A Versatile Method for Encapsulating Large-Sized DNA into Small

Mar 21, 2014 - The cloud point of the polymer solutions were measured using a Beckman DU 640 UV spectrophotometer equipped with a digital temperature ...
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A Versatile Method for Encapsulating Large-Sized DNA into SmallSized Bioreducible Nanocapsules Long-Hai Wang,† Sheng-Gang Ding,‡ Jun-Jie Yan,† and Ye-Zi You*,† †

CAS Key Lab of Soft Matter Chemistry, Department of Polymer Science and Engineering, University of Science and Technology of China, Hefei 230026, Anhui, People’s Republic of China ‡ Department of Pediatrics, The First Affiliated Hospital of Anhui Medical University, Hefei, Anhui 230022, People’s Republic of China

ABSTRACT: Encapsulation of negatively charged plasmid DNA into a small-sized nanocapsule without using any condensing agent is very challenging up to now. Here we report a versatile method for encapsulating large-sized plasmid DNAs into smallsized bioreducible nanocapsules in which shearing force and surfactant can fold large-sized plasmid DNAs into small-sized emulsion droplets containing bioreducible branched polymers. Subsequently, temperature triggers the bioreducible branched polymers to aggregate and cross-link at the water/oil interface of the emulsion nanodroplet, forming a bioreducible shell around the nanodroplet. Thus, a small-sized nanocapsule (∼110 nm) containing large-sized plasmid DNA (∼1900 nm long) forms by removal of the surfactant.

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maximize the rate of cell uptake.14,15 For polymeric nanoparticles, it has been found that ∼100 nm-sized particles have a 2.3-fold greater cell uptake compared with 50 nm-sized particles, ∼1.8 fold greater compared with 1000 nm-sized particles.17 At the present time, though DNA can be encapsulated into capsules, most of these capsules are among 1−5 μm; therefore it is very difficult to encapsulate DNA into small-sized capsules.9,11,13 Schroeder et al. succeed in encapsulating DNA with 4 kb into vesicles with size of ∼200 nm or above, but they failed to encapsulate DNA with 4 kb into ∼100 nm-sized nanocapsules.13 It is generally believed that the spontaneous coiling of the negatively charged DNA to fit the dimensions of the nanocapsule (∼100 nm) is inefficient without using a condensing agent.13 Though viruses can easily compact the negatively charged DNA into their small-sized procapsids shell, encapsulation of DNA into small-sized manmade nanocapsules without using any condensing agent has been very challenging up to now. Recently, it has been found that the emulsion droplets can exert sufficient force to bend the nanofilaments into nanorings;21 intuitively, one might deduce that shearing force and emulsion droplets may exert sufficient force to fold large-sized DNA to fit the size of the emulsion droplets during the formation of the nanoemulsion droplet. Here, we report a new method for easily encapsulating largesized plasmid DNA into small-sized man-made bioreducible nanocapsules without using any condensing agent in which

INTRODUCTION DNA is an important molecule with many applications in gene therapy, diagnostics, and molecular evolution. The major challenge for the effective and efficient use of DNA in these areas is to prevent its degradation.1 In biosystem, viruses can assemble empty procapsids with a size of ∼100 nm into which the large-sized DNA can be subsequently packaged. This DNA packaging process compacts the highly charged DNA to a density similar to that of crystalline DNA. This packaging is not spontaneous but is driven into the shell by a translocating motor powered by ATP hydrolysis.2 Different from the biosystem, in artificial systems DNA degradation is generally prevented via condensing DNA with polycations,1 cationic lipids,3−6 or confining DNA within nanogel,7 polymeric microparticles,8 and nano- or microcapsules.9,10 Recently, many researchers devoted their effort to mimic a virus by encapsulating DNA into a bioreducible nanocapsule.1,11,12 For example, Shchukin et al. developed an approach for encapsulating DNA inside polyelectrolyte microshell (with a size of ∼4 μm) retaining the natural double-helix structure of DNA via layer-by-layer method,11 Caruso et al. encapsulated DNA into degradable polymer microcapsules (∼1 μm) via layer-by-layer method,9,10 Anderson et al. encapsulated DNA with 4 kb (∼1360 nm long) into lipid vesicles.13 It has been proposed that the size of particles plays a key role in the particle’s adhesion and interaction with the biological cells.14−16 Creation of small-sized capsulate is essential for many aspects of the drug or gene delivery process. For spherical gold nanoparticles, silica nanoparticles, single-walled carbon nanotubes, and quantum dots, a 50 nm diameter is optimal to © 2014 American Chemical Society

Received: January 20, 2014 Revised: March 12, 2014 Published: March 21, 2014 3893

dx.doi.org/10.1021/jp500683n | J. Phys. Chem. B 2014, 118, 3893−3898

The Journal of Physical Chemistry B

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salt was removed by filtration, and then methanol was removed. Water (100 mL) was added to the residue, and washed three times with diethyl ether. Diethyl ether and 1.0 M HCl (250 mL) were added, dithiobenzoic acid (DTBA) was extracted into the ether layer, and the ether layer was washed three times with water. Water (200 mL) and 1.0 M NaOH (250 mL) were added, and sodium dithiobenzoate was extracted into the aqueous layer and washed three times with diethyl ether. Then, diethyl ether and 1.0 M HCl (250 mL) were added, DTBA was extracted into the ether layer, washed the ether layer with water three times, and dried by anhydrous Na2SO4, and the residual ether was removed via rotary evaporator. Dithiobenzoic acid was obtained as oil. 1H NMR (300 MHz, CDCl3): δ 7.40−8.00 (m, 5H), 6.3(s, 1H). A mixture of DTBA (10.59 g), α-methylstyrene (10.0 g), and carbon tetrachloride (40 mL) was heated to 70 °C. After 6 h, the solvent was removed to give the rude product, pure cumyl dithiobenzoate as a dark purple oil was obtained by passing through a column chromatography with n-hexane as an eluent. The yield is 32.0%. 1H NMR (300 MHz, CDCl3): δ 7.86 (m, 2H), 7.20−7.60 (m, 8H), 2.03 (s, 6H). Synthesis of PEG-Based Branched Polymer via Reversible Addition−Fragmentation Chain Transfer (RAFT) Polymerization. In a typical procedure, MEO2MA (655.0 mg, 3.5 mmol), OEGMA (711.9 mg, 1.5 mmol), CBA (261.0 mg, 1.0 mmol), CDB (16.3 mg, 0.075 mmol), AIBN (2.8 mg, 0.015 mmol), and THF (8.0 mL) were added into a polymerization tube, and then the solution was degassed by three freeze−pump−thaw cycles. After the tube was sealed under vacuum, it was placed in a preheated oil bath at 60 °C. After 30 h, the polymerization was stopped, and the resulting polymer was isolated by precipitation in hexane and filtration. The obtained PEG-based branched polymer has molecular weight of 48 700 and PDI of 1.9. Encapsulation of DNA into Small-Sized Nanocapsules. For the encapsulation of DNA, the aqueous solution containing branched polymer (50.0 mg), NaCl (10.0 mg), DNA (0.2 mL, 5.0 mg/mL), and deionized water (0.175 mL) was prepared. The continuous phase consisted of Span 80 (150.0 mg), Tween 80 (50.0 mg), and cyclohexane (7.5 mL). Then the aqueous solution was added to the continuous phase, and the mixture was stirred vigorously. The formed emulsion droplet was ultrasonicated under ice cooling to form emulsion nanodroplet. Subsequently, the nanodroplets were transferred to a flask and kept at 50 °C while stirring for 3 h. After cooling to room temperature, the nanocapsules were separated from the oil phase by centrifugation. The obtained nanocapsules were washed with cyclohexane three times and PBS buffer three times. Transfection. HeLa cells were seeded in a 4-chamber glass bottom dish at a density of 80 000 cells per chamber and incubated overnight in 500 μL DMEM supplemented with 10% FBS. After the media was refreshed, DNA loaded nanocapsules were added into chambers. After 12 h incubation, the medium was replaced with 500 μL of fresh medium, followed by another 48 h incubation. Then the media was removed, and the cells were washed three times with PBS. The confocal laser scanning microscopy observation was performed using a confocal laser scanning microscopy (Leica TCS SP5 microscope) at excitation wavelength of 488 nm (Ar laser), and the emission detection channel was set to 500−600 nm. Agarose Gel Electrophoresis Assay. To confirm that the DNA was loaded into nanocapsules, agarose gel electrophoresis

large-sized plasmid DNA is first folded into the small-sized nanoemulsion droplets containing bioreducible branched polymers. Subsequently, temperature triggers the bioreducible branched polymer to aggregate and cross-link at the water/oil interface of nanodroplets, forming a bioreducible shell around nanodroplet. Hence, a small-sized nanocapsule with compacted DNA forms by removal of surfactant.

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EXPERIMENTAL SECTION Chemicals. Dithiothreitol (DTT, ≥98%, Sigma), Dulbecco’s modified Eagle’s medium (DMEM, Hyclone), and fetal bovine serum (FBS, Hyclone) were used as obtained. 2-(2Methoxyethoxy)ethyl methacrylate (MEO2MA, Mn ∼ 188 g/ mol, 95%, Aldrich) and oligo(ethylene glycol) methacrylate (OEGMA, Mn ∼ 475 g/mol, 95%, Aldrich) were deinhibited by passing through a column of activated basic alumina before use. Acryloyl chloride (technical grade) was freshly distilled before use. Azobisisobutyronitrile (AIBN, 99%, Aladdin) was recrystallized twice from ethanol. Cysteamine hydrochloride (97%), sodium methoxide (≥50%, solution in methanol), elemental sulfur (≥99.5%), benzyl chloride (99%), Span 80 (AR), Tween 80 (AR), ρ-toluenesulfonic acid (≥98%), and α-methylstyrene (99%) were purchased from Sinopharm Chemical Reagent Co., LTD, and were used as received. Water was deionized to 18 MΩ·cm resistivity using the Nanopure system. Circular gWizGFP DNA with 5757 bp was purchased from Aldevron Company (U.S.A.). Characterizations. 1H NMR was recorded on a Bruker AV 300 (300 MHz) instrument. Molecular weight and polydispersity index (PDI) were determined by gel permeation chromatography (GPC) performed with three linear Styragel 15 columns and a Waters 2414 differential refractive index (RI) detector (flow rate of 1.0 mL/min, DMF as eluent). Polystyrenes were used as standard. The cloud point of the polymer solutions were measured using a Beckman DU 640 UV spectrophotometer equipped with a digital temperature controller. Transmission electron microscopy (TEM) was performed on a JEM-2100F field emission transmission electron microscope with an accelerating voltage of 200 KV. Energy dispersive X-ray spectroscopy (EDS) was also performed on a JEM-2100F field emission TEM microscope equipped with an energy dispersive spectrometer probe. The zeta potential measurements were performed on a Malvern Zetasizer Nano ZS90 using an aqueous dip cell. Synthesis of N,N′-Cystaminebisacrylamide (CBA).18 Cystamine dihydrochloride (11.6 g, 50 mmol) was dissolved in 100 mL water. Aqueous solution of sodium hydroxide (20 mL, 10 M) and solution of acryloyl chloride (9.4 g, 100 mmol) in dichloromethane (10 mL) were added dropwise simultaneously under stirring at 0 °C. The reaction was performed for 3 h at room temperature. Then the reaction mixture was filtered and washed three times with deionized water. The product was obtained by crystallization from ethyl acetate (yield 51%). 1H NMR (300 MHz, DMSO-d6): δ 8.28 (s, 1H), 6.11 (m, 2H), 5.61 (m, 1H), 3.43 (m, 2H), 2.82 (m, 2H). Synthesis of Cumyl Dithiobenzoate (CDB).19 Sodium methoxide (≥50% solution in methanol, 55.00 g, ∼0.50 mol), elemental sulfur (16.00 g, 0.50 mol) and anhydrous methanol (160.00 mL) were added to a 500 mL, three-neck roundbottomed flask, followed by dropwise addition of benzyl chloride (31.50 g, 0.25 mol) in 60 min at room temperature. The reaction mixture was then heated to 70 °C. After 18 h, the mixture was cooled to 5 °C using an ice bath, the precipitated 3894

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Figure 1. The outline for encapsulation of large-sized DNA into small-sized nanocapsules and the release of DNA from the nanocapsules in the presence of glutathione (GSH).

became poor due to the liberation of water at high temperature. The branched polymers in the nanodroplet moved to the interface of oil/water slowly, but when they reached the interface of oil/water, they could not dissolve in the oil phase; thus the branched polymers stayed at the interface of oil/water. These branched macromolecules packed closely together and were ready to undergo intermolecular disulfide-exchange to cross-link the branched macromolecules,20−22 forming a bioreducible nanoshell around nanodroplet; bioreducible nanocapsules formed by removing the surfactant (Figure 1). The smallest nanocapsule prepared via this method is ∼90 nm. Figure 2 shows the nanocapsules with size of ∼110 nm prepared via this method without DNA. The shell for the prepared nanocapsule is cross-linked by disulfide bonds, hence these nanocapsules are redox-responsive. Here, we choose circular gWiz-GFP DNA with 5757 bps (gWIZ-GFP) because it has a large size (∼1900 nm long)9 and it can express green fluorescent protein after it moves in the cell. In order to encapsulate the highly charged DNA into small-sized nanocapsules, DNA and the PEG-based branched polymer were first dissolved in water; then the aqueous solution was added into cyclohexane under stirring using Span 80 and Tween 80 as a surfactant. The nanodroplets with the size of ∼110 nm formed after 20 min of stirring. Before stirring, there are two phases: oil phase and aqueous phase containing PEGbranched polymer and DNA. DNA and the branched polymer are hydrophilic and insoluble in cyclohexane, therefore the branched polymer and DNA go inside the emulsion droplet under stirring. To fit DNA of ∼1900 nm long into the 110-nm

assay was performed. The gel was prepared with 0.9% of agarose in Tris-acetate-EDTA (TAE) buffer. The obtained nanocapsules loaded with gWiz-GFP DNA were redispersed in an aqueous solution of DTT at different concentrations, and allowed to stand for 30 min at room temperature. Subsequently, the mixtures were added into wells of agarose gel. Electrophoresis was performed using TAE running buffer at 90 V for 50 min. The migrated DNA was visualized by soaking the gel into 0.5 μg/mL ethidium bromide aqueous solution for 20 min, then the electrophoresis gel was scanned on a UVP ED3 Imaging System.

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RESULTS AND DISCUSSION In order to address the above issue, we prepared PEG-based branched polymer via reversible addition−fragmentation chain transfer (RAFT) polymerization of 2-(2-methoxyethoxy)ethyl methacrylate (655.0 mg) and oligo(ethylene glycol) methacrylate (711.9 mg) using N,N′-cystaminebisacrylamide (260.0 mg) as branching unit and cumyl dithiobenzoate (16.3 mg) as RAFT agent. The produced PEG-based branched polymer has a molecular weight of 48 700 and PDI of 1.9. This branched polymer has disulfide bonds in the backbone and lower critical solution temperature (LCST) of 45 °C. We prepared emulsion nanodroplets by stirring the mixture of cyclohexane and water containing DNA and branched polymer using Span 80 and Tween 80 as surfactant. The nanodroplets were dispersed in cyclohexane, DNA, and PEG-based branched polymer that was inside the nanodroplets. After we heated the dispersion to 50 °C, the solubility of branched polymers in the nanodroplet 3895

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branched macromolecules at the interface, forming a bioreducible nanoshell around nanodroplets. Removal of the surfactant gave nanocapsules containing circular gWiz-GFP DNA (as shown in Figure 1). To ensure that the highly charged DNA is inside the nanocapsules, we checked the cyclohexane phase and did not find DNA in cyclohexane. On the other hand, DNA may be at the surface of the nanocapsules. Because circular gWiz-GFP DNA is the negatively charged and PEG polymer is neutral, the surface charge value will be negative if DNA is at the surface of the nanocapsules. The zeta-potential value corresponds to the surface charge value of nanoparticles. Therefore, we used a zetapotential meter to check the zeta-potential values of the formed nanocapsules; the zeta-potential value of nanocapsules containing negatively charged DNA is almost neutral, which is the same as that of the empty nanocapsules (Figure 3), indicating that DNA is not at the surface of nanocapsules but inside the nanocapsules. Furthermore, based on the TEM images, it is clear that the nanocapsules containing DNA had almost no empty space (Figure 4A), as opposed to the blank nanocapsules, which indicates that the cavity of the nanocapsule has been occupied by DNA. Energy-dispersive X-ray spectroscopy (EDS) spectrum can give the chemical element information of the nanocapsule and those loaded in nanocapsules. There is P element from DNA in the nanocapsules containing DNA while there is no P element in the empty nanocapsules. Therefore, in order to further verify the negatively charged DNA in these nanocapsules, EDS spectra of nanocapsules were recorded; it is clear that there was a P atom signal detected in the EDS spectrum of nanocapsules containing DNA while there was no P atom signal detected in the empty nanocapsules (Figure 4B).

Figure 2. The TEM images of emulsion nanodroplets (top) and the formed bioreducible nanocapsules (bottom).

nanodroplets, the shearing force and surfactant can exert significant force to fold the highly charged DNA several times to fit the small-sized emulsion droplet during the stirring. Then, the nanodroplets were heated and the PEG branched polymer liberated water; they collapsed together and became insoluble in the aqueous phase. Subsequently, they moved to the interface of oil/water slowly, but when they reached at the interface they were not soluble in the organic phase either. Therefore, they stayed at the interface of oil/water while the DNA remained in the nanodroplet. These branched macromolecules packed closely together, and they were ready to undergo intermolecular disulfide-exchange to cross-link the

Figure 3. Zeta-potential distributions of nanocapsules with and without DNA. 3896

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that there is not any signal from the empty nanocapsules and no DNA signal from nanocapsules without being treated with GSH, but DNAs signals are detected from the nanocapsules when these nanocapsules are treated with GSH of 5 mM and 10 mM, indicating that there is DNA inside nanocapsules (Figure 5). Moreover, the released DNA is similar to the DNA marker (Figure 5), indicating that the surfactant and shear forces have not destroyed the DNA chain structure. To evaluate the biocompatibility of this branched polymer, the cytotoxicity experiment was carried out. It is clear the cell viability remains at ∼100% even at the polymer concentration of 1000 μg/mL, indicating that this PEG-based branched polymer has almost no cytotoxicity; hence, the PEG-based branched polymer is biocompatible, and the formed nanocapsules are biocompatible and bioreducible (Figure 6A).

Figure 4. (A) TEM images of nanocapsules with DNA. (B) Energy dispersive X-ray spectroscopy (EDS) spectrum of the nanocapsules with and without DNA. Figure 6. (A) The cytotoxicity of the PEG-based branched polymer and (B) confocal fluorescence images of DNA loaded nanocapsules in Hela cells after 3 days incubation.

Moreover, the formed nanocapsules have a disulfide-crosslinked shell, and this shell can be easily degraded in the presence of glutathione (GSH) or dithiothreitol (DTT). If the nanocapsules have DNA inside, the DNA will be released when these nanocapsules are treated with GSH. Agarose gel electrophoresis analysis can be used to detect DNA released from the nanocapsules. Agarose gel electrophoresis results show

These nanocapsules can thereby be used as biocompatible gene delivery vector. We used the nanocapsules as delivery vector to delivery gWiz-GFP DNA with the gene for green fluorescent protein into the cell. There will be green fluorescent protein in

Figure 5. Agarose gel electrophoresis results of empty nanocapsules and nanocapsules containing DNA treated with 0, 5, and 10 mM glutathione (GSH). 3897

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the cell if gWiz-GFP DNA with the gene for green fluorescent protein has been successfully delivered into the cell; otherwise, there is no green fluorescent protein in the cell. HeLa cells were seeded in a 4-chamber glass bottom dish at a density of 80 000 cells per chamber and incubated overnight in 500 μL DMEM supplemented with 10% FBS. Subsequently, the bioreducible nanocapsules containing DNA were added in chambers. After incubation, it is clear that the nanocapsules inside the cell can release the loaded DNA as the GSH inside the cell breaks the nanocapsules. GFP expression was observed in Hela cell (Figure 6B), which indicates that these bioreducible and biocompatible nanocapsules can be used as a vector for DNA.

(10) Zelikin, A. N.; Li, Q.; Caruso, F. Degradable polyelectrolyte capsules filled with oligonucleotide sequences. Angew. Chem., Int. Ed. 2006, 45 (46), 7743−7745. (11) Shchukin, D. G.; Patel, A. A.; Sukhorukov, G. B.; Lvov, Y. M. Nanoassembly of Biodegradable Microcapsules for DNA Encasing. J. Am. Chem. Soc. 2004, 126 (11), 3374−3375. (12) Miyata, K.; Gouda, N.; Takemoto, H.; Oba, M.; Lee, Y.; Koyama, H.; Yamasaki, Y.; Itake, K.; Nishiyama, N.; Kataoka, K. Enhanced transfection with silica-coated polyplexes loading plasmid DNA. Biomaterials 2010, 31 (17), 4764−4770. (13) Schroeder, A.; Goldberg, M. S.; Kastrup, C.; Wang, Y. X.; Jiang, S.; Joseph, B. J.; Levins, C. G.; Kannan, S. T.; Langer, R.; Anderson, D. G. Remotely Activated Protein-Producing Nanoparticles. Nano Lett 2012, 12 (6), 2685−2689. (14) Zhang, S. L.; Li, J.; Lykotrafitis, G.; Bao, G.; Suresh, S. Sizedependent endocytosis of nanoparticles. Adv. Mater. 2009, 21 (4), 419−+. (15) Lu, F.; Wu, S. H.; Hung, Y.; Mou, C. Y. Size effect on cell uptake in well-suspended, uniform mesoporous silica nanoparticles. Small 2009, 5 (12), 1408−1413. (16) Oh, W. K.; Kim, S.; Choi, M.; Kim, C.; Jeong, Y. S.; Cho, B. R.; Hahn, J. S.; Jang, J. Cellular Uptake, Cytotoxicity, and Innate Immune Response of Silica−Titania Hollow Nanoparticles Based on Size and Surface Functionality. ACS Nano 2010, 4 (9), 5301−5313. (17) Win, K. Y.; Feng, S. S. Effects of particle size and surface coating on cellular uptake of polymeric nanoparticles for oral delivery of anticancer drugs. Biomaterials 2005, 26 (15), 2713−2722. (18) Emilitri, E.; Ranucci, E.; Ferruti, P. New poly(amidoamine)s containing disulfide linkages in their main chain. J. Polym. Sci., Part A: Polym. Chem. 2005, 43, 1404−1416. (19) Liu, Y.; He, J.; Xu, J.; Fan, D.; Tang, W.; Yang, Y. Thermal Decomposition of Cumyl Dithiobenzoate. Macromolecules 2005, 38, 10332−10335. (20) Chen, L. Y.; Yu, S. Z.; Wang, H.; Xu, J.; Liu, C. C.; Chong, W. H.; Chen, H. Y. General Methodology of Using Oil-in-Water and Water-in-Oil Emulsions for Coiling Nanofilaments. J. Am. Chem. Soc. 2013, 135 (2), 835−843. (21) Yu, Z. Q.; Sun, J. T.; Pan, C. Y.; Hong, C. Y. Bioreducible nanogels/microgels easily prepared via temperature induced selfassembly and self-crosslinking. Chem. Commun. 2012, 48 (45), 5623− 5625. (22) Wang, Z. K.; Wang, L. H.; Sun, J. T.; Han, L. F.; Hong, C. Y. In situ generation of bioreducible and acid labile nanogels/microgels simply via adding water into the polymerization system. Polym. Chem. 2013, 4 (5), 1694−1699.

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CONCLUSION We successfully use shear force and surfactant to fold largesized DNA into a small-sized nanoemulsion droplet, subsequently using temperature to trigger the formation of bioreducible nanoshell around nanoemulsion droplet. Removing the surfactant gives bioreducible nanocapsules containing DNA. The nanocapsules are bioreducible and biocompatible and can act as a vector to deliver DNA into cell.

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AUTHOR INFORMATION

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Financial support from National Natural Science Foundation of China (51033005, 51273187, and 21090354), the Fundamental Research Funds for the Central Universities (WK2060200012) and the Program for New Century Excellent Talents in Universities (NCET-11-0882) is gratefully acknowledged.

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REFERENCES

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dx.doi.org/10.1021/jp500683n | J. Phys. Chem. B 2014, 118, 3893−3898