Biomacromolecules 2003, 4, 1759-1762
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Degradable Poly(amino alcohol esters) As Potential DNA Vectors with Low Cytotoxicity Sangyong Jon, Daniel G. Anderson, and Robert Langer* Department of Chemical Engineering, E25-342, Massachusetts Institute of Technology, 77 Mass Ave, Cambridge, Massachusetts 02139 Received June 5, 2003; Revised Manuscript Received August 6, 2003
The synthesis of a new degradable polymer system, poly(amino alcohol esters) and the resulting polymers’ potential for use in gene transfection vectors are reported. The polymerization proceeded in a one step reaction from commercially available bis(secondary amines) monomers (N,N′-dimethyl-1,3-propanediamine and N,N′-dimethyl-1,6-hexanediamine, respectively) through nucleophilic addition to the diglycidyl ester of dicarboxylic acid (diglycidyl adipate). Poly(amino alcohol ester) 1 and 2 were synthesized with a yield of 89% and 91% with Mn ) 24 800 and Mn ) 36 400, respectively. Poly(amino alcohol ester) 1 degraded hydrolytically in phosphate buffer at pH 7.4 with a half-life of approximately 5 days. Both polymers readily self-assembled with plasmid DNA into nanometer-sized DNA/polymer complexes less than 180 nm diameter and are significantly less cytotoxic than the commonly used DNA delivery polymer, poly(ethylene imine) (PEI). Introduction The efficient and safe delivery of DNA remains a fundamental challenge to the success of gene therapy.1 Although viral vectors are efficient gene carriers, growing safety concerns have focused much attention on nonviral, synthetic carriers as attractive alternatives.2 In particular, much progress has been made with cationic polymers such as poly(lysine),3 poly(ethylene imine),4 starburst PAMAM dendrimers,5 and cationic liposomes.6 These systems, however have significant cytotoxicity issues, in part due to the poor biocompatibility of nondegradable polymers.7 Consequently, recent attention has focused on the development of biodegradable cationic polymers. Several examples of degradable gene carriers reported by us and others include: linear poly(β-amino ester),8 poly(4-hydroxy-L-proline ester),9 hyperbranched poly(amino ester),10 poly[R-(4-aminobutyl)L-glycolic acid] (PAGA),11 and poly(2-aminoethyl propylene phosphate) (PPE-EA).12 Here we describe a new degradable polymer system, poly(amino alcohol esters), with many of the attributes characteristic of ideal gene delivery vectors: Poly(amino alcohol esters) are degradable via their polyester backbone, condense DNA into nanometer-sized particles in physiological pH, possess hydroxy groups that enhance water solubility, and both polymer and degradation products are nontoxic. These polymers are easily synthesized without the generation of toxic products and offer great potential as the next generation of gene delivery vectors. Experimental Section General Considerations. All manipulations involving live cells or sterile materials were performed in a laminar flow * To whom correspondence should be addressed. Phone: 1-617-2533107. Fax: 1-617-258-8827. E-mail:
[email protected].
hood using standard sterile technique. 1H NMR (400 MHz) and 13C NMR (100 MHz) spectra were recorded on a Bruker instrument (Avance DPX 400). Aqueous phase GPC was performed by American Polymer Standards (Mentor, OH) using Ultrahydrogels L and 120A columns in series (Waters Corporation, Milford, MA). Water (1% acetic acid, 0.3 M NaCl) was used as the eluent at a flow rate of 1.0 mL/min. Data were collected using a Knauer differential refractometer and processed using an IBM/PC GPC-PRO 3.13 software package (Viscotek Corporation, Houston, TX). Thermogravimetric analysis (TGA) was made using a PerkinElmer instrument (model: TGA 7) (Shelton, CT). Samples were heated at 20 °C min-1 from 30 to 900 °C in a nitrogen atmosphere. Differential scanning calorimetry (DSC) was performed using a PerkinElmer instrument (model: Diamond DSC): heating from -30 to 200 °C at 10 °C min-1, holding at 200 °C for 2 min, cooling from 200 to -30 °C in a nitrogen atmosphere, and finally repeat the process. Materials. N,N′-Dimethyl-1,3-propanediamine, N,N′-dimethyl-1,6-hexanediamine, dimethyl adipate (DBE-6 dibasic ester), glycidol, and thalium(III)acetate sesquihydrate were purchased from Aldrich Chemical Co. (Milwaukee, WI). 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) was purchased from Sigma Chemical Co. (St. Louis, MO). Plasmid DNA (pCMV-Luc) was purchased from Elim Biopharmaceuticals, Inc (Hayward, CA). NIH 3T3 cells were purchased from American Type Culture Collection and grown at 37 °C, 5% CO2 in Dulbecco’s modified Eagle’s medium (DMEM), 90%; fetal bovine serum, 10%; penicillin, 100 units/mL; streptomycin, 100 µg/mL. All other materials and solvents were used as received without further purification. Synthesis of Diglycidyl Adipate (a). This compound was prepared from transesterification of dimethyl adipate with
10.1021/bm034176f CCC: $25.00 © 2003 American Chemical Society Published on Web 08/30/2003
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glycidol as described elsewhere.13 1H NMR (CDCl3, 400 MHz) δ 4.44 (dd, J ) 12.3 Hz, 2H), 3.92 (dd, J ) 6.38 Hz, 2H), 3.22 (m, 2H), 2.86 (t, J ) 4.25 Hz, 2H), 2.66 (m, 2H), 2.41 (br, 4H), 1.69 (br, 4H). 13C NMR (CDCl3, 100 MHz) δ 173.4, 65.3, 49.7, 45.1, 34.0, 24.6. General Polymerization Procedure. The diamine reagent (1 mmol) was added to a solution of diglycidyl adipate (a) (259 mg, 1 mmol) in anhydrous dichloromethane (1 mL) in a vial at room temperature. The vial was sealed with a Teflon-lined screw cap, and the reaction mixture was stirred at 42 °C for 72 h. After cooling the reaction mixture to room temperature, anhydrous tetrahydrofuran was added to the resulting highly viscous reaction mixture to precipitate the polymer. The remained gummy material was washed several times with tetrahydrofuran to remove unreacted monomers and dried under vacuum for several days. Poly-1: 1H NMR (D2O, 400 MHz) δ 4.25-3.97 (br m, 4H), 3.60-3.41 (br m, 2H), 3.21 (br, 8H), 2.88 (br s, 6H), 2.35 (br, 4H), 2.13 (br, 2H), 1.52 (br s, 4H). FTIR (cm-1, neat): 3340, 2939, 2861, 1737, 1555, 1450, 1411, 1062, 853. Poly-2: 1H NMR (D2O, 400 MHz) δ 4.29-3.99 (br m, 4H), 3.75-3.45 (br m, 4H), 3.26 (br, 2H), 2.90-2.51 (br, 8H), 2.51-2.29 (br, 6H), 2.30-2.15 (br, 2H), 1.82-1.34 (br, 12H). FTIR (cm-1, neat): 3304, 2935, 2862, 1733, 1562, 1459, 1402, 1053, 873. Thermogravimetric analysis (TGA) showed that poly-1 and poly-2 started to decompose at 234.5 °C and 225.4 °C, respectively. Although poly-2 showed the glass transition temperature (Tg) at 10.9 °C by differential scanning calorimetry (DSC), no Tg was observed for poly-1 over temperature range studied (-30 to +200 °C). Degradation Study. The polymer 1 was dissolved in phosphate buffer (1X, pH ) 7.4) at a concentration of 10 mg/mL. The resulting solution was incubated at 37 °C and aliquots (0.1 mL) were removed at appropriate time intervals for aqueous GPC analysis. Agarose Gel Retardation Assay. DNA/Polymer complexes were formed by adding 50 µL of each polymer solution in 25 mM of acetate buffer, pH 5.1 (concentrations adjusted to yield desired DNA/Polymer weight ratios) to 50 µL of a plasmid DNA solution (0.1 µg/µL in water) in a tube. The resulting mixtures were gently vortexed and left to stand at room temperature for 30 min, after which 15 µL of the complex was loaded on a 0.8% agarose gel with loading buffer (not including bromophenol blue) consisting of 10% Ficoll 400 (Amersham Pharmacia Biotech, Uppsala, Sweden) in TAE buffer. The gel was run at 50 V for 1 h using Embi Tec (model: RunOne Electrophoresis Cell) (San Diego, CA) and DNA bands were visualized by ethidium bromide staining. Particle Sizing and Zeta Potential Measurements. Quasi-elastic laser light scattering (QELS) experiments were carried out to measure particle size using a ZetaPALS dynamic light scattering detector (Brookhaven Instruments Corporation, Holtsville, NY, 15 mW laser, incident beam ) 676 nm). DNA/polymer complexes were formed as described above for the agarose gel retardation assay. Samples were diluted with 3 mL of HEPES buffer (25 mM, pH 7.0). Particle sizes were measured at 25 °C. Correlation functions were collected at a scattering angle of 90°, and particle sizes
Jon et al. Scheme 1. Synthetic Scheme of Linear Poly(amino alcohol esters)
were calculated using the MAS option of the company’s particle sizing software (version 2.30) under the viscosity and refractive index of pure water at 25 °C. Particle sizes expressed as effective diameters assuming a log-normal distribution. Three measurements were made on each sample, and the results are reported as average diameters. Electrophoretic mobilities were measured at 25 °C using BIC PALS zeta potential analysis software, and zeta potentials were calculated using the Smoluchowsky model. Cytotoxicity Assay. Immortalized NIH 3T3 cells were grown in 96-well plates at an initial seeding density of 10 000 cells/well in 200 µL of growth medium. Cells were grown for 24 h after which the growth medium was removed and replaced with 180 µL of serum-free DMEM. Appropriate amounts of polymers were added in 20 µL aliquots. Samples were incubated at 37 °C, 5% CO2 for 4 h, and the metabolic activity of each well was determined by using a MTT/ thiazolyl blue assay: to each well was added 25 µL of a 5 mg/mL solution of MTT stock solution in sterile PBS buffer. The samples were incubated at 37 °C for 2 h, and 100 µL of extraction buffer (20% w/v sodium dodecyl sulfate in DMF/water (1/1), pH 4.7) was added to each well. Samples were incubated at 37 °C, 5% CO2 for 24 h. Optical absorbance was measured at 560 nm using a microplate reader (model: SPECTRAmax PLUS) (Molecular Devices Corporation, Sunnyvale, CA) and expressed as a percent relative to control cells. Results and Discussion Synthesis and Characterization of Poly(amino alcohol esters). Although poly(amino alcohol esters) made from reaction between diglycidyl esters of dicarboxylic acids and bis(alkylamines) had been reported for the first time in the late 1960s,14 these polymers have received little attention to date. In general, poly(amino alcohol esters) are formed from the nucleophilic addition of diamines to diepoxy esters (diglycidyl esters) under mild conditions. In this paper, linear poly(amino alcohol esters) were obtained using bis(secondary amines) as diamine nucleophiles and diglycidyl adipate as a model of a diepoxy ester (Scheme 1). We avoided using bis(primary amines) because they could form cross-linked
Degradable Poly(amino alcohol esters)
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Figure 2. Agarose gel electrophoresis retardation of pCMV-Luc DNA by poly-1 and poly-2. Lane numbers correspond to different DNA/ polymer weight ratios and insect denotes zeta potentials of such complexes. Poly-1: (1) DNA only, (2) 1:1, (3) 1:2, (4) 1:3, (5) 1:5, (6) 1:7.5. Poly-2: (7) 1:1, (8) 1:2, (9) 1:3, (10) 1:5, and (11) 1:7.5. Figure 1. Degradation profile of polymer 1 at 37 °C in phosphate buffer (pH 7.4). A percent degradation (Mw,time(t)/Mw,time(0) × 100) is expressed as a function of time based on GPC data.
polymer networks through further nucleophilic addition of secondary amines generated.15 There are several attributes of linear poly(amino alcohol esters) that make them particularly suitable as gene carriers: (1) the polymers contain readily degradable polyester linkages by hydrolysis, (2) amines for self-assembly with DNA in physiological pH, (3) polar hydroxy groups at each repeating unit for high water solubility, and (4) a variety of the polymer analogues may be easily synthesized from the library of diacids and diamines and screened for highly efficient gene vectors.8b Polymers 1 and 2 were synthesized from the reactions of N,N′-dimethyl-1,3-propanediamine and N,N′-dimethyl-1,6hexanediamine, respectively, with diglycidyl adipate (a) in CH2Cl2 at 42 °C for 3 days with a yield of 89% and 91%, respectively (Scheme 1). Polymer 2 is expected to be more flexible than polymer 1 because it has a longer alkyl chain between two tertiary amine units. Because of the presence of polar OH groups at each repeating unit, the polymers were soluble in highly polar solvents such as DMSO and water but insoluble in most of organic solvents such as THF, CH2Cl2, and ethyl acetate. Although polymer 1 was highly soluble in water regardless of pH, polymer 2 was more soluble in rather acidic water (< pH 6). Molecular weights of polymer 1 and 2 were measured by aqueous gel permeation chromatography (GPC) using water (1% acetic acid, 0.3 M NaCl solution) as eluent. Molecular weights (Mn) and polydispersities (PDI) were obtained relative to poly(2-vinylpyridine) standards: Poly-1, Mn ) 24 800 (PDI ) 1.22); Poly-2, Mn ) 36 400 (PDI ) 1.93). When other organic solvents such as THF, CHCl3, and DMF were used, much lower molecular weights of polymers than those synthesized in CH2Cl2 were generally obtained and polymerization proceeded very slowly. To examine degradability of such poly(amino alcohol esters), poly-1 was dissolved in phosphate buffer (1X, pH ) 7.4), incubated at 37 °C, and aliquots were removed at appropriate times. Aqueous GPC analysis was performed with the aliquots. A percentage of degradation was calculated from relative change in Mw of the aliquot at a certain time point to the initial Mw. Figure 1 shows the degradation profile of poly-1 as a function of time. The result showed that poly-1 degraded slowly and the half-life of degradation was approximately 5 days. Because both poly-1 and poly-2 have
Figure 3. Average effective diameters of DNA/polymer complexes as a function of polymer concentration.
the same general backbone structure (differing only in length), we expect that poly-2 is also degradable, but with a different degradation rate. Self-Assembly of Polymer 1 and 2 with Plasmid DNA. The ability of polymers 1 and 2 to complex with plasmid DNA (pCMV-Luc) was demonstrated using agarose gel electrophoresis, quasi-elastic laser scattering (QELS), and zeta potential analysis. Complete gel retardation of DNA/ polymer complexes indicates that charge neutralization of DNA is achieved because agarose gel electrophoresis separates molecules based on charge and on size as well. Both polymers were dissolved in sodium acetate buffer (25 mM, pH 5.1) at the concentration of 1 µg/µL. A fixed amount of plasmid DNA (0.1 µg/µL) was incubated with polymer solutions at desired DNA/polymer weight ratios and vortexed for a few seconds followed by agarose gel electrophoresis (Figure 2). Although complete gel retardation occurred effectively at the DNA/polymer ratio of 1:1 (w:w) with a zeta potential of 3.9 ( 0.8 mV in the case of polymer 2 (Figure 2, lane 7), complete neutralization with polymer 1 was observed at the 1:3 DNA/polymer ratio (Figure 2, lane 4) with a zeta potential of 2.4 ( 0.5 mV. The higher efficiency of polymer 2 in DNA complexation relative to polymer 1 may be due to higher flexibility of the polymer 2 backbone structure than that of the polymer 1: hexanediamine versus propanediamine.16 The size of DNA/polymer complexes as a function of increasing polymer ratios was determined by quasi-elastic laser scattering (QELS) (Figure 3). Previously published data suggests that polymers must be able to compact DNA into nanometer-sized particles on the order of 80% viability even at a higher dose of the polymers (100 µg/mL) with no significant change in cell morphology and proliferation relative to controls. Summary A new nontoxic, degradable, and water-soluble polymeric vector for gene transfection is reported. Poly(amino alcohol esters) 1 and 2 were easily prepared in high yields through nucleophilic addition of N,N′-dimethyl-1,3-propanediamine and N,N′-dimethyl-1,6-hexanediamine to diglycidyl adipate.
Jon et al.
This synthetic strategy is expected to be applicable to the preparation of a variety of polyesters containing tertiary amines as well as hydroxy functional groups from commercially available starting materials in one step. Polymers 1 and 2 readily self-assembled with DNA into nanometersized DNA/polymer complexes on the order of 150-200 nm, which was determined by agarose gel electrophoresis and quasi-elastic dynamic light scattering (QELS). These polymers are relatively noncytotoxic in vitro, as measured by MTT. Based on the results reported herein, we concluded that poly(amino alcohol esters) have good potential for use as gene transfection vectors. We are currently investigating transfection ability of these polymers in vitro. Acknowledgment. This work was supported in part by the Postdoctoral Fellowship Program of Korea Science & Engineering Foundation (KOSEF) and by the NSF (through the MIT Biotechnology Process and Engineering Center) and by NIH (Grant No. EB-00244). D.G.A also thanks the NIH for his postdoctoral fellowship. References and Notes (1) Pouton, C. W.; Seymour, L. W. AdV. Drug DeliVery ReV. 2001, 46, 187-203. (2) Luo, D.; Saltzman, W. M. Nat. Biotechnol. 2000, 18, 33-37. (3) Zauner, W.; Ogris, M.; Wagner, E. AdV. Drug DeliVery ReV. 1998, 30, 98-113. (4) Kircheis, R.; Wightman, L.; Wagner, E. AdV. Drug DeliVery ReV. 2001, 53, 341-358. (5) (a) Bielinska, A. U.; Chen, C. L.; Johnson, J.; Baker, J. R. Bioconjugate Chem. 1999, 10, 843-850. (b) Tang, M. X.; Redemann, C. T.; Szoka, F. C., Jr. Bioconjugate Chem. 1996, 7, 703-714. (6) Miller, A. D. Angew. Chem., Int. Ed. Engl. 1998, 37, 1768-1785. (7) (a) Godbey, W. T.; Wu, K. K.; Mikos, A. G. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 5177-5181. (b) Zabner, J. AdV. Drug DeliVery ReV. 1997, 27, 17-28. (8) (a) Lynn, D. M.; Langer, R. J. Am. Chem. Soc. 2000, 122, 1076110768. (b) Lynn, D. M.; Anderson, D. G.; Putnam, D.; Langer, R. J. Am. Chem. Soc. 2001, 123, 8155-8156. (c) Akinc, A.; Lynn, D. M.; Anderson, D. G.; Langer, R. J. Am. Chem. Soc. 2003, 125, 53165323. (9) Putnam, D.; Langer, R. Macromolecules 1999, 32, 3658-3662. (10) Lim, Y.; Kim, S.-M.; Lee, Y.; Lee, W.; Yang, T.; Lee, M.; Suh, H.; Park, J. J. Am. Chem. Soc. 2001, 123, 2460-2461. (11) Lim, Y.; Kim, C.; Kim, K.; Kim, S. W.; Park, J. J. Am. Chem. Soc. 2000, 122, 6524-6525. (12) Wang, J.; Mao, H.-Q.; Leong, K. W. J. Am. Chem. Soc. 2001, 123, 9480-9481. (13) Zondler, H.; Trachsler, D.; Lohse, F. HelV. Chim. Acta 1977, 60, 1845-1860. (14) Kato, F.; Yoshihara, M. U. S. Patent U.S. 3,535,289, 1967. (15) When we carried out the reaction with bis(primary amines) such as 1,n-diaminoalkane, we observed considerable extent of cross-linking in polymers backbone, and as a result, these polymers are insoluble in aqueous solution as well as most organic solvents. (16) Hwang, S. J.; Bellocq, N. C.; Davis, M. E. Bioconjugate Chem. 2001, 12, 280-290. (17) Kennedy, G. L., Jr. Drug Chem. Toxicol. 2002, 25 (2), 191-202. (18) (a) Smith, D. J.; Patel, S.; Rowland, E. C. U.S. Patent U.S. 4,778,825, 1988. (b) Bhide, M. V.; Patel, S.; Rowland, E. C.; Smith, D. J. J. Immunopharmacol. 1985, 7(3), 303-312.
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