
sizes of the Ca2+@DNA nanoparticles were all about 100 nm in diameter, and at 100 mM Ca2+ the particle size was much smaller (around 72 nm) (Figures S5g and S6). Meanwhile, the formed nanoparticles show negative surface potential (Figure S6). Due to the negative charges carried by nanoparticles, Ca2+@DNA nanoparticles can attach to the positively charged SN-PDM surface by electrostatic interaction (Figure S5g). Results in Figure 3a indicated that, at 100 mM Ca 2+ concentration, the size and the potential of Ca2+@DNA nanoparticles are appropriate and favorable for excellent transfection. When the concentrations were larger than 100 mM, the transfection efficiency decreased. The possible reason might be that stable and efficient Ca2+@DNA complex could be well formed only in the presence of adequate Ca2+; under the condition of very high Ca2 concentration, the surface potential of nanoparticles is more close to zero (Figure S6), and this would result in the poor attachment of nanoparticle to SNPDM and thus reduction in DNA loading on the material 15141
DOI: 10.1021/acsami.6b04689 ACS Appl. Mater. Interfaces 2016, 8, 15138−15144
Research Article
ACS Applied Materials & Interfaces surface. So very high concentrations of Ca2+ are unfavorable for gene transfection. In addition, excess Ca2+ also could affect the normal concentration of Ca2+ in the cells, causing the disorder of cellular physiological activities such as cell communication and material transport.56 Moreover, the incubation time of Ca2+@DNA complex with SN-PDM on transfection could affect the efficiency directly by influencing the condense and release process of DNA.57 The effect of the incubation time of Ca2+@DNA complex with SNPDM on transfection was studied by preparation of Ca2+@ DNA complex at 100 mM Ca2+ and then incubation with SNPDM 3 for different periods. As shown by Figure 3b, it could be seen that the transfection efficiency increased as the incubation time extended from 10 to 20 min and reached 1.2 × 107 RLU/ (mg of protein) at 20 min; after 20 min, transfection efficiency decreased when the incubation time continued to increase. The results were consistent with those reported by Sang et al.58 It illustrated that the appropriate incubation time is important for better transfection and this might be because it promises both the maximal amount of pDNA loading outside cells and the sufficient release of pDNA inside cells. If the incubation time is too short, the binding and compacting of Ca2+@DNA complex on SN-PDM would not be enough for efficient and maximal loading outside the cells; however, if the incubation time is too long, the tight and strong binding of Ca2+@DNA complex with polymers’ chains would inhibit the release of pDNA inside the cells and lead to the lower transfection efficiency. Biocompatibility of the SN-PDM Assisted Ca2+Dependent Transfection System. The cytotoxicity of some gene transfection systems limits them in biomedical applications. L929 fibroblast is a standard cell line in evaluating the biocompatibility of materials. In order to know cell growth and attachment on SN-PDM, L929 cells were cultured on the SN-PDM 3 surface at a density of 104 cells/well for 24 and 72 h. The results showed that the density of cells on SN-PDM was significantly higher than that on unmodified SiNWAs (Figure 4a−c), which may be due to the improvement of SiNWAs by
environment for cell growth, which is of crucial importance in guiding the design of gene delivery vectors. In the future more biocompatible polymers could also be attempted and used in this gene transfection system, for example, poly(glycidyl methacrylate) (PGMA).59 Silicon nanostructured substrates, after being modified with multifunctional polymers, could be competent for many tasks in various medical applications, for instance, efficient capture of special tumor cells44 and artificial cell capture−release systems capable of responding to dual- or multistimulation.43,60 Then the polymer-modified SiNWAs have great potential to be widely applied for in vitro gene therapy of biologically defective cells or stem cells.
4. CONCLUSION In conclusion, the SN-PDM assisted Ca2+-dependent transfection system was successfully obtained via surface-initiated ATRP of DMAEMA on SiNWAs, followed by absorbing the complex of calcium ions and DNA for enhancing the cell adhesion and gene transfection efficiency. The gene transfection efficiency achieved by this system is determined by several factors. First, proper time for the surface-initiated polymerization of DMAEMA on SiNWAs should be controlled to gain high coverage of polymers on the surface, which was shown to be 24 h in this study. Second, adequate calcium ions were necessary to form the Ca2+@DNA complex, while more than 100 mM Ca2+ might lead to some negative effect on the cells due to the cation shock. Third, the optimum incubation time for the loading of Ca2+@DNA complex on SN-PDM would benefit the compaction of DNA on materials and release of it in the cells, which was found to be around 20 min. Moreover, the inherent advantage in the clustered nanostructures of SiNWAs, the biocompatibility of PDMAEMA, and the bioactivity of calcium play an indispensable role in the improvement of gene transfection by the SN-PDM assisted Ca2+-dependent transfection system. It could be further developed for application in drug delivery, clinical detection, and biotechnology.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.6b04689. Surface modification of PDMAEMA on SiNWAs, characterization of the amino groups density of SNPDM with different polymerization times, fluorescence intensity of negative charged fluorescein disodium salt on SN-PDM with different polymerization times, transfection efficiency of SN-PDM compared to SN-PEI, size, ζ potential, and stability of nanoparticles, TEM pictures of SN-PDM, and effect of Ca2+@DNA amount on transfection efficiency (PDF)
Figure 4. (a) Density of L929 cells on SN-PDM compared to the cells cultured on SN for 24 or 72 h; (b, c) immunofluorescence images by DAPI staining of L929 cells after culture on SiNWAs (b) and SNPDM (c) for 24 h; (d−f) SEM images of L929 cells after culture on SN-PDM (d, e) and SiNWAs (f) for 24 h. Data are presented as the mean ± SE (n = 3).
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AUTHOR INFORMATION
Corresponding Authors
*(H.W.) Tel.: +86-512-65880567. Fax: +86-512-65880583. Email:
[email protected]. *(L.Y.) Tel.: +86-512-65880567. Fax: +86-512-65880583. Email:
[email protected].
PDMAEMA in attaching to the cells. Moreover, the cells maintained their normal morphology and biological activity on SN-PDM (Figure 4d,e and Figure S8), while the cells on SiNWAs shrank and lost the spindle shape (Figure 4f). It could be concluded that the modification of PDMAEMA on SiNWAs eliminated the cytotoxicity of SiNWAs and provided a suitable
Notes
The authors declare no competing financial interest. 15142
DOI: 10.1021/acsami.6b04689 ACS Appl. Mater. Interfaces 2016, 8, 15138−15144
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ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (Grant 21334004 for H.C., Grant 21374070 for L.Y., and Grant 21474072 for H.W.), the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD), and the Jiangsu clinical research center for cardiovascular surgery.
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