Bioconjugate Chem. 2007, 18, 2037–2044
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Synthesis and in Vitro Evaluation of Novel Star-Shaped Block Copolymers (Blocked Star Vectors) for Efficient Gene Delivery Yasuhide Nakayama,*,†,‡ Chiaki Kakei,† Ayaka Ishikawa,† Yue-Min Zhou,† Yasushi Nemoto,†,‡,§ and Kingo Uchida| Department of Bioengineering, Advanced Medical Engineering Center National Cardiovascular Center Research Institute, Division of Biotechnology and Macromolecular Chemistry, Faculty of Engineering, Hokkaido University, Chemical Products Division, Development Department, Bridgestone Company, and Department of Materials Chemistry, Faculty of Science and Technology Ryukoku University. Received February 11, 2007; Revised Manuscript Received July 29, 2007
Novel 4-branched diblock copolymers consisting of cationic chains as an inner domain and nonionic chains as an outer domain were prepared by iniferter-based living radial polymerization and evaluated as a polymeric transfectant. The cationic polymerization of 3-(N,N-dimethylamino)propyl acrylamide (DMAPAAm) using 1,2,4,5-tetrakis(N,Ndiethyldithiocarbamylmethyl)benzene as a 4-functional iniferter followed by the nonionic block polymerization of N,N-dimethylacrylamide (DMAAm) afforded 4-branched diblock copolymers with controlled compositions. By changing the solution or irradiation conditions, 4-branched PDMAPAAms with molecular weights of 10 000, 20 000, and 50 000 were synthesized. In addition, by graft polymerization, PDMAPAAm–PDMAAm blocked copolymers with copolymer composition (unit ratio of DMAAm/DMAPAAm) ranging from 0.18 to 1.0 for each cationic polymer were synthesized. All polymers were shown to interact with and condense plasmid DNA to yield polymer/DNA complexes (polyplexes). A transfection study on COS-1 cells showed that the polyplexes from block copolymers with cationic chain length of approximately 50 000 and a nonionic chain length of 30 000, which were approximately 200 nm in diameter and very stable in aqueous media, had the most efficient luciferase activity with minimal cellular cytotoxicity under a charge ratio of 20 (vector/pDNA). The PDMAPAAm–PDMAAmblocked, star-shaped polymers are an attractive novel class of nonviral gene delivery systems.
INTRODUCTION Cationic polymers such as poly(ethylenimine) (PEI) (1–4) and poly(N,N-dimethylaminopropyl acrylamide) (PDMAPAAm) (5, 6), which can generate nanoparticles by the formation of polyion complexes, i.e., “polyplexes” with DNA, are highly expected as one of the major carriers in nonviral gene delivery systems due to the many advantages they offer over viral systems (7–11). These advantages include blocking of specific immune responses, no restrictions in the size of DNA, and ease of large-scale production. However, the primary obstacle toward implementing an effective gene therapy using cationic polymers remains their relatively inefficient gene transfection in ViVo when compared to virus vectors. Recently, to improve gene transfection efficiency using cationic polymers, we designed a series of branched cationic polymers, linear and 3-, 4-, or 6-branched PDMAPAAm as a novel high-performance gene carrier called star vector (5), prepared to have the same molecular weights (Mn 18 000) by iniferter (initiator–transfer agent–terminator)-based photoliving radical polymerization (12, 13) from the respective multidithiocarbamate-derivatized benzenes (multifunctional iniferters) and DMAPAAm as a cationic monomer. This experiment revealed that the relative gene expression efficiency increased in the degree of branching that may affect the cationic charge density, * To whom correspondence should be addressed. Department of Bioengineering, Advanced Medical Engineering Center, National Cardiovascular Center Research Institute, 5-7-1 Fujishiro-dai, Suita, Osaka 565-8565, Japan. Telephone: (+81) 6-6833-5012(ex. 2624). Fax: (+81) 6-6872-8090 E-mail:
[email protected]. † National Cardiovascular Center Research Institute. ‡ Hokkaido University. § Bridgestone Company. | Ryukoku University.
and that the compaction of DNA polyplexes was stabilized on an increase in the branch number (5). However, the in ViVo transfection efficiency was not satisfactory because of the high interaction between DNA polyplexes, which was caused by the low colloidal stability of the polyplex particles. On the other hand, the conjugation of cationic polymers with a hydrophilic and biocompatible polymer such as poly(ethylene glycol) (PEG) (14–18) has been reported as a major vector modification strategy to improve transfection efficiency (19–22). Surface modification of the cationic polymers with PEG was effective in increasing the long circulation time for DNA delivery system, and PEGylation effect for PEI was studied in detail by Kissel and co-workers (23, 24). Kim et al. reported that triblocking by PEG of PEI caused the lack of serum inhibition of transfection activity with low cytotoxicity (19). Therefore, it is highly expected that, by linking the nonionic hydrophilic chains with PDMAPAAm-branched polymers, DNA condensation and gene transfection efficiency will be improved by shielding the particles from unspecific interactions and conferring them stability in a similar fashion as PEGylation of polyplexes. To this end, in this study, we report novel structurally well-defined PDMAPAAm-branched polymers blocked with poly(N,N-dimethyl acrylamide) (PDMAAm), which is a biocompatible nonionic polymer, as a nonionic chain for in Vitro gene delivery. The blocked, branched copolymers were synthesized by photoliving radical polymerization of DMAPAAm using benzene derivatized from four dithiocarbamate groups as a 4-functional iniferter followed by block copolymerization of DMAAm. Their DNA-binding properties were studied using dynamic light scattering (DLS). The in Vitro transfection activity
10.1021/bc070045q CCC: $37.00 2007 American Chemical Society Published on Web 10/09/2007
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as well as the cytotoxicity of the block copolymer-based polyplexes was evaluated using COS-1 cells.
EXPERIMENTAL PROCEDURES Materials. 1,2,4,5-Tetrakis(bromomethyl)benzene was obtained from Sigma-Aldrich (Milwaukee, WI). Sodium N,Ndiethyldithiocarbamate and N,N-dimethylacrylamide (DMAAm) were purchased from Wako Pure Chemical Ind., Ltd. (Osaka, Japan), and 3-(N,N-dimethylamino)propyl acrylamide (DMAPAAm) was from Tokyo Kasei Kogyo Co., Ltd. (Tokyo, Japan). Other chemical reagents were commercially obtained from Wako Pure Chemical Ind., Ltd. DMAAm and DMAPAAm were distilled under reduced pressure before use to remove the stabilizer, and other reagents were purified before use according to the requirements. The luciferase reporter vector plasmid pGL3 control (3.5 MDa, 5256 bp) was purchased from Promega Inc. (Tokyo, Japan) and was amplified with a competent E. coli strain DH5R (Promega) and purified using Maxiprep according to a protocol from QIAGEN (Tokyo, Japan). The purity of the plasmid was checked by electrophoresis on a 1% agarose gel, and the concentration was determined by measuring UV absorbance at 260 and 280 nm. Exgen 500 (liner 22 kDa PEI) was obtained from COSMO BIO Co., Ltd. (Tokyo, Japan). Polymer Synthesis. PDMAPAAm–PDMAAm 4-branched block copolymers were synthesized by iniferter-based photoliving radical polymerization (12, 13) from 1,2,4,5-tetrakis(N,Ndiethyldithiocarbamylmethyl)benzene as a 4-functional iniferter with DMAPAAm and DMAAm as cationic and nonionic monomers, respectively. Synthesis of 4-Functional Iniferter (2). We dissolved 1,2,4,5tetrakis(bromomethyl)benzene (1, 5 g) in ethanol (250 mL) and added sodium N,N-diethyldithiocarbamate (4.8 equiv for 1) at 0 °C, followed by stirring at room temperature for 24 h. After filtering out the resulting sodium chloride, the filtrate was concentrated, dissolved in water (200 mL), and the extraction was repeated with ether (50 mL × 3). The organic layer was dried using MgSO4 and condensed to yield iniferter 2. 1H NMR
Nakayama et al.
(in D2O): δ 1.30–1.26 (t, 24H, -CH2-CH3), 3.77–3.69 (q, 8H, -N-CH2-), 4.07–3.99 (q, 8H, -N-CH2-), 4.57 (s, 8H, Ar-CH2S), 7.49 (s, 2H, Ar-H). Synthesis of PDAPAAm 4-Branched Polymer. The iniferterbased photoliving radical reaction of 2 with DMAPAAm was carried out as follows. A methanol solution (20 mL) of 2 (72 mg, 0.1 mmol) and DMAPAAm (3.43 g, 220 equiv) was placed into a 50 mL quartz crystal tube; subsequently, dry N2 gas was bubbled into the tube to preclude the air gas from solution for 5 min. The mixture solution was then irradiated for 30 min with a 200 W high-pressure mercury lamp (SPOT CURE, USHIO, Tokyo, Japan) under N2 atmosphere at room temperature, and the light intensity was set to 1 mW/cm2 at a wavelength of 250 nm (UVR-1; TOPCON, Tokyo, Japan). The reaction mixture was concentrated and adjusted to an appropriate concentration to purify it by precipitation in a large amount of ether. Reprecipitation was carried out in a methanol–ether system. The last precipitate was dried under vacuum to yield PDMAPAAm 4-branched polymer (conv. 40%). The molecular weight was determined by gel permeation chromatography (GPC) analysis: Mn 50 000. 1H NMR: δ 1.7–1.5 (br, 3H, -CH2-CH- and -CH2CH2-CH2-), 2.0–1.8 (br, 1H, -CH-CO), 2.2–2.1 (br, 6H, -NCH3), 2.4–2.2 (br, 2H, -CH2-N(CH3)2), 3.2–3.0 (br, 2H, -NHCH2-), 7.8–7.4 (br, 1H, -NH). Other branched polymers with different number average molecular weights were also synthesized using a similar procedure by changing the monomer concentration (see Table 1). Synthesis of PDMAPAAm-PDMAAm 4-Branched Copolymer. The general procedure of the synthesis of PDMAPAAm–PDMAAm 4-branched copolymer is described below. A methanol solution (20 mL) of PDMAPAAm 4-branched polymer (Mn 50 000, 125 mg, 2.5 µmol) and DMAAm (2.97 g, 1200 equiv) was placed into a 50 mL quartz crystal tube and deoxygenated by bubbling with dry N2 gas for 5 min. The mixture solution was then irradiated for 30 min under the above-mentioned conditions. The reaction mixture was concentrated and precipitated in 500 mL of ether. Reprecipitation was carried out in a methanol–ether system. The last precipitate was dried under
Figure 1. Syntheses of PDMAPAAm 4-branched polymers and PDMAPAAm–PDMAAm 4-branched block copolymers.
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Table 1. Reaction Conditionsa and Molecular Weights of the PDMAPAAm 4-Branched Polymers PDMAPAAm 4-branched polymer [2] (mM)
[DMAPAAm] (M)
irradiation timeb (min)
conv. (%)
Mnc (g/mol)
Mw/Mnc
no of DCd
5 5 5
0.34 0.50 1.10
30 30 30
21 34 40
10000 20000 50000
1.3 1.4 1.4
3.7 4.2 3.6
a Solvent: methanol. b Irradiation intensity: 1 mW/cm2. c Setermined by GPC (PEO standard). d DC means dithiocarbamate group, and the number of DC was determined by UV spectroscopy.
Figure 2. GPC elution curves after polymerization of DMAPAAm from 4-functional iniferter (2) as a function of monomer concentration (polymerization conditions were summarized in Table 1) and PDMAPAAm–PDMAAm 4-branched block copolymer (Mn 83 000) obtained after block copolymerization from PDMAPAAm 4-branched polymer (Mn 50 000) under polymerization conditions in Table 2.
vacuum to yield the PDMAPAAm–PDMAAm 4-branched block copolymer (conv. 19%). The total molecular weight was estimated from the integral value of 1H NMR spectra between PDMAPAAm and PDMAAm (see Figure 2): Mn 83 000. 1H NMR (in D2O): δ 1.0–1.7 (br, 1922H, -CH2CH- and -CH2CH2CH2-), 1.9 (br, 315H, -CH2CH- of PDMAPAAm), 2.1 (s, 1890H, -N(CH3)2 of PDMAPAAm), 2.3 (br, 630H, -CH2N(CH3)2), 2.4–2.7 (br, 331H, -CH2CH- of PDMAAm), 2.8–2.9 (br, 1986H, -N(CH3)2 of PDMAAm), 3.0 (br, 630H, -CONHCH2-). Other 4-branched block copolymers with different compositions were also synthesized using a similar procedure by changing the concentrations of PDMAPAAm 4-branched polymer and DMAAm or reaction times (see Table 2). General Methods. 1H NMR spectra were recorded in D2O with a 300 MHz NMR spectrometer (Gemini-300; Varian, Palo Alto, CA) at room temperature. GPC analyses in N,N-dimethylformamide were carried out with a HPLC-8020 instrument (Tosoh, Tokyo, Japan) (column: Tosoh TSKgel R-3000 and R-5000). The columns were calibrated with narrow weight distribution PEG standards (Tosoh). UV–vis spectra in H2O were recorded with a UV–vis spectrophotometer (UV-1700, Shimadzu Co., Kyoto, Japan). Preparation of Polymer/Plasmid Complexes. The PDMAPAAm–PDMAAm 4-branched copolymer was dissolved in a saline solution. Aliquots of these solutions (60 µL) were added to the plasmid dissolved in 90 µL of Tris-HCl buffer (pH7.4) to obtain polymer/DNA ratios of 1–20, which correspond to cation/anion (C/A) ratios. The solutions (total volume, 150 µL; plasmid concentration, 20 µg/mL) were mixed using a pipette. After 10 min of complex formation, 25 µL of the complex solutions were added into each well of 24-multiwell dishes for transfection (the amount of DNA added to a well, 0.5 µg/well). Biophysical Characterization of Polymer/Plasmid Complexes. The ζ-potentials and mean diameters of the polymer/ plasmid complexes in a saline solution having the same
concentration that was used for transfection at a C/A ratio of 5 were obtained by employing dynamic light scattering (DLS) on an ELS-8000 system (Otuska Co., Osaka, Japan) equipped with a 10 mW He–Ne laser. The mean diameters were determined by cumurant analysis in multimodal setting. The colloidal stability of polymer/plasmid complexes was studied using DLS at 37 °C and pH 7.4 and 2.0, which was adjusted by 0.1 N HCl solution. The polyplexes were prepared as described above with a C/A ratio of 5. The ionic strength of the suspension was adjusted to 150 mM NaCl. In Vitro Transfection and Cell Viability Assays. COS-1 cells (approximately 3 × 104 cells per well) were seeded prior to treatment in 24-well plates and grown for 24 h in DMEM (Gibco, Invitrogen Corp., Carlsbad, CA) containing 10% fetal calf serum (Hyclone Laboratories Inc., Logan, UT), penicillin (200 units/mL; ICN Biomedicals Inc., Aurora, OH), and streptomycin (200 mg/mL; ICN) in an atmosphere of 5% CO2 at 37 °C. Transfections were performed with 0.5 µg of plasmid DNA (pGL3-control) in 24-multiwell dishes in 0.2 mL of OPTIMEM I (Gibco). After 3 h of incubation, the cells were washed once with PBS(–) and cultured in 1 mL of DMEM containing 10% fetal calf serum for an additional 48 h. The medium was removed and the cells were washed twice with PBS(–). The cells were lysed with 0.2 mL of cell lysis buffer (Promega, Madison, WI) and mixed by vortexing. The lysate was centrifuged at 15 000 rpm for 1 min at 4 °C, and 5 µL of the supernatant was analyzed for luciferase activity using a Luminous CT-9000D (Dia-Iatron, Tokyo, Japan) luminometer. The relative light unit/s (RLU) were converted into the amount of luciferase (pg) using a luciferase standard curve, which was obtained by diluting recombinant luciferase (Promega) in lysis buffer. The protein concentrations of cell lysates were measured by performing a Bio-Rad protein assay (BIO-RAD, Hercules, CA) using bovine serum albumin as a standard. The expressed luciferase represented the amount (mole quantity), which is standardized for the total protein content of the cell lysate. The data are presented as means ( SD (n ) 5). Cytotoxicity was assessed by performing a cell viability assay using the WST-8 method (Dojindo, Kumamoto, Japan). COS-1 cells were seeded 24 h prior to treatment in 96-well plates at 5000 cells per well. Cells were treated under the same conditions used for luciferase assay with 6.2 µL of the transfection mixture including 0.124 µg of pDNA added to each well. They were treated under appropriate conditions for 3 h, washed once with PBS, and cultured in 50 µL of DMEM (Gibco) containing 10% fetal calf serum for an additional 24 h. To each well, 10 µL of WST-8 reagent (5 mmol/L) was added. After 2 h of incubation at 37 °C, absorbance at 450 nm was read in a BIO-RAD microplate reader (model 680). The data are presented as means ( SD (n ) 5).
RESULTS AND DISCUSSION Synthesis and Characterization of the Block Copolymers. The reaction scheme for the preparation of PDMAPAAm–PDMAAm 4-branched block copolymers is depicted in Figure 1. In this reaction, iniferter-based photoliving radical polymerization (12, 13), in which chain length can be easily controlled by changing irradiation conditions such as time or
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Table 2. Reaction Conditionsa, Molecular Weights, And Composition of PDMAPAAm-PDMAAm 4-Branched Block Copolymers PDMAPAAm
PDMAPAAm–PDMAAm
Mnb (g/mol)
(mM)
[DMAAm] (M)
irradiation timec (min)
conv. (%)
Mnd (g/mol)
DMAAm/DMAPAAmd
10 000 10 000 10 000 18 000 18 000 18 000 50 000 50 000 50 000
0.63 1.25 2.50 1.25 1.25 1.25 1.25 1.25 1.25
0.5 0.5 0.5 0.5 0.5 0.5 0.5 1.0 1.5
30 30 30 10 15 20 10 10 10
6 5 3 7 8 9 13 15 19
15 000 13 000 11 000 22 000 25 000 27 000 56 000 65 000 83 000
0.86 0.51 0.17 0.36 0.64 0.82 0.19 0.48 1.05
a
Solvent: methanol. b Determined by GPC (PEO standard). c Irradiation intensity: 1 mW/cm2. d Polymerization ratios were determined by 1H NMR.
Figure 3. 1H NMR spectra (300 MHz) in D2O of (A) PDMAPAAm 4-branched polymer and (C) PDMAPAAm–PDMAAm 4-branched block copolymer. (B) Absorption spectrum of PDMAPAAm 4-branched polymer in H2O. PDMAPAAm 4-branched polymer was prepared by iniferterbased living radical polymerization of 3-(N,N-dimethylamino)propyl acrylamide initiated from 1,2,4,5-tetrakis(N,N-diethyldithiocarbamylmethyl)benzene as a 4-functional iniferter. PDMAPAAm–PDMAAm 4-branched block copolymer was obtained by similar living radical polymerization of N,N-dimethylacrylamide from PDMAPAAm 4-branched polymer.
light intensity, and solution conditions such as the concentration of iniferter or monomer, was repeated for two different monomers of DMAPAAm and DMAAm. The iniferter-based photoliving radical polymerization was a powerful tool for precise structural design in macromolecules (5, 18, 25). In a previous paper, we reported that coating materials and surface modification for the improvement of biocompatibility in medical devices were precisely developed (26, 27). At first, the polymerization of a cationic monomer (DMAPAAm) from 4-functional iniferter (2), which was synthesized by the dithiocarbamylation of 1,2,4,5-tetrakis(bromomethyl)benzene (1), was performed using a different monomer concentration to obtain PDMAPAAm 4-branched polymers. The preparation conditions are listed in Table 1. Upon 30 min of UV light irradiation at a DMAPAAm concentration of 0.34 M, the initial GPC trace of the iniferter (2) with an Mn of 720 g mol-1 was completely absent (Figure 2). An increase in the DMAPAAm concentration at a fixed irradiation condition resulted in an increase in the molecular weight with complete shift of the GPC curves toward a high-molecular-weight region, with a narrow polydispersity at 1.3–1.4.
On the other hand, a 1H NMR spectrum showed that no nonreactive monomer and iniferter were detected completely in the all polymers obtained (Figure 3A). In addition, the broad peaks of N-methyl protons originating from the cationic chains were observed at approximately 2.1 ppm. However, the peak of ethyl protons corresponding to the dithiocarbamate group was not detected in the spectrum due to an extremely low existence ratio of the dithiocarbamate group against cationic chains, whereas a UV spectrum showed the remaining dithiocarbamate group (Figure 3B). The number of dithiocarbamate groups in one 4-branched PDMAPAAm molecule ranged from 3.6 to 4.2, which was close to the theoretical value of 4, irrespective of the molecular weigh of PDMAPAAm (Table 1). These results, combined with the afore-mentioned GPC data, indicated that the polymerization of DMAPAAm occurred from iniferter (2) via a living radical polymerization mechanism, resulting in the preparation of PDMAPAAm 4-branched cationic polymers. In this reaction, each branch was end-capped almost completely with the dithiocarbamate group, with molecular weights of approximately 10 000, 20 000, and 50 000.
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Figure 4. Changes in number average molecular weight (Mn) by changing (A) concentration of macroiniferter (PDMAPAAm 4-branched polymer), (B) irradiation time, and (C) concentration of monomer (N,N-dimethylacrylamide). Polymerization conditions were summarized in Table 2.
Figure 5. Scattering intensity and cumulant diameter of aqueous solutions of polyplexes prepared by mixing DNA (pGL3-control plasmid, 3 µg) and (A) PDMAPAAm 4-branched polymer with molecular weight of approximately 50 000 or (B) PDMAPAAm–PDMAAm 4-branched block copolymer with molecular weight of approximately 83 000 in different charge ratios.
Second, the photopolymerization of the nonionic monomer, DMAAm, from the obtained PDMAPAAm 4-branched polymers was conducted under a similar UV irradiation condition. Upon UV irradiation, the GPC curves of the polymers were completely shifted to a high-molecular-weight region as shown in Figure 2, indicating that block copolymerization occurred from dithiocarbamated groups located at the terminals of each branch in PDMAPAAm. Narrow polydispersity around 1.4 remained after block copolymerization. Table 2 summarizes the preparation conditions, molecular weight defined by 1H NMR spectra, and the composition of PDMAPAAm–PDMAAm block copolymers. Because the polymerization reactivity of the macroiniferters was very low, it was necessary to raise monomer concentration. Therefore, monomer conversion was controlled at a very low level under 20%. The 1H NMR spectrum of the obtained polymers showed that the peaks of N-methyl protons at approximately 2.8 ppm, carbonyl methyl protons at approximately 2.5 ppm, and methylene protons at approximately 1.3 ppm, all of which originated from the nonionic monomer, were newly detected in the obtained polymers. This indicates that the PDMAPAAm 4-branched polymers could function as a macroiniferter, and the block polymerization of DMAAm occurred from the terminals of each chain of the PDMAPAAm (Fig. 3C). The dependency of molecular weight on the concentration of the macroiniferters, i.e., PDMAPAAm 4-branched polymers, at a fixed monomer concentration and irradiation time is shown in Figure 4A. The molecular weight of PDMAPAAm–PDMAAm block copolymers that were obtained tended to decrease with an increase in the concentration of the macroiniferter. The molecular weight also increased with irradiation time at a fixed concentration of the macroiniferter and monomer (Figure 4B),
and with the concentration of the monomer at a fixed irradiation time and concentration of macroiniferter (Figure 4C). Thus, it can be said that the molecular weight of the PDMAPAAm–PDMAAm block copolymers was precisely controlled by changing the irradiation time and the concentration of the macroiniferter, i.e., PDMAPAAm, and monomer, i.e., DMAAm. Formation of Polymer/Plasmid Complexes and Their Stability. When a Tris–HCl-buffered solution of pDNA (pGL3control plasmid) was mixed with a saline solution of the cationic polymers, namely, PDMAPAAm 4-branched polymers, the scattering intensity in DLS measurements was immediately observed regardless of the molecular weight of the cationic polymers, indicating that polymer/plasmid complexes (polyplexes) were formed. Figure 5A shows the typical behavior of the scattering intensity obtained from the cationic polymer with a molecular weight of 50 000, by changing the charge ratio (polymer/pDNA). The intensity increased with the charge ratio and reached a plateau over the ratio of 5, indicating that, for sufficient complex formation, a minimum charge ratio of 5 was required. It was considered that the formation was due to nonspecific electrostatic interaction between the cationic polymers and anionic DNA as previously described in reports (28, 29). The effective diameters of the polyplexes prepared from the cationic polymer (Mn ) 50 000), measured by DLS, were approximately 250 nm irrespective of the charge ratio (Figure 5A). The surface charge as revealed by ζ-potential measurements was approximately +30 mV, which was similar to the value observed for the PEI with an Mn of approximately 25 000 (19). The size of the formed polyplexes increased upon reduction of the molecular weight of the cationic polymers as shown in Figure 6A. With molecular weight of 10 000, the complex size was approximately 900 nm at a charge ratio of 5. It was
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Figure 6. Incubation time dependent cumulant diameter changes in aqueous solutions of polyplexes prepared by mixing DNA (pGL3-control, 3 µg) and (A) PDMAPAAm 4-branched polymer with molecular weights of approximately 10 000, 20 000, and 50 000 at a charge ratio of 5 at pH 7.4 or (B) PDMAPAAm–PDMAAm 4-branched block copolymer with molecular weight of approximately 83 000 at a charge ratio of 5 at pH 7.4 (b) and 2.0 (O).
considered that, in polymers with higher molecular weights, a higher compaction of the polyplexes might occur. In addition, the polyplexes were gradually aggregated with increasing incubation time; that is, the size of the polyplexes formed from cationic polymers with the lowest molecular weight increased to approximately 2000 nm until 20 min after mixing. On the other hand, in the polymer with the highest molecular weight, the increase in the polyplex size was more or less prevented. The stability was higher in the polyplexes formed from polymers with higher molecular weight. Since in those studies the charge ratio was fixed at 5, the outermost surface of the complexes must be completely covered with the cationic polymers. The higher the molecular weigh is, the higher the charge density is. Therefore, it was considered that the electrostatic repulsion power rises with molecular weight, by which the aggregation of the polyplexes might be suppressed in the polymer with highest molecular weight. On the other hand, in PDMAPAAm–PDMAAm 4-branched block copolymer a similar increase in the scattering intensity was observed immediately after mixing the polymer and DNA. A stable intensity was obtained for charge ratios greater than 2. The formed polyplexes were smaller in size (approximately 180 nm) than the nonblocked polymer. Interestingly, the size remained small even 1 month after complex formation (Figure 6B). The polyplexes were very stable under acidic conditions (Figure 6B) with moderate ζ-potentials of approximately +10 mV, which remained constant over a broad range of charge ratios between 2 and 20. This value was significantly lower than that observed for the cationic homopolymers (∼30 mV). This reduced ζ-potential can be ascribed to shielding of the positive surface charge of the polyplexes by the nonionic PDMAAm chains. In addition, there was little change in the size of polyplexes after dilution to 1/20 with saline solution (Figure 7), which may be a great advantage when used for animal application. Obviously, the presence of PDMAAm leads to good colloidal stability of the PDMAPAAm–PDMAAm 4-branched block copolymers. In Vitro Transfection Efficiency. The transfection efficiency of the two types of cationic polymers—PDMAPAAm 4-branched polymer and PDMAPAAm–PDMAAm 4-branched block copolymer—at the charge ratio of 5 was tested on the model cell line of COS-1 monkey kidney cells. In our previous study, the transfection efficiency was higher in a branching polymer than a linear one (5). Therefore, in this study, optimization in 4-branched polymers was performed.
Figure 7. Dilution-dependent cumulant diameter changes in aqueous solutions of polyplexes prepared by mixing DNA (pGL3-control, 3 µg) and PDMAPAAm–PDMAAm 4-branched block copolymer with molecular weight of approximately 83 000 at a charge ratio of 5.
The cells transfected with the luciferase-encoding plasmid (pGL3), in the absence of any polymer, exhibited negligible luciferase expression. On the other hand, in cationic homopolymers the luciferase expression increased with the molecular weight of the polymers (Figure 8). The highest luciferase expression consistently occurred at the highest molecular weight, which may be due to the stability of the polyplexes as shown in Figure 6A. Figure 9 shows the dependency of the molecular weight of the blocked polymers, which had the same core cationic polymer chain with molecular weight of approximately 50 000 but a nonionic polymer chain with different molecular weights ranging from 0 to approximately 33 000. Blocking resulted in an increase in luciferase activity with the peak transfection activity observed at a total molecular weight of approximately 83 000. The highest activity was approximately 3 times higher than that in PEI (ExGen 500) (30), which is a major commercially available typical polymeric vector used as a positive control. It is considered that the DNA expression using the polyplex was observed by the following 5 sequential steps: (1) attachment of the polyplex onto the cell surface, (2) internalization of the polyplex into the cell, (3) endosomal escape of the polyplex, (4) DNA release from the polyplex, and (5) internalization of DNA into the nucleus. Almost all of the cationic homopolymers
Novel Blocked, Star-Shaped Polymeric Gene Carrier
Figure 8. Transfection efficiency of polyplexes prepared by mixing DNA (pGL3-control, 3 µg) and PDMAPAAm 4-branched polymers at a charge ratio of 5.
Figure 9. Transfection efficiency of polyplexes prepared by mixing DNA (pGL3-control, 3 µg) and PDMAPAAm–PDMAAm 4-branched block copolymers at a charge ratio of 5 at different preservation times of polyplexes after preparation. For comparison, transfection efficiency data of polyplexes of linear PEI (ExGen 500) at the charge ratio of 5 is also given.
including PEI can conjugate with DNA to prepare nanosized polyplexes immediately after mixing by electrostatic affinity. However, the polyplexes are gradually aggregated with an increase in incubation time to grow to micrometer size by similar electrostatic affinity as demonstrated in Figure 6A. In general, it was reported that transfection efficiency was low in large particles due to difficulty internalizing polyplexes into the cells, which is the afore-mentioned second step. In addition, polyplexes formed from DNA and cationic polymer were relatively rigid compared to those from DNA and block copolymer with a combination of cationic and nonionic chains, because a nonionic chain in the block copolymer can function as a buffer layer to weaken the electrostatic affinity between DNA and polymer. Therefore, it is considered that the only cationic polymer based polyplexes may be not dissociated easily. That is, this may prevent DNA release from the polyplex, which is the afore-mentioned fourth step. For these two reasons it is considered that the 4-branched block copolymer had higher transfection efficiency than linear PEI. Interestingly, the high activity using the block copolymer was maintained at least 1 month after polyplex formation though the activity markedly decreased with time in nonblocked cationic polymers where approximately 60% of the activity was lost after
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Figure 10. Cytotoxicity of polyplexes prepared by mixing DNA (pGL3control, 3 µg) and PDMAPAAm–PDMAAm 4-branched block copolymers in different charge ratios. Cell viability was determined by the WST assay.
1 day (Figure 9). In PEI, little activity remained even 1 day after polyplex formation. In addition, the block copolymer could work as a gene delivery vector in the presence of serum. When serum was present, the transfection activity decreased to approximately 60% at serum concentration of 10%, and approximately 50% at 20%. Even at the serum concentration of 50%, approximately 30% of the initial activity remained. The in Vitro cytotoxicity of polyplexes based on the block copolymers was studied as a function of the charge ratio by using the WST assay. The block copolymer with a cationic chain of 50 000 and nonionic chain of 30 000 had little nontoxicity at charge ratios up to 20 (Figure 9B). At high charge ratios of 10 and 20, permissible viability, which was higher than that in PEI at a charge ratio of 6, was observed. In conclusion, we have demonstrated that nonionic polymer blocked, 4-branched cationic copolymers are a novel class of efficient polymeric carriers for DNA delivery in Vitro. Excellent colloidal stability with low cytotoxicity of polyplexes based on PDMAPAAm–PDMAAm block copolymer with a cationic chain length of approximately 50 000 and nonionic chain length of approximately 33 000 makes it very promising for in ViVo gene transfer. The most important advantage of the 4-branched block copolymer is high stability in the formed polyplexes, in which, however, DNA and the polymer were moderately binded. That is, excellent balance between polymer formation and dissociation may be realized in the branched blocked copolymer. In addition, the branched blocked copolymer was applied to an animal model (31). Even though the study was limited to only preliminary stages, mice injected with the polyplexes showed a high level of gene expression in liver, kidney, or spleen without any tissue damage. Therefore, in the near future, our research will be directed at verifying the macromolecular structure and molecular weights and the balance of the ratio in the cationic block and the nonionic block with detailed molecular characterization, including mass spectroscopy for several types of cells, for detailed study in in Vitro and in ViVo gene delivery.
ACKNOWLEDGMENT We thank Dr. Mariko Shiba for offering advice regarding this study and Dr. Takeshi Masuda for his excellent technical work.
LITERATURE CITED (1) Boussif, O., Lezoualc’h, F., Zanta, M., Mergny, M. D., Scherman, D., Demeneix, B., and Behr, J. P. (1995) A versatile vector for gene and oligonucleotide transfer into cells in culture
2044 Bioconjugate Chem., Vol. 18, No. 6, 2007 and in ViVo: polyethylenimine. Proc. Natl. Acad. Sci. U.S.A. 92, 7297–7301. (2) Godbey, W. T., Wu, K. K., and Mikos, A. G. (1999) Poly(ethylenimine) and its role in gene delivery. J. Controlled Release 60, 149–160. (3) Kichler, A. (2004) Gene transfer with modified polyethylenimines. J. Gene Med. 6 (Suppl 1), S3–10. (4) Demeneix, B., Hassani, Z., and Behr, J. P. (2004) Towards multifunctional synthetic vectors. Curr. Gene Ther. 4, 445–455. (5) Nakayama, Y., Masuda, T., Nagaishi, M., Hayashi, M., Ohira, M., and Harada-Shiba, M. (2005) High performance gene delivery polymeric vector: nano-structured cationic star polymers (star vector). Curr. Drug DeliVery 2, 53–57. (6) Mizuguchi, I., Ooe, Y., Hoshino, S., Shimura, M., Kasahara, T., Kano, S., Ohta, T., Takaku, F., Nakayama, Y., and Ishizaka, Y. (2005) Improved gene expression in resting macrophages using an oligopeptide derived from Vpr of human immunodeficiency virus type-1. Biochem. Biophys. Res. Commun. 338, 1499–1506. (7) Li, S., and Huang, L. (2000) Gene therapy progress and prospects: non-viral gene therapy by systemic delivery. Gene Ther. 7, 31–34. (8) Luo, D., and Saltzman, W. M. (2000) Enhancement of transfection by physical concentration of DNA at the cell surface. Nat. Biotechnol. 18, 33–37. (9) Gebhart, C. L., and Kabanov, A. K. (2001) Evaluation of polyplexes as gene transfer agents. J. Controlled Release 73, 401–416. (10) Thomas, M., and Klibanov, A. M. (2003) Non-viral gene therapy: polycation-mediated DNA delivery. Appl. Microbiol. Biotechnol. 62, 27–34. (11) Dubruel, P., and Schacht, E. (2006) Vinyl polymers as nonviral gene delivery carriers: current status and prospects. Macromol. Biosci. 6, 789–810. (12) Otsu, T., Yoshida, M., and Tazaki, T. (1982) A model for living radical polymerization. Makromol. Chem. Rapid Commun. 3, 133–140. (13) Otsu, T., Matsunaga, T., Doi, T., and Matsumoto, A. (1995) Features of living radical polymerization of vinyl monomers in homogeneous system using N,N-diethyldithiocarbamate derivatives as photoiniferters. Eur. Polym. J. 31, 67–78. (14) Lee, J. K., Kopecek, J., and Andrade, J. D. (1989) Proteinresistant surfaces prepared by PEO-containing block copolymer surfactants. J. Biomed. Mater. Res. 23, 351–368. (15) Maechling-Strasser, C., Dejardin, P., Galin, J. C., and Schmitt, A. (1989) Preadsorption of polymers on glass and silica to reduce fibinogen adsorption. J. Biomed. Mater. Res. 23, 1385–1393. (16) Amiji, M., and Park, K. (1992) Prevention of protein adsorption and platelet adhesion on surfaces by PEO/PPO/PEO triblock copolymers. Biomaterials 13, 682–692. (17) Grainger, D. W., Nojiri, C., Okano, T., and Kim, S. W. (1989) In Vitro and ex ViVo platelet interactions with hydrophilichydrophobic poly(ethylene oxide)-polystyrene multiblock copolymers. J. Biomed. Mater. Res. 23, 979–1005.
Nakayama et al. (18) Nakayama, Y., Miyamura, M., Hirano, Y., Goto, K., and Matsuda, T. (1999) Preparation of poly(ethylene glycol)polystyrene block copolymers using photochemistry of dithiocarbamate as a reduced cell-adhesive coating material. Biomaterials 20, 963–970. (19) Zhong, Z., Feijen, J., Lok, M. C., Hennink, W. E., Christensen, L. V., Yockmen, J. W., Kim, Y., and Kim, S. W. (2005) Low molecular weight linear polyethyleninime-b-poly(ethylene glycol)b-polyethylenimine triblock copolymers; synthesis, characterization, and in vitro gene transfer properties. Biomacromolecules 6, 3440–3448. (20) Brissault, B., Kichler, A., Leborgne, C., Danos, O., Cheradame, H., Gau, J., Auvray, L., and Guis, C. (2006) Synthesis, characterization, and gene transfer application of poly(ethylene glycol-bethylenimine) with high molar mass polyamine block. Biomacromolecules 7, 2863–2870. (21) Avgoustakis, K. (2004) Pegylated poly(lactide) and poly(lactid-co-glycolide) nanoparticles: preparation, properties and possible applications in drug delivery. Curr. Drug. DeliVery. 1, 321– 333. (22) Otsuka, H., Nagasaki, Y., and Kataoka, K. (2003) PEGylated nanoparticles for biological and pharmaceutical applications. AdV. Drug DeliVery ReV. 24, 403–419. (23) Patersen, H., Kunath, K., Martin, A. L., Stolnik, S., Roberts, C. J., Davies, M. C., and Kissel, T. (2002) Star-shaped poly(ethylene glycol)-block-polyethylenimine copolymers enhance DNA condensation of low molecular weight polyethylenimines. Biomacromolecules 3, 926–936. (24) Shuai, X., Merdan, T., Unger, F., and Kissel, T. (2005) Supramolecular gene delivery vectors showing enhanced transgene expression and good biocompatibility. Bioconjugate Chem. 16, 322–329. (25) Matsuda, T., Nagase, J., Ghoda, A., Hirano, Y., Kidoaki, S., and Nakayama, Y. (2003) Phosphorylcholine-endcapped oligomer and block co-polymer and surface biological reactivity. Biomaterials 24, 4517–4527. (26) Nakayama, Y., and Matsuda, T. (1996) Surface macromolecular architectural designs using photo-graft copolymerization based on photochemistry of benzyl N,N-diethyldithiocarbamate. Macromolecules 29, 8622–8630. (27) Nakayama, Y., and Matsuda, T. (1999) Surface macromolecular microarchitecture design: Biocompatible surfaces via photo-block-graft copolymerization using N,N-diethyldithiocarbamate. Langmuir 15, 5560–5566. (28) Xu, B., Wiehle, S., Roth, J. A., and Cristiano, R. J. (1998) The contribution of poly-L-lysine, epidermal growth factor and streptavidin to EGF/PLL/DNA polyplex formation. Gene Ther. 5, 1235–1243. (29) Benns, J. M., and Kim, S. W. (2000) Tailoring new gene delivery designs for specific targets. J. Drug Target. 8, 1–12. (30) Ferrari, S., Moro, E., Pettenazzo, A., Behr, J. P., Zacchello, F., and Scarpa, M. (1997) ExGen 500 is an efficient vector for gene delivery to lung epithelial cells in Vitro and in ViVo. Gene Ther. 4, 1100–1106. (31) Zhou, Y., and Nakayama, Y. (2005) Highly effective in vivo gene transfection by blocked star vector. Conf. Proc. IEEE Eng. Med. Biol. Soc. 1, 501–503. BC070045Q
