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A Novel Cationic Microbubble Coated with Stearic Acid-Modified Polyethylenimine to Enhance DNA Loading and Gene Delivery by Ultrasound

A Novel Cationic Microbubble Coated with Stearic Acid-Modified Polyethylenimine to Enhance DNA... A novel cationic microbubble (MB) for improvement of the DNA loading capacity and the ultrasound-mediated gene delivery efficiency has been developed; it has been prepared with commercial lipids and a stearic acid modified polyethylenimine 600 (Stearic-PEI600) polymer synthesized via acylation reaction of branched PEI600 and stearic acid mediated by N, N’-carbonyldiimidazole (CDI). The MBs’ concentration, size distribution, stability and zeta potential (f- potential) were measured and the DNA loading capacity was examined as a function of the amount of Stearic-PEI600. The gene transfection efficiency and cytotoxicity were also examined using breast cancer MCF-7 cells via the reporter plasmid pCMV-Luc, encoding the firefly luciferase gene. The results showed that the Stearic-PEI600 polymer caused a significant increase in magnitude of f-potential of MBs. The addition of DNA into cationic MBs can shift f-potentials from positive to 23 2 23 2 negative values. The DNA loading capacity of the MBs grew linearly from (560.2)610 pg/mm to (2061.8)610 pg/mm when Stearic-PEI600 was increased from 5 mol% to 30 mol%. Transfection of MCF-7 cells using 5% PEI600 MBs plus 3 2 ultrasound exposure yielded 5.7662.58610 p/s/cm /sr average radiance intensity, was 8.97- and 7.53-fold higher than 2 2 those treated with plain MBs plus ultrasound (6.4165.82) 610 p/s/cm /sr, (P,0.01) and PEI600 MBs without ultrasound 2 2 (7.6566.18)610 p/s/cm /sr, (P,0.01), respectively. However, the PEI600 MBs showed slightly higher cytotoxicity than plain MBs. The cells treated with PEI600-MBs and plain MBs plus ultrasound showed 59.566.1% and 71.467.1% cell viability, respectively. In conclusion, our study demonstrated that the novel cationic MBs were able to increase DNA loading capacity and gene transfection efficiency and could be potentially applied in targeted gene delivery and therapy. Citation: Jin Q, Wang Z, Yan F, Deng Z, Ni F, et al. (2013) A Novel Cationic Microbubble Coated with Stearic Acid-Modified Polyethylenimine to Enhance DNA Loading and Gene Delivery by Ultrasound. PLoS ONE 8(9): e76544. doi:10.1371/journal.pone.0076544 Editor: Efstathios Karathanasis, Case Western Reserve University, United States of America Received April 3, 2013; Accepted August 27, 2013; Published September 26, 2013 Copyright:  2013 Jin et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: The work was supported by National Basic Research Program 973 (Grant Nos. 2012CB733800, 2011CB707903, and 2010CB732604) from Ministry of Science and Technology, China, National Science Foundation Grants (Grant Nos. 61020106008 and 30900749). The work of J. Wu was partially supported by HAS fund of the University of Vermont, USA. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: hr.zheng@siat.ac.cn (HZ); fei.yan@siat.ac.cn (FY) scale production and simplicity of usage [2]. Both gene therapies Introduction via viruses and non-viral vectors have potential to be treatment The success of gene therapy largely depends on the develop- techniques particularly for gene-diseases, but the development of a ment of vectors or vehicles that can selectively and efficiently safe and efficient gene delivery system is a long process which deliver genes to targeted cells with minimal toxicity. Generally, the necessarily involves clinical trials [3,4,5]. gene delivery vectors can be divided into two categories: viral and Ultrasound targeted microbubble (MB) destruction (UTMD) is non-viral. The former which uses replication-deficient viruses a physical gene transfection technique, known for being safe, (such as retrovirus, adenovirus, adeno-associated virus and herpes effective, and non-invasive [6,7,8]. The MB, in addition to its well- simplex virus) has the advantage of high gene delivery efficiency, known application as a contrast agent, has also been used as a but is handicapped in clinical applications due to their immuno- drug/gene carrier, can be visualized and monitored in real time genicity, potential mutagenicity, low transgene size and high cost with assistance of ultrasound imaging. Cargo-loaded MBs can [1]. The non-viral vectors usually includ cationic liposomes, circulate easily within the vascular system until they reach a cationic polymers, synthetic peptides and naturally occurring specific region of interest, and then they can be cavitated locally compounds. Although the non-viral vectors have shown to be with high intensity focused ultrasound, causing site-specific significantly less effective in vivo in comparison with the viral delivery of the bioactive materials into cells through a process vectors, they are believed to attractive alternatives to viral vectors called sonoporation [9]. Excited by ultrasound, gas-filled MBs may for their lack of specific immune response, versatility, ease of large- oscillate drastically and eventually collapse via a process called PLOS ONE | www.plosone.org 1 September 2013 | Volume 8 | Issue 9 | e76544 A New Cationic Microbubble as a Non-Viral Vector inertial cavitation, releasing the energy necessary to induce Huahe New-technology Development Company (Tianjin, China). transient cell membrane permeabilization [9]. Microstreaming Hoechst 33258 fluorescent dye for DNA labeling was obtained and acoustic radiation force are also thought to contribute to gene from Beyotime (Shanghai, China). All other chemicals were uptakes [10,11]. UTMD has been proposed as an innovative prepared with analytical grade reagents dissolved in 18.2 mV method for noninvasive gene delivery for different kinds of tissue. deionized water prepared by Milipore (Milli-Q Reference). Recently, the therapeutic effects of ultrasound-mediated gene delivery with MBs have been demonstrated both in cell culture Synthesis and characterization of stearic acid modied [12,13] and in vivo studies [14,15,16,17]; however, the transfec- polyethylenimine 600 (Stearic-PEI600) tion efficiency was found to be low. One of the main reasons to low The Stearic-PEI600 was synthesized according to a previous efficiency is the low DNA loading capacity of MBs. Simple report described by Wan et al[28]. In brief, 0.35 g (2.16 mmol) N, blending of plasmid DNA with plain MBs, a method being most N’-carbonyldiimidazole (CDI) was dissolved in 10 ml anhydrous commonly performed, cannot upload enough DNA to MBs. chloroform. 0.6 g (2.1 mmol) stearic acid was dissolved in dry Therefore it is difficult to achieve sufficient concentration of chloroform (10 ml) and then added dropwise into upper CDI genetic material at the sonoporation site. Many strategies and solution under magnetic stirring. The mixture was reacted at room formulations have been proposed to prepare DNA loading MBs. temperature for 2 h under argon protection. The activated stearic The methods include (1) preparing polymer MBs by using double- acid was further added drop by drop to the dry branched PEI emulsion solvent evaporation method (w1/o/w2) and adding solution (0.7 g, 1.17 mmol, in 20 ml dry chloroform). The DNA to the inner water (w1) phase during the primary suspension was kept stirring at room temperature for further 24 emulsification [18], (2) layer-by-layer (LBL) assembly technique h under argon protection. The resulting product was purified by to deposit multi-layers of cationic polymer on the MB shell to precipitation in cold ether and collected by centrifuge at 3000 rpm electrostatically bind DNA [19], (3) non-covalent coupling of RNA for 10 min. The purified Stearic-PEI600 was further dried under loaded cationic liposomes onto the MB surface via avidin-biotin high vacuum condition to remove trace amount of solvent. interactions [20], (4) preparing cationic MBs by incorporating some cationic lipids such as DMTAP, DOTAP, DPTAP, DSTAP, Preparation of plain and cationic MBs DOTMA, or DDAB into the lipid MB shell to electrostatically The plain MBs were prepared by using mechanical agitation bind DNA [12,15,21,22]. Most experiments using the above- method reported in a previous publication [29]. As for the cationic mentioned strategies have increased effectiveness of DNA loading. MBs, the Stearic-PEI600 polymer was introduced. A lipid film, However, those methods were usually complicated and less- with molar percentages of 10% DSPE-PEG2000, N% Stearic- convenient in preparation or still not enough to promote PEI600, and (90-N) % DSPC, was formed by removing the intracellular delivery and trafficking to the nucleus. chloroform in the phospholipid solution under nitrogen flow, The cationic polymer, polyethylenimine (PEI), has been widely where N was variable. Residual chloroform was further eliminated used for gene transfection due to its strong DNA compaction under high vacuum for at least 2 h. A Liposome (3 mg/ml) capacity and intrinsic endosomolytic activity [23,24,25]. Also, the suspension was produced by hydrating the dry lipid films with a ‘‘proton-sponge’’ effect makes DNA/PEI complex escape from the given buffer consisting of 0.1 M Tris (pH 7.4, glycerol and phagolysosomes into the cytoplasm to minimize the enzymatic propylene glycol (80:10:10 by volume). The suspension was then degradation in the lysosomes [25,26]. During transfection, cDNA sonicated at 60uC for 5 min by a bath sonicator (40 kHz, 240 W). is released in the cytoplasm and is then trafficked uncoated by an The resulting solution was sealed in a 3 ml serum vial (1 ml each) inefficient mechanism into the nucleus. It has been demonstrated with a rubber cap and an aluminum seal. Finally, air in the vial that polyethylenimine promote transgene delivery to the nucleus in was exchanged with C F using a homemade apparatus. MBs 3 8 mammalian cells [27]. were formed by shaking the vial with a vibrator for 45 s. The In this in vitro study, we introduce a novel cationic lipid MB to FITC-labeled Stearic-PEI600 was used to prepare fluorescent enhance the DNA loading capacity of the MBs by coupling PEI MBs and to show that the Stearic-PEI600 was incorporated into onto the shell of the MBs. The concentration, size distributions, the shell of the MBs. stability, zeta potentials and DNA loading capacity of the MBs were measured. The gene transfection efficiency and the Plasmid DNA and salmon sperm DNA preparation cytotoxicity were also examined using breast cancer MCF-7 cells The reporter plasmid pCMV-Luc, encoding the firefly lucifer- via the reporter plasmid pCMV-Luc, encoding the firefly ase gene under the control of the CMV promoter, was propagated luciferase gene. in Escherichia coli TOP10, extracted and purified using plasmid extraction kit (NucleoBondH Xtra Midi EF) according to the Materials and Methods manufacturer’s instructions. The concentration and purity were determined by measuring UV absorbance at 260/280 nm with a Materials BioPhotometer (Eppendorf, Germany). Salmon sperm DNA was 1,2-distearoyl-sn-glycero-3-phosphocholine(DSPC) and 1,2-dis- dispersed in deionized water by using a bath sonicator (40 kHz, tearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethy- 240 W), and the concentration was measured by UV spectropho- leneglycol)-2000] (ammonium salt) (DSPE-PEG2000) were pur- tometry. chased from Avanti Polar Lipids (Alabaster, AL). Branched polyethylenimine (molecular weight = 600 Dalton, PEI600), Concentration and size distribution of MBs stearic acid and N, N’-carbonyldiimidazole (CDI) were purchased from Aladdin (Shanghai, China). FITC and Salmon sperm DNA After shaking the vial for 45 s using a vibrator, the obtained were purchased from Sigma-Aldrich (St. Louis, MO). SYBR green milky MB suspension was drawn into a 10-ml syringe and diluted was purchased from Invitrogen (USA). The human MCF-7 cancer to a final volume of 4 ml. MBs were washed with PBS three times cells were obtained from the American Type Culture Collection in a bucket rotor centrifuge (ALLEGRAX-12R, Beckman Coulter, (ATCC). Cell Counting Kit-8 was purchased from Dojindo USA) at 400 g for 3 min at 4uC to remove excess free (Kumamoto Japan). Perfluoropropane (C F ) was purchased from unincorporated lipids. The size distribution and concentration of 3 8 PLOS ONE | www.plosone.org 2 September 2013 | Volume 8 | Issue 9 | e76544 A New Cationic Microbubble as a Non-Viral Vector MBs were measured using an Accusizer 780A (Particle Sizing Ultrasound-mediated gene transfer with MBs System, Santa Barbara, USA). The freshly prepared MBs that Human breast cancer MCF-7 cells were seeded in 96-well plates were directly drawn from the vials right after shaking were also and transfection experiments were performed when the cell sampled. confluence reached 70–80%. Plain MBs/DNA and PEI600-MB/ DNA complexes were prepared by premixing and incubating 10 ml DNA with 10 ml plain MBs or 5 mol% PEI600 MBs (20 ml Zeta potential of the MBs total mixture volume in 0.9% NaCl) for 30 min at room Zeta potential of the MBs was measured using a Zetasizer temperature (25 uC) to allow for spontaneous binding of the NANO ZS system (Malvern, UK). Before measurement, the MBs anionic DNA and MBs. Then the complexes were added into each were washed with 10 mM sodium chloride solution or deionized well, and the final concentrations of the DNA and MBs were water thrice as described above. Diluted MBs with a concentration maintained at 2 mg/ml and 10 /ml, respectively. 10 ml DNA and of 1610 bubbles/ml were measured. All samples were measured 10 ml MBs without premix were added to each well of a control three times. group successively. After the 96-wells plate was sealed firmly and turned upside down for 15 min to allow the DNA/MBs complexes Characterization of plasmid DNA-loaded MBs to float and adhere to the cell monolayer, ultrasound exposure was In order to determine the optimal ratio of MBs to plasmid performed. DNA, the DNA-loaded MBs were resolved with 1% agarose gel The ultrasound system used in this experiment includes an stained with SYBR green (Invitrogen). In each experiment, arbitrary waveform generator (model AFG3102, Tektronix, USA), 6 6 6 different amounts of PEI600 MBs (1.0610 , 2.0610 , 4.0610 , an RF power amplifier (model AR150A100B, AR, USA), and a 6 7 7 7 8.0610 , 1.2610 , 1.6610 , 2.0610 cationic MBs) were incu- weakly focused transducer (Valpey Fisher, MA, USA) whose bated with 0.36 mg DNA for 15 min to form the DNA/MB center frequency is 1.2 MHz, focal length is 5.0 cm, diameter is complexes. To maintain the same final volume, an appropriate 5.1 cm, and f-number is close to unity. A 120-cycle sinusoidal amount of DNA loading buffer was added to each sample. Gel tone-burst with 1 kHz pulse repetition frequency and an acoustic electrophoresis was carried out at 110 V for 20 min. Image of the pressure amplitude of 0.6 MPa, which was measured in situ using gel was captured using a Gel Imaging System (Dolphin-Doc Plus). a needle hydrophone of resolution 1 mm, was used to treat each To verify the coupling of the plasmid DNA and cationic MBs, sample for 15 s. Then the treated plates were incubated for 6 h in a Hoechst 33258 working solution was used to stain the DNA on the humidified atmosphere containing 5% CO at 37uC. After that, surface of MBs. The images were captured using a inverted the medium was replaced with fresh DMEM with 10% FBS and fluorescence microscope (Leica DMI 3000B). 1% antibiotics. 24 h after transfection, 100 ml DMEM containing D-luciferin (150 mg/mL final concentration) was added to each DNA loading capacity of MBs well and the luciferase expression of these MCF-7 cells was Salmon sperm DNA (Sigma-Aldrich) was used to measure the evaluated using a Xenogen IVIS-100 system (Caliper Life DNA loading capacity of cationic MBs as previous described [30]. Sciences). Briefly, 2 mg/ml salmon sperm DNA was prepared by dissolving a proper amount of DNA in deionized water using a bath sonicator. Cell viability assay An amount of 500 ml PEI600 MBs (resulting in 10 MBs/mL) was The cytotoxicities of PEI600 MBs with or without ultrasound slowly injected into 1 ml of DNA solution. The suspension was exposure were measured and compared with Plain MBs. 24 h after incubated by gentle rotation for 1 h to speed up the process of transfection, the cell viability was evaluated using Cell Counting DNA adsorption onto MBs; the uncoupled free DNA was then Kit-8 (Dojindo, Kumamoto, Japan) according to the manufactur- removed by centrifugation and washing thrice in a centrifugal tube er’s protocol. The absorbance at 450 nm was measured by using a (400 g, 3 min). After the residual DNA loaded MBs were collected, multimode plate reader (Synergy 4, BioTek). the zeta potentials and size distributions were measured as mentioned. Statistical analysis The MBs were then destroyed in a bath sonicator heated about Statistical analysis was performed using the two-tailed t-Test 65uC for several minutes until the suspension became transparent. method assuming unequal variances. A p value of ,0.05 was The concentration of DNA in the suspension was measured using considered to be statistically significant. a BioPhotometer (Eppendorf, Germany), basing on 1 OD (i.e. a solution having an absorbance of one unit at 260 nm with a path Results length of 1 cm) corresponds to a concentration of 50 mg/ml for double-stranded DNA. All the measurements were made in PEI modification and PEI600 MBs triplicate. To reduce the discrepancies caused by the varied size As schematically presented in Fig. 1, branched PEI600 (MW = 600 Da) was modified with stearic acid via acylation reaction at a distributions between samples, the DNA loading capacity of MBs were normalized by their total MB surface area. Assuming that molar ratio of 1:2. Hydrophobic stearic chains were introduced onto the branched PEI by CDI. The molecular structure of the MBs were all sphere shapes, and their total surface area of the 1 1 product was confirmed by H NMR in CDCl . H NMR analysis measured sample could be estimated by the summation of surface 3 indicates that 14% amino-groups of the PEI600 were acylated area of all MBs in all channels sized by Accusizer 780A. (Fig. 2). Each PEI600 molecule is connected with nearly two stearic chains and has a very similar structure with the Cell culture phospholipids used to fabricate the shell of MBs [31]. Human breast cancer MCF-7 cells were employed to evaluate As shown in Fig. 1, the modified PEI was incorporated into the gene transfer and expression. The cells were maintained in shell of the MBs by hydrophobic-hydrophilic interactions. Dulbecco’s Modified Eagle Medium (DMEM), supplemented with Fluorescence images confirm that the Stearic-PEI600 was partially 10% FBS and 1% penicillin-streptomycin solution and maintained embedded into the shell of the MBs (Fig. 3). The bright field image in a humidified atmosphere containing 5% CO at 37 uC. shows bright gas cores of the MBs surround by dark circular rings PLOS ONE | www.plosone.org 3 September 2013 | Volume 8 | Issue 9 | e76544 A New Cationic Microbubble as a Non-Viral Vector Figure 1. Illustration of the PEI modification by stearic acid for preparing of cationic microbubbles (MBs) to load DNA. The stearic acid modified polyethylenimine 600 (Stearic-PEI) polymer was synthesized. The resulting Stearic-PEI, combined with DSPC and DSPE-PEG2k, was used to fabricate the cationic MBs. The PEI endows the cationic MBs with more amino groups to couple DNA than plain MBs. doi:10.1371/journal.pone.0076544.g001 (Fig. 3A). Some of the MBs seem to be a bit fuzzy because they Concentration, size distribution of PEI600 MBs were out of the focus. The fluorescence image shows the opposite Fig. 4A shows that the concentration and size distribution of the pattern with dark cores and bright green shells (Fig. 3B). They resulting cationic MBs. The concentrations, the number of MBs demonstrate the FITC-labeled Stearic-PEI600 cationic MBs have per ml, of both freshly prepared MBs without wash and that been established. washed with 10 mM NaCl, gradually decreased with the increase of Stearic-PEI600 in cationic MB formulations. Compared with the concentration of 5 mol% PEI600 MBs, the concentration of 30 mol% PEI600 MBs had a nearly 90% decrease in MB Figure 2. H NMR spectrum of Stearic-PEI. (CDCl ), d (ppm) 0.86–0.89 (t, -CH CH (CH ) CH ), 1.25 (br, -CH CH (CH ) CH ), 1.62 (br, - 3 2 2 2 15 3 2 2 2 15 3 CH CH (CH ) CH ), 2.18 (br, -CH CH (CH ) CH ), 2.39–3.3 (m, -CH CH NH-, -CH CH N-, -CH CH NHCO-, -CH CH NHCO-). 2 2 2 9 3 2 2 2 9 3 2 2 2 2 2 2 2 2 doi:10.1371/journal.pone.0076544.g002 PLOS ONE | www.plosone.org 4 September 2013 | Volume 8 | Issue 9 | e76544 A New Cationic Microbubble as a Non-Viral Vector achieving decreases of 42.20 mV (P,0.01), 61.60 mV (P,0.01), 52.00 mV (P,0.01) and 53.50 mV (P,0.01) respectively. However, there was no significant decrease of the magnitude of surface potentials for plain MBs without Stearic-PEI600 (P . 0.05). Plasmid DNA binding onto PEI600 MBs In order to visually examine whether the PEI600 MBs could bind with plasmid DNA, Hoechst 33258 was used to stain plasmid DNA on the cationic MBs. The bright field image showed the Figure 3. The bright field and fluorescence images of FITC- shape of DNA-MB complexes were not affected by DNA-binding labelled Stearic-PEI600 cationic MBs. (A) Bright field image and (B) (Fig. 6A). The blue surface of the DNA-binding MBs observed fluorescent image of MBs containing 5% Stearic-PEI600. Scale bars: under fluorescent microscope (Fig. 6B), which indicated successful 10 mm. doi:10.1371/journal.pone.0076544.g003 adhesion of the negatively charged plasmids to the surface of cationic MBs. Agarose gel electrophoresis of cationic MB/DNA complexes at concentration (P, 0.01) for freshly prepared MBs and a 75% various MB/DNA ratios was presented in Fig. 6C. Reduced or no decrease for NaCl-washed MBs respectively. The size distributions migration of the DNA into the gel at $ 2.0610 MB/mg DNA of the freshly prepared and NaCl-washed MBs were presented in ratio indicated the complexes formation. 0.36 mg DNA was Fig. 4B and Fig. 4C. The submicron bubbles (size ,1 mm) completely captured by 2.0610 MBs, responding to the ratio dominated in the freshly prepared MBs when the amount of 5.5610 MBs coupled 1 mg plasmid DNA. Stearic-PEI600 used for the cationic MB fabrication was less than 20 mol%. The size distributions of NaCl-washed MBs showed a similar trend (Fig. 4C). The ratios of the submicron bubbles were DNA loading capacity of PEI600 MBs reduced from 68.3% to 27.7% for fleshly prepared MBs and from The DNA-loading capacity was quantitatively determined 45.9% to 20.8% for NaCL washed MBs with the increased through calculating the DNA density on the MBs according to amount of Stearic-PEI600 from 5% to 30%, which were in the previous publication [30,32]. From Fig. 7 we can see that the agreement with the previously reported results [30]. The mean DNA density on the MBs increased nearly linearly from 23 2 23 2 and median sizes of freshly prepared MBs and washed MBs are (560.2)610 pg/mm to (2061.8) 610 pg/mm when Stearic-PEI600 increased from 5 mol% Stearic-PEI600 to 30 shown in Fig. 4D and which show a gradually increase in the mean and median diameters of MBs with the increased amount of mol%. Since the average molecular weight of one DNA base pair Stearic-PEI600 from 0% to 30%. Moreover, NaCl-washed MBs is about 670 Daltons, a 2 mm 5% PEI600 MB would couple 7 7 0.0628 pg DNA, corresponding to about 6610 base pairs. 10 also have larger mean and median diameters than freshly prepared MBs (P,0.05). MBs would bind 0.68 mg DNA, which was in the same order of magnitude with the estimated data from gel electrophoresis. Zeta potential of the PEI600 MBs Ultrasound-mediated transfection in cell culture Zeta potential of the cationic MBs is significant for DNA binding through the electrostatic interactions. The detection of Transfection of MCF-7 cells using DNA/5% PEI600 MBs laser Doppler anemometry showed that Stearic-PEI600 had a complex plus ultrasound exposure yielded 5.7662.58610 p/s/ cm /sr average radiance intensity, was 8.97- and 7.53-fold higher significant effect on f-potential of MBs. Plain MBs without Stearic- PEI600 (0%) had a f-potential of –28.164.3 mV in deionized than those treated by plain MBs with ultrasound exposure 2 2 (6.4165.82610 p/s/cm /sr, P,0.01) and by PEI600 MBs water due to the presence of negatively charged phosphate groups 2 2 without ultrasound (7.6566.18610 , p/s/cm /sr, P,0.01), re- in the DSPE-PEG2000. Addition of 5%, 10%, 20% or 30% spectively. And the cells treated by adding PEI600 MBs and DNA Stearic-PEI600 during MB preparation resulted in a dramatic (without premix) with ultrasound yielded an average radiance increase of the MB surface f-potentials from negative to positive; 3 2 5.6762.46610 p/s/cm /sr, is was 8.84-fold higher than treated they reached 42.164.65 mV, 42.7065.34 mV, 54.3064.70 mV, by plain MBs and ultrasound exposure. The samples treated by 57.8067.41 mV for MBs in deionized water, respectively. It was ultrasound exposure with or without premixing PEI600 MBs and noted that the f-potentials of all kinds of MBs in 10 mM NaCl DNA made no difference in the plasmid DNA transfection decreased in comparison with those of MBs dispersed in deionized efficiency (Fig. 8). water (Fig. 5A). MBs without Stearic-PEI600 had a negative f- potential of –8.6164.26 mV when dispersed in 10 mM NaCl, with a 19.49 mV decrease of the magnitude of MB surface Cytotoxicities potential (P,0.01), compared with sample without Stearic- The cell viability was measured by using CCK-8 assay. The cells PEI600. Similarly, there were 26.3 mV (P,0.01), 15.4 mV treated with DNA/PEI600-MBs and DNA/plain MBs complexes (P,0.01), 35.7 mV (P,0.01) and 40 mV (P,0.01) decreases of under ultrasound exposure showed 59.566.1% and 71.467.1% the magnitude of f-potential for 5%, 10%, 20% and 30% PEI600 cell viability, respectively. No matter with and without ultrasound MBs, respectively. Fig. 5B showed the f-potentials of MBs coated exposure, the cytotoxicitiy of PEI600 MB was slightly larger than with the Stearic-PEI600 and that saturated by excess salmon plain MBs (P,0.01), and ultrasound exposure showed a strong sperm DNA. Interestingly, the addition of DNA into cationic MB toxicity in every groups (P,0.001). There were not significant suspension in 10 mM NaCl dramatically reversed the f-potentials differences in cell viability whether the DNA and PEI600MB were of MBs from positive to negative (Fig. 5B). when saturated by premixed or not (Fig. 9). Thus, it was indicated that the excess salmon sperm DNA, the surface potentials of 5%, 10%, cytotoxicity may be mainly caused by the MB cavition induced 20% or 30% Stearic-PEI600 cationic MBs were –26.4068.93 mV, by ultrasound exposure, however, the PEI600 MBs showed higher –34.3065.95 mV, –33.4066.49 mV and –35.7065.35 mV, cytotoxicity than plain MBs. PLOS ONE | www.plosone.org 5 September 2013 | Volume 8 | Issue 9 | e76544 A New Cationic Microbubble as a Non-Viral Vector Figure 4. Concentration and size distribution of fresh prepared and washed MBs. (A) Concentrations of freshly prepared and washed MBs with various amounts of Stearic-PEI600. (B) Size distribution of freshly prepared MBs with various amounts of Stearic-PEI600. (C) Size distributionof 10 mM NaCl washed MBs with various amounts of Stearic-PEI600. (D) Mean and median size of fresh prepared and 10 mM NaCl washed versus the Stearic-PEI600 fraction. The ‘‘freshly prepared’’ MBs were taken straight from the vial within 1 h of formation. The ‘‘washed’’ MBs were obtained by three cycles of flotation-centrifugation with infranatant exchange to remove the submicrometer bubbles. doi:10.1371/journal.pone.0076544.g004 Figure 5. The zeta potential of PEI600 MBs. (A) Zeta potential of MBs dispersed in deionized water and 10 mM NaCl. (B) Zeta potential of MBs binding with and without DNA. doi:10.1371/journal.pone.0076544.g005 PLOS ONE | www.plosone.org 6 September 2013 | Volume 8 | Issue 9 | e76544 A New Cationic Microbubble as a Non-Viral Vector Figure 6. Binding of plasmid DNA onto PEI600 MBs. (A) The bright field image and (B) the corresponding fluorescent image of plasmid DNA loaded MBs. (C) Agarose gel electrophoresis of 0.36 mg plasmid DNA mixed with different number of MBs with 5% Stearic-PEI600. doi:10.1371/journal.pone.0076544.g006 can increase effectiveness of DNA loading, but are often Discussion complicated and less-convenient during preparation. The latter Recently, most MB-mediated gene delivery experiments were also has some drawbacks. For example, the DNA loading capacity performed by co-injection of MBs and DNA vectors. In those of the MBs would easily be saturated at a low concentration level studies, circulating MBs were insonified to increase local vascular due to the limited surface area of MBs. Contrarily, in our study, permeability, allowing DNA vectors to passively extravasate into DNA vectors were attached to the surfaces of the PEI600-modified tissue. However, circulating genetic agents may also extravasate cationic MBs, forming MB-DNA hybrid vectors. In consideration into fenestrated organs (such as the lung, liver, and spleen), of its relatively low cytotoxicity compared with that of mostly used resulting in off-target effects [33]. Strategies such as DNA-loaded PEI 25000 [31], PEI600 was selected to fabricate the cationic polymer MBs using double-emulsion solvent evaporation method MBs. By attaching DNA onto the MB surface, their release can be (w /o/w ), layer-by-layer (LBL) assembly technique and DNA- 1 2 mediated by acoustic cavitation around the ultrasound focal zone, loaded liposome-MB complexes have been proposed to overcome thus providing more specific control in tissue-targeting applica- this problem [30,24,13]. Another strategy is to incorporate directly tions [34]. Furthermore, attachment of DNA to the MB surface cationic polymer such as DSTAP into the MB shell. The former has potentially provided some protection, preventing rapid clearance of DNA [21,35]. Several studies have demonstrated that DNA molecules loaded onto MBs improves their intracellular uptake in vitro [36,37] and deposition into target tissue in vivo [38]. Wang et al. showed that plasmid-binding cationic MBs had enhanced ultrasound-mediated gene delivery efficiency relative to neutral MBs in both cell culture and mouse hind limb tumors [21,39]. In the current study, we found that the MB size, concentration and size distributions were significantly impacted by the molar amount of Stearic-PEI600 in preparing the cationic MBs. Our results were in agreement with the previous reports. Borden et al. have reported the lateral phase separation in lipid-coated MBs [40]. The ordered domains in the shell of MBs are composed primarily of DSPC, while the disordered interdomain regions are mainly lipopolymer. Thus, increasing the cationic polymer concentration would generally result in disorder domains increase and condensed domains reduction. These disorder domains would increase the mass transfer, so that stability was weaken. In another study, Feshitan et al. found that higher concentration MBs, in Figure 7. DNA loading capacity vs Stearic-PEI600 (%). The relationship between DNA loading capacity (y) and Stearic-PEI600% (x) general, tended to be more stable, regardless of MB size [41]. can be fitted to a linear rewlation : y = 0.0566x+0.0013 with R = 0.9576. Surface charge of the cationic MBs indicated a significant Salmon sperm DNA surface concentrations were obtained by analyzing change in the surface of MBs after addition of the cationic polymer the 260 nm absorbance and corresponding total surface area of sized Stearic-PEI600 (Fig. 5A). This is mainly because that the ionic MBs. strength of the polyelectrolyte such as PEI is determined not only doi:10.1371/journal.pone.0076544.g007 PLOS ONE | www.plosone.org 7 September 2013 | Volume 8 | Issue 9 | e76544 A New Cationic Microbubble as a Non-Viral Vector Figure 8. DNA transfection in MCF-7 cells. (A) Optical images of luciferase gene expression. (B) Bar graph shows the luciferase activity in MCF-7 cells after ultrasound-mediated transfection of pCMV-Luc with complex of DNA/Plain MBs, complex of DNA/5% PEI600 MBs by premix DNA and MB for 15minutes, and with 5% PEI600 MB and DNA without premixing and added separately. pCMV-Luc activities were evaluated 24 h after gene transfection. doi:10.1371/journal.pone.0076544.g008 by the concentration of the polyelectrolyte itself, but also the ionic character of the suspending medium. Nomikou et al. concentration of small molecule electrolytes such as NaCl. The measured a lipid-shelled MB with a 8% molar ratio of DSTAP protonated amine group of the PEI is shielded to some extent by and described a zeta potential of approximately 4–5 mV in Opti- the counter-ions in the NaCl solution. It was demonstrated that DMEM [42], while Borden et al. described a MB zeta potential of the zeta potential was sensitive to both pH and the presence of Figure 9. Cell cytotoxicity assay. The cell viability was measured by using the CCK-8 assay after 24 h transfection of pCMV-Luc with with complex of DNA/Plain MBs, complex of DNA/5% PEI600 MBs by premix DNA and MB for 15minutes, and with 5% PEI600 MB and DNA without premixing and added respectively, with or without ultrasound exposure. doi:10.1371/journal.pone.0076544.g009 PLOS ONE | www.plosone.org 8 September 2013 | Volume 8 | Issue 9 | e76544 A New Cationic Microbubble as a Non-Viral Vector approximately 33 mV with a 20% molar ratio of DSTAP in ly, as for the UTMD strategy for DNA transfection, it seems that 10 mM NaCl, which was closed to our results [30]. the cell viability and transfection efficiency are contradictory. For It is notable that a significant improvement of DNA loading example, transfection efficiency increased approximately linearly capacity was achieved in our new designed cationic MBs. The with MB concentration, but cell viability inversely correlated with improvement of DNA loading capacity may contribute to the MB concentration. The relationship between acoustic intensity abundant amino group in PEI molecules. Each PEI600 molecule and transfection efficiency was highly nonlinear. Clearly, the goal has approximately 14 nitrogen atoms, and at most 12 residual of this technology is to obtain high transfection efficiency and low nitrogen atoms of which could be protonated to adsorb DNA after cell death. However, the acoustic intensity at site, MB concentra- being modified by two stearic acid molecules. So, ideally, the tion and pulsing sequence may need to be carefully adjusted to maximum DNA loading capacity should be ten folds more than obtain an optimal conditions, considering the tradeoffs between that of the traditional cationic MBs which consist of the same the transfection efficiency and cell viability [12]. molar ratio of polymers such as DSTAP. Herein, DNA loading capacity of PEI600 MB we measured is about five times higher Conclusions than that of the reported DSTAP MBs, which may be because of the steric hindrance. Borden et al. applied a layer-by-layer (LBL) We have developed a cationic MB by modifying PEI with assembly technique to adsorb multiple layers of DNA and poly-L- stearic acid and coating it onto the shell of MBs. The addition of lysine (PLL) onto lipid-coated MBs [30]. The DNA loading the modified cationic polymer may affect the yield and size capacity was enhanced by over 10-fold by using five paired layers. distribution of the MBs by forming a disorder domain in the shell Nevertheless, LBL assembly technique is somewhat complex as the of the MBs. Also, the buffer used to dilute and wash the MBs will fragility of the MBs. affect both the size distribution and the zeta potential of the MBs It has been proved that DNA could be effectively protected due to the shielding effect of the small molecular electrolyte. The from degradation by coupling them to the cationic MBs via plasmid DNA can be effectively coupled onto the surface of the electrostatic interactions [35,43]. In recent studies, the gene cationic MBs. Additionally, our results demonstrated an increasing transfer by using UTMD with injecting MBs and DNA/PEI DNA-loading capacity with increase of the Stearic-PEI600. DNA complex simultaneously have shown that UTMD could improve transfection of MCF-7 cells using the Stearic-PEI600 MBs and the gene transfection efficiency of DNA/PEI complex in vitro and ultrasound is significantly higher than that of treatment with plain in vivo [17,44]. Sirsi et al. also reported that DNA/PEI-MBs could MBs with ultrasound, PEI600 MBs without ultrasound respec- transfect tumor tissue in a site-specific manner by virtue of tively. And premix the PEI600 MBs with DNA or not doesn’t ultrasound. In their study, the branched polyethylenimine (PEI) affect the transfection efficiency significantly in vitro. Our study was first modified with polyethylene glycol and hydrosulfide group, may have laid down a foundation for image-guided gene therapy and then covalently attached to the maleimide groups on the shell which we plan to explore in future. of the MBs that contain a functionalized PEG lipid. Thus, DNA could be adsorbed by the PEI coated MBs. Acknowledgments In this study, the DNA transfection was carried out under the We thank to Fei Li, Chengzhi Zeng for advices and comments regarding following conditions: frequency = 1.2 MHz, the acoustic pressure setup of ultrasound apparatus and parameters, Qian Wan for technical amplitude = 0.6 MPa, a 120-cycle sinusoidal tone-burst with 1 assistance and support with using of the Xenogen IVIS-100 system. kHz pulse repetition frequency, MB concentration = 1610 MBs/ml, DNA concentration = 2 mg/ml. In fact, the UTMD for DNA transfection in vitro and in vivo has been studied Author Contributions in similar conditions. It has been demonstrated that the DNA Conceived and designed the experiments: QJ FY HZ. Performed the transfection efficiency depends on multiple factors, such as experiments: QJ ZW FN ZD. Analyzed the data: QJ ZW FY XL HZ. experimental systems, MB concentration, DNA concentration, Contributed reagents/materials/analysis tools: ZW FY HZ. Wrote the acoustic intensity and pulse sequence, etc. [45,46,47]. Interesting- paper: QJ FY JW RS. References 1. Manno CS, Arruda VR, Pierce GF, Glader B, Ragni M, et al. (2006) Successful 8. Ulasov I, Hsu P-H, Wei K-C, Huang C-Y, Wen C-J, et al. (2013) Noninvasive transduction of liver in hemophilia by AAV-Factor IX and limitations imposed and Targeted Gene Delivery into the Brain Using Microbubble-Facilitated by the host immune response (vol 12, pg 342, 2006). Nature Medicine 12: 592– Focused Ultrasound. Plos One 8: e57682. 592. 9. Wu J, Nyborg WL (2008) Ultrasound, cavitation bubbles and their interaction with cells. 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Zhou Q, Yu Z-Q, Yan J-J, You Y-Z (2012) Bioreducible and acid-labile Analysis of in vitro Transfection by Sonoporation Using Cationic and Neutral Microbubbles. Ultrasound in Medicine & Biology 36: 1907–1918. poly(amido amine)s for efficient gene delivery. International Journal of 13. De Temmerman M-L, Dewitte H, Vandenbroucke RE, Lucas B, Libert C, et al. Nanomedicine: 5819. (2011) mRNA-Lipoplex loaded microbubble contrast agents for ultrasound- 6. Daigeler A, Chromik AM, Haendschke K, Emmelmann S, Siepmann M, et al. assisted transfection of dendritic cells. Biomaterials 32: 9128–9135. (2010) Synergistic effects of sonoporation and taurolidin/TRAIL on apoptosis in 14. Bekeredjian R (2003) Ultrasound-Targeted Microbubble Destruction Can human fibrosarcoma. Ultrasound in Medicine and Biology 36: 1893–1906. Repeatedly Direct Highly Specific Plasmid Expression to the Heart. Circulation 7. Suzuki J-i, Ogawa M, Takayama K, Taniyama Y, Morishita R, et al. (2010) 108: 1022–1026. 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(2012) Polyplex-microbubble hybrids for ultrasound-guided plasmid DNA delivery to States of America 103: 8469–8474. solid tumors. Journal of Controlled Release 157: 224–234. 17. Chen Z-Y, Liang K, Qiu R-X (2010) Targeted gene delivery in tumor xenografts 33. Sirsi SR, Borden MA (2012) Advances in ultrasound mediated gene therapy by the combination of ultrasound-targeted microbubble destruction and using microbubble contrast agents. Theranostics 2: 1208–1222. polyethylenimine to inhibit survivin gene expression and induce apoptosis. 34. Lum A, Borden M, Dayton P, Kruse D, Simon S, et al. (2006) Ultrasound Journal of Experimental & Clinical Cancer Research 29: 152. radiation force enables targeted deposition of model drug carriers loaded on 18. Tse MT, Blatchford C, Oya Alpar H (2009) Evaluation of different buffers on microbubbles. Journal of Controlled Release 111: 128–134. plasmid DNA encapsulation into PLGA microparticles. Int J Pharm 370: 33–40. 35. Lentacker I, De Geest BG, Vandenbroucke RE, Peeters L, Demeester J, et al. 19. Borden MA, Caskey CF, Little E, Gillies RJ, Ferrara KW (2007) DNA and (2006) Ultrasound-responsive polymer-coated microbubbles that bind and polylysine adsorption and multilayer construction onto cationic lipid-coated protect DNA. Langmuir 22: 7273–7278. microbubbles. Langmuir 23: 9401–9408. 36. Frenkel PA, Chen S, Thai T, Shohet RV, Grayburn PA (2002) DNA-loaded 20. De Temmerman ML, Dewitte H, Vandenbroucke RE, Lucas B, Libert C, et al. albumin microbubbles enhance ultrasound-mediated transfection in vitro. (2011) mRNA-Lipoplex loaded microbubble contrast agents for ultrasound- Ultrasound in Medicine and Biology 28: 817–822. assisted transfection of dendritic cells. Biomaterials 32: 9128–9135. 37. Lentacker I, Geers B, Demeester J, De Smedt SC, Sanders NN (2010) Design 21. Wang DS, Panje C, Pysz MA, Paulmurugan R, Rosenberg J, et al. (2012) and evaluation of doxorubicin-containing microbubbles for ultrasound-triggered Cationic versus neutral microbubbles for ultrasound-mediated gene delivery in doxorubicin delivery: cytotoxicity and mechanisms involved. Molecular Therapy cancer. Radiology 264: 721–732. 18: 101–108. 22. Sun L, Huang C-W, Wu J, Chen K-J, Li S-H, et al. (2013) The use of cationic 38. Bekeredjian R, Chen S, Grayburn P, Shohet R (2005) Augmentation of cardiac microbubbles to improve ultrasound-targeted gene delivery to the ischemic protein delivery using ultrasound targeted microbubble destruction. Ultrasound myocardium. Biomaterials 34: 2107–2116. in Medicine & Biology 31: 687–691. 23. Lungwitz U, Breunig M, Blunk T, Go¨pferich A (2005) Polyethylenimine-based 39. Lawrie A, Brisken AF, Francis SE, Cumberland DC, Crossman DC, et al. (2000) non-viral gene delivery systems. European Journal of Pharmaceutics and Microbubble-enhanced ultrasound for vascular gene delivery. Gene Ther 7: Biopharmaceutics 60: 247–266. 2023–2027. 24. Min S-H, Lee DC, Lim MJ, Park HS, Kim DM, et al. (2006) A composite gene 40. Borden MA, Martinez GV, Ricker J, Tsvetkova N, Longo M, et al. (2006) delivery system consisting of polyethylenimine and an amphipathic peptide Lateral Phase Separation in Lipid-Coated Microbubbles. Langmuir 22: 4291– KALA. The Journal of Gene Medicine 8: 1425–1434. 25. Chandrashekhar C, Pons B, Muller CD, Tounsi N, Mulherkar R, et al. (2013) 41. Feshitan JA, Chen CC, Kwan JJ, Borden MA (2009) Microbubble size isolation Oligobenzylethylenimine enriches linear polyethylenimine with a pH-sensitive by differential centrifugation. Journal of Colloid and Interface Science 329: 316– membrane-disruptive property and leads to enhanced gene delivery activity. Acta Biomaterialia 9: 4985–4993. 42. Nomikou N, Tiwari P, Trehan T, Gulati K, McHale AP (2012) Studies on 26. Godbey WT, Barry MA, Saggau P, Wu KK, Mikos AG (2000) Poly(ethyleni- neutral, cationic and biotinylated cationic microbubbles in enhancing ultra- sound-mediated gene delivery in vitro and in vivo. Acta Biomaterialia 8: 1273– mine)-mediated transfection: A new paradigm for gene delivery. Journal of Biomedical Materials Research 51: 321–328. 43. Deshpande MC, Prausnitz MR (2007) Synergistic effect of ultrasound and PEI 27. Pollard H, Remy JS, Loussouarn G, Demolombe S, Behr JP, et al. (1998) on DNA transfection in vitro. Journal of Controlled Release 118: 126–135. Polyethylenimine but not cationic lipids promotes transgene delivery to the 44. Qiu YY, Luo Y, Zhang YL, Cui WC, Zhang D, et al. (2010) The correlation nucleus in mammalian cells. Journal of Biological Chemistry 273: 7507–7511. between acoustic cavitation and sonoporation involved in ultrasound-mediated 28. Wan Q, Xie L, Gao L, Wang Z, Nan X, et al. (2013) Self-assembled magnetic DNA transfection with polyethylenimine (PEI) in vitro. Journal of Controlled theranostic nanoparticles for highly sensitive MRI of minicircle DNA delivery. Release 145: 40–48. Nanoscale 5: 744–752. 45. Bao S, Thrall BD, Miller DL (1997) Transfection of a reporter plasmid into 29. Yan F, Li X, Jin Q, Chen J, Shandas R, et al. (2012) Ultrasonic Imaging of cultured cells by sonoporation in vitro. Ultrasound in Medicine and Biology 23: Endothelial CD81 Expression Using CD81-Targeted Contrast Agents in In 953–959. Vitro and In Vivo Studies. Ultrasound in Medicine & Biology 38: 670–680. 46. Zarnitsyn VG, Prausnitz MR (2004) Physical parameters influencing optimiza- 30. Borden MA, Caskey CF, Little E, Gillies RJ, Ferrara KW (2007) DNA and tion of ultrasound-mediated DNA transfection. Ultrasound in Medicine and Polylysine Adsorption and Multilayer Construction onto Cationic Lipid-Coated Biology 30: 527–538. Microbubbles. Langmuir 23: 9401–9408. 47. Karshafian R, Bevan PD, Williams R, Samac S, Burns PN (2009) Sonoporation 31. Borden MA, Kruse DE, Caskey CF, Zhao S, Dayton PA, et al. (2005) Influence by Ultrasound-Activated Microbubble Contrast Agents: Effect of Acoustic of lipid shell physicochemical properties on ultrasound-induced microbubble Exposure Parameters on Cell Membrane Permeability and Cell Viability. destruction. IEEE Trans Ultrason Ferroelectr Freq Control 52: 1992–2002. Ultrasound in Medicine and Biology 35: 847–860. PLOS ONE | www.plosone.org 10 September 2013 | Volume 8 | Issue 9 | e76544 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png PLoS ONE Pubmed Central

A Novel Cationic Microbubble Coated with Stearic Acid-Modified Polyethylenimine to Enhance DNA Loading and Gene Delivery by Ultrasound

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1932-6203
DOI
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Abstract

A novel cationic microbubble (MB) for improvement of the DNA loading capacity and the ultrasound-mediated gene delivery efficiency has been developed; it has been prepared with commercial lipids and a stearic acid modified polyethylenimine 600 (Stearic-PEI600) polymer synthesized via acylation reaction of branched PEI600 and stearic acid mediated by N, N’-carbonyldiimidazole (CDI). The MBs’ concentration, size distribution, stability and zeta potential (f- potential) were measured and the DNA loading capacity was examined as a function of the amount of Stearic-PEI600. The gene transfection efficiency and cytotoxicity were also examined using breast cancer MCF-7 cells via the reporter plasmid pCMV-Luc, encoding the firefly luciferase gene. The results showed that the Stearic-PEI600 polymer caused a significant increase in magnitude of f-potential of MBs. The addition of DNA into cationic MBs can shift f-potentials from positive to 23 2 23 2 negative values. The DNA loading capacity of the MBs grew linearly from (560.2)610 pg/mm to (2061.8)610 pg/mm when Stearic-PEI600 was increased from 5 mol% to 30 mol%. Transfection of MCF-7 cells using 5% PEI600 MBs plus 3 2 ultrasound exposure yielded 5.7662.58610 p/s/cm /sr average radiance intensity, was 8.97- and 7.53-fold higher than 2 2 those treated with plain MBs plus ultrasound (6.4165.82) 610 p/s/cm /sr, (P,0.01) and PEI600 MBs without ultrasound 2 2 (7.6566.18)610 p/s/cm /sr, (P,0.01), respectively. However, the PEI600 MBs showed slightly higher cytotoxicity than plain MBs. The cells treated with PEI600-MBs and plain MBs plus ultrasound showed 59.566.1% and 71.467.1% cell viability, respectively. In conclusion, our study demonstrated that the novel cationic MBs were able to increase DNA loading capacity and gene transfection efficiency and could be potentially applied in targeted gene delivery and therapy. Citation: Jin Q, Wang Z, Yan F, Deng Z, Ni F, et al. (2013) A Novel Cationic Microbubble Coated with Stearic Acid-Modified Polyethylenimine to Enhance DNA Loading and Gene Delivery by Ultrasound. PLoS ONE 8(9): e76544. doi:10.1371/journal.pone.0076544 Editor: Efstathios Karathanasis, Case Western Reserve University, United States of America Received April 3, 2013; Accepted August 27, 2013; Published September 26, 2013 Copyright:  2013 Jin et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: The work was supported by National Basic Research Program 973 (Grant Nos. 2012CB733800, 2011CB707903, and 2010CB732604) from Ministry of Science and Technology, China, National Science Foundation Grants (Grant Nos. 61020106008 and 30900749). The work of J. Wu was partially supported by HAS fund of the University of Vermont, USA. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: hr.zheng@siat.ac.cn (HZ); fei.yan@siat.ac.cn (FY) scale production and simplicity of usage [2]. Both gene therapies Introduction via viruses and non-viral vectors have potential to be treatment The success of gene therapy largely depends on the develop- techniques particularly for gene-diseases, but the development of a ment of vectors or vehicles that can selectively and efficiently safe and efficient gene delivery system is a long process which deliver genes to targeted cells with minimal toxicity. Generally, the necessarily involves clinical trials [3,4,5]. gene delivery vectors can be divided into two categories: viral and Ultrasound targeted microbubble (MB) destruction (UTMD) is non-viral. The former which uses replication-deficient viruses a physical gene transfection technique, known for being safe, (such as retrovirus, adenovirus, adeno-associated virus and herpes effective, and non-invasive [6,7,8]. The MB, in addition to its well- simplex virus) has the advantage of high gene delivery efficiency, known application as a contrast agent, has also been used as a but is handicapped in clinical applications due to their immuno- drug/gene carrier, can be visualized and monitored in real time genicity, potential mutagenicity, low transgene size and high cost with assistance of ultrasound imaging. Cargo-loaded MBs can [1]. The non-viral vectors usually includ cationic liposomes, circulate easily within the vascular system until they reach a cationic polymers, synthetic peptides and naturally occurring specific region of interest, and then they can be cavitated locally compounds. Although the non-viral vectors have shown to be with high intensity focused ultrasound, causing site-specific significantly less effective in vivo in comparison with the viral delivery of the bioactive materials into cells through a process vectors, they are believed to attractive alternatives to viral vectors called sonoporation [9]. Excited by ultrasound, gas-filled MBs may for their lack of specific immune response, versatility, ease of large- oscillate drastically and eventually collapse via a process called PLOS ONE | www.plosone.org 1 September 2013 | Volume 8 | Issue 9 | e76544 A New Cationic Microbubble as a Non-Viral Vector inertial cavitation, releasing the energy necessary to induce Huahe New-technology Development Company (Tianjin, China). transient cell membrane permeabilization [9]. Microstreaming Hoechst 33258 fluorescent dye for DNA labeling was obtained and acoustic radiation force are also thought to contribute to gene from Beyotime (Shanghai, China). All other chemicals were uptakes [10,11]. UTMD has been proposed as an innovative prepared with analytical grade reagents dissolved in 18.2 mV method for noninvasive gene delivery for different kinds of tissue. deionized water prepared by Milipore (Milli-Q Reference). Recently, the therapeutic effects of ultrasound-mediated gene delivery with MBs have been demonstrated both in cell culture Synthesis and characterization of stearic acid modied [12,13] and in vivo studies [14,15,16,17]; however, the transfec- polyethylenimine 600 (Stearic-PEI600) tion efficiency was found to be low. One of the main reasons to low The Stearic-PEI600 was synthesized according to a previous efficiency is the low DNA loading capacity of MBs. Simple report described by Wan et al[28]. In brief, 0.35 g (2.16 mmol) N, blending of plasmid DNA with plain MBs, a method being most N’-carbonyldiimidazole (CDI) was dissolved in 10 ml anhydrous commonly performed, cannot upload enough DNA to MBs. chloroform. 0.6 g (2.1 mmol) stearic acid was dissolved in dry Therefore it is difficult to achieve sufficient concentration of chloroform (10 ml) and then added dropwise into upper CDI genetic material at the sonoporation site. Many strategies and solution under magnetic stirring. The mixture was reacted at room formulations have been proposed to prepare DNA loading MBs. temperature for 2 h under argon protection. The activated stearic The methods include (1) preparing polymer MBs by using double- acid was further added drop by drop to the dry branched PEI emulsion solvent evaporation method (w1/o/w2) and adding solution (0.7 g, 1.17 mmol, in 20 ml dry chloroform). The DNA to the inner water (w1) phase during the primary suspension was kept stirring at room temperature for further 24 emulsification [18], (2) layer-by-layer (LBL) assembly technique h under argon protection. The resulting product was purified by to deposit multi-layers of cationic polymer on the MB shell to precipitation in cold ether and collected by centrifuge at 3000 rpm electrostatically bind DNA [19], (3) non-covalent coupling of RNA for 10 min. The purified Stearic-PEI600 was further dried under loaded cationic liposomes onto the MB surface via avidin-biotin high vacuum condition to remove trace amount of solvent. interactions [20], (4) preparing cationic MBs by incorporating some cationic lipids such as DMTAP, DOTAP, DPTAP, DSTAP, Preparation of plain and cationic MBs DOTMA, or DDAB into the lipid MB shell to electrostatically The plain MBs were prepared by using mechanical agitation bind DNA [12,15,21,22]. Most experiments using the above- method reported in a previous publication [29]. As for the cationic mentioned strategies have increased effectiveness of DNA loading. MBs, the Stearic-PEI600 polymer was introduced. A lipid film, However, those methods were usually complicated and less- with molar percentages of 10% DSPE-PEG2000, N% Stearic- convenient in preparation or still not enough to promote PEI600, and (90-N) % DSPC, was formed by removing the intracellular delivery and trafficking to the nucleus. chloroform in the phospholipid solution under nitrogen flow, The cationic polymer, polyethylenimine (PEI), has been widely where N was variable. Residual chloroform was further eliminated used for gene transfection due to its strong DNA compaction under high vacuum for at least 2 h. A Liposome (3 mg/ml) capacity and intrinsic endosomolytic activity [23,24,25]. Also, the suspension was produced by hydrating the dry lipid films with a ‘‘proton-sponge’’ effect makes DNA/PEI complex escape from the given buffer consisting of 0.1 M Tris (pH 7.4, glycerol and phagolysosomes into the cytoplasm to minimize the enzymatic propylene glycol (80:10:10 by volume). The suspension was then degradation in the lysosomes [25,26]. During transfection, cDNA sonicated at 60uC for 5 min by a bath sonicator (40 kHz, 240 W). is released in the cytoplasm and is then trafficked uncoated by an The resulting solution was sealed in a 3 ml serum vial (1 ml each) inefficient mechanism into the nucleus. It has been demonstrated with a rubber cap and an aluminum seal. Finally, air in the vial that polyethylenimine promote transgene delivery to the nucleus in was exchanged with C F using a homemade apparatus. MBs 3 8 mammalian cells [27]. were formed by shaking the vial with a vibrator for 45 s. The In this in vitro study, we introduce a novel cationic lipid MB to FITC-labeled Stearic-PEI600 was used to prepare fluorescent enhance the DNA loading capacity of the MBs by coupling PEI MBs and to show that the Stearic-PEI600 was incorporated into onto the shell of the MBs. The concentration, size distributions, the shell of the MBs. stability, zeta potentials and DNA loading capacity of the MBs were measured. The gene transfection efficiency and the Plasmid DNA and salmon sperm DNA preparation cytotoxicity were also examined using breast cancer MCF-7 cells The reporter plasmid pCMV-Luc, encoding the firefly lucifer- via the reporter plasmid pCMV-Luc, encoding the firefly ase gene under the control of the CMV promoter, was propagated luciferase gene. in Escherichia coli TOP10, extracted and purified using plasmid extraction kit (NucleoBondH Xtra Midi EF) according to the Materials and Methods manufacturer’s instructions. The concentration and purity were determined by measuring UV absorbance at 260/280 nm with a Materials BioPhotometer (Eppendorf, Germany). Salmon sperm DNA was 1,2-distearoyl-sn-glycero-3-phosphocholine(DSPC) and 1,2-dis- dispersed in deionized water by using a bath sonicator (40 kHz, tearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethy- 240 W), and the concentration was measured by UV spectropho- leneglycol)-2000] (ammonium salt) (DSPE-PEG2000) were pur- tometry. chased from Avanti Polar Lipids (Alabaster, AL). Branched polyethylenimine (molecular weight = 600 Dalton, PEI600), Concentration and size distribution of MBs stearic acid and N, N’-carbonyldiimidazole (CDI) were purchased from Aladdin (Shanghai, China). FITC and Salmon sperm DNA After shaking the vial for 45 s using a vibrator, the obtained were purchased from Sigma-Aldrich (St. Louis, MO). SYBR green milky MB suspension was drawn into a 10-ml syringe and diluted was purchased from Invitrogen (USA). The human MCF-7 cancer to a final volume of 4 ml. MBs were washed with PBS three times cells were obtained from the American Type Culture Collection in a bucket rotor centrifuge (ALLEGRAX-12R, Beckman Coulter, (ATCC). Cell Counting Kit-8 was purchased from Dojindo USA) at 400 g for 3 min at 4uC to remove excess free (Kumamoto Japan). Perfluoropropane (C F ) was purchased from unincorporated lipids. The size distribution and concentration of 3 8 PLOS ONE | www.plosone.org 2 September 2013 | Volume 8 | Issue 9 | e76544 A New Cationic Microbubble as a Non-Viral Vector MBs were measured using an Accusizer 780A (Particle Sizing Ultrasound-mediated gene transfer with MBs System, Santa Barbara, USA). The freshly prepared MBs that Human breast cancer MCF-7 cells were seeded in 96-well plates were directly drawn from the vials right after shaking were also and transfection experiments were performed when the cell sampled. confluence reached 70–80%. Plain MBs/DNA and PEI600-MB/ DNA complexes were prepared by premixing and incubating 10 ml DNA with 10 ml plain MBs or 5 mol% PEI600 MBs (20 ml Zeta potential of the MBs total mixture volume in 0.9% NaCl) for 30 min at room Zeta potential of the MBs was measured using a Zetasizer temperature (25 uC) to allow for spontaneous binding of the NANO ZS system (Malvern, UK). Before measurement, the MBs anionic DNA and MBs. Then the complexes were added into each were washed with 10 mM sodium chloride solution or deionized well, and the final concentrations of the DNA and MBs were water thrice as described above. Diluted MBs with a concentration maintained at 2 mg/ml and 10 /ml, respectively. 10 ml DNA and of 1610 bubbles/ml were measured. All samples were measured 10 ml MBs without premix were added to each well of a control three times. group successively. After the 96-wells plate was sealed firmly and turned upside down for 15 min to allow the DNA/MBs complexes Characterization of plasmid DNA-loaded MBs to float and adhere to the cell monolayer, ultrasound exposure was In order to determine the optimal ratio of MBs to plasmid performed. DNA, the DNA-loaded MBs were resolved with 1% agarose gel The ultrasound system used in this experiment includes an stained with SYBR green (Invitrogen). In each experiment, arbitrary waveform generator (model AFG3102, Tektronix, USA), 6 6 6 different amounts of PEI600 MBs (1.0610 , 2.0610 , 4.0610 , an RF power amplifier (model AR150A100B, AR, USA), and a 6 7 7 7 8.0610 , 1.2610 , 1.6610 , 2.0610 cationic MBs) were incu- weakly focused transducer (Valpey Fisher, MA, USA) whose bated with 0.36 mg DNA for 15 min to form the DNA/MB center frequency is 1.2 MHz, focal length is 5.0 cm, diameter is complexes. To maintain the same final volume, an appropriate 5.1 cm, and f-number is close to unity. A 120-cycle sinusoidal amount of DNA loading buffer was added to each sample. Gel tone-burst with 1 kHz pulse repetition frequency and an acoustic electrophoresis was carried out at 110 V for 20 min. Image of the pressure amplitude of 0.6 MPa, which was measured in situ using gel was captured using a Gel Imaging System (Dolphin-Doc Plus). a needle hydrophone of resolution 1 mm, was used to treat each To verify the coupling of the plasmid DNA and cationic MBs, sample for 15 s. Then the treated plates were incubated for 6 h in a Hoechst 33258 working solution was used to stain the DNA on the humidified atmosphere containing 5% CO at 37uC. After that, surface of MBs. The images were captured using a inverted the medium was replaced with fresh DMEM with 10% FBS and fluorescence microscope (Leica DMI 3000B). 1% antibiotics. 24 h after transfection, 100 ml DMEM containing D-luciferin (150 mg/mL final concentration) was added to each DNA loading capacity of MBs well and the luciferase expression of these MCF-7 cells was Salmon sperm DNA (Sigma-Aldrich) was used to measure the evaluated using a Xenogen IVIS-100 system (Caliper Life DNA loading capacity of cationic MBs as previous described [30]. Sciences). Briefly, 2 mg/ml salmon sperm DNA was prepared by dissolving a proper amount of DNA in deionized water using a bath sonicator. Cell viability assay An amount of 500 ml PEI600 MBs (resulting in 10 MBs/mL) was The cytotoxicities of PEI600 MBs with or without ultrasound slowly injected into 1 ml of DNA solution. The suspension was exposure were measured and compared with Plain MBs. 24 h after incubated by gentle rotation for 1 h to speed up the process of transfection, the cell viability was evaluated using Cell Counting DNA adsorption onto MBs; the uncoupled free DNA was then Kit-8 (Dojindo, Kumamoto, Japan) according to the manufactur- removed by centrifugation and washing thrice in a centrifugal tube er’s protocol. The absorbance at 450 nm was measured by using a (400 g, 3 min). After the residual DNA loaded MBs were collected, multimode plate reader (Synergy 4, BioTek). the zeta potentials and size distributions were measured as mentioned. Statistical analysis The MBs were then destroyed in a bath sonicator heated about Statistical analysis was performed using the two-tailed t-Test 65uC for several minutes until the suspension became transparent. method assuming unequal variances. A p value of ,0.05 was The concentration of DNA in the suspension was measured using considered to be statistically significant. a BioPhotometer (Eppendorf, Germany), basing on 1 OD (i.e. a solution having an absorbance of one unit at 260 nm with a path Results length of 1 cm) corresponds to a concentration of 50 mg/ml for double-stranded DNA. All the measurements were made in PEI modification and PEI600 MBs triplicate. To reduce the discrepancies caused by the varied size As schematically presented in Fig. 1, branched PEI600 (MW = 600 Da) was modified with stearic acid via acylation reaction at a distributions between samples, the DNA loading capacity of MBs were normalized by their total MB surface area. Assuming that molar ratio of 1:2. Hydrophobic stearic chains were introduced onto the branched PEI by CDI. The molecular structure of the MBs were all sphere shapes, and their total surface area of the 1 1 product was confirmed by H NMR in CDCl . H NMR analysis measured sample could be estimated by the summation of surface 3 indicates that 14% amino-groups of the PEI600 were acylated area of all MBs in all channels sized by Accusizer 780A. (Fig. 2). Each PEI600 molecule is connected with nearly two stearic chains and has a very similar structure with the Cell culture phospholipids used to fabricate the shell of MBs [31]. Human breast cancer MCF-7 cells were employed to evaluate As shown in Fig. 1, the modified PEI was incorporated into the gene transfer and expression. The cells were maintained in shell of the MBs by hydrophobic-hydrophilic interactions. Dulbecco’s Modified Eagle Medium (DMEM), supplemented with Fluorescence images confirm that the Stearic-PEI600 was partially 10% FBS and 1% penicillin-streptomycin solution and maintained embedded into the shell of the MBs (Fig. 3). The bright field image in a humidified atmosphere containing 5% CO at 37 uC. shows bright gas cores of the MBs surround by dark circular rings PLOS ONE | www.plosone.org 3 September 2013 | Volume 8 | Issue 9 | e76544 A New Cationic Microbubble as a Non-Viral Vector Figure 1. Illustration of the PEI modification by stearic acid for preparing of cationic microbubbles (MBs) to load DNA. The stearic acid modified polyethylenimine 600 (Stearic-PEI) polymer was synthesized. The resulting Stearic-PEI, combined with DSPC and DSPE-PEG2k, was used to fabricate the cationic MBs. The PEI endows the cationic MBs with more amino groups to couple DNA than plain MBs. doi:10.1371/journal.pone.0076544.g001 (Fig. 3A). Some of the MBs seem to be a bit fuzzy because they Concentration, size distribution of PEI600 MBs were out of the focus. The fluorescence image shows the opposite Fig. 4A shows that the concentration and size distribution of the pattern with dark cores and bright green shells (Fig. 3B). They resulting cationic MBs. The concentrations, the number of MBs demonstrate the FITC-labeled Stearic-PEI600 cationic MBs have per ml, of both freshly prepared MBs without wash and that been established. washed with 10 mM NaCl, gradually decreased with the increase of Stearic-PEI600 in cationic MB formulations. Compared with the concentration of 5 mol% PEI600 MBs, the concentration of 30 mol% PEI600 MBs had a nearly 90% decrease in MB Figure 2. H NMR spectrum of Stearic-PEI. (CDCl ), d (ppm) 0.86–0.89 (t, -CH CH (CH ) CH ), 1.25 (br, -CH CH (CH ) CH ), 1.62 (br, - 3 2 2 2 15 3 2 2 2 15 3 CH CH (CH ) CH ), 2.18 (br, -CH CH (CH ) CH ), 2.39–3.3 (m, -CH CH NH-, -CH CH N-, -CH CH NHCO-, -CH CH NHCO-). 2 2 2 9 3 2 2 2 9 3 2 2 2 2 2 2 2 2 doi:10.1371/journal.pone.0076544.g002 PLOS ONE | www.plosone.org 4 September 2013 | Volume 8 | Issue 9 | e76544 A New Cationic Microbubble as a Non-Viral Vector achieving decreases of 42.20 mV (P,0.01), 61.60 mV (P,0.01), 52.00 mV (P,0.01) and 53.50 mV (P,0.01) respectively. However, there was no significant decrease of the magnitude of surface potentials for plain MBs without Stearic-PEI600 (P . 0.05). Plasmid DNA binding onto PEI600 MBs In order to visually examine whether the PEI600 MBs could bind with plasmid DNA, Hoechst 33258 was used to stain plasmid DNA on the cationic MBs. The bright field image showed the Figure 3. The bright field and fluorescence images of FITC- shape of DNA-MB complexes were not affected by DNA-binding labelled Stearic-PEI600 cationic MBs. (A) Bright field image and (B) (Fig. 6A). The blue surface of the DNA-binding MBs observed fluorescent image of MBs containing 5% Stearic-PEI600. Scale bars: under fluorescent microscope (Fig. 6B), which indicated successful 10 mm. doi:10.1371/journal.pone.0076544.g003 adhesion of the negatively charged plasmids to the surface of cationic MBs. Agarose gel electrophoresis of cationic MB/DNA complexes at concentration (P, 0.01) for freshly prepared MBs and a 75% various MB/DNA ratios was presented in Fig. 6C. Reduced or no decrease for NaCl-washed MBs respectively. The size distributions migration of the DNA into the gel at $ 2.0610 MB/mg DNA of the freshly prepared and NaCl-washed MBs were presented in ratio indicated the complexes formation. 0.36 mg DNA was Fig. 4B and Fig. 4C. The submicron bubbles (size ,1 mm) completely captured by 2.0610 MBs, responding to the ratio dominated in the freshly prepared MBs when the amount of 5.5610 MBs coupled 1 mg plasmid DNA. Stearic-PEI600 used for the cationic MB fabrication was less than 20 mol%. The size distributions of NaCl-washed MBs showed a similar trend (Fig. 4C). The ratios of the submicron bubbles were DNA loading capacity of PEI600 MBs reduced from 68.3% to 27.7% for fleshly prepared MBs and from The DNA-loading capacity was quantitatively determined 45.9% to 20.8% for NaCL washed MBs with the increased through calculating the DNA density on the MBs according to amount of Stearic-PEI600 from 5% to 30%, which were in the previous publication [30,32]. From Fig. 7 we can see that the agreement with the previously reported results [30]. The mean DNA density on the MBs increased nearly linearly from 23 2 23 2 and median sizes of freshly prepared MBs and washed MBs are (560.2)610 pg/mm to (2061.8) 610 pg/mm when Stearic-PEI600 increased from 5 mol% Stearic-PEI600 to 30 shown in Fig. 4D and which show a gradually increase in the mean and median diameters of MBs with the increased amount of mol%. Since the average molecular weight of one DNA base pair Stearic-PEI600 from 0% to 30%. Moreover, NaCl-washed MBs is about 670 Daltons, a 2 mm 5% PEI600 MB would couple 7 7 0.0628 pg DNA, corresponding to about 6610 base pairs. 10 also have larger mean and median diameters than freshly prepared MBs (P,0.05). MBs would bind 0.68 mg DNA, which was in the same order of magnitude with the estimated data from gel electrophoresis. Zeta potential of the PEI600 MBs Ultrasound-mediated transfection in cell culture Zeta potential of the cationic MBs is significant for DNA binding through the electrostatic interactions. The detection of Transfection of MCF-7 cells using DNA/5% PEI600 MBs laser Doppler anemometry showed that Stearic-PEI600 had a complex plus ultrasound exposure yielded 5.7662.58610 p/s/ cm /sr average radiance intensity, was 8.97- and 7.53-fold higher significant effect on f-potential of MBs. Plain MBs without Stearic- PEI600 (0%) had a f-potential of –28.164.3 mV in deionized than those treated by plain MBs with ultrasound exposure 2 2 (6.4165.82610 p/s/cm /sr, P,0.01) and by PEI600 MBs water due to the presence of negatively charged phosphate groups 2 2 without ultrasound (7.6566.18610 , p/s/cm /sr, P,0.01), re- in the DSPE-PEG2000. Addition of 5%, 10%, 20% or 30% spectively. And the cells treated by adding PEI600 MBs and DNA Stearic-PEI600 during MB preparation resulted in a dramatic (without premix) with ultrasound yielded an average radiance increase of the MB surface f-potentials from negative to positive; 3 2 5.6762.46610 p/s/cm /sr, is was 8.84-fold higher than treated they reached 42.164.65 mV, 42.7065.34 mV, 54.3064.70 mV, by plain MBs and ultrasound exposure. The samples treated by 57.8067.41 mV for MBs in deionized water, respectively. It was ultrasound exposure with or without premixing PEI600 MBs and noted that the f-potentials of all kinds of MBs in 10 mM NaCl DNA made no difference in the plasmid DNA transfection decreased in comparison with those of MBs dispersed in deionized efficiency (Fig. 8). water (Fig. 5A). MBs without Stearic-PEI600 had a negative f- potential of –8.6164.26 mV when dispersed in 10 mM NaCl, with a 19.49 mV decrease of the magnitude of MB surface Cytotoxicities potential (P,0.01), compared with sample without Stearic- The cell viability was measured by using CCK-8 assay. The cells PEI600. Similarly, there were 26.3 mV (P,0.01), 15.4 mV treated with DNA/PEI600-MBs and DNA/plain MBs complexes (P,0.01), 35.7 mV (P,0.01) and 40 mV (P,0.01) decreases of under ultrasound exposure showed 59.566.1% and 71.467.1% the magnitude of f-potential for 5%, 10%, 20% and 30% PEI600 cell viability, respectively. No matter with and without ultrasound MBs, respectively. Fig. 5B showed the f-potentials of MBs coated exposure, the cytotoxicitiy of PEI600 MB was slightly larger than with the Stearic-PEI600 and that saturated by excess salmon plain MBs (P,0.01), and ultrasound exposure showed a strong sperm DNA. Interestingly, the addition of DNA into cationic MB toxicity in every groups (P,0.001). There were not significant suspension in 10 mM NaCl dramatically reversed the f-potentials differences in cell viability whether the DNA and PEI600MB were of MBs from positive to negative (Fig. 5B). when saturated by premixed or not (Fig. 9). Thus, it was indicated that the excess salmon sperm DNA, the surface potentials of 5%, 10%, cytotoxicity may be mainly caused by the MB cavition induced 20% or 30% Stearic-PEI600 cationic MBs were –26.4068.93 mV, by ultrasound exposure, however, the PEI600 MBs showed higher –34.3065.95 mV, –33.4066.49 mV and –35.7065.35 mV, cytotoxicity than plain MBs. PLOS ONE | www.plosone.org 5 September 2013 | Volume 8 | Issue 9 | e76544 A New Cationic Microbubble as a Non-Viral Vector Figure 4. Concentration and size distribution of fresh prepared and washed MBs. (A) Concentrations of freshly prepared and washed MBs with various amounts of Stearic-PEI600. (B) Size distribution of freshly prepared MBs with various amounts of Stearic-PEI600. (C) Size distributionof 10 mM NaCl washed MBs with various amounts of Stearic-PEI600. (D) Mean and median size of fresh prepared and 10 mM NaCl washed versus the Stearic-PEI600 fraction. The ‘‘freshly prepared’’ MBs were taken straight from the vial within 1 h of formation. The ‘‘washed’’ MBs were obtained by three cycles of flotation-centrifugation with infranatant exchange to remove the submicrometer bubbles. doi:10.1371/journal.pone.0076544.g004 Figure 5. The zeta potential of PEI600 MBs. (A) Zeta potential of MBs dispersed in deionized water and 10 mM NaCl. (B) Zeta potential of MBs binding with and without DNA. doi:10.1371/journal.pone.0076544.g005 PLOS ONE | www.plosone.org 6 September 2013 | Volume 8 | Issue 9 | e76544 A New Cationic Microbubble as a Non-Viral Vector Figure 6. Binding of plasmid DNA onto PEI600 MBs. (A) The bright field image and (B) the corresponding fluorescent image of plasmid DNA loaded MBs. (C) Agarose gel electrophoresis of 0.36 mg plasmid DNA mixed with different number of MBs with 5% Stearic-PEI600. doi:10.1371/journal.pone.0076544.g006 can increase effectiveness of DNA loading, but are often Discussion complicated and less-convenient during preparation. The latter Recently, most MB-mediated gene delivery experiments were also has some drawbacks. For example, the DNA loading capacity performed by co-injection of MBs and DNA vectors. In those of the MBs would easily be saturated at a low concentration level studies, circulating MBs were insonified to increase local vascular due to the limited surface area of MBs. Contrarily, in our study, permeability, allowing DNA vectors to passively extravasate into DNA vectors were attached to the surfaces of the PEI600-modified tissue. However, circulating genetic agents may also extravasate cationic MBs, forming MB-DNA hybrid vectors. In consideration into fenestrated organs (such as the lung, liver, and spleen), of its relatively low cytotoxicity compared with that of mostly used resulting in off-target effects [33]. Strategies such as DNA-loaded PEI 25000 [31], PEI600 was selected to fabricate the cationic polymer MBs using double-emulsion solvent evaporation method MBs. By attaching DNA onto the MB surface, their release can be (w /o/w ), layer-by-layer (LBL) assembly technique and DNA- 1 2 mediated by acoustic cavitation around the ultrasound focal zone, loaded liposome-MB complexes have been proposed to overcome thus providing more specific control in tissue-targeting applica- this problem [30,24,13]. Another strategy is to incorporate directly tions [34]. Furthermore, attachment of DNA to the MB surface cationic polymer such as DSTAP into the MB shell. The former has potentially provided some protection, preventing rapid clearance of DNA [21,35]. Several studies have demonstrated that DNA molecules loaded onto MBs improves their intracellular uptake in vitro [36,37] and deposition into target tissue in vivo [38]. Wang et al. showed that plasmid-binding cationic MBs had enhanced ultrasound-mediated gene delivery efficiency relative to neutral MBs in both cell culture and mouse hind limb tumors [21,39]. In the current study, we found that the MB size, concentration and size distributions were significantly impacted by the molar amount of Stearic-PEI600 in preparing the cationic MBs. Our results were in agreement with the previous reports. Borden et al. have reported the lateral phase separation in lipid-coated MBs [40]. The ordered domains in the shell of MBs are composed primarily of DSPC, while the disordered interdomain regions are mainly lipopolymer. Thus, increasing the cationic polymer concentration would generally result in disorder domains increase and condensed domains reduction. These disorder domains would increase the mass transfer, so that stability was weaken. In another study, Feshitan et al. found that higher concentration MBs, in Figure 7. DNA loading capacity vs Stearic-PEI600 (%). The relationship between DNA loading capacity (y) and Stearic-PEI600% (x) general, tended to be more stable, regardless of MB size [41]. can be fitted to a linear rewlation : y = 0.0566x+0.0013 with R = 0.9576. Surface charge of the cationic MBs indicated a significant Salmon sperm DNA surface concentrations were obtained by analyzing change in the surface of MBs after addition of the cationic polymer the 260 nm absorbance and corresponding total surface area of sized Stearic-PEI600 (Fig. 5A). This is mainly because that the ionic MBs. strength of the polyelectrolyte such as PEI is determined not only doi:10.1371/journal.pone.0076544.g007 PLOS ONE | www.plosone.org 7 September 2013 | Volume 8 | Issue 9 | e76544 A New Cationic Microbubble as a Non-Viral Vector Figure 8. DNA transfection in MCF-7 cells. (A) Optical images of luciferase gene expression. (B) Bar graph shows the luciferase activity in MCF-7 cells after ultrasound-mediated transfection of pCMV-Luc with complex of DNA/Plain MBs, complex of DNA/5% PEI600 MBs by premix DNA and MB for 15minutes, and with 5% PEI600 MB and DNA without premixing and added separately. pCMV-Luc activities were evaluated 24 h after gene transfection. doi:10.1371/journal.pone.0076544.g008 by the concentration of the polyelectrolyte itself, but also the ionic character of the suspending medium. Nomikou et al. concentration of small molecule electrolytes such as NaCl. The measured a lipid-shelled MB with a 8% molar ratio of DSTAP protonated amine group of the PEI is shielded to some extent by and described a zeta potential of approximately 4–5 mV in Opti- the counter-ions in the NaCl solution. It was demonstrated that DMEM [42], while Borden et al. described a MB zeta potential of the zeta potential was sensitive to both pH and the presence of Figure 9. Cell cytotoxicity assay. The cell viability was measured by using the CCK-8 assay after 24 h transfection of pCMV-Luc with with complex of DNA/Plain MBs, complex of DNA/5% PEI600 MBs by premix DNA and MB for 15minutes, and with 5% PEI600 MB and DNA without premixing and added respectively, with or without ultrasound exposure. doi:10.1371/journal.pone.0076544.g009 PLOS ONE | www.plosone.org 8 September 2013 | Volume 8 | Issue 9 | e76544 A New Cationic Microbubble as a Non-Viral Vector approximately 33 mV with a 20% molar ratio of DSTAP in ly, as for the UTMD strategy for DNA transfection, it seems that 10 mM NaCl, which was closed to our results [30]. the cell viability and transfection efficiency are contradictory. For It is notable that a significant improvement of DNA loading example, transfection efficiency increased approximately linearly capacity was achieved in our new designed cationic MBs. The with MB concentration, but cell viability inversely correlated with improvement of DNA loading capacity may contribute to the MB concentration. The relationship between acoustic intensity abundant amino group in PEI molecules. Each PEI600 molecule and transfection efficiency was highly nonlinear. Clearly, the goal has approximately 14 nitrogen atoms, and at most 12 residual of this technology is to obtain high transfection efficiency and low nitrogen atoms of which could be protonated to adsorb DNA after cell death. However, the acoustic intensity at site, MB concentra- being modified by two stearic acid molecules. So, ideally, the tion and pulsing sequence may need to be carefully adjusted to maximum DNA loading capacity should be ten folds more than obtain an optimal conditions, considering the tradeoffs between that of the traditional cationic MBs which consist of the same the transfection efficiency and cell viability [12]. molar ratio of polymers such as DSTAP. Herein, DNA loading capacity of PEI600 MB we measured is about five times higher Conclusions than that of the reported DSTAP MBs, which may be because of the steric hindrance. Borden et al. applied a layer-by-layer (LBL) We have developed a cationic MB by modifying PEI with assembly technique to adsorb multiple layers of DNA and poly-L- stearic acid and coating it onto the shell of MBs. The addition of lysine (PLL) onto lipid-coated MBs [30]. The DNA loading the modified cationic polymer may affect the yield and size capacity was enhanced by over 10-fold by using five paired layers. distribution of the MBs by forming a disorder domain in the shell Nevertheless, LBL assembly technique is somewhat complex as the of the MBs. Also, the buffer used to dilute and wash the MBs will fragility of the MBs. affect both the size distribution and the zeta potential of the MBs It has been proved that DNA could be effectively protected due to the shielding effect of the small molecular electrolyte. The from degradation by coupling them to the cationic MBs via plasmid DNA can be effectively coupled onto the surface of the electrostatic interactions [35,43]. In recent studies, the gene cationic MBs. Additionally, our results demonstrated an increasing transfer by using UTMD with injecting MBs and DNA/PEI DNA-loading capacity with increase of the Stearic-PEI600. DNA complex simultaneously have shown that UTMD could improve transfection of MCF-7 cells using the Stearic-PEI600 MBs and the gene transfection efficiency of DNA/PEI complex in vitro and ultrasound is significantly higher than that of treatment with plain in vivo [17,44]. Sirsi et al. also reported that DNA/PEI-MBs could MBs with ultrasound, PEI600 MBs without ultrasound respec- transfect tumor tissue in a site-specific manner by virtue of tively. And premix the PEI600 MBs with DNA or not doesn’t ultrasound. In their study, the branched polyethylenimine (PEI) affect the transfection efficiency significantly in vitro. Our study was first modified with polyethylene glycol and hydrosulfide group, may have laid down a foundation for image-guided gene therapy and then covalently attached to the maleimide groups on the shell which we plan to explore in future. of the MBs that contain a functionalized PEG lipid. Thus, DNA could be adsorbed by the PEI coated MBs. Acknowledgments In this study, the DNA transfection was carried out under the We thank to Fei Li, Chengzhi Zeng for advices and comments regarding following conditions: frequency = 1.2 MHz, the acoustic pressure setup of ultrasound apparatus and parameters, Qian Wan for technical amplitude = 0.6 MPa, a 120-cycle sinusoidal tone-burst with 1 assistance and support with using of the Xenogen IVIS-100 system. kHz pulse repetition frequency, MB concentration = 1610 MBs/ml, DNA concentration = 2 mg/ml. In fact, the UTMD for DNA transfection in vitro and in vivo has been studied Author Contributions in similar conditions. It has been demonstrated that the DNA Conceived and designed the experiments: QJ FY HZ. Performed the transfection efficiency depends on multiple factors, such as experiments: QJ ZW FN ZD. Analyzed the data: QJ ZW FY XL HZ. experimental systems, MB concentration, DNA concentration, Contributed reagents/materials/analysis tools: ZW FY HZ. Wrote the acoustic intensity and pulse sequence, etc. [45,46,47]. Interesting- paper: QJ FY JW RS. References 1. Manno CS, Arruda VR, Pierce GF, Glader B, Ragni M, et al. (2006) Successful 8. Ulasov I, Hsu P-H, Wei K-C, Huang C-Y, Wen C-J, et al. (2013) Noninvasive transduction of liver in hemophilia by AAV-Factor IX and limitations imposed and Targeted Gene Delivery into the Brain Using Microbubble-Facilitated by the host immune response (vol 12, pg 342, 2006). Nature Medicine 12: 592– Focused Ultrasound. Plos One 8: e57682. 592. 9. Wu J, Nyborg WL (2008) Ultrasound, cavitation bubbles and their interaction with cells. 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