pubs.acs.org/Langmuir © 2009 American Chemical Society
Facile Approach for DNA Encapsulation in Functional Polyion Complex for Triggered Intracellular Gene Delivery: Design, Synthesis, and Mechanism Zixu Gu, Yuan Yuan, Jinlin He, Mingzu Zhang, and Peihong Ni* Key Laboratory of Organic Chemistry of Jiangsu Province, College of Chemistry, Chemical Engineering and Materials Science, Soochow University, Suzhou 215123, China Received December 8, 2008. Revised Manuscript Received January 30, 2009 A facile route for DNA encapsulation in triggered intracellular degradable polymer microcapsules has been achieved via electrostatic interaction, using a polycation, that is, poly[(dimethylamino)ethyl methacrylate] end-capped with cholesterol moiety (Chol-PDMAEMA30), along with a polyanion named MePEG2000-block-poly(methacrylic acid) carring partial thiol groups (MePEG2000-b-PMAASH). The encapsulation procedure involves three steps: (i) DNA was first complexed with the polycation (Chol-PDMAEMA30); (ii) the complex was then further set into interaction with the anion-containing MePEG2000-b-PMAASH; and (iii) the compound carrier was subsequently obtained by cross-linking the thiol groups of the MePEG2000-b-PMAASH to form disulfide linkages. The interactions between every pair among calf thymus DNA, Chol-PDMAEMA30, and MePEG2000-b-PMAASH were studied by agarose gel retardation assay and ethidium bromide displacement assay. The results indicate that the prepared microcapsules may remain stable during systemic circulation, but degrade and release the carried DNA in a cellular reducing environment. Furthermore, the biophysical properties of the microcapsule have been investigated by ζ-potential, laser light scattering, and transmission electron microscopy (TEM) measurements.
1.
Introduction
Gene therapy has been recognized as an alternative approach to overcome the drawbacks of protein therapy due to its enormous potential in curing a broad range of genetic diseases including infectious disease, gene-related disorders, and cancer. However, most nucleic acid drugs are susceptible to nucleases, especially when delivered through systemic administration. Therefore, efficient delivery becomes the key to gene therapy, and appropriate carriers are requisite for successful gene delivery aimed at bringing out maximum therapeutic efficacy.1 Basically, there are two kinds of gene vectors, classified on the basis of the properties of the carriers, viral and nonviral ones. Viral vectors have been predominantly employed in clinical examinations due to their high efficiency. However, safety concerns such as immune response or gene mutation limit their practical use and accelerate research on nonviral vectors based on liposomes or cationic polymers.2 Among them, polycation-based nonviral gene carriers are the most widely investigated due to their potential advantages such as higher degree of biosafety, greater flexibility, facile manufacturing, and easy modification.3-6 Typical cationic polymers reported for the function in gene delivery include poly(ethylenimine) (PEI), poly(amidoamine) (PAMAM) dendrimer,
*To whom correspondence should be addressed. E-mail: phni@suda. edu.cn. (1) Jeong, J. H.; Kim, S. W.; Park, T. G. Prog. Polym. Sci. 2007, 32, 1239– 1274. (2) Glover, D. J.; Lipps, H. J.; Jans, D. A. Nat. Rev. Genet. 2005, 6, 299– 310. (3) Lin, S.; Du, F. S.; Wang, Y.; Ji, S. P.; Liang, D. H.; Yu, L.; Li, Z. C. Biomacromolecules 2008, 9, 109–115. (4) Pack, D. W.; Hoffman, A. S.; Pun, S.; Stayton, P. S. Nat. Rev. Drug Discovery 2005, 4, 581–593. (5) Wagner, E.; Kloeckner, J. Adv. Polym. Sci. 2006, 192, 135–173. (6) Merdan, T.; Kopecek, J.; Kissel, T. Adv. Drug Delivery Rev. 2002, 54, 715–758.
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poly(L-lysine), chitosan, and poly[2-(dimethylamino)ethyl methacrylate] (PDMAEMA).1 In general, a large number of positively charged amino groups of these cationic polymers seem to be extremely important for realizing high DNA binding efficiency.5 However, it has been discovered that high molecular weight polycations would eventually lead to increased cytotoxicity.5,7 On the other hand, polycations with low molecular weight will generally exhibit much lower cytotoxicity,8,9 but they are not considered as appropriate for transfection purposes because of their poor DNA binding affinity.10 In order to solve this paradox, researchers have recently designed a water-soluble lipopolymer (WSLP) consisting of a hydrophobic lipid anchor group and a low molecular weight polycation chain for gene delivery because of their low cytotoxicity and high DNA binding efficiency.11-14 As a hydrophobic lipid anchor, cholesterol is a natural lipid and can be metabolized in the body. The early success of the 3-β-[N-(N0 , N0 -dimethylaminoethane) carbamoyl] cholesterol (DC-Chol) lipid-based gene delivery system spurred interest in the development of novel cholesterol-based cationic lipids.15,16 Recent (7) Ahn, C. H.; Chae, S. Y.; Bae, Y. H.; Kim, S. W. J. Controlled Release 2004, 97, 567–574. (8) Li, Y.; Cui, L.; Li, Q. B.; Jia, L.; Xu, Y. H.; Fang, Q.; Cao, A. M. Biomacromolecules 2007, 8, 1409–1416. (9) Kunath, K.; Harpe, A.; Fischer, D.; Petersen, H.; Bickel, U.; Voigt, K.; Kissel, T. J. Controlled Release 2003, 89, 113–125. (10) Godbey, W. T.; Wu, K. K.; Mikos, A. G. J. Biomed. Mater. Res. 1999, 45, 268–275. (11) Janat-Amsbury, M. M.; Yockman, J. W.; Lee, M.; Kerna, S.; Furgeson, D. Y.; Bikram, M.; Kim, S. W. J. Controlled Release 2005, 101, 273–285. (12) Wang, D. A.; Narang, A. S.; Kotb, M.; Gaber, A. O.; Miller, D. D.; Kim, S. W.; Mahato, R. I. Biomacromolecules 2002, 3, 1197–1207. (13) Wang, Y.; Wang, L. S.; Goh, S. H.; Yang, Y. Y. Biomacromolecules 2007, 8, 1028–1037. (14) Mahato, R. I. Adv. Drug Delivery Rev. 2005, 57, 699–712. (15) Furgeson, D. Y.; Chan, W. S.; Yockman, J. W.; Kim, S. W. Bioconjugate Chem. 2003, 14, 840–847. (16) Vigneron, J. P.; Oudrhiri, N.; Fauquet, M.; Vergely, L.; Bradley, J. C.; Basseville, M.; Lehn, P.; Lehn, J. M. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 9682–9686.
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studies indicated that cholesterol has a lot of advantages for polycation gene carriers such as strong tendency for self-association,17 improved stability of lipid rafts,18 and increased transfection efficiency.12,19,20 Many methods have been utilized to combine cholesterol with cationic polymers to obtain WSLPs. However, most of the synthetic routes involved a series of procedures with a tendency to result in low yields and complicated purification of polycations. Therefore, it is urgent to develop a more facile synthetic route which enables one-step synthesis of a well-defined WSLP gene carrier. To get high transfection efficiency, positively charged complexes are favorable because the excess positive charges facilitate interaction of complexes with negatively charged cell membranes and thus accelerate both the cellular uptake21-23 and endosomal escape.24 However, when systemically administered, the positively charged complexes readily interact with negatively charged components including serum albumin and other opsonins to form larger aggregates and lead to a rapid clearance of the complexes by the reticuloendothelial system (RES) or phagocytes, sometimes causing significant toxicity.25,26 Development of a neutral nanoparticle which shields the polycation/DNA complexes during systemic circulation would overcome the limitations of a polycation gene delivery system. And to achieve this purpose, one strategy is to introduce hydrophilic neutral polymers such as poly(ethylene glycol) (PEG) and poly[N-(2-hydroxypropyl) methacrylamide] (PHPMA) in the outer layer of the carrier, which can prolong blood circulation time and reduce side interaction with blood components.1,6 Compared with the chemosynthesis method, coating the functional polymers on the surface of polycation/ DNA complexes by electrostatic interaction is relatively simple, which significantly improves the serum stability of polycation/DNA complexes and considerably raises gene expression levels and reduction of cytotoxicity.27-30 For example, poly(propylacrylic acid) (PPAA) has been used to coat the 1,2-dioleoyl-3-trimethylammoniumpropane/DNA (DOTAP/DNA) complexes proposed by Hoffman et al.;27 poly(methacryloyl sulfadimethoxine)-b-PEG (PSD-b-PEG) has been applied to bind with PEI/DNA complexes reported by Bae et al.;28 and human serum albumin (HSA) has been (17) Yusa, S. I.; Kamachi, M.; Morishima, Y. Macromolecules 2000, 33, 1224–1231. (18) Klok, H. A.; Hwang, J. J.; Iyer, S. N.; Stupp, S. I. Macromolecules 2002, 35, 746–759. (19) Azzam, T.; Eliyahu, H.; Makovitzki, A.; Linial, M.; Domb, A. J. J. Controlled Release 2004, 96, 309–323. (20) Kim, W. J.; Chang, C. W.; Lee, M.; Kim, S. W. J. Controlled Release 2007, 118, 357–363. (21) Deshpande, M. C.; Davies, M. C.; Garnett, M. C.; Williams, P. M.; Armitage, D.; Bailey, L.; Vamvakaki, M.; Armes, S. P.; Stolnik, S. J. Controlled Release 2004, 97, 143–156. (22) Merdan, T.; Kunath, K.; Fischer, D.; Kopecek, J.; Kissel, T. Pharm. Res. 2002, 19, 140–146. (23) Mislick, K. A.; Baldeschwieler, J. D. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 12349–12354. (24) Zhang, Z. Y.; Smith, B. D. Bioconjugate Chem. 2000, 11, 805–814. (25) Dash, P. R.; Read, M. L.; Barrett, L. B.; Wolfert, M. A.; Seymour, L. W. Gene Ther. 1999, 6, 643–650. (26) Zou, S. M.; Erbacher, P.; Remy, J. S.; Behr, J. P. J. Gene Med. 2000, 2, 128–134. (27) Cheung, C. Y.; Murthy, N.; Stayton, P. S.; Hoffman, A. S. Bioconjugate Chem. 2001, 12, 906–910. (28) Sethuraman, V. A.; Na, K.; Bae, Y. H. Biomacromolecules 2006, 7, 64–70. (29) Gioia, S. D.; Rejman, J.; Carrabino, S.; Fino, I. D.; Rudolph, C.; Doherty, A.; Hyndman, L.; Cicco, M. D.; Copreni, E.; Bragonzi, A.; Colombo, C.; Boyd, A. C.; Conese, M. Biomacromolecules 2008, 9, 859–866. (30) Trubetskoy, V. S.; Wong, S. C.; Subbotin, V.; Budker, V. G.; Loomis, A.; Hagstrom, J. E.; Wolff, J. A. Gene Ther. 2003, 10, 261–271.
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explored to encapsulate PEI/DNA complexes by Conese et al.29 However, we have noted that there still remains a critical problem when using this method; that is, simple dissociation of an ion complex between a pair of oppositely charged macromolecules with sufficient chain length may not occur because of the integrated stabilization through multisite interaction. On the other hand, an exchange reaction of complex with other charged components is known to take place, as described by Kataoka and Katayose,31 and Bakeev et al.32 So, it is necessary to investigate chain-exchanging reaction through qualitative and quantitative analysis when applying this method to the preparation of gene carriers. Moreover, the above-mentioned gene carrier model is usually influenced by a large dilution effect, for example,, blood circulation after administration, and/or the presence of high concentration of salts, and it has been well-proved that both cases might disintegrate the complexes.33,34 In solving this problem and increasing the stability of complexes, shell cross-linking (SCL) or core cross-linking (CCL) by reversible bonds is found to be a promising method when the bond is cleavable in response to certain physical and/ or chemical stimuli. Several research groups34-36 have reported that there is a stabilization of the complexes through disulfide cross-linking of the core or the shell, and that the cleavage of the disulfide bond would occur within the cell because the intracellular compartment has a stronger reducing environment than the extracellular fluid,34,37 which is supposed to allow the controlled release of gene inside the cells. In the present study, a new type of well-defined WSLP gene carrier consisting of hydrophilic PDMAEMA and hydrophobic cholesterol (Chol-PDMAEMA30) was prepared by a onestep synthesis route via oxyanion-initiated polymerization and the representative synthesis route is outlined in Scheme 1. To overcome the limitations of the positively charged complexes, we have developed an intelligent delivery system: MePEG2000-b-PMAASH, which was also prepared via oxyanion-initiated polymerization (as shown in Scheme 2), was first used to coat the Chol-PDMAEMA30/DNA complexes by electrostatic interaction. Subsequently, the complexes were stabilized by oxidizing the thiol groups to form bridging disulfide linkages between the MePEG2000-b-PMAASH chains. We believe the cross-linking shell would keep the complexes stable during the systemic circulation but degrade and release the carried DNA once the disulfide linkages are destroyed in the cell reducing environment. We have studied the properties of Chol-PDMAEMA30 and complexes formed by electrostatic interactions between DNA, Chol-PDMAEMA30, and MePEG2000-b-PMAASH. Biophysical properties of these complexes were also investigated by using ζ-potential, laser light scattering, and transmission electron microscopy measurements.
(31) Katayose, S.; Kataoka, K. Bioconjugate Chem. 1997, 8, 702–707. (32) Bakeev, K. N.; Izumrudov, V. A.; Kuchanov, S. I.; Zezin, A. B.; Kabanov, V. A. Macromolecules 1992, 25, 4249–4254. (33) Zhang, J. Y.; Zhou, Y. M.; Zhu, Z. Y.; Ge, Z. S.; Liu, S. Y. Macromolecules 2008, 41, 1444–1454. (34) Kakizawa, Y.; Harada, A.; Kataoka, K. J. Am. Chem. Soc. 1999, 121, 11247–11248. (35) Zelikin, A. N.; Li, Q.; Caruso, F. Angew. Chem., Int. Ed. 2006, 45, 7743–7745. (36) Zelikin, A. N.; Becker, A. L.; Johnston, A. P. R.; Wark, K. L.; Turatti, F.; Caruso, F. ACS Nano 2007, 1, 63–69. (37) Huang, S. Y.; Pooyan, S.; Wang, J.; Choudhury, I.; Leibowitz, M. J.; Stein, S. Bioconjugate Chem. 1998, 9, 612–617.
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Scheme 1. Representative Reaction Route for the Synthesis of Chol-PDMAEMA30 via Oxyanion-Initiated Polymerization Using Cholesterol as the Initiator Precursor
Scheme 2. Reaction Procedures for the Synthesis of MePEG2000-b-PMAASH Copolymer via the Following Steps: (1) Oxyanion-Initiated Polymerization of tert-Butyl Methacrylate Using MePEG2000 as the Macroinitiator Precursor to Yield Well-Defined MePEG-b-PtBMA Diblock Copolymer, (2) Hydrolysis of MePEG-b-PtBMA to Produce MePEG-b-PMAA Diblock Copolymer, and (3) Partial Mercapto Substitution of Carboxyl Groups in MePEG-b-PMAA to Yield MePEG-b-PMAASH Copolymer
2.
Experimental Section
2.1. Materials. 2-(N,N-Dimethylamino)ethyl methacrylate (DMAEMA, Aldrich) and tert-butyl methacrylate (tBMA, TCI) were each dried over calcium hydride (CaH2) and distilled in vacuum immediately before use. Cholesterol (Sinopharm Chemical Reagent Co.) was purified by recrystallization from acetone three times and dried in a vacuum oven at 40 °C for at least 12 h. Potassium hydride (KH, Aldrich, 35 wt % dispersion in mineral oil) was washed three times with anhydrous tetrahydrofuran (THF) in an inert atmosphere before use. THF was initially dried over potassium hydroxide for at least 2 days and then refluxed over sodium wire with benzophenone as indicator until the color turned to purple. Polyethylene glycol 2000 monomethyl ether (MePEG2000) and ethidium bromide (EtBr) purchased from Fluka were used as received. Phosphate buffers saline (PBS) tablets and tris-borate-EDTA buffer (TBE) were from Medicago. Cysteamine hydrochloride and dithiothreitol (DTT) were from Alfa Aesar. 4-Dimethylaminopyridine (DMAP) and N,N0 -dicyclohexylcarbodiimide (DCC) were provided by Shanghai Medpep Co. Calf thymus DNA (Sigma) and plasmid pUC18 (Takara) were used for ζ-potential, particle size, and TEM measurements. Other reagents used as received were purchased from Sinopharm Chemical Reagent Co.
2.2. Preparation of Novel Water-Soluble Lipopolymer Chol-PDMAEMA30. The PDMAEMA with a cholesterol lipid Langmuir 2009, 25(9), 5199–5208
anchor group was prepared through oxyanion-initiated polymerization using cholesterol as the initiator precursor as shown in Scheme 1. The detailed polymerization process has been described in previous literature.38,39 A representative synthesis procedure is described as follows: All flasks and magnetic stirring bars used in the experiments were heated overnight at 120 °C, and then an exhausting-refilling argon process was operated several times until room temperature. Potassium hydride, stored in mineral oil, was washed with anhydrous THF three times in an argon atmosphere in a round-bottom flask, and then anhydrous THF was injected into the flask with KH powder. A specified weight of dry cholesterol was dissolved in anhydrous THF in a round-bottom flask with an argon atmosphere. The cholesterol/THF solution was then transferred via a cannula into the flask containing KH to produce potassium alcoholate of cholesterol as the initiator. After the complete reaction, the upper clear liquid was transferred into the other dry flask with the same method. A certain amount of DMAEMA monomer was then added to the initiator solution by syringe, and the reaction was carried out at 25 °C with stirring for 1 h. The living polymer chains were terminated with methanol, and the solvent was afterward removed with a rotary (38) Xu, J.; Ni, P. H.; Mao, J. e-Polymers 2006, 1–15. (39) He, J. L.; Ni, P. H.; Liu, C. C. J. Polym. Sci., Part A: Polym. Chem. 2008, 46, 3029–3041.
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vacuum distillatory. The resulting product was purified through repeated precipitation into cold n-hexane and dried in a vacuum oven at 30 °C for at least 12 h.
2.3. Preparation of Functional Double Hydrophilic Polyanion MePEG2000-b-PMAASH. In this study, functional double hydrophilic polyanion MePEG2000-b-PMAASH was designed and synthesized via three-step reactions as indicated in Scheme 2. (1). Synthesis of MePEG2000-b-PtBMA30. MePEG2000-b-PtBMA30 was also prepared by oxyanion-initiated polymerization using the similar polymerization procedure described above. Functional macroinitiator MePEG2000O-K+ was prepared by the reaction of MePEG2000 with potassium hydride in anhydrous THF at 45 °C for 12 h. A certain amount of tBMA monomer was then added to the initiator solution while stirring at 25 °C for another 1 h. The living polymer chains were terminated with methanol, and the solvent was afterward removed with a rotary vacuum distillatory. The resulting product was purified through repeated precipitation into cold ether and dried in a vacuum oven at 30 °C for at least 12 h.
(2). Synthesis of MePEG2000-b-PMAA via the Hydrolysis of MePEG2000-b-PtBMA30. To a CH2Cl2 solution of MePEG2000-b-PtBMA30 (1 g), trifluoroacetic acid (TFA, 3 mL) was slowly added at 0 °C under magnetic stirring. After the addition was completed, the reaction mixture was kept at room temperature for 12 h. The reaction mixture was concentrated, precipitated into 50 mL of cold n-hexane, filtered, and dried in a vacuum oven to give an off white solid product MePEG2000-b-PMAA (0.68 g). Yield: 93%. 1H NMR (DMSO-d6): δ ∼3.2 (H of -O-CH3 from MePEG2000), ∼3.5 (H of -O-CH2-CH2- from MePEG2000), 1.7-2.0 (H of -CH2- from PMAA), ∼1.0 (H of CH3 from PMAA), ∼12.2 (H of -COOH from PMAA). (3). Synthesis of MePEG2000-b-PMAASH. To a mixture of cysteamine hydrochloride (0.12 g, target modification = 30 mol %), MePEG2000-b-PMAA (0.5 g), and 4-dimethylaminopyridine (DMAP) (0.13 g) in a mixed solvent CH2Cl2/ DMF (16 mL) at 0 °C was added a solution of N,N0 -dicyclohexylcarbodiimide (DCC) (0.22 g) in CH2Cl2 (4 mL) over 30 min, and the reaction was then continued for 6 h with stirring at room temperature. After removing the insoluble N,N0 -dicyclohexylurea by filtration, the filtrate was concentrated, precipitated into 50 mL of cold ether, filtered, and dried in a vacuum oven at room temperature to yield a white solid, that is, polyanion MePEG2000-b-PMAASH. The resulting product was then further purified by precipitation from ethanol into cold ether. The degree of mercapto substitution was estimated from elemental analysis of the polymer (C, 51%; S, 3.7%), corresponding to 21 mol % modification of carboxyl groups in MePEG2000-b-PMAA. 2.4. Formation of Complexes. The stock solutions of DNA (1 mg mL-1) and polymers (polycation and polyanion, 1 mg mL-1) were prepared in PBS buffer (pH 7.4, 10 mM) and filtered through 0.45 μm filter (Shanghai CAIENFU Technology). All the complexes were prepared immediately prior to use. The typical procedures for complexes formation are elucidated in Scheme 3: First, DNA was mixed with different amounts of Chol-PDMAEMA30 to form Chol-PDMAEMA30/DNA complexes with various N/P ratios, which represent the molar ratios of nitrogen atoms in the lipopolymer to phosphates in the testing DNA. Here, the DNA content was controlled to ensure positive charging of Chol-PDMAEMA30/DNA complexes. After they were mixed for 20 s using a vortex mixer, the samples were incubated for 30 min at room temperature before measurements. Second, the excess positive charge on the Chol-PDMAEMA30/DNA complexes (N/P ratio = 20) were utilized to interact with the negatively charged MePEG2000-b-PMAASH
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to prepare MePEG2000-b-PMAASH/Chol-PDMAEMA30/ DNA complexes with different [MAA]/[DMAEMA] molar ratios, which were based on the units of PMAA block and PDMAEMA segment. The samples were further agitated for 10 s using a vortex mixer at a low speed and incubated for another 30 min at room temperature. Here, DTT was added into the stock solution of MePEG2000-b-PMAASH (1 mg mL-1) beforehand at a concentration of 100 mg mL-1, and then they were incubated at least 12 h to ensure separation of the polymer chains.36 Finally, hydrogen peroxide solution (10 mM) was added without stirring to stabilize the complexes by oxidizing the mercapto groups and forming bridging disulfide linkages between the MePEG2000-b-PMAASH chains.
2.5. Nuclear Magnetic Resonance (1H NMR) Measurements. The chemical structures of Chol-PDMAEMA30, Me-
PEG2000-b-PtBMA30, and MePEG2000-b-PMAA were determined by using a 400 MHz 1H NMR instrument (INOVA-400) using CDCl3 or DMSO-d6 as solvent and tetramethylsilane (TMS) as the internal standard.
2.6. Gel Permeation Chromatography (GPC) Measurements. The number-average molecular weight (Mn) and polydispersity index (Mw/Mn) of Chol-PDMAEMA30 and MePEG2000-b-PtBMA30 were determined by using a Waters 1515 gel permeation chromatograph (GPC) instrument using a PLgel 5.0 μm bead size guard column (50 7.5 mm2), followed by two linear PLgel columns (500 A˚ and Mixed-C) and a differential refractive index detector. THF was used as the eluent at 30 °C with a flow rate of 1.0 mL min-1, and a series of standard poly(methyl methacrylate) was applied as the calibration.
2.7. Critical Micelle Concentration (cmc) Determination. To estimate the cmc of the Chol-PDMAEMA30, steady-state fluorescence measurements were carried out and pyrene was used as the probe. A predetermined amount of pyrene in acetone was added into a series of ampules, and the acetone was then removed by vacuum. Ten milliliters of the polymer solution in PBS buffer (pH 7.4, 10 mM) was then added into each ampule to get solutions with different concentrations ranging from 1.0 10-3 to 20.0 g L-1, while the concentration of pyrene in each solution was fixed at 5.93 10-6 M. The polymer solutions were allowed to stir for 24 h at room temperature before measurement. Fluorescence spectra were recorded on a FLS920 fluorescence spectrofluorometer (Edinburgh Co. U.K.) at 335 nm excitation wavelength, and the emission spectra were recorded from 350 to 500 nm. Both the excitation and emission slit width were set at 1 nm. From the pyrene emission spectra, the intensity ratio (I3/I1) of the third band (383 nm, I3) to the first band (372 nm, I1) was analyzed as a function of polymer concentration. The cmc value was defined as the point of intersection of the two lines in the plot of I3/I1 ratio versus polymeric concentration. The experiments were conducted in triplicate, and the average values were recorded. 2.8. Competition Binding Assay. The interactions among calf thymus DNA, Chol-PDMAEMA30, and MePEG2000-bPMAASH were investigated with competition binding assay. Ethidium bromide (2 μg) was added to 1 mL of PBS buffer (pH 7.4, 1 mM) in a fluorimetry cuvette and mixed with a vortex agitator. The fluorescence of the solution was recorded on a FLS920 fluorescence spectrofluorometer (Edinburgh Co. UK) at 560 nm excitation wavelength and 610 nm emission wavelength. Calf thymus DNA (10 μg) was then added to the solution, and the fluorescence was measured as described above. Aliquots of the Chol-PDMAEMA30 solution were then added in a stepwise manner, mixed vigorously with the vortex agitator and incubated for 30 min, and the fluorescence was measured after each addition. To study the chain exchange reaction of Chol-PDMAEMA30/DNA complexes on the addition of MePEG2000-b-PMAASH, aliquots of the MePEG2000-bPMAASH solution were added into the Chol-PDMAEMA30/ DNA complexes in a stepwise manner (N/P ratio = 20), mixed Langmuir 2009, 25(9), 5199–5208
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Scheme 3. Facile Procedure for the formation of MePEG2000-b-PMAASH/Chol-PDMAEMA30/DNA Complexes Stabilized by Disulfide Linkages in the Shell Layer
vigorously with the vortex agitator, and incubated for another 30 min, and the fluorescence was then measured after each addition. The fluorescence readings for every sample were conducted in triplicate. The relative fluorescence was calculated as follows:40 %relative fluorescence ¼ fluorescence ðobsÞ -fluorescence ðEtBrÞ 100 fluorescence ðDNA þ EtBrÞ -fluorescence ðEtBrÞ where fluorescence (obs) = fluorescence of (DNA + EtBr + polymer); fluorescence (EtBr) = fluorescence of EtBr; and fluorescence (DNA + EtBr) = fluorescence of (DNA + EtBr).
2.9. Gel Retardation Assay. Before loading into the gel, various formulations of complexes were prepared as described in the competition binding assay. The complex solution (8 μL) was mixed with 2 μL of loading buffer (85% glycerol and 15% bromophenol blue) and loaded onto an 0.8% agarose gel containing ethidium bromide (0.5 μg mL-1). Electrophoresis was conducted at a voltage of 70 V for 1 h in Tris-borate-EDTA buffer (TBE: 40 mM trisborate, 1 mM EDTA, and pH 7.4). The gel was visualized on a UV illuminator (M-15E, UVP Inc., Upland, CA) to indicate the location of DNA. 2.10. Zeta (ζ) Potential Measurement. The determination of aqueous microelectrophoresis of the complexes was carried out in a JS94J microeletrophoresis instrument (Shanghai Zhongchen Co., China). The device was equipped with a CCD camera, frame grabber, and software to capture the image of the moving particles. The ζ-potential data were directly provided by the instrument. 2.11. Particle Size Measurement. The average particle sizes of the complexes were measured using a laser light scattering technique on a Malvern Zetasizer HPPS5001 instrument equipped with a He-Ne laser (633 nm). All complex solutions were made by mixing various components in PBS buffer (pH 7.4, 10 mM). All measurements were carried out at 25 °C, and the data were analyzed with Malvern Dispersion Technology software 3.00. 2.12. Transmission Electron Microscopy (TEM). To confirm the morphologies of complexes, samples were visualized with a TEM instrument (TECNAI G2 20, FEI Co.) at an acceleration voltage of 200 kV. The carbon-coated copper grid (400 meshes) was immersed into the complexes solution, (40) Rungsardthong, U.; Deshpande, M.; Bailey, L.; Vamvakaki, M.; Armes, S. P.; Garnett, M. C.; Stolnik, S. J. Controlled Release 2001, 73, 359– 380.
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taken out, and air-dried overnight prior to the measurements.
3. Results and Discussion 3.1. Synthesis of Novel Water-Soluble Lipopolymer (WSLP) Chol-PDMAEMA30. In this paper, a novel watersoluble lipopolymer Chol-PDMAEMA30 was synthesized by oxyanion-initiated polymerization of DMAEMA using cholesterol as the initiator precursor as shown in Scheme 1. In our previous reports, many functional initiator precursors have been studied in oxyanion-initiated polymerization including PEO,38 Pluronic F127,39 fluorinated alcohols,41 and hydroxyl-capped hyperbranched polyether.42 In this study, we first indicate that potassium alcoholate of cholesterol can be used as the initiator in oxyanion-initiated polymerization, which provides an efficient and environmentally friendly route for the incorporation of synthetic polymers and biomolecules. The chemical structure and molecular weight of Chol-PDMAEMA30 were confirmed with 1H NMR and GPC measurements, respectively. Figure 1 shows the 1H NMR spectrum of Chol-PDMAEMA30, from which we can find out all the characteristic peaks corresponding to the lipopolymer structure, indicating that the synthesis was successful. 1H NMR (CDCl3): d ∼0.68 (signal a, H of CH3 from cholesterol), ∼5.4 (signal b, H of dCH- from cholesterol), ∼3.5 (signal c, H of -O-CH< from cholesterol), 1.8-2.0 (signal d, H of -CH2- from PDMAEMA), ∼1.0 (signal e, H of -CH3 from PDMAEMA), ∼4.1 (signal f, H of -O-CH2- from PDMAEMA), ∼2.6 (signal g, H of -CH2-N< from PDMAEMA), ∼2.3 (signal h, H of CH3-N< from PDMAEMA). GPC: PDI (Mw/Mn) = 1.27; Mn(actual) = 5070 g mol-1; Mn(theory) = 5100 g mol-1. 3.2. Synthesis of Amphiphilic Block Copolymer MePEG2000-b-PtBMA30. We have reported that tertiary amine methacrylates,38 methyl methacrylate, and fluoroalkyl methacrylate39,41,42 are proved as suitable for oxyanioninitiated polymerization. In order to obtain a diblock copolymer containing both PEG block and polyanion moiety, we first prepared MePEG2000-b-PtBMA diblock copolymer via oxyanion-initiated polymerization using tert-butyl methacrylate (tBMA) as the monomer and potassium alcoholate of MePEG2000 as the macroinitiator. The polyanion (MePEG2000-b-PMAA) was then obtained by the (41) Zhang, H.; Ni, P. H.; He, J. L.; Liu, C. C. Langmuir 2008, 24, 4647– 4654. (42) Mao, J.; Ni, P. H.; Mai, Y. Y.; Yan, D. Y. Langmuir 2007, 23, 5127– 5134.
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Figure 3. Intensity ratio I3/I1 obtained from the fluorescence ex1
Figure 1. H NMR spectrum of water-soluble lipopolymer CholPDMAEMA30.
Figure 2. 1H NMR spectrum of amphiphilic block copolymer MePEG2000-b-PtBMA30. hydrolysis of PtBMA as shown in Scheme 2. The chemical structure, composition, and molecular weights of MePEG2000-b-PtBMA were verified with 1H NMR in CDCl3 and GPC measurements, respectively. Figure 2 shows the 1H NMR spectrum of MePEG2000-b-PtBMA30 and the characteristic signals are identified as follows: ∼3.4 (signal a, H of -O-CH3 from MePEG2000), ∼3.6 (signal b, H of -OCH2-CH2- from MePEG2000), 1.8-2.0 (signal c, H of CH2- from PtBMA), ∼1.0 (signal d, H of -CH3 from PtBMA), ∼1.4 (signal e, H of -O-C(CH3)3 from PtBMA). GPC: PDI (Mw/Mn) = 1.28; Mn(actual) = 6500 g mol-1; Mn(theory) = 6300 g mol-1. 3.3. Critical Micelle Concentration (cmc) Determination. The cmc value of polymers is not only strong evidence to the formation of micelles but also an important parameter in evaluating the stability of the micelles in the blood post administration. Amphiphilic lipopolymers with a high cmc value seem to be unsuitable for gene delivery because the forming micelles may be dissociated after being administered into the body due to the dilution effect. In addition, the micellar cores have a tendency to dissociate after adsorbing proteins in plasma, leading to rapid clearance from systemic circulation.1 From this point of view, low cmc values are a necessary requisite for efficient gene carriers.13 Considering cholesterol has a rigid structure and strong hydrophobicity, 5204
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citation spectra of pyrene plotted versus the polymer concentrations of (a) Chol-PDMAEMA30 and (b) PDMAEMA30, measured at pH 7.4 in 10 mM PBS buffer and 25 °C.
we designed the cholesterol-capped PDMAEMA lipopolymer and expected that this kind of polycation can provide stable micelles with a low cmc value. The fluorescence probe method was used to determine the cmc value of amphiphilic polycation in this study. Pyrene is a commonly used probe to monitor micropolarity because the relative peak intensity ratio of the third vibronic peak to the first one (I3/I1) in the pyrene fluorescence spectrum is sensitive to the polarity environment, with the I3/I1 ratio being larger in less polar media.43 Figure 3 shows the comparative fluorescence spectra of PDMAEMA in the presence and absence of hydrophobic cholesterol end groups in PBS buffer (pH 7.4, 10 mM). The I3/I1 ratios of CholPDMAEMA30 and PDMAEMA30 are estimated to be 0.89 and 0.52, respectively, at the polymer concentration of about 10 g L-1. According to previous publications, the I3/I1 ratio for pyrene solubilized in micelles formed by sodium dodecyl sulfate (SDS) in water is 0.88,44 while the ratio in pure water is 0.54.43 Herein, we can deduce that almost no micelles form in the aqueous solution of PDMAEMA30. However, for the Chol-PDMAEMA30 system, it can be considered that pyrene is prone to be caught by the hydrophobic micellar core formed by the terminal cholesterol groups, whereas the hydrophilic PDMAEMA chains can extend in water to stabilize the micelles. This result is in good agreement with the report by Yusa et al.43 More importantly, the cmc value of Chol-PDMAEMA30 in PBS buffer (pH 7.4, 10 mM) is only 0.35 g L-1, which is much lower compared with those of the reported lipopolymers,45 and this is what we anticipated for the design of an efficient gene carrier. 3.4. Studies on DNA Binding Affinity of the Polycation Chol-PDMAEMA30. The DNA binding affinity of CholPDMAEMA30 has been studied via gel retardation assay and ethidium bromide (EtBr) displacement assay. Gel Retardation Assay. Figure 4a shows the electrophoresis images of Chol-PDMAEMA30/calf thymus DNA complexes at various N/P ratios. Lane 1 represents the DNA intercalated with EtBr. Lanes 2-6 (0 < N/P ratio < 2) represent the polycation/DNA complexes at lower N/P ratios, which indicate that the proportion of DNA in these (43) Yusa, S. I.; Kamachi, M.; Morishima, Y. Macromolecules 2000, 33, 1224–1231. (44) Kalyanasundaram, K.; Thomas, J. K. J. Am. Chem. Soc. 1977, 99, 2039–2044. (45) Han, S. O.; Mahato, R. I.; Kim, S. W. Bioconjugate Chem. 2001, 12, 337–345.
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Figure 4. (a) Gel retardation assay results for Chol-PDMAEMA30/calf thymus DNA complexes at various N/P ratios. Lane 1 is the DNA control; lanes 2-11 correspond to 0.2, 0.4, 0.6, 0.8, 1.0, 2.0, 4.0, 6.0, 8.0, and 10.0 N/P ratios, respectively. (b) Ethidium bromide displacement by Chol-PDMAEMA30 interacting with calf thymus DNA in PBS buffer (pH 7.4, 1 mM) at various N/P ratios.
wells increases with increasing N/P ratios and an excess of DNA migration toward the anode. The migration of DNA in the gel was completely retarded when the N/P ratio was equal to or above 2 (N/P ratio g 2, lanes 7-11), which shows that the DNA is saturated with the lipopolymer. At an N/P ratio of 2 (lane 7), DNA did not migrate and this can be explained as follows: the ionization of Chol-PDMAEMA30 is approximately 50%,46 and the value for calf thymus DNA is 100% at pH 7.4, so the positive charge of Chol-PDMAEMA30 is equal to the negative charge of calf thymus DNA when the N/P ratio is 2, and almost all of the calf thymus DNA participates in binding with Chol-PDMAEMA30. Ethidium Bromide (EtBr) Displacement Assay. This technique was used to measure the decrease in fluorescence of ethidium bromide/DNA complexes when the CholPDMAEMA30 was added and thus to further assess the binding strength between the polycation and DNA. As shown in Figure 4b, the initial fluorescence level was nearly 100% before the addition of Chol-PDMAEMA30. As the N/ P ratio was increased from 0 to 2, there was a steep fall in fluorescence, which clearly indicates that the affinity between the Chol-PDMAEMA30 and DNA was so strong that the EtBr was displaced by the polycation. Moreover, when the N/P ratio was above 2, the fluorescence reached a plateau at about 20% , and this implies that about 20% of the intercalated EtBr was not displaced. In this study, the fluorescence declined to 20% for Chol-PDMAEMA30 (N/ P ratio = 2, pH 7.4). However, there are other reports showing that the fluorescence can be decreased only to 40% or 50% at the same measurement conditions for the systems of PDMAEMA,47 poly{[2-(dimethylamino)ethyl methacrylate]-block-2-[(methacryloyloxyethyl phosphorylcholine)]} (PDMAEMA-b-PMPC),48 and PEO-b-PDEAEMA.49 These results indicate that the cholesterol groups would enhance the solubilization of the Chol-PDMAEMA30/DNA complex in the micellar core, resulting in the prevention of EtBr intercalation. (46) Van de Wetering, P.; Zuidam, N. J.; Van Steenbergen, M. J.; Van der Houwen, O. A. G. J.; Underberg, W. J. M.; Hennink, W. E. Macromolecules 1998, 31, 8063–8068. (47) Rungsardthong, U.; Ehtezazi, T.; Bailey, L.; Armes, S. P.; Garnett, M. C.; Stolnik, S. Biomacromolecules 2003, 4, 683–690. (48) Lam, J. K. W.; Ma, Y.; Armes, S. P.; Lewisc, A. L.; Baldwin, T.; Stolnik, S. J. Controlled Release 2004, 100, 293–312. (49) Tan, J. F.; Too, H. P.; Hatton, T. A.; Tam, K. C. Langmuir 2006, 22, 3744–3750.
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3.5. Interaction between MePEG2000-b-PMAASH and Chol-PDMAEMA30/DNA Complexes. To minimize the nonspecific interactions of complexes with serum proteins, MePEG2000-b-PMAASH was selected in our study to shield the surface positive charges of Chol-PDMAEMA30/DNA complexes through electrostatic interaction between PDMAEMA and PMAA moieties.50 In order to gain better insight into the exact interaction between MePEG2000-bPMAASH and Chol-PDMAEMA30/DNA complexes, we have examined the interaction via gel retardation assay and ethidium bromide displacement assay at 25 °C. Gel Retardation Assay. The effect of MePEG2000-bPMAASH on the electrophoretic migration of DNA from Chol-PDMAEMA30/DNA complexes is shown in Figure 5a. For Chol-PDMAEMA30/calf thymus DNA complexes (N/P ratio = 20), neither migration from the slot of the agarose gel nor illumination was observed, which indicates that the DNA had been saturated with excess polycations and thereby preventing EtBr intercalation (lane 1). With the addition of MePEG2000-b-PMAASH into the solution of CholPDMAEMA30/DNA complexes (N/P ratio = 20), in which the molar ratios of units [MAA]/[DMAEMA] were kept lower than 1.5, the illumination and slight migration of DNA could be observed from lanes 2 to 6 marked as range A in Figure 5a. When the MePEG2000-b-PMAASH was increased and the [MAA]/[PDMAEMA] molar ratio was above 1.5, complete migration of DNA from the complexes was observed from lanes 8 to 12 marked as range B in Figure 5a, which is due to the varying degrees of cooperative chain-exchanging reaction of MePEG2000-b-PMAASH with DNA in the complexes. Kataoka and Katayose have discussed a similar phenomenon via a mechanism of exchange reaction between the polyelectrolytes.31 Here, we propose a possible mechanism to explain the above-mentioned phenomena (as shown in Figure 6). When the [MAA]/[DMAEMA] molar ratio is lower than 1.5, the cooperative chain-exchanging reaction is weak and only the segments of the DNA chains are free as shown in area A in Figure 6, resulting in limited DNA migration. However, the cooperative chain-exchanging reaction is sufficient when the [MAA]/[DMAEMA] molar ratio is above 1.5 and most of the DNA chains are replaced as shown in area B in Figure 6, leading to the complete (50) Gohy, J. F.; Creutz, S.; Garcia, M.; Mahltig, B.; Stamm, M.; Jerome, R. Macromolecules 2000, 33, 6378–6387.
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Figure 5. (a) Effect of MePEG2000-b-PMAASH on the electrophoretic migration of DNA from Chol-PDMAEMA30/calf thymus DNA complexes. Lane 1 corresponds to Chol-PDMAEMA30/Calf thymus DNA complexes (N/P ratio = 20); lanes 2-12 correspond to CholPDMAEMA30/calf thymus DNA complexes (N/P ratio = 20) with progressively increasing proportions of MePEG2000-b-PMAASH ([MAA]/ [DMAEMA] molar ratios: 0.25, 0.5, 0.75, 1.0, 1.25, 1.5, 1.75, 2.0, 2.25, 2.5, and 2.75, respectively). (b) Ethidium bromide displacement by MePEG2000-b-PMAASH interacting with Chol-PDMAEMA30/calf thymus DNA complexes (N/P ratio = 20) in PBS buffer (pH 7.4, 1 mM) at various [MAA]/[DMAEMA] ratios.
Figure 6. Schematic mechanism of the cooperative chain-exchanging reaction between MePEG2000-b-PMAASH and Chol-PDMAEMA30/DNA complexes. migration of DNA from the complexes. Furthermore, we can observe from lane 7 that these two types of DNA migration happen simultaneously at the [MAA]/[DMAEMA] molar ratio of 1.5. Ethidium Bromide Displacement Assay. As shown in Figure 5b, the fluorescence level of Chol-PDMAEMA30/calf thymus DNA complexes was 3.3% when the N/P ratio was fixed at 20. As the [MAA]/[DMAEMA] molar ratio was increased from 0 to 1, the fluorescence increased slowly from 3.3% to 22%. When it was further increased above 1, the fluorescence rose sharply to 72% and reached a plateau, which is consistent with the results given by gel retardation assay and this can be explained using the same mechanism as shown in Figure 6. When the [MAA]/[DMAEMA] molar ratio is lower than 1 or equal to 1, the surfaces of CholPDMAEMA30/DNA complexes are coated by MePEG2000-b-PMAASH and the segment of the DNA chains can be released due to the electrostatic interaction between the PMAASH block and PDMAEMA segment, as shown in area A in Figure 6. The released DNA combines with EtBr and brings on the gradually increased fluorescence intensity. Moreover, when the [MAA]/[DMAEMA] molar ratio is above 1.5, DNA is released completely and binds with EtBr as shown in area B in Figure 6, resulting in the rapid increase of the fluorescence. Based on these two assays, we can conclude that the binding of Chol-PDMAEMA30 with DNA can be destroyed by the excess MePEG2000-b-PMAASH and then DNA would be released from the complexes. The appropriate [MAA]/[DMAEMA] molar ratio is in the range of 1-1.5 for the fabrication of stable MePEG2000-b-PMAASH/CholPDMAEMA30/DNA complexes. 5206
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Figure 7. Gel retardation assay assays. Lane 1 is the DNA control. Lanes 2-11 represent Chol-PDMAEMA30/DNA complexes (N/P ratio = 20) with progressively increasing proportions of MePEG2000-b-PMAASH ([MAA]/[DMAEMA] molar ratios: 0.25, 0.5, 0.75, 1.0, 1.25, 1.5, 1.75, 2.0, 2.25, and 2.5, respectively), and the hydrogen peroxide solution was added 30 min later without stirring to form bridging disulfide linkages between the MePEG2000-b-PMAASH chains. 3.6. Forming Bridging Disulfide Linkages. To cross-link and stabilize the MePEG2000-b-PMAASH/Chol-PDMAEMA30/DNA complexes, hydrogen peroxide solution was added to oxidize the shells of complexes to form bridging disulfide linkages between the MePEG2000-b-PMAASH chains, and the electrophoretic image of complexes is shown in Figure 7. Lane 1 represents naked DNA intercalated with EtBr. Lanes 2-11 represent MePEG2000-b-PMAASH/ Chol-PDMAEMA30/DNA complexes at the [MAA]/ [DMAEMA] molar ratio from 0.25 to 2.5. As expected, DNA did not migrate even when the [MAA]/[DMAEMA] molar ratio was higher than 1.5 as shown in lanes 8-11 in Figure 7. In contrast, DNA migration was remarkable for the same complexes without disulfide cross-linking, as indicated in Figure 5a from lanes 8 to 12. It may be deduced that although the DNA was released by chain-exchanging reaction, the DNA chains were still limited within the capsule as shown in Figure 8A, in which the core and the shell are composed of DNA chains and MePEG2000-b-PMAASH/ Chol-PDMAEMA30 complexes, respectively. The capsule was not stable and would be dissociated due to the dilution effect and/or the shearing effect, as shown in Figure 8B. If the Langmuir 2009, 25(9), 5199–5208
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Figure 8. Schematic illustration concerning the effect of disulfide cross-linking on the stability of MePEG2000-b-PMAASH/Chol-PDMAEMA30/DNA complexes.
Figure 9. Gel retardation assay. Lane 1 is the DNA control. Lanes 2-12 correspond to the in vitro degradation of complexes, which were the disulfide cross-linked MePEG2000-b-PMAASH/CholPDMAEMA30/DNA complexes (N/P ratio=20, [MAA]/[DMAEMA] molar ratios: 0.25, 0.5, 0.75, 1.0, 1.25, 1.5, 1.75, 2.0, 2.25 and 2.5, respectively). capsule was not disulfide cross-linked, then it would be disintegrated and DNA would hence be released from the complexes, as illustrated in Figure 8C. This is confirmed by gel retardation assay shown in Figure 5. Otherwise, the capsule would remain integrated after the disulfide crosslinking and the migration of DNA was retarded, as shown in Figure 7 from lanes 8 to 11. This deduction is in agreement with the TEM measurement as shown in Figure 10d, in which the obvious capsule structure can be observed. 3.7. In Vitro Degradation of the Complexes. Gel retardation assay was utilized to evaluate the in vitro degradation of the complexes, and the electrophoretic image is shown in Figure 9. Lane 1 represents naked DNA intercalated with EtBr. Lanes 2-11 represent disulfide cross-linked MecomPEG2000-b-PMAASH/Chol-PDMAEMA30/DNA plexes with various [MAA]/[DMAEMA] molar ratios (N/P ratio = 20). It was obvious that DNA began to migrate when the [MAA]/[DMAEMA] molar ratio was above 1.0, as shown in Figure 9 from lanes 5 to 11. This indicates that the disulfide linkages (S-S) between PMAASH chains were successfully destroyed and DNA was hence released. It is worth mentioning that the chain-exchanging reaction may take place when DNA is released from the complexes with polycations in the intracellular environment, because various types of negatively charged macromolecules including mRNA sulfated sugars and nuclear chromatin exist as essential cellular components.31,51 From this perspective, the results would be beneficial for the release of DNA because once the disulfide linkages (S-S) are destroyed in cells, the cooperative chain-exchanging reaction between MePEG2000-b-PMAASH and DNA in the complexes will take place and lead to easier release of DNA.
3.8. Biophysical Properties of the Complexes. In order to further assess the biophysical properties of MePEG2000-bPMAASH/Chol-PDMAEMA30/DNA complexes, we measured the ζ-potential and particle size of various particles, and the results are listed in Table 1. The lipopolymer CholPDMAEMA30 could self-assemble into micelles with cholesterol as the core and water-soluble PDMAEMA as the corona, and the ζ-potential of these micelles was 27 ( 2 mV. After combining with DNA that possessed negative charges, the Chol-PDMAEMA30/DNA complexes (N/P ratio = 20) showed positive ζ-potential of about 20 ( 4 mV. Subsequently, the negative-charged MePEG2000-b-PMAASH was coated onto the above-mentioned complexes with positive surface charges, resulting in MePEG2000-b-PMAASH/CholPDMAEMA30/DNA complexes with slightly negative potentials (-5 ( 3 mV). It can be concluded that the MePEG2000-bPMAASH was effectively adsorbed onto the surface of the Chol-PDMAEMA30/DNA complexes via electrostatic interaction and shielded the positive charges. On the other hand, the average particle sizes of particles formed by Chol-PDMAEMA30 and naked DNA in the solution were 195 and 129 nm, respectively. When they were mixed together, the average particle size of the complexes (N/P ratio = 20) increased sharply to 316 nm. It should be taken into account that the aggregation of the Chol-PDMAEMA30/DNA complexes is dominant at pH 7.4 in 10 mM PBS buffer despite the presence of excess polycations, probably due to the relatively low charge density of PDMAEMA chains and they cannot provide sufficient electrostatic repulsion in the buffer media.47 When the MePEG2000-b-PMAASH was added into the Chol-PDMAEMA30/DNA complex, the MePEG2000-b-PMAASH/CholPDMAEMA30/DNA complexes were obtained and their average particle size decreased to 230 nm. These results indicate that hydrophilic MePEG2000 chains can stabilize the complexes and reduce the aggregation of Chol-PDMAEMA30/DNA complexes. TEM analysis can also present evidence for the above explanations. Figure 10 shows the TEM images of the particles for Chol-PDMAEMA30 micelles, naked DNA, Chol-PDMAEMA30/DNA complexes, and MePEG2000b-PMAASH/Chol-PDMAEMA30/DNA complexes. It can be found from Figure 10d that the aggregation between the particles of MePEG2000-b-PMAASH/Chol-PDMAEMA30/ DNA complexes was greatly reduced after the addition of MePEG2000-b-PMAASH, which is quite different from the aggregation between the particles of Chol-PDMAEMA30/ DNA complexes observed from Figure 10c.
4. (51) Erbacher, P.; Roche, A. C.; Monsigny, M.; Midoux, P. Bioconjugate Chem. 1995, 6, 401–410.
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ζ-potential (mV)
Dz (nm)
Chol-PDMAEMA30 DNA (plasmid pUC 18) Chol-PDMAEMA30/DNAa MePEG2000-b-PMAASH/Chol-PDMAEMA30/DNAb
27 ( 2 -26 ( 3 20 ( 4 -5 ( 3
195 ( 20 129 ( 12 316 ( 27 230 ( 15
a
N/P ratio = 20. b N/P ratio = 20; [MAA]/[DMAEMA] molar ratio = 2.
Figure 10. TEM micrographs of various particles formed in 10 mM PBS buffer at pH 7.4: (a) micelles self-assembled from CholPDMAEMA30 of 1 g L-1; (b) naked DNA particles from plasmid pUC 18 DNA of 1 g L-1; (c) Chol-PDMAEMA30/DNA complexes at N/P ratio = 20; and (d) MePEG2000-b-PMAASH/Chol-PDMAEMA30/DNA complexes at a [MAA]/[DMAEMA] molar ratio of 1 and N/P ratio = 20. (In each TEM image, bar = 0.2 μm.)
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anion-initiated polymerization. The cmc measurement and competition binding assay indicated that the Chol-PDMAEMA30 could be an effective nonviral gene carrier. Subsequently, an intelligent delivery system has been developed to overcome the defects of the positively charged complexes, in which MePEG2000-b-PMAASH with partial mercapto groups was used to coat the Chol-PDMAEMA30/DNA complexes via electrostatic interaction between PDMAEMA and PMAASH. Hydrogen peroxide solution was then added to oxidize the mercapto groups to form bridging disulfide linkages between the MePEG2000-b-PMAASH chains and to stabilize the complexes. This system can provide a better stability during systemic circulation, and it will disintegrate and release DNA once the disulfide linkages are destroyed in a cell reducing environment. The studies on chain-exchanging reaction indicate that the excess MePEG2000-b-PMAASH will lead to the aborted binding of Chol-PDMAEMA30 with DNA and the amount of MePEG2000-b-PMAASH is appropriate when the [MAA]/[DMAEMA] molar ratio is in the range of 1.0-1.5. However, the results of further investigation showed that the exchange reaction may be beneficial for the DNA release when the disulfide linkages are destroyed in cells. Presently, follow-up studies on the cytotoxicity and transfection efficiency of this gene vector are underway in our laboratory. Acknowledgment. The authors gratefully acknowledge the financial support from the Natural Science Foundation of Jiangsu Province (BK2008157), the National Natural Science Foundation of China (20474041), and Qing Lan Project for Innovation Team of Jiangsu Province.
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