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Biomedical Applications
Anionic Lipid, pH-Sensitive Liposome-Gold Nanoparticle Hybrids for Gene Delivery – Quantitative Research of the Mechanism Baoji Du, Li Tian, Xiaoxiao Gu, Dan Li,* Erkang Wang,* and Jin Wang*
Gene therapy is a potential method for treating a large range of diseases. Gene vectors are widely used in gene therapy for promoting the gene delivery efficiency to the target cells. Here, gold nanoparticles (AuNPs) coated with dimethyldioctadecylammonium bromide (DODAB)/dioleoylphosphatidylethanolamine (DOPE) are synthesized using a facile method for a new gene vector (DODAB/DOPE-AuNPs), which possess 3- and 1.5-fold higher transfection efficiency than those of DODABAuNPs and a commercial transfection agent, respectively. Meanwhile, it is nontoxic with concentrations required for effective gene delivery. Imaging and quantification studies of cellular uptake reveal that DOPE increases gene copies in cells, which may be attributed to the smaller size of AuNPs/DNA complexes. The dissociation efficiency of DNA from the endocytic pathway is quantified by incubating with different buffers and investigated directly in the cells. The results suggest that DOPE increases the internalization of AuNPs/DNA complexes and promotes DNA release from early endosomes for the vector is sensitive to the anionic lipid membrane and the decreasing pH along the endocytic pathway. The new vector contains the potential to be the new alternative as gene delivery vector for biomedical applications.
Dr. B. Du, Dr. L. Tian, X. Gu, Prof. D. Li, Prof. E. Wang, Prof. J. Wang State Key Laboratory of Electroanalytical Chemistry Changchun Institute of Applied Chemistry Changchun, Jilin 130022, China E-mail: [email protected]; [email protected]; [email protected] Dr. B. Du University of Chinese Academy of Sciences Beijing 100039, China Prof. J. Wang College of Physics Jilin University Changchun, Jilin 130012, China Prof. J. Wang Department of Chemistry and Physics State University of New York at Stony Brook, Stony Brook, New York, 11794–3400, USA DOI: 10.1002/smll.201402470 small 2015, DOI: 10.1002/smll.201402470
1. Introduction Gene therapy is a potential method in treating cancers and genetic diseases by repairing the damaged DNA or inserting new functional genes into the living cells.[1,2] It is hard for negatively charged genes to traverse the negative cell membrane efficiently without the help of gene delivery vectors, such as nonviral[3–8] and viral vectors.[9–12] Considering that the viral vectors are unsafe for human and limited in DNA package,[13] nonviral vectors have been studied rapidly.[14–17] Cationic lipid-based gene vector is one of the most successful nonviral vectors[18–21] and some kinds of cationic lipids have been commercialized and applied for gene therapy. However, the lower stability and transfection efficiency hinder them to be used widely in clinical application.[14] With the development of nanotechnology, some nanomaterials including gold nanoparticles (AuNPs), quantum dots
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(QDs), superparamagnetic iron oxides (SPIOs) as well as viral-nanoparticles have been hybridized with bilayer lipids for gene delivery in order to increase the efficiency or controllability of gene delivery.[22–26] We have also synthesized AuNPs coated by dimethyldioctadecylammonium bromide (DODAB) served as the gene vector (DODAB-AuNPs),[23] which could significantly increase the transfection efficiency and decrease the cytotoxic effect of DODAB to the cells. To further increase the transfection efficiency and to meet with the desired features of gene vectors, such as good biocompatibility, high gene loading capability, terrific gene protective capability as well as sensitive and effective gene release ability,[27–29] a neutral lipid, dioleoylphosphatidylethanolamine (DOPE) was added to synthesize a new gene vector (DODAB/DOPE-AuNPs). DOPE, with unsaturated long tails, has been used as a helper lipid for conventional liposome formation.[30–32] However, the function of DOPE after being directly coated on nanoparticles has not yet been studied. Whether the addition of DOPE will affect the interaction performance with the genes and the release efficiency of bound nucleic acid from the endocytic pathway are worthy to be studied. When using green fluorescent protein (GFP) and luciferase as the reporters, the transfection efficiency of DODAB/ DOPE (1:1)-AuNPs was found to be 3- and 200-fold higher than that of DODAB-AuNPs, respectively. The function of DOPE was systematically studied by comparing the size, zeta potential, cellular internalization of AuNPs/DNA complexes, and DNA release efficiency from the endocytic pathway, and the results demonstrated that the excellent internalization and DNA release capability were the key factors for the higher transfection efficiency of DODAB/DOPE (1:1)AuNPs. It is expected that the new vector will be more suitable for the future biomedical application.
2. Results and Discussion 2.1. Preparation of DODAB/DOPE-AuNPs for Gene Transfection Cationic lipids mixing with various proportions of DOPE exhibit different performances in gene delivery efficiency.[3,33,34] Different DODAB/DOPE-AuNPs were thus prepared by using the mixtures of DODAB and DOPE with molar ratios of 3:1, 2:1, 1:1, 1:2, and 1:3.[23] Results from the confocal laser scanning microscopy (CLSM) and the flow cytometry (FCM) simultaneously demonstrated the highest transfection efficiency was obtained at the molar ratio of 1:1 (Figure 1). The transfection efficiency of DODAB/DOPE (1:1)-AuNPs was over 30% (Figure 1a), which was 3- and 1.5-fold higher than that of DODAB-AuNPs and Lipotap (a commercial gene vector). Moreover, the transfection efficiency of each vector was further investigated using another plasmid (pGL3-Control) expressing the luciferase as the reporter protein, which was relatively sensitive in determining the expression of exogenous genes in each transfected cell.[35] As shown in Figure 1b, the relative luciferase activity (RLU/mg protein) of DODAB/DOPE (1:1)-AuNPs was 1.2 × 108, which was 200-fold higher than that of DODAB-AuNPs (5.8 × 105). Around the best ratio of AuNPs to DNA, the cells transfected with DODAB/DOPE (1:1)-AuNPs possessed higher GFP expression in no more than 24 h (Figure S1, Supporting Information), much less than that of DODAB-AuNPs, which was nearly 48 h to obtain the highest expression.[36] In addition, compared with DODAB/DOPE alone or Lipotap, DODAB/DOPE (1:1)-AuNPs achieved the most quick and highest gene expression after being transfected for 12, 24, and 48 h. The above results indicated that DODAB/DOPE (1:1)AuNPs as a gene vector was very effective.
Figure 1. Transfection efficiency measurement. The CLSM images of HEK 293 cells transfected with pEGFP-N1 and DODAB-AuNPs A); DODAB/DOPE (3:1)-AuNPs B); DODAB/DOPE (2:1)-AuNPs C); DODAB/DOPE (1:1)-AuNPs D); DODAB/DOPE (1:2)-AuNPs E); DODAB/DOPE (1:3)-AuNPs F); and Lipotap G). Proportions of fluorescent cells in above samples quantified by FCM (a). Transfection efficiency measured by luciferase activity assay (b). Intensity of the chemiluminescence was normalized to the amount of the total protein. Error bars represented standard deviations (SD) for at least three independent experiments. Data were shown as mean ± SD. Scale bar was 300 µm.
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www.MaterialsViews.com Table 1. Zeta potentials, sizes (measured by DLS), and polydispersity indexes (PDIs) of DODAB-AuNPs and DODAB/DOPE (1:1)-AuNPs before and after being complexed with DNA. Vectors/complexes
Zeta potential [mV]
Size [nm]
PDI
DODAB-AuNPs
+35.8 ± 1.2
22.7 ± 3.6
0.384
DODAB/DOPE (1:1)-AuNPs
+49.8 ± 0.8
12.4 ± 2.5
0.434
DODAB-AuNPs/DNA complex
+26.7 ± 1.8
556.7 ± 13.8a)
0.306a)
DODAB/DOPE (1:1)-AuNPs/DNA complex
+34.9 ± 0.2
135.4 ± 9.2
0.213
a)
Size of DODAB-AuNPs/DNA complexes in the suspension.
2.2. Characterization of the AuNPs and Measurement of DNA Loading Capability Higher gene delivery efficiency is one of the most important standards for choosing a gene vector. So we selected the DODAB/DOPE (1:1)-AuNPs as the optimized vector and DODAB-AuNPs as a contrast to investigate the effects of DOPE on gene delivery. As shown in Figure S2 (Supporting Information), the UV–vis absorption peak of DODAB/ DOPE (1:1)-AuNPs (535 nm, B) was slightly blueshift compared with that of DODAB-AuNPs (540 nm, A), which indicated the smaller size of the former nanoparticles.[37] The transmission electron microscopy (TEM) images confirmed that the average diameter of DODAB/DOPE (1:1)-AuNPs (E, 6.2 ± 1.3 nm) was smaller than that of DODAB-AuNPs (B, 14.5 ± 4.2 nm). Dynamic light scattering (DLS) data suggested that the average diameters of DODAB-AuNPs and DODAB/DOPE (1:1)-AuNPs were 22.7 ± 3.6 and 12.4 ± 2.5 nm (Figure S2C and F), respectively. The larger sizes measured by DLS than those analyzed by TEM might be attributed to DODAB or DODAB/DOPE lipid coated on the AuNPs.[38] The zeta potentials of DODAB-AuNPs and DODAB/DOPE (1:1)-AuNPs were +35.8 ± 1.2 and +49.8 ± 0.8 mV, respectively (Table 1). The higher zeta potential of DODAB/DOPE (1:1)-AuNPs was beneficial to interact with negatively charged DNA.[39] As seen in Figure S3 (Supporting Information), there was no significant redshift in the absorption peak of the AuNPs after 4 h incubation in either phosphate buffer saline (PBS) or 10% fetal bovine serum (FBS) solution compared with ddH2O.[40] The broadening of the absorption peak of DODAB-AuNPs in PBS was much more obvious than that of DODAB/DOPE (1:1)-AuNPs, which indicated that the addition of DOPE avoided the aggregation of the AuNPs in PBS.[41] The above results demonstrated that DOPE decreased the size, increased the zeta potential, and improved the stability of the gold nanoparticles, which made them more suitable to be used as a transfection vector. The best ratio of DODAB/DOPE (1:1)-AuNPs to DNA can easily be investigated by electrophoretic mobility shift assay (EMSA).[42] As the results shown in Figure S4 (Supporting Information), the best weight ratios of DODAB/ DOPE (1:1)-AuNPs and DODAB-AuNPs to DNA were 2.5:1 and 5:1, respectively, which indicated that the loading capability of DODAB/DOPE (1:1)-AuNPs was twofold higher than that of DODAB-AuNPs. The better DNA loading capability might be attributed to the higher zeta potential of DODAB/DOPE (1:1)-AuNPs.[39] The high DNA small 2015, DOI: 10.1002/smll.201402470
loading capability can decrease the number of vectors in need for the same quantity of nucleic acids, and the reduction of vectors will decrease the toxicity to cells. The protection of DNA from being degraded by exonuclease after forming complexes with DODAB-AuNPs and DODAB/DOPE (1:1)AuNPs was also studied. As shown in Figure S5 (Supporting Information), DNA had been protected well by both of them and did not be hydrolyzed when exposed in the exonuclease solution.
2.3. Transfection In Vivo and Cytotoxicity Measurement The ultimate purpose of studying gene delivery is to be used in clinical applications. So we investigated the gene transfection efficiency of the above vectors in vivo. We injected AuNPs/DNA or Naked DNA (control) into the posterior tibialis muscles of six-week-old female BALB/c mice.[43] After 72 h, we observed the green fluorescence on the cryostat section of the muscle by CLSM. Similar to the in vitro result, the fluorescence intensity of muscles transfected with DODAB/DOPE (1:1)-AuNPs/DNA (Figure 2C) was the highest, and quantitative research showed that GFP expression was about twofold higher than that of DODAB-AuNPs (Figure 2D). Such result proved that the DODAB/DOPE (1:1)-AuNPs had better performance in vivo. The cytotoxicity of the AuNPs vectors had also been measured by MTT assay (Figure 2E) and the relatively lower cytotoxicity of DODAB/ DOPE (1:1)-AuNPs used in the transfection for the same quantity of DNA indicated that it was a better alternative for gene delivery.
2.4. Quantitative Researching the Cellular Uptake of AuNPs/ DNA Complexes In general, the cellular uptake efficiency of vector/DNA complexes is critical to the transfection efficiency. Allogeneic materials will take different accesses to enter eukaryotic cells,[2] depending on cell type and the nature of materials. The main cellular uptake pathways include phagocytosis, macropinocytosis, clathrin-dependent, caveolae-dependent, clathrin- and caveolae-independent endocytosis.[44] To figure out which pathway is involved in the internalization of AuNPs/DNA complexes, we first incubated groups of cells with AuNPs/DNA at 4 or 37 °C. As shown in Figure 3A, the cell uptake for the two vectors were both significantly
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of clathrin-coated vesicles and stop the clathrin-mediated pathway.[45] The quantitative result measured by FCM showed that MβCD but not sucrose strongly prevented the transverse of FITC-DNA (Figure 3A), which indicated that both of the AuNPs/DNA complexes were internalized through the caveolae-mediated pathway. As shown in Figure 3B, the entry of FITC-DNA carried by DODAB/DOPE (1:1)-AuNPs was two times higher than that by DODAB-AuNPs. This result was also confirmed by the TEM image of cells after being incubated with AuNPs/DNA for 7 h. As shown in Figure 3C–-F, the number of AuNPs/DNA complexes in cells treated by DODAB/DOPE (1:1)-AuNPs (Figure 3E, F) was far more than those treated by DODAB-AuNPs (Figure 3C, D). In order to study the reason of difFigure 2. EGFP expression in mouse muscles 3 d after intramuscular injections. CLSM images of naked DNA A), DODAB-AuNPs/DNA B), and DODAB/DOPE (1:1)-AuNPs/DNA C). ferent internalization ability for these EGFP expression level analyzed by quantifying the fluorescence in the CLSM images using the two vectors, we characterized both comImage J software D). Indicated values were mean ± SD of at least four experiments. Cytotoxicity plexes. It was found that the two vectors of AuNPs complexed with equal quantity of DNA to HEK 293 cells E). Scale bar was 100 µm. showed different physical appearance after being mixed with DNA. The soluinhibited at 4 °C, which indicated the internalization was tion of DODAB/DOPE (1:1)-AuNPs/DNA complexes was an energy- and receptor-dependent endocytosis. Then, we more homogeneous and clearer with red color (Figure 4b). pretreated the cells with methyl-β-cyclodextrin (MβCD) or However, the DODAB-AuNPs/DNA complexes seemed to sucrose.[45] It is known that MβCD will deplete or inhibit the flock together, and the aggregations of which were visible to synthesis of cholesterol and repress the caveolae-mediated naked-eye with purple color (Figure 4a). The CLSM images pathway.[46] In contrast, sucrose will disrupt the formation of the above samples also proved that the DODAB/DOPE (1:1)-AuNPs/DNA was well-distributed (Figure 4B) compared to DODABAuNPs/DNA (Figure 4A). The UV–vis data showed the absorption peaks of both AuNPs were broadened after complexed with DNA, which indicated the formation of AuNPs/DNA complexes (Figure 4C). However, the aggregation level of DODAB/DOPE (1:1)-AuNPs was much lower than that of DODAB-AuNPs, which was consistent with the optical and fluorescent images of the AuNPs/DNA complexes. Furthermore, as shown in the result of DLS measurement, the average sizes of DODAB/DOPE (1:1)-AuNPs/ DNA and DODAB-AuNPs/DNA (in the suspension) were 135.4 ± 9.2 and 556.7 ± 13.8 nm, respectively (Table 1). It is known that caveolae-mediated internalization is a size-dependent pathway.[4] The smaller and more homogeneous DODAB/DOPE Figure 3. Percentage of fluorescent cells after transfected with AuNPs/DNA complexes for (1:1)-AuNPs/DNA complexes would pos4 h A). Cells cultured at 37 °C (control), 4 °C or pretreated with MβCD and sucrose. The sess the higher cellular uptake efficiency,[4] fluorescence intensity per HEK 293 cell after incubated with AuNPs/FITC-DNA for 4 h or 7 h which led to the more effective internaliB). TEM images of HEK 293 cells after incubated with DODAB-AuNPs/DNA C, D) or DODAB/ DOPE (1:1)-AuNPs/-DNA E, F) for 7 h. Panels D) and F) showed the higher magnification of the zation of complexes. Zeta potential can be used to charrectangle area in panels C) and E), respectively. Arrows in each panel pointed to the AuNPs/ acterize the surface charge density of DNA complexes. Bars indicated 200 nm.
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DNA release. So the following timedependent DNA release assays were processed in PB buffer of different pH containing 0.175 mM SDS (Figure S6B–D, Supporting Information). As shown in Figure 5, the released DNA increased as time goes on (30, 60, and 120 min) in the same PB buffer, and the DNA released from DODAB/DOPE (1:1)-AuNPs/DNA was always more than that from DODABAuNPs/DNA complexes, especially in PB of pH 6.0 and 5.0. It seemed there was no significant difference in the DNA release efficiency from DODAB/DOPE (1:1)AuNPs/DNA complexes in pH 6.0 and 5.0 PB buffers, which indicated that the genes could be released from the early endosomes (pH 6.0–6.5) during transfecFigure 4. Optical and CLSM images of DODAB-AuNPs/DNA a, A) and DODAB/DOPE (1:1)- tion, avoiding being digested by hydrolytic AuNPs/DNA complexes b, B). UV-vis spectra of DODAB-AuNPs and DODAB/DOPE (1:1)-AuNPs enzymes in the lysosomes (pH 4.5–5.5). before and after complexed with DNA C). Scale bar was 5 µm. As a result, the transfection efficiency of DODAB/DOPE (1:1)-AuNPs was higher. vectors/DNA complexes, which is very critical to the transDNA release capability in cells was also investigated. As fection efficiency.[47] The zeta potential of DODAB-AuNPs/ shown in Figure 6A, the release efficiency (RE) of DNA was DNA and DODAB/DOPE (1:1)-AuNPs/DNA were +26.7 ± quantified through dividing the FITC fluorescence that is 1.8 and +34.9 ± 0.2 mV, respectively (Table 1). The higher zeta not co-locating with the lysosome (red) by the whole FITC potential of DODAB/DOPE (1:1)-AuNPs/DNA would fluorescence in the cell (Figure S7, Supporting Information). increase their electrostatic attraction to cell surfaces, which As shown in Figure 6B, the average DNA release efficiency might promote the internalization. from DODAB/DOPE (1:1)-AuNPs/DNA was about 1.5-fold higher than that from DODAB-AuNPs/DNA, which was consistent with the measurement in above PB buffers. All 2.5. Quantitative Investigation of DNA Release from AuNPs/ the data indicated that DODAB/DOPE (1:1)-AuNPs show DNA Complex in Phosphate Buffer (PB) and Cells excellent release capability and the superior performance might be related to the special structure: inverted hexagonal Another factor involved in the gene delivery efficiency is the (HII) of DOPE, which could make the complexes destabilize DNA release from the endocytic pathway to the cells. Many and promote DNA release when meeting with endosomal methods have been applied to promote the DNA release, membrane.[3,32,52] It was found that the relative ratios of the internalizasuch as light-,[5,48,49] pH-,[50] and micromolecule-dependent release.[15] DOPE is sensitive to anionic lipids,[18] so we tion and the release of DNA for DODAB/DOPE (1:1)expect that it can promote DNA release when meeting with AuNPs/DNA to DODAB-AuNPs/DNA were about 2 endosomal vesicles in cells. Considering that the intracellular (Figure 3B) and 1.5 (Figure 6B), respectively. The product environment along the endocytic pathway changes from pH of the first two terms was just about 3, which was in 6.0–6.5 (early endosomes) to pH 4.5–5.5 (late endosomes accordance with the enhancement ratio of the GFP expresand lysosomes),[51] we imitated DNA release in different pH (7.4, 6.0 and 5.0) before and after meeting with anionic micelles. We used an anionic lipid, sodium dodecyl sulfate (SDS) as a substitute for endosomal vesicles.[18] First, we tested the DNA disassociation capability from the AuNPs/DNA complexes in different concentrations of SDS at pH = 7.4. Quantitative measurement results from the EMSA image showed that DNA release efficiency of DODAB/DOPE (1:1)-AuNPs was higher than that of DODAB-AuNPs at each SDS concentration (Figure S6A, Supporting Information) and 0.175 mM Figure 5. Quantified measurement of DNA released from AuNPs/DNA complexes induced SDS was almost enough for the abundant by 0.175 mM SDS for 10, 30, 60, and 120 min in pH 5.0, 6.0, and 7.4 PB. small 2015, DOI: 10.1002/smll.201402470
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hybrids (Scheme 1). Meanwhile, it also confirmed that the data of our study on internalization and release of DNA were reliable.
3. Conclusion In summary, we synthesized DODAB/ DOPE-AuNPs as a new gene vector. When the molar ratio of DODAB and DOPE was 1:1, the gene delivery efficiency was much higher than pure DODAB-AuNPs and a similar result was achieved in vivo. Our following experiment gave the reasons of such excellent performance: (I) smaller size and higher zeta potential of the DODAB/ DOPE (1:1)-AuNPs donated the higher DNA loading; (II) homogeneity, smaller size, and higher zeta potential of the DODAB/DOPE (1:1)-AuNPs/DNA complexes resulted in higher internalization; and (III) sensitivity to the anion in endosomal membrane and the decreasing pH along the endocytic pathway resulted in fast and effective DNA release to Figure 6. CLSM images showed the distribution of FITC-DNA in HEK 293 cells after incubated the cytoplasm. Moreover, AuNPs and with DODAB-AuNPs/FITC-DNA or DODAB/DOPE (1:1)-AuNPs/FITC-DNA for 7 h A). LysoTracker Red DODAB/DOPE complemented each channel showed the lysosomes of the cell and the green channel showed the FITC-DNA. DNA other. AuNPs could decrease the size release efficiency of the two vectors quantified by Image J software B). Scale bar was 20 µm. of DODAB/DOPE liposome, while the sion (Figure 1). These results proved that the internaliza- DODAB/DOPE gave AuNPs the ability of gene delivery. tion and release efficiency were the two principal factors Such method and mechanism can be used to guide the for tuning the transfection efficiency for AuNPs-liposome design of new gene vectors.
Scheme 1. Comparison of DODAB/DOPE (1:1)-AuNPs/DNA (smaller complex) and DODAB-AuNPs/DNA (bigger complex) internalized into HEK 293 cells (spotted rectangle area) and DNA released from the AuNPs/DNA complexes (spotted circle area) during the endocytic pathway.
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4. Experimental Section Materials: Dimethyldioctadecylammonium bromide (DODAB), NaBH4, and HAuCl4 were purchased from Sigma–Aldrich (USA). 2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) was purchased from Shanghai Advanced Vehicle Technology, China. Plasmid DNA pEGFP-N1 (Clontech, Mountain View, CA, USA) and pGL3-Control (Promega Corp. USA) were purified by Purification System (Promega Corp. USA). Lipotap, a cationic liposome transfection agent bought from Beyotime Institute of Biotechnology (China). Fluorescein isothiocyanate (FITC) labeled DNA was synthesized by TaKaRa Biotechnology Co., Ltd. (Dalian, China), the sequence of which is FITC-5’-ATGGTGAGCAAGGGCGAGGAGCTGTTCAC-3’. The line-EGFP gene was cloned by PCR reaction using the pEGFP-N1 plasmid as a template.[28] The primer sequence pairs were 5’-CCCCTGATTCTGTGGATAACCG-3’ and 5’-CGCCTTAAGATACATTGATGAGTTTGG-3’. Human embryonic kidney 293 cells (HEK 293 cells) were obtained from Kunming Institute of Zoology, Chinese Academy of Sciences. BCA protein assay system was obtained from Pierce (France). Six weeks old female BALB/c mice were purchased from the Animal Centre of Jilin University, and Animal handling was in accordance with the guidelines of the Animal Care and Use Committee of Jilin University. Synthesis and Characterization of AuNPs: DODAB-AuNPs were synthesized according to the reported method with modifications.[23] First, totally 3.0 mmol DODAB or DODAB/DOPE at each molar ratio (3:1, 2:1, 1:1, 1:2, 1:3) was first mixed in chloroform. Mixtures were dried by blowing nitrogen and then by vacuum for 2 h. Sterile Milli-Q water (20 mL) was added and the liposome solutions were prepared by sonication at 50 °C. HAuCl4 aqueous solution (62.5 µL, 0.1 M) was added into the above as-prepared vesicle solution under vigorous stirring. Then, a freshly prepared NaBH4 aqueous solution (67 µL, 0.4 M) was added. The reaction was maintained for 1 h at room temperature by stirring. The AuNPs solution was condensed and purified by centrifugation at 15 000 × g for 10 min at for two times. The AuNPs were characterized by UV-vis-NIR spectrophotometer (CARY 500). The TEM images were obtained from a Hitachi H-8100 TEM. The zeta-potential and the size of the AuNPs were measured by a Malvern Zetasizer Nano ZS (United Kingdom).[53] Cell Culture and Transfection: HEK 293 cells were cultured in DMEM medium, containing 10% FBS at 37 °C in a 5% CO2 incubator. Ten thousand cells were seeded per well in 96-well tissue culture plate and the cells were grown about 60%–70% confluence on the day of transfection. The medium was changed by fresh DMEM with 10% FBS, AuNPs or Lipotap and plasmid DNA (200 ng) complexes at a nitrogen/phosphate (N/P) ratio of 1.0 were added and incubated with the cells for 12, 24, or 48 h. The fluorescence photographs were taken using a CLSM (Leica TCS sp2) with an argon laser source at 488 nm and emission was collected between 500 and 550 nm. The gene delivery efficiency was measured by FCM (FACSAria, BD Biosciences). The gene delivery efficiency measured by luciferase assay (Promega, USA) either according to the user manual and the luminescence intensity was measured by BERTHOLD Centro XS3 LB 960. Luciferase activity was normalized to the protein content of each sample measured by Pierce BCA assay (Pierce, France). Transfection in Vivo and MTT Assay: AuNPs and 5 µg of pEGFPN1 complexed with AuNPs or naked DNA alone were injected to small 2015, DOI: 10.1002/smll.201402470
the posterior tibialis muscles of six-week-old female BALB/c mice. Three days later, the muscles were isolated and embedded with Optimal Cutting Temperature (OCT) compound (Sakura Finetek U. S. A) and froze at −70 °C. Frozen sections of 5 µm thick were cut by Cryotome E (Thermo Scientific, USA). The fluorescence of EGFP was observed by CLSM and analyzed by Image J software (National Institute of Health, USA). MTT assay was done according to the published method.[36] HEK 293 cells were plated in a 96-well plate with 10 000 cells in each well. After 12 h, the AuNPs/DNA complex with the N/P ratio of 1:1 was added to the medium. The final concentrations of DNA in each well were 0.5, 1.0, 1.5, 2.0, and 2.5 ng/µL. DNA Loading Capability Measurement: DOAB-AuNPs or DODAB/DOPE-AuNPs were mixed with DNA separately at room temperature for 15 min and then performed agarose gel electrophoresis.[23] The weight ratios of DODAB-AuNPs and DODAB/DOPEAuNPs to DNA were 2.5:1, 5:1, 7.5:1, and 10:1 and 1.25:1, 2.5:1, 5:1, and 7.5:1, respectively. AuNPs/DNA Complexes Characterization: The AuNPs and DNA were mixed with the N/P ratio of 1:1. The mixture was first photographed using a camera, and then added onto a 35 mm glass bottom dish (NEST Biotechnology, China) and taken photos by CLSM with the excitation at 488 nm and emission at 510–530 nm. The UV-vis spectra, zeta-potentials and DLS of the AuNPs were all measured. Considering that there were already visible sediments in sample of DDAB-AuNPs/DNA complexes, so we tested the complexes in the suspension by DLS measurement. AuNPs/DNA Cellular Uptake Pathway and Internalization Efficiency Measurement: The FCM was used to assess the cellular uptake by counting the cells internalized by the FITC-DNA. To inhibit clathrin or caveolae-dependent endocytosis, the cells were pre-incubated with 0.45 M sucrose or 10 mM methyl-β-cyclodextrin (Sigma) for 30 min, respectively.[45] Then the AuNPs/FITC-DNA (200 ng) complexes were added to the medium and incubated at 37 °C for 4 h or 7 h. After that, the cells were trypsinized and washed with PBS for three times before the fluorescence intensity of FITC was measured by FCM with 488 nm excitation. For each experiment, the fluorescence of 10 000 single cells were measured and the mean ± SD of three independent experiments was calculated. Time-Dependent DNA Release Measurement by Incubating in SDS and PB Buffer of Different pH: Line-EGFP was firstly mixed with the desired amount of AuNPs with NP ratio of 1:1. After incubation at room temperature for 15 min, the mixtures were incubated with SDS with the concentration of 0.0875. 0.175, 0.35 and 0.7 mM before being loaded into a 2.0% agarose gel. The gel was allowed to run for 15 min at 150 V (7.5 v/cm) in Tris-acetate-EDTA (TAE) buffer. After that, the gel was photographed under UV light using a Vilber lourmat fluorescence imaging system. Time- and pH-dependent DNA release was done by incubating AuNPs/DNA complexes with 0.175 mM SDS in different pH (5.0, 6.0 and 7.4) PB (0.1 m) for 10, 30, 60 and 120 min before the agarose gel electrophoresis. Intracellular AuNPs/DNA Complexes Distribution Study by CLSM and TEM: HEK-293 cells were seeded on a 35 mm glass bottom dish with the concentration of 1 × 105 cells/well. AuNPs/DNA complexes were added to the cell culture and incubated with the cells for 6 h. Then, 50 pM LysoTracker Red (Molecular Probes, Eugene, OR, U.S.A.) was added to the medium and incubated at 37 °C for another 1 h. The cells were observed by CLSM and thereafter quantitatively analyzed. The Image J 1.45s software was used to
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quantify the total areas of free FITC-DNA, which was not associated with lysosomes in an individual cell. DNA dissociation efficiency was defined as the ratio of total fluorescence of free FITC-DNA to the total of green fluorescence of the cell.[54] TEM was also used to study the intracellular location of the vector/DNA complexes, after being transfected for 7 h according to the published method.[36]
Supporting Information Supporting Information is available from the Wiley Online Library or from the author.
Acknowledgements This work was supported by the National Natural Science Foundation of China (Nos. 21190040, 91227114 and 31301177), State Key Instrument Developing Special Project of Ministry of Science and Technology of China (2012YQ17000303), and the Instrument Developing Project of the Chinese Academy of Sciences (YZ201203). J.W. appreciated NSF.
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Received: August 18, 2014 Revised: November 13, 2014 Published online:
small 2015, DOI: 10.1002/smll.201402470
Biomedical Applications
Anionic Lipid, pH-Sensitive Liposome-Gold Nanoparticle Hybrids for Gene Delivery – Quantitative Research of the Mechanism Baoji Du, Li Tian, Xiaoxiao Gu, Dan Li,* Erkang Wang,* and Jin Wang*
Gene therapy is a potential method for treating a large range of diseases. Gene vectors are widely used in gene therapy for promoting the gene delivery efficiency to the target cells. Here, gold nanoparticles (AuNPs) coated with dimethyldioctadecylammonium bromide (DODAB)/dioleoylphosphatidylethanolamine (DOPE) are synthesized using a facile method for a new gene vector (DODAB/DOPE-AuNPs), which possess 3- and 1.5-fold higher transfection efficiency than those of DODABAuNPs and a commercial transfection agent, respectively. Meanwhile, it is nontoxic with concentrations required for effective gene delivery. Imaging and quantification studies of cellular uptake reveal that DOPE increases gene copies in cells, which may be attributed to the smaller size of AuNPs/DNA complexes. The dissociation efficiency of DNA from the endocytic pathway is quantified by incubating with different buffers and investigated directly in the cells. The results suggest that DOPE increases the internalization of AuNPs/DNA complexes and promotes DNA release from early endosomes for the vector is sensitive to the anionic lipid membrane and the decreasing pH along the endocytic pathway. The new vector contains the potential to be the new alternative as gene delivery vector for biomedical applications.
Dr. B. Du, Dr. L. Tian, X. Gu, Prof. D. Li, Prof. E. Wang, Prof. J. Wang State Key Laboratory of Electroanalytical Chemistry Changchun Institute of Applied Chemistry Changchun, Jilin 130022, China E-mail: [email protected]; [email protected]; [email protected] Dr. B. Du University of Chinese Academy of Sciences Beijing 100039, China Prof. J. Wang College of Physics Jilin University Changchun, Jilin 130012, China Prof. J. Wang Department of Chemistry and Physics State University of New York at Stony Brook, Stony Brook, New York, 11794–3400, USA DOI: 10.1002/smll.201402470 small 2015, DOI: 10.1002/smll.201402470
1. Introduction Gene therapy is a potential method in treating cancers and genetic diseases by repairing the damaged DNA or inserting new functional genes into the living cells.[1,2] It is hard for negatively charged genes to traverse the negative cell membrane efficiently without the help of gene delivery vectors, such as nonviral[3–8] and viral vectors.[9–12] Considering that the viral vectors are unsafe for human and limited in DNA package,[13] nonviral vectors have been studied rapidly.[14–17] Cationic lipid-based gene vector is one of the most successful nonviral vectors[18–21] and some kinds of cationic lipids have been commercialized and applied for gene therapy. However, the lower stability and transfection efficiency hinder them to be used widely in clinical application.[14] With the development of nanotechnology, some nanomaterials including gold nanoparticles (AuNPs), quantum dots
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(QDs), superparamagnetic iron oxides (SPIOs) as well as viral-nanoparticles have been hybridized with bilayer lipids for gene delivery in order to increase the efficiency or controllability of gene delivery.[22–26] We have also synthesized AuNPs coated by dimethyldioctadecylammonium bromide (DODAB) served as the gene vector (DODAB-AuNPs),[23] which could significantly increase the transfection efficiency and decrease the cytotoxic effect of DODAB to the cells. To further increase the transfection efficiency and to meet with the desired features of gene vectors, such as good biocompatibility, high gene loading capability, terrific gene protective capability as well as sensitive and effective gene release ability,[27–29] a neutral lipid, dioleoylphosphatidylethanolamine (DOPE) was added to synthesize a new gene vector (DODAB/DOPE-AuNPs). DOPE, with unsaturated long tails, has been used as a helper lipid for conventional liposome formation.[30–32] However, the function of DOPE after being directly coated on nanoparticles has not yet been studied. Whether the addition of DOPE will affect the interaction performance with the genes and the release efficiency of bound nucleic acid from the endocytic pathway are worthy to be studied. When using green fluorescent protein (GFP) and luciferase as the reporters, the transfection efficiency of DODAB/ DOPE (1:1)-AuNPs was found to be 3- and 200-fold higher than that of DODAB-AuNPs, respectively. The function of DOPE was systematically studied by comparing the size, zeta potential, cellular internalization of AuNPs/DNA complexes, and DNA release efficiency from the endocytic pathway, and the results demonstrated that the excellent internalization and DNA release capability were the key factors for the higher transfection efficiency of DODAB/DOPE (1:1)AuNPs. It is expected that the new vector will be more suitable for the future biomedical application.
2. Results and Discussion 2.1. Preparation of DODAB/DOPE-AuNPs for Gene Transfection Cationic lipids mixing with various proportions of DOPE exhibit different performances in gene delivery efficiency.[3,33,34] Different DODAB/DOPE-AuNPs were thus prepared by using the mixtures of DODAB and DOPE with molar ratios of 3:1, 2:1, 1:1, 1:2, and 1:3.[23] Results from the confocal laser scanning microscopy (CLSM) and the flow cytometry (FCM) simultaneously demonstrated the highest transfection efficiency was obtained at the molar ratio of 1:1 (Figure 1). The transfection efficiency of DODAB/DOPE (1:1)-AuNPs was over 30% (Figure 1a), which was 3- and 1.5-fold higher than that of DODAB-AuNPs and Lipotap (a commercial gene vector). Moreover, the transfection efficiency of each vector was further investigated using another plasmid (pGL3-Control) expressing the luciferase as the reporter protein, which was relatively sensitive in determining the expression of exogenous genes in each transfected cell.[35] As shown in Figure 1b, the relative luciferase activity (RLU/mg protein) of DODAB/DOPE (1:1)-AuNPs was 1.2 × 108, which was 200-fold higher than that of DODAB-AuNPs (5.8 × 105). Around the best ratio of AuNPs to DNA, the cells transfected with DODAB/DOPE (1:1)-AuNPs possessed higher GFP expression in no more than 24 h (Figure S1, Supporting Information), much less than that of DODAB-AuNPs, which was nearly 48 h to obtain the highest expression.[36] In addition, compared with DODAB/DOPE alone or Lipotap, DODAB/DOPE (1:1)-AuNPs achieved the most quick and highest gene expression after being transfected for 12, 24, and 48 h. The above results indicated that DODAB/DOPE (1:1)AuNPs as a gene vector was very effective.
Figure 1. Transfection efficiency measurement. The CLSM images of HEK 293 cells transfected with pEGFP-N1 and DODAB-AuNPs A); DODAB/DOPE (3:1)-AuNPs B); DODAB/DOPE (2:1)-AuNPs C); DODAB/DOPE (1:1)-AuNPs D); DODAB/DOPE (1:2)-AuNPs E); DODAB/DOPE (1:3)-AuNPs F); and Lipotap G). Proportions of fluorescent cells in above samples quantified by FCM (a). Transfection efficiency measured by luciferase activity assay (b). Intensity of the chemiluminescence was normalized to the amount of the total protein. Error bars represented standard deviations (SD) for at least three independent experiments. Data were shown as mean ± SD. Scale bar was 300 µm.
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www.MaterialsViews.com Table 1. Zeta potentials, sizes (measured by DLS), and polydispersity indexes (PDIs) of DODAB-AuNPs and DODAB/DOPE (1:1)-AuNPs before and after being complexed with DNA. Vectors/complexes
Zeta potential [mV]
Size [nm]
PDI
DODAB-AuNPs
+35.8 ± 1.2
22.7 ± 3.6
0.384
DODAB/DOPE (1:1)-AuNPs
+49.8 ± 0.8
12.4 ± 2.5
0.434
DODAB-AuNPs/DNA complex
+26.7 ± 1.8
556.7 ± 13.8a)
0.306a)
DODAB/DOPE (1:1)-AuNPs/DNA complex
+34.9 ± 0.2
135.4 ± 9.2
0.213
a)
Size of DODAB-AuNPs/DNA complexes in the suspension.
2.2. Characterization of the AuNPs and Measurement of DNA Loading Capability Higher gene delivery efficiency is one of the most important standards for choosing a gene vector. So we selected the DODAB/DOPE (1:1)-AuNPs as the optimized vector and DODAB-AuNPs as a contrast to investigate the effects of DOPE on gene delivery. As shown in Figure S2 (Supporting Information), the UV–vis absorption peak of DODAB/ DOPE (1:1)-AuNPs (535 nm, B) was slightly blueshift compared with that of DODAB-AuNPs (540 nm, A), which indicated the smaller size of the former nanoparticles.[37] The transmission electron microscopy (TEM) images confirmed that the average diameter of DODAB/DOPE (1:1)-AuNPs (E, 6.2 ± 1.3 nm) was smaller than that of DODAB-AuNPs (B, 14.5 ± 4.2 nm). Dynamic light scattering (DLS) data suggested that the average diameters of DODAB-AuNPs and DODAB/DOPE (1:1)-AuNPs were 22.7 ± 3.6 and 12.4 ± 2.5 nm (Figure S2C and F), respectively. The larger sizes measured by DLS than those analyzed by TEM might be attributed to DODAB or DODAB/DOPE lipid coated on the AuNPs.[38] The zeta potentials of DODAB-AuNPs and DODAB/DOPE (1:1)-AuNPs were +35.8 ± 1.2 and +49.8 ± 0.8 mV, respectively (Table 1). The higher zeta potential of DODAB/DOPE (1:1)-AuNPs was beneficial to interact with negatively charged DNA.[39] As seen in Figure S3 (Supporting Information), there was no significant redshift in the absorption peak of the AuNPs after 4 h incubation in either phosphate buffer saline (PBS) or 10% fetal bovine serum (FBS) solution compared with ddH2O.[40] The broadening of the absorption peak of DODAB-AuNPs in PBS was much more obvious than that of DODAB/DOPE (1:1)-AuNPs, which indicated that the addition of DOPE avoided the aggregation of the AuNPs in PBS.[41] The above results demonstrated that DOPE decreased the size, increased the zeta potential, and improved the stability of the gold nanoparticles, which made them more suitable to be used as a transfection vector. The best ratio of DODAB/DOPE (1:1)-AuNPs to DNA can easily be investigated by electrophoretic mobility shift assay (EMSA).[42] As the results shown in Figure S4 (Supporting Information), the best weight ratios of DODAB/ DOPE (1:1)-AuNPs and DODAB-AuNPs to DNA were 2.5:1 and 5:1, respectively, which indicated that the loading capability of DODAB/DOPE (1:1)-AuNPs was twofold higher than that of DODAB-AuNPs. The better DNA loading capability might be attributed to the higher zeta potential of DODAB/DOPE (1:1)-AuNPs.[39] The high DNA small 2015, DOI: 10.1002/smll.201402470
loading capability can decrease the number of vectors in need for the same quantity of nucleic acids, and the reduction of vectors will decrease the toxicity to cells. The protection of DNA from being degraded by exonuclease after forming complexes with DODAB-AuNPs and DODAB/DOPE (1:1)AuNPs was also studied. As shown in Figure S5 (Supporting Information), DNA had been protected well by both of them and did not be hydrolyzed when exposed in the exonuclease solution.
2.3. Transfection In Vivo and Cytotoxicity Measurement The ultimate purpose of studying gene delivery is to be used in clinical applications. So we investigated the gene transfection efficiency of the above vectors in vivo. We injected AuNPs/DNA or Naked DNA (control) into the posterior tibialis muscles of six-week-old female BALB/c mice.[43] After 72 h, we observed the green fluorescence on the cryostat section of the muscle by CLSM. Similar to the in vitro result, the fluorescence intensity of muscles transfected with DODAB/DOPE (1:1)-AuNPs/DNA (Figure 2C) was the highest, and quantitative research showed that GFP expression was about twofold higher than that of DODAB-AuNPs (Figure 2D). Such result proved that the DODAB/DOPE (1:1)-AuNPs had better performance in vivo. The cytotoxicity of the AuNPs vectors had also been measured by MTT assay (Figure 2E) and the relatively lower cytotoxicity of DODAB/ DOPE (1:1)-AuNPs used in the transfection for the same quantity of DNA indicated that it was a better alternative for gene delivery.
2.4. Quantitative Researching the Cellular Uptake of AuNPs/ DNA Complexes In general, the cellular uptake efficiency of vector/DNA complexes is critical to the transfection efficiency. Allogeneic materials will take different accesses to enter eukaryotic cells,[2] depending on cell type and the nature of materials. The main cellular uptake pathways include phagocytosis, macropinocytosis, clathrin-dependent, caveolae-dependent, clathrin- and caveolae-independent endocytosis.[44] To figure out which pathway is involved in the internalization of AuNPs/DNA complexes, we first incubated groups of cells with AuNPs/DNA at 4 or 37 °C. As shown in Figure 3A, the cell uptake for the two vectors were both significantly
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of clathrin-coated vesicles and stop the clathrin-mediated pathway.[45] The quantitative result measured by FCM showed that MβCD but not sucrose strongly prevented the transverse of FITC-DNA (Figure 3A), which indicated that both of the AuNPs/DNA complexes were internalized through the caveolae-mediated pathway. As shown in Figure 3B, the entry of FITC-DNA carried by DODAB/DOPE (1:1)-AuNPs was two times higher than that by DODAB-AuNPs. This result was also confirmed by the TEM image of cells after being incubated with AuNPs/DNA for 7 h. As shown in Figure 3C–-F, the number of AuNPs/DNA complexes in cells treated by DODAB/DOPE (1:1)-AuNPs (Figure 3E, F) was far more than those treated by DODAB-AuNPs (Figure 3C, D). In order to study the reason of difFigure 2. EGFP expression in mouse muscles 3 d after intramuscular injections. CLSM images of naked DNA A), DODAB-AuNPs/DNA B), and DODAB/DOPE (1:1)-AuNPs/DNA C). ferent internalization ability for these EGFP expression level analyzed by quantifying the fluorescence in the CLSM images using the two vectors, we characterized both comImage J software D). Indicated values were mean ± SD of at least four experiments. Cytotoxicity plexes. It was found that the two vectors of AuNPs complexed with equal quantity of DNA to HEK 293 cells E). Scale bar was 100 µm. showed different physical appearance after being mixed with DNA. The soluinhibited at 4 °C, which indicated the internalization was tion of DODAB/DOPE (1:1)-AuNPs/DNA complexes was an energy- and receptor-dependent endocytosis. Then, we more homogeneous and clearer with red color (Figure 4b). pretreated the cells with methyl-β-cyclodextrin (MβCD) or However, the DODAB-AuNPs/DNA complexes seemed to sucrose.[45] It is known that MβCD will deplete or inhibit the flock together, and the aggregations of which were visible to synthesis of cholesterol and repress the caveolae-mediated naked-eye with purple color (Figure 4a). The CLSM images pathway.[46] In contrast, sucrose will disrupt the formation of the above samples also proved that the DODAB/DOPE (1:1)-AuNPs/DNA was well-distributed (Figure 4B) compared to DODABAuNPs/DNA (Figure 4A). The UV–vis data showed the absorption peaks of both AuNPs were broadened after complexed with DNA, which indicated the formation of AuNPs/DNA complexes (Figure 4C). However, the aggregation level of DODAB/DOPE (1:1)-AuNPs was much lower than that of DODAB-AuNPs, which was consistent with the optical and fluorescent images of the AuNPs/DNA complexes. Furthermore, as shown in the result of DLS measurement, the average sizes of DODAB/DOPE (1:1)-AuNPs/ DNA and DODAB-AuNPs/DNA (in the suspension) were 135.4 ± 9.2 and 556.7 ± 13.8 nm, respectively (Table 1). It is known that caveolae-mediated internalization is a size-dependent pathway.[4] The smaller and more homogeneous DODAB/DOPE Figure 3. Percentage of fluorescent cells after transfected with AuNPs/DNA complexes for (1:1)-AuNPs/DNA complexes would pos4 h A). Cells cultured at 37 °C (control), 4 °C or pretreated with MβCD and sucrose. The sess the higher cellular uptake efficiency,[4] fluorescence intensity per HEK 293 cell after incubated with AuNPs/FITC-DNA for 4 h or 7 h which led to the more effective internaliB). TEM images of HEK 293 cells after incubated with DODAB-AuNPs/DNA C, D) or DODAB/ DOPE (1:1)-AuNPs/-DNA E, F) for 7 h. Panels D) and F) showed the higher magnification of the zation of complexes. Zeta potential can be used to charrectangle area in panels C) and E), respectively. Arrows in each panel pointed to the AuNPs/ acterize the surface charge density of DNA complexes. Bars indicated 200 nm.
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DNA release. So the following timedependent DNA release assays were processed in PB buffer of different pH containing 0.175 mM SDS (Figure S6B–D, Supporting Information). As shown in Figure 5, the released DNA increased as time goes on (30, 60, and 120 min) in the same PB buffer, and the DNA released from DODAB/DOPE (1:1)-AuNPs/DNA was always more than that from DODABAuNPs/DNA complexes, especially in PB of pH 6.0 and 5.0. It seemed there was no significant difference in the DNA release efficiency from DODAB/DOPE (1:1)AuNPs/DNA complexes in pH 6.0 and 5.0 PB buffers, which indicated that the genes could be released from the early endosomes (pH 6.0–6.5) during transfecFigure 4. Optical and CLSM images of DODAB-AuNPs/DNA a, A) and DODAB/DOPE (1:1)- tion, avoiding being digested by hydrolytic AuNPs/DNA complexes b, B). UV-vis spectra of DODAB-AuNPs and DODAB/DOPE (1:1)-AuNPs enzymes in the lysosomes (pH 4.5–5.5). before and after complexed with DNA C). Scale bar was 5 µm. As a result, the transfection efficiency of DODAB/DOPE (1:1)-AuNPs was higher. vectors/DNA complexes, which is very critical to the transDNA release capability in cells was also investigated. As fection efficiency.[47] The zeta potential of DODAB-AuNPs/ shown in Figure 6A, the release efficiency (RE) of DNA was DNA and DODAB/DOPE (1:1)-AuNPs/DNA were +26.7 ± quantified through dividing the FITC fluorescence that is 1.8 and +34.9 ± 0.2 mV, respectively (Table 1). The higher zeta not co-locating with the lysosome (red) by the whole FITC potential of DODAB/DOPE (1:1)-AuNPs/DNA would fluorescence in the cell (Figure S7, Supporting Information). increase their electrostatic attraction to cell surfaces, which As shown in Figure 6B, the average DNA release efficiency might promote the internalization. from DODAB/DOPE (1:1)-AuNPs/DNA was about 1.5-fold higher than that from DODAB-AuNPs/DNA, which was consistent with the measurement in above PB buffers. All 2.5. Quantitative Investigation of DNA Release from AuNPs/ the data indicated that DODAB/DOPE (1:1)-AuNPs show DNA Complex in Phosphate Buffer (PB) and Cells excellent release capability and the superior performance might be related to the special structure: inverted hexagonal Another factor involved in the gene delivery efficiency is the (HII) of DOPE, which could make the complexes destabilize DNA release from the endocytic pathway to the cells. Many and promote DNA release when meeting with endosomal methods have been applied to promote the DNA release, membrane.[3,32,52] It was found that the relative ratios of the internalizasuch as light-,[5,48,49] pH-,[50] and micromolecule-dependent release.[15] DOPE is sensitive to anionic lipids,[18] so we tion and the release of DNA for DODAB/DOPE (1:1)expect that it can promote DNA release when meeting with AuNPs/DNA to DODAB-AuNPs/DNA were about 2 endosomal vesicles in cells. Considering that the intracellular (Figure 3B) and 1.5 (Figure 6B), respectively. The product environment along the endocytic pathway changes from pH of the first two terms was just about 3, which was in 6.0–6.5 (early endosomes) to pH 4.5–5.5 (late endosomes accordance with the enhancement ratio of the GFP expresand lysosomes),[51] we imitated DNA release in different pH (7.4, 6.0 and 5.0) before and after meeting with anionic micelles. We used an anionic lipid, sodium dodecyl sulfate (SDS) as a substitute for endosomal vesicles.[18] First, we tested the DNA disassociation capability from the AuNPs/DNA complexes in different concentrations of SDS at pH = 7.4. Quantitative measurement results from the EMSA image showed that DNA release efficiency of DODAB/DOPE (1:1)-AuNPs was higher than that of DODAB-AuNPs at each SDS concentration (Figure S6A, Supporting Information) and 0.175 mM Figure 5. Quantified measurement of DNA released from AuNPs/DNA complexes induced SDS was almost enough for the abundant by 0.175 mM SDS for 10, 30, 60, and 120 min in pH 5.0, 6.0, and 7.4 PB. small 2015, DOI: 10.1002/smll.201402470
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hybrids (Scheme 1). Meanwhile, it also confirmed that the data of our study on internalization and release of DNA were reliable.
3. Conclusion In summary, we synthesized DODAB/ DOPE-AuNPs as a new gene vector. When the molar ratio of DODAB and DOPE was 1:1, the gene delivery efficiency was much higher than pure DODAB-AuNPs and a similar result was achieved in vivo. Our following experiment gave the reasons of such excellent performance: (I) smaller size and higher zeta potential of the DODAB/ DOPE (1:1)-AuNPs donated the higher DNA loading; (II) homogeneity, smaller size, and higher zeta potential of the DODAB/DOPE (1:1)-AuNPs/DNA complexes resulted in higher internalization; and (III) sensitivity to the anion in endosomal membrane and the decreasing pH along the endocytic pathway resulted in fast and effective DNA release to Figure 6. CLSM images showed the distribution of FITC-DNA in HEK 293 cells after incubated the cytoplasm. Moreover, AuNPs and with DODAB-AuNPs/FITC-DNA or DODAB/DOPE (1:1)-AuNPs/FITC-DNA for 7 h A). LysoTracker Red DODAB/DOPE complemented each channel showed the lysosomes of the cell and the green channel showed the FITC-DNA. DNA other. AuNPs could decrease the size release efficiency of the two vectors quantified by Image J software B). Scale bar was 20 µm. of DODAB/DOPE liposome, while the sion (Figure 1). These results proved that the internaliza- DODAB/DOPE gave AuNPs the ability of gene delivery. tion and release efficiency were the two principal factors Such method and mechanism can be used to guide the for tuning the transfection efficiency for AuNPs-liposome design of new gene vectors.
Scheme 1. Comparison of DODAB/DOPE (1:1)-AuNPs/DNA (smaller complex) and DODAB-AuNPs/DNA (bigger complex) internalized into HEK 293 cells (spotted rectangle area) and DNA released from the AuNPs/DNA complexes (spotted circle area) during the endocytic pathway.
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4. Experimental Section Materials: Dimethyldioctadecylammonium bromide (DODAB), NaBH4, and HAuCl4 were purchased from Sigma–Aldrich (USA). 2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) was purchased from Shanghai Advanced Vehicle Technology, China. Plasmid DNA pEGFP-N1 (Clontech, Mountain View, CA, USA) and pGL3-Control (Promega Corp. USA) were purified by Purification System (Promega Corp. USA). Lipotap, a cationic liposome transfection agent bought from Beyotime Institute of Biotechnology (China). Fluorescein isothiocyanate (FITC) labeled DNA was synthesized by TaKaRa Biotechnology Co., Ltd. (Dalian, China), the sequence of which is FITC-5’-ATGGTGAGCAAGGGCGAGGAGCTGTTCAC-3’. The line-EGFP gene was cloned by PCR reaction using the pEGFP-N1 plasmid as a template.[28] The primer sequence pairs were 5’-CCCCTGATTCTGTGGATAACCG-3’ and 5’-CGCCTTAAGATACATTGATGAGTTTGG-3’. Human embryonic kidney 293 cells (HEK 293 cells) were obtained from Kunming Institute of Zoology, Chinese Academy of Sciences. BCA protein assay system was obtained from Pierce (France). Six weeks old female BALB/c mice were purchased from the Animal Centre of Jilin University, and Animal handling was in accordance with the guidelines of the Animal Care and Use Committee of Jilin University. Synthesis and Characterization of AuNPs: DODAB-AuNPs were synthesized according to the reported method with modifications.[23] First, totally 3.0 mmol DODAB or DODAB/DOPE at each molar ratio (3:1, 2:1, 1:1, 1:2, 1:3) was first mixed in chloroform. Mixtures were dried by blowing nitrogen and then by vacuum for 2 h. Sterile Milli-Q water (20 mL) was added and the liposome solutions were prepared by sonication at 50 °C. HAuCl4 aqueous solution (62.5 µL, 0.1 M) was added into the above as-prepared vesicle solution under vigorous stirring. Then, a freshly prepared NaBH4 aqueous solution (67 µL, 0.4 M) was added. The reaction was maintained for 1 h at room temperature by stirring. The AuNPs solution was condensed and purified by centrifugation at 15 000 × g for 10 min at for two times. The AuNPs were characterized by UV-vis-NIR spectrophotometer (CARY 500). The TEM images were obtained from a Hitachi H-8100 TEM. The zeta-potential and the size of the AuNPs were measured by a Malvern Zetasizer Nano ZS (United Kingdom).[53] Cell Culture and Transfection: HEK 293 cells were cultured in DMEM medium, containing 10% FBS at 37 °C in a 5% CO2 incubator. Ten thousand cells were seeded per well in 96-well tissue culture plate and the cells were grown about 60%–70% confluence on the day of transfection. The medium was changed by fresh DMEM with 10% FBS, AuNPs or Lipotap and plasmid DNA (200 ng) complexes at a nitrogen/phosphate (N/P) ratio of 1.0 were added and incubated with the cells for 12, 24, or 48 h. The fluorescence photographs were taken using a CLSM (Leica TCS sp2) with an argon laser source at 488 nm and emission was collected between 500 and 550 nm. The gene delivery efficiency was measured by FCM (FACSAria, BD Biosciences). The gene delivery efficiency measured by luciferase assay (Promega, USA) either according to the user manual and the luminescence intensity was measured by BERTHOLD Centro XS3 LB 960. Luciferase activity was normalized to the protein content of each sample measured by Pierce BCA assay (Pierce, France). Transfection in Vivo and MTT Assay: AuNPs and 5 µg of pEGFPN1 complexed with AuNPs or naked DNA alone were injected to small 2015, DOI: 10.1002/smll.201402470
the posterior tibialis muscles of six-week-old female BALB/c mice. Three days later, the muscles were isolated and embedded with Optimal Cutting Temperature (OCT) compound (Sakura Finetek U. S. A) and froze at −70 °C. Frozen sections of 5 µm thick were cut by Cryotome E (Thermo Scientific, USA). The fluorescence of EGFP was observed by CLSM and analyzed by Image J software (National Institute of Health, USA). MTT assay was done according to the published method.[36] HEK 293 cells were plated in a 96-well plate with 10 000 cells in each well. After 12 h, the AuNPs/DNA complex with the N/P ratio of 1:1 was added to the medium. The final concentrations of DNA in each well were 0.5, 1.0, 1.5, 2.0, and 2.5 ng/µL. DNA Loading Capability Measurement: DOAB-AuNPs or DODAB/DOPE-AuNPs were mixed with DNA separately at room temperature for 15 min and then performed agarose gel electrophoresis.[23] The weight ratios of DODAB-AuNPs and DODAB/DOPEAuNPs to DNA were 2.5:1, 5:1, 7.5:1, and 10:1 and 1.25:1, 2.5:1, 5:1, and 7.5:1, respectively. AuNPs/DNA Complexes Characterization: The AuNPs and DNA were mixed with the N/P ratio of 1:1. The mixture was first photographed using a camera, and then added onto a 35 mm glass bottom dish (NEST Biotechnology, China) and taken photos by CLSM with the excitation at 488 nm and emission at 510–530 nm. The UV-vis spectra, zeta-potentials and DLS of the AuNPs were all measured. Considering that there were already visible sediments in sample of DDAB-AuNPs/DNA complexes, so we tested the complexes in the suspension by DLS measurement. AuNPs/DNA Cellular Uptake Pathway and Internalization Efficiency Measurement: The FCM was used to assess the cellular uptake by counting the cells internalized by the FITC-DNA. To inhibit clathrin or caveolae-dependent endocytosis, the cells were pre-incubated with 0.45 M sucrose or 10 mM methyl-β-cyclodextrin (Sigma) for 30 min, respectively.[45] Then the AuNPs/FITC-DNA (200 ng) complexes were added to the medium and incubated at 37 °C for 4 h or 7 h. After that, the cells were trypsinized and washed with PBS for three times before the fluorescence intensity of FITC was measured by FCM with 488 nm excitation. For each experiment, the fluorescence of 10 000 single cells were measured and the mean ± SD of three independent experiments was calculated. Time-Dependent DNA Release Measurement by Incubating in SDS and PB Buffer of Different pH: Line-EGFP was firstly mixed with the desired amount of AuNPs with NP ratio of 1:1. After incubation at room temperature for 15 min, the mixtures were incubated with SDS with the concentration of 0.0875. 0.175, 0.35 and 0.7 mM before being loaded into a 2.0% agarose gel. The gel was allowed to run for 15 min at 150 V (7.5 v/cm) in Tris-acetate-EDTA (TAE) buffer. After that, the gel was photographed under UV light using a Vilber lourmat fluorescence imaging system. Time- and pH-dependent DNA release was done by incubating AuNPs/DNA complexes with 0.175 mM SDS in different pH (5.0, 6.0 and 7.4) PB (0.1 m) for 10, 30, 60 and 120 min before the agarose gel electrophoresis. Intracellular AuNPs/DNA Complexes Distribution Study by CLSM and TEM: HEK-293 cells were seeded on a 35 mm glass bottom dish with the concentration of 1 × 105 cells/well. AuNPs/DNA complexes were added to the cell culture and incubated with the cells for 6 h. Then, 50 pM LysoTracker Red (Molecular Probes, Eugene, OR, U.S.A.) was added to the medium and incubated at 37 °C for another 1 h. The cells were observed by CLSM and thereafter quantitatively analyzed. The Image J 1.45s software was used to
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quantify the total areas of free FITC-DNA, which was not associated with lysosomes in an individual cell. DNA dissociation efficiency was defined as the ratio of total fluorescence of free FITC-DNA to the total of green fluorescence of the cell.[54] TEM was also used to study the intracellular location of the vector/DNA complexes, after being transfected for 7 h according to the published method.[36]
Supporting Information Supporting Information is available from the Wiley Online Library or from the author.
Acknowledgements This work was supported by the National Natural Science Foundation of China (Nos. 21190040, 91227114 and 31301177), State Key Instrument Developing Special Project of Ministry of Science and Technology of China (2012YQ17000303), and the Instrument Developing Project of the Chinese Academy of Sciences (YZ201203). J.W. appreciated NSF.
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Received: August 18, 2014 Revised: November 13, 2014 Published online:
small 2015, DOI: 10.1002/smll.201402470
