Star-Shaped Cationic Polymers by Atom Transfer Radical

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Star-Shaped Cationic Polymers by Atom Transfer Radical Polymerization from β-Cyclodextrin Cores for Nonviral Gene Delivery F. J. Xu,*,†,‡ Z. X. Zhang,§ Y. Ping,§ J. Li,§,| E. T. Kang,‡ and K. G. Neoh‡ College of Materials Science and Engineering, Beijing University of Chemical Technology, Beijing 100029 China, Department of Chemical and Biomolecular Engineering, National University of Singapore, Kent Ridge, Singapore 119260, Division of Bioengineering, National University of Singapore, 7 Engineering Drive 1, Singapore 117574, and Institute of Materials Research and Engineering (IMRE), 3 Research Link, Singapore 117602 Received September 11, 2008; Revised Manuscript Received November 28, 2008

Cationic polymers with low cytotoxicity and high transfection efficiency have attracted considerable attention as nonviral carriers for gene delivery. Herein, well-defined and star-shaped CDPD consisting of β-CD cores and P(DMAEMA) arms, and CDPDPE consisting of CDPD and P(PEGEEMA) end blocks (where CD ) cyclodextrin, P(DMAEMA) ) poly(2-(dimethylamino)ethyl methacrylate), P(PEGEEMA) ) poly(poly(ethylene glycol)ethyl ether methacrylate)) for gene delivery were prepared via atom transfer radical polymerization (ATRP) from the bromoisobutyryl-terminated β-CD core. The CDPD and CDPDPE exhibit good ability to condense plasmid DNA (pDNA) into 100-200 nm size nanoparticles with positive zeta potentials of 25-40 mV at nitrogen/phosphate (N/P) ratios of 10 or higher. CDPD and CDPDPE exhibit much lower cytotoxicity and higher gene transfection efficiency than high molecular weight P(DMAEMA) homopolymers. A comparison of the transfection efficiencies between CDPD and P(DMAEMA) homopolymer indicates that the unique star-shaped architecture involving the CD core can enhance the gene transfection efficiency. In addition to reducing cytotoxicity, the introduction of a biocompatible P(PEGEEMA) end block to the P(DMAEMA) arms in CDPDPE can further enhance the gene transfection efficiency.

1. Introduction The most challenging task in gene therapy is the design of gene delivery vectors with low cytotoxicity and high transfection efficiency.1,2 In comparison with viral vectors and cationic lipids, cationic polymers as the major type of nonviral gene delivery vectors show low host immunogenicity and can be produced on a large scale. A large number of polycations, including polyethylenimine (PEI),3 poly(tertiary amine methacrylate),4-6 poly(L-lysine),7,8 polyamidoamine,9 chitosan,10,11 and cyclodextrin (CD)-based cationic carriers,2,12-14 have been reported to deliver nucleic acids. CDs are a series of cyclic oligosaccharides composed of six, seven, or eight D(+)-glucose units linked by R-1,4-linkages and named R-, β- or γ-CD, respectively. They generally exhibit excellent biocompatibility, nonimmunogenicity, and low toxicity in animal and human bodies.15 A class of linear, CD-based cationic polymers has been introduced by Davis’s group for the efficient delivery of nucleic acids.13,14 CDs have also been incorporated into polyamidoamine dendrimers16,17 and PEI18-21 to enhance gene transfection. Further improvement in transfection efficiency and reduction in cytotoxicity of these cationic polymers will facilitate their application in gene therapy. Star-shaped cationic polymers have recently attracted considerable attention as nonviral gene carriers due to their dense * To whom correspondence should be addressed. E-mail: xufj@ mail.buct.edu.cn; [email protected]. † Beijing University of Chemical Technology. ‡ Department of Chemical and Biomolecular Engineering, National University of Singapore. § Division of Bioengineering, National University of Singapore. | Institute of Materials Research and Engineering.

molecular architecture with moderate flexibility.22,23 CDs have the unique steric structure of a truncated cone.24 Novel starshaped gene carriers using CDs as cores could be developed if the hydroxyl groups on the outside surfaces of CDs could be derivatized to serve as initiation sites for growing cationic branches. In the present work, well-defined star-shaped cationic polymers, CDPD and CDPDPE, consisting of the β-CD core and the respective poly(2-(dimethylamino)ethyl methacrylate) (P(DMAEMA)) and P(DMAEMA)-block-poly(poly(ethylene glycol)ethyl ether methacrylate) (P(PEGEEMA)) arms, are prepared via atom transfer radical polymerization (ATRP)25-27 from the bromoisobutyryl-terminated β-CD (CD-Br) core (Scheme 1). The CDPD and CDPDPE have good ability to condense plasmid DNA (pDNA) into complex nanoparticles of 100-200 nm in sizes with positive zeta potentials of 25-40 mV at nitrogen/phosphate (N/P) ratios of 10 or higher. The CDPD and CDPDPE exhibit very low cytotoxicity and good gene transfection efficiency. In addition to reducing the cytotoxicity, the incorporated biocompatible P(PEGEEMA) end blocks on the P(DMAEMA) arms can further enhance the gene transfection efficiency. The novel CDPD and CDPDPE structures are thus attractive as nonviral gene vectors for future gene therapy applications.

2. Experimental Section 2.1. Materials. 2-(Dimethylamino)ethyl methacrylate (DMAEMA, >98%) and poly(ethylene glycol)ethyl ether methacrylate) (PEGEEMA, Mn ∼ 246) were obtained from Sigma-Aldrich Chemical Co., Milwaukee, WI. They were used after removal of the inhibitors in a readyto-use disposable inhibitor-removal column (Sigma-Aldrich). β-Cyclodextrin (β-CD, >98%, vacuum-dried at 100 °C overnight before

10.1021/bm8010165 CCC: $40.75  2009 American Chemical Society Published on Web 01/07/2009

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Scheme 1. Processes for the Preparation of Star-Shaped Cationic Copolymers via Atom Transfer Radical Polymerization (ATRP) from the 2-Bromoisobutyryl-Terminated β-CD Macroinitiator (CD-Br)a

a CD ) cyclodextrin, DMAEMA ) 2-(dimethylamino)ethyl methacrylate, PEGEEMA ) poly(ethylene glycol)ethyl ether methacrylate, m ) DMAEMA repeat units, and n ) PEGEEMA repeat units.

use), branched polyethylenimine (PEI, Mw ∼ 25000 Da), 1,4,7,10,10,hexamethyl triethylenetetramine (HMTETA, 99%), copper(I) bromide (CuBr, 99%), and 2-bromoisobutyryl bromide (BIBB, 98%) were also obtained from Sigma-Aldrich Chemical Co. 3-(4,5-Dimethylthiazol2-yl)-2,5-diphenyl tetrazolium bromide (MTT), penicillin, and streptomycin were purchased from Sigma Chemical Co., St. Louis, MO. HEK293 cell lines were purchased from the American type Culture Collection (ATCC, Rockville, MD). The control sample of P(DMAEMA) homopolymer with about 120 repeat units of DMAEMA was prepared via ATRP, using the reported ethylene glycol di-2bromoisobutyrate as the initiator.27 2.2. Synthesis of Star-Shaped Cationic Polymers (CDPD and CDPDPE) via ATRP. In this work, the target arm number of the starlike polymers from the CD core is four. The starting bromoisobutyryl-

terminated CD (CD-Br), with about four initiation sites, was synthesized using the following procedures. β-CD (4.5 mmol) was dissolved completely in 30 mL of anhydrous N,N-dimethylacetamide with stirring and then kept in an ice bath. BIBB (18.0 mmol) in anhydrous N,Ndimethylacetamide (10 mL) was added dropwise to the β-CD solution over a period of 1 h at 0 °C under a N2 atmosphere. After this addition, the reaction mixture was gently stirred at 0 °C for another 2 h and then at room temperature for 24 h. The final reaction mixture was precipitated in 900 mL of diethyl ether. The white powder precipitate was collected by centrifugation and washed with acetone (2 × 30 mL). The crude product was purified by suspending it in 100 mL of DI water at room temperature overnight. The purified CD-Br (yield, 4.25 g, 54.6%) was collected by centrifugation, washed with acetone (2 × 30 mL), and dried under reduced pressure.

Star-Shaped Cationic Polymers The CD-g-P(DMAEMA) star polymers (CDPDs, Scheme 1) were synthesized using a molar feed ratio [DMAEMA]/[CD-Br (0.2 g)]/ [CuBr]/[HMTETA] of 120:1.0:4.0:4.8. The reaction was performed in a 25 mL flask equipped with a magnetic stirrer and under the typical conditions for ATRP.27 DMAEMA, CD-Br, and HMTETA were introduced into the flask containing 8 mL of methanol/water (20/1, v/v) mixture. After CD-Br had dissolved completely, the reaction mixture was degassed by bubbling argon for 30 min. Then, CuBr was added into the mixture under an argon atmosphere. The reaction mixture was purged with argon for another 10 min. The flask was then sealed with a rubber stopper under an argon atmosphere. The polymerization was allowed to proceed under continuous stirring at room temperature for 0.5-8 h. The reaction was stopped by diluting with THF. The catalyst complex was removed by passing the blue dilute polymer solution through a short aluminum oxide column. A colorless solution was obtained. After removal of THF in a rotary evaporator, the CDPDs were precipitated in excess n-hexane. The crude polymer was purified by reprecipitation twice in hexane to remove the reactant residues, prior to being dried under reduced pressure. The CDPD yields (and the conversion of DMAEMA) from ATRP time of 0.5, 1.5, and 8 h are 0.5 g (13%), 0.9 g (39%), and 1.7 g (65%), respectively. One of the unique characteristics of polymers synthesized by ATRP is the preservation of the “dormant” alkyl halide chain ends throughout the polymerization reaction.5,27 Thus, the CDPD can be used as the macroinitiators for the subsequent ATRP of PEGEEMA to produce the CD-g-P(DMAEMA)b-P(PEGEEMA) copolymers (CDPDPE, Scheme 1). The CDPDPE (yield, 1.05 g, 50%) was synthesized using a molar feed ratio [PEGEEMA (0.5 mL)]/[CuBr]/[HMTETA] of 10:1:1.2 at room temperature for 12 h in 8 mL of ethanol/water (20/1, v/v) mixture containing 0.8 g of CDPD (from 8 h of ATRP). The ATRP reaction and product purification were performed using the same procedures as those described above. 2.3. Polymer Characterization. The molecular weights of polymers were determined by gel permeation chromatography (GPC), chemical composition by X-ray photoelectron spectroscopy (XPS), and chemical structure by nuclear magnetic resonance (NMR) spectroscopy. GPC measurements were performed on a Waters GPC system equipped with Waters Styragel columns, a Waters-2487 dual wavelength (λ) UV detector, and a Waters-2414 refractive index detector. THF containing 2 vol % triethylamine was used as the eluent at a low flow rate of 1.0 mL/min. Monodispersed polystyrene standards were used to generate the calibration curve. The XPS measurements were performed on a Kratos AXIS HSi spectrometer equipped with a monochromatized Al KR X-ray source (1486.6 eV photons), using the same procedures as those described earlier.27 The 1H NMR spectra were recorded on a 400 MHz Bruker AV-400 NMR spectrometer at room temperature. The 1 H NMR spectral measurements were carried out using an acquisition time of 3.2 s, a pulse repetition time of 2.0 s, and a 30° pulse width. Chemical shifts were referenced to the DMSO-d6 solvent peak at 2.50 ppm. 2.4. Characterization of Polymer/Plasmid DNA Complexes. The plasmid (encoding Renilla luciferase) used in this work was pRL-CMV (Promega Co., Cergy Pontoise, France), which was cloned originally from the marine organism Renilla reniformis. The plasmid DNA (pDNA) was amplified in Escherichia coli and purified according to the supplier’s protocol (Qiagen GmbH, Hilden, Germany). The purity and concentration of the purified DNA were determined by absorption at 260 and 280 nm and by agrose gel electrophoresis.5 The purified pDNA was resuspended in the tris-EDTA (TE) buffer at pH 7.5, containing 10 mM Tris-Cl (prepared from tris base and hydrochloric acid) and 1 mM ethylenediaminetetraacetic acid (EDTA), and kept in aliquots of 0.5 mg/mL in concentration. All CDPDs and CDPDPE prepared in this work are readily soluble in water at the concentration of 10 mg/mL. All polymer stock solutions were prepared based on a nitrogen component concentration of 10 mM in distilled water and the pH was adjusted to 7.4. Solutions were filtered through sterile membranes of 0.2 µm in average pore size and stored at 4 °C. Star


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polymer to DNA ratios are expressed as molar ratios of nitrogen (N) in the CDPD and CDPDPE to phosphate (P) in DNA (or as N/P ratios). An average mass weight of 325 per phosphate group of DNA was assumed.3 All polymer/pDNA complexes were formed by mixing equal volumes of polymer and pDNA solutions to achieve the desired N/P ratio. Each mixture was vortexed and incubated for 30 min at room temperature. Each cationic polymer was examined for its ability to bind pDNA through agarose gel electrophoresis using procedures similar to those described earlier.5 The polymer/pDNA complexes at various N/P ratios were investigated. Gel electrophoresis was carried out in the TAE running buffer (containing 40 mM Tris-acetate and 1 mM EDTA) at a voltage of 110 V for 30 min in a Sub-Cell system (Bio-Rad Laboratory, Hercules, CA). DNA bands were visualized and photographed by a UV transilluminator and BioDco-It imaging system (UVP Inc., Upland, CA). The particle sizes and zeta potentials of the polymer/pDNA complexes were measured using a Zetasizer Nano ZS (Malvern Instruments, Southborough, MA) with a laser of wavelength of 633 nm at a 173° scattering angle and procedures similar to those described earlier.5 The polymer/pDNA complexes were also imaged with a fieldemission scanning electron microscope, equipped with a scanning transmission electron microscopy (STEM) detector (JEOL JSM-6700F), using procedures similar to those described earlier.5 The complexes were visualized by transmitted electrons (STEM-in-SEM mode) at a low voltage of 15 kV, in comparison to about 200 kV for the conventional TEM. Such low voltage can minimize damage to the polymer/pDNA complex and allow visualizing of the complex in the TEM mode at high spatial resolution. 2.5. Cell Viability and In Vitro Transfection Assay. The cytotoxicity of the star-like polymers was evaluated via MTT assay in the HEK293 cell line. The cells were cultured in Dulbecco’s modified eagle medium (DMEM), supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100 units/mL of penicillin, and 100 µg/mL of streptomycin at 37 °C, under an atmosphere of 5% CO2 and 95% relative humidity. The cells were seeded in a 96-well microtiter plate (Nunc Co., Wiesbaden, Germany) at a density of 104 cells/well and incubated in 100 µL of DMEM/well for 24 h. The culture media was replaced with fresh culture media containing serial dilutions of polymers, and the cells were incubated for 24 h. Then, 10 µL of sterile-filtered MTT stock solution in PBS (5 mg/mL) was added to each well, giving a final MTT concentration of 0.5 mg/mL. After 5 h, the unreacted dye was removed by aspiration. The so-produced formazan crystals were solubilized in DMSO (100 µL/well). The absorbance was measured using a microplate reader (Spectra Plus, Tecan, Zurich, Switzerland) at a wavelength of 570 nm. The cell viability (%), relative to that of the control cells cultured in a medium without the polymers, was calculated from [A]test/[A]control × 100%, where [A]test and [A]control are the absorbance values of the wells (with the polymers) and control wells (without the polymers), respectively. For each sample, the final absorbance was the average of those measured from six wells in parallel. Transfection assays were performed using plasmid pRL-CMV as the reporter gene in the HEK293 cell line. The cells were seeded in 24-well plates at a density of 5 × 104 cells in 500 µL of medium/well and incubated for 24 h. The star polymer/pDNA complexes of N/P ratios from 5 to 30 were prepared a priori by adding the polymer into the DNA solutions, followed by vortexing and incubation for 30 min at room temperature. At the time of transfection, the medium in each well was replaced with 300 µL of serum-free medium or fresh normal medium (supplemented with 10% FBS). The complexes were added (20 µL/well containing 1.0 µg of pDNA) into the transfection medium and incubated with the cells for 4 h under the standard incubation conditions. Then, the medium was replaced with 500 µL of the fresh normal medium (supplemented with 10% FBS). The cells were further incubated for an additional 20 h under the same conditions, giving rise to a total transfection time of 24 h.21 The cultured cells were washed with PBS twice and lysed in 100 µL of the cell culture lysis reagent (Promega Co., Cergy Pontoise, France) using procedures similar to those

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Figure 1. XPS C 1s core-level spectra of the (a) CD, (b) CD-Br, (c) CDPD1, (d) CDPD2, (e) CDPD3, and (f) CDPD3PE polymers, and Br 3d core-level spectra of the (b′) CD-Br, (c′) CDPD1, (d′) CDPD2, and (e′) CDPD3 polymers.

described earlier.5 Luciferase gene expression was quantified using a commercial kit (Promega Co., Cergy Pontoise, France) and a luminometer (Berthold Lumat LB 9507, Berthold Technologies GmbH. KG, Bad Wildbad, Germany). Protein concentration in the cell samples was analyzed using a bicinchoninic acid assay (Biorad Laboratory, Hercules, CA). Gene expression results were expressed as relative light units (RLUs) per milligram of cell protein lysate (RLU/mg protein).

3. Results and Discussion 3.1. Synthesis and Characterization of Star-Shaped Cationic Polymers (CDPD and CDPDPE) via ATRP. To prepare the star polymers via ATRP from the β-cyclodextrin (CD) cores, it is essential to introduce alkyl halide initiators onto the CDs. In this work, the bromoisobutyryl-terminated CD (CD-Br) was prepared via the reaction of hydroxyl groups on the outside surface of β-CD with 2-bromoisobutyrl bromide (BIBB) (Scheme 1). It has been reported that all the 21 hydroxyl groups of β-CD can be converted into 21 initiation sites.25,26 When ATRP is carried out from a multifunctional core with a high local concentration of initiation sites, radical-radical coupling of the propagating chains will probably occur and result in gelation. To avoid the gelation effect and to allow some flexibility on the star-like polymers for securing a more compact complex structure with DNA,22,23,28 a starting CD-Br with about

four initiation sites was prepared for forming the well-defined CDPD and CDPDPE with four arms via ATRP. The XPS C 1s core-level spectra of the pristine β-CD and CD-Br samples are shown in Figure 1a and b, respectively. The C 1s core-level spectrum of β-CD can be curve-fitted into three peak components with binding energies (BEs) at about 284.6, 286.2, and 287.6 eV, attributable to the C-H/C-C, C-O, and O-C-O species, respectively.5 The C 1s core-level spectrum of CD-Br can be curve-fitted by four peak components with BEs at about 284.6, 286.2, 287.6, and 288.4 eV, attributable to the C-H/C-C, C-O/C-Br, O-C-O, and O)C-O species, respectively.5 The unambiguous O-C)O peak component is associated with the bromide-capped ester groups of CD-Br. The corresponding Br 3d core-level spectrum is shown in Figure 1b′. From the [Br]/[C] ratio of 0.072 (determined from the sensitivity factorcorrected Br 3d and C 1s core-level spectral area ratio), the average degree of substitution of the hydroxyl groups on the outside surface of β-CD is about 4.3. The chemical structure of CD-Br is also characterized by 1H NMR spectroscopy in DMSO (Figure 2a). The peak located at a chemical shift of δ ) 1.92 ppm is associated with the methyl protons (a, C(Br)CH3) of the 2-bromoisobutyryl groups.5 The signals located at broad chemical shifts in the region of 3.35-3.9 ppm are mainly associated with the inner methylidyne and methylene protons between the oxygen and carbon moieties (b, O-CH-C and

Star-Shaped Cationic Polymers

Figure 2. 1H NMR (400 MHz) spectra of (a) CD-Br (in DMSO-d6), (b) CDPD3 (in D2O), and (c) CDPD3PE (in D2O).

O-CH2-C) on the glucose units of β-CD. The peak located at a chemical shift of δ ) 4.88 ppm is attributable to the inner methylidyne protons between the oxygen moieties (b′, O-CHO). The signals located at the chemical shifts in the region of 4.19-4.4 ppm are mainly attributable to the hydroxyl protons adjacent to the methylene moieties (c, CH2-OH). The peak at δ ) 5.75 ppm corresponds to the hydroxyl protons adjacent to the methylidyne moieties (c′, CH-OH) of glucose units. From the area ratio of peak a and peak b′, the degree of substitution of the hydroxyl groups on the outside surface of CD is determined to be about 4.0. The XPS and NMR results thus indicate that a CD-Br core with about four alkyl halide initiation sites has been successfully prepared. The well-defined CDPD star polymer, consisting of a CD core and four P(DMAEMA) arms, or CD-g-P(DMAEMA), was subsequently synthesized via ATRP of DMAEMA from CDBr (Scheme 1). The CDPDs with different arm lengths of P(DMAEMA) were synthesized by varying the ATRP time. Table 1 summarizes the GPC results of CDPD1 (from 0.5 h of ATRP), CDPD2 (from 1.5 h of ATRP), and CDPD3 (from 8 h of ATRP). With the increase in reaction time from 0.5 to 8 h, the number average molecular weight (Mn) of CDPD increases from 4.2 × 103 to 1.7 × 104 g/mol, and the total number of


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DMAEMA repeat units increases accordingly from about 16 to 99. The polydispersity index (PDI) of CDPDs remains at about 1.1, indicating that the ATRP of DMAEMA from the CD-Br core is well-controlled. One of the unique characteristics of polymers prepared by ATRP is that the dormant alkyl halide chain ends can be reactivated.5 In this work, CDPD3 was used as the macroinitiator for the subsequent ATRP of PEGEEMA to produce the CD-g-P(DMAEMA)-b-P(PEGEEMA) polymer (CDPD3PE, Scheme 1). CDPD3PE from 12 h of ATRP of PEGEEMA possesses relative short P(PEGEEMA) end blocks. From the GPC result of CDPD3PE, the total number of PEGEEMA repeat units is about 15 (Table 1). In addition, the short P(PEGEEMA) end blocks did not affect the solubility in water. The C 1s core-level spectra of CDPDs and CDPD3PE (Figure 1c-f) can be curve-fitted by five peak components with BE’s at about 284.6, 285.5, 286.2, 287.6, and 288.4 eV, attributable to the C-H/C-C, C-N, C-O, O-C-O, and O)C-O species, respectively. The C-N and O-C-O peak components are associated with the P(DMAEMA) arms and the CD core, respectively. For the CDPDs, the increase (and decrease) in intensities of the C-N (and O-C-O) species with ATRP time is consistent with the increase in P(DMAEMA) contents. In comparison with the C 1s peak components of the corresponding CDPD3 (Figure 1e), the relative intensities of C-N (and C-O) species in the C 1s core-level spectrum of CDPD3PE (Figure 1f) have decreased (and increased), indicating that the additional P(PEGEEMA) end blocks have been successfully block copolymerized from the “dormant” chain ends of CDPD3PE. The presence of “dormant” CDPD chain ends is also ascertained by the persistence of Br 3d signals at the BE of about 70 eV,5 associated with the covalent bromine species (Figure 1c′,d′,e′), after the ATRP of DMAEMA. The P(DMAEMA) and P(PEGEEMA) contents (derived from XPS results) are higher than those obtained from GPC with reference to linear polystyrene standards (Table 1). This discrepancy is likely to be due to the difference in hydrodynamic volumes between star and linear polymers.25 Figure 2b and 2c shows the respective 1H NMR spectra of CDPD3 and CDPD3PE in D2O. The peaks located at chemical shifts of 0.81-0.10 ppm and about 2.2 ppm are mainly attributable to the C-CH3 (a′) and N-CH3 (a′′) methyl protons, respectively, of the P(DMAEMA) arms. The chemical shift associated with the methylene protons (d, N-CH2) of P(DMAEMA) arms is about 2.65 ppm. The signal located at a chemical shift of 1.86 ppm is mainly associated with the methylene protons (d′, C-CH2) of the DMAEMA and PEGEEMA units. The signal at δ ) 4.07 ppm corresponds to the methylene protons adjacent to the oxygen moieties of the ester linkages (d′′, CH2-O-CdO). The peaks located at chemical shifts of about 1.12 and 3.61 ppm are attributable to the methyl (a′′′, C-CH3) and methylene (adjacent to the oxygen moieties, d′′′, O-CH2) protons of the P(PEGEEMA) end blocks, respectively. The signal associated with the CD core (b) has become less obvious, due to the minor contribution of CD to the overall star polymer structure. From the peak area ratios of d′′/b and d′′/(a′ + a′′′), the contributions of CD, P(DMAEMA), and P(PEGEEMA) segments to the star polymer are also summarized in Table 1. 3.2. Polymer/Plasmid DNA Complexes. For cellular transfection, DNA has to be condensed by cationic polymers into polymer/plasmid nanoparticles suitable for cellular uptake. The ability to condense DNA is a prerequisite for polymeric gene vectors. In this work, the ability of CDPDs and CDPD3PE to

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Table 1. Characterization of the Star-Shaped Cationic Polymers chemical composition (monomer repeat units) c

c

d

sample

reaction time (h)

Mn (g/mol)

PDI

[N]/[C]

DMAEMA

PEGEEMA

CD-g-P(DMAEMA)1 or CDPD1a CD-g-P(DMAEMA)2 or CDPD2a CD-g-P(DMAEMA)3 or CDPD3a CD-g-P(DMAEMA)3-b-P(PEGEEMA) or CDPD3PEb

0.5 1.5 8 12

4.2 × 103 1.1 × 104 1.7 × 104 2.1 × 104

1.05 1.10 1.13 1.20

0.092 0.112 0.117 0.095

16e 20f 30g 56e 63f 77g 99e 106f 110g 99e 106f 110g

15e 18f 23g

a Synthesized using a [DMAEMA]/[CD-Br (0.2 g)]/[CuBr]/[HMTETA] molar feed ratio of 120:1.0:4.0:4.8 in 8 mL of methanol/water (20/1, v/v) mixture at room temperature. The calculated molecular composition of CD-Br is C58H90Br4O39, and the arm number is four. b Synthesized using a [0.5 mL of PEGEEMA]/[CuBr]/[HMTETA] molar feed ratio of 10:1:1.2 at room temperature in 8 mL of methanol/water (20/1, v/v) mixture containing 0.8 g of CDPD3. c Determined from GPC results. PDI ) weight average molecular weight/number average molecular weight or Mw/Mn. d Determined from XPS N 1s and C 1s core-level spectral area ratio. e Determined from Mn and the molecular weights of CD-Br (1731 g/mol), DMAEMA (157 g/mol), and PEGEEMA (246 g/mol). f Determined from the XPS [N]/[C] ratios, where the number of DMAEMA units was determined from the [N/C] ratio of CDPD ([N]/[C] ≈ n[DMAEMA]/ (58 + 8n[DMAEMA])), and the number of PEGEEMA units was determined from the [N/C] ratio of CDPD3PE ([N]/[C] ≈ 106/(58 + 8 × 106 + 12m[PEGEEMA])). g Determined from 1H NMR data.

Figure 4. (a) Particle size and (b) zeta potential of the complexes between the cationic polymers (CDPD1, CDPD2, CDPD3, CDPD3PE, P(DMAEMA), and PEI) and pDNA at various N/P ratios.

Figure 3. Electrophoretic mobility of plasmid DNA (pDNA) in the complexes of the cationic polymers ((a) CDPD1, (b) CDPD2, (c) CDPD3, (d) CDPD3PE, (e) P(DMAEMA), and (f) PEI) at various N/P ratios.

condense plasmid DNA (pDNA) into particulate structures was confirmed by agarose gel electrophoresis and particle size and zeta potential measurements, as well, by STEM imaging. Figure 3 shows the gel retardation results of the cationic star polymer (CDPD1, CDPD2, CDPD3, and CDPD3PE)/pDNA complexes with increasing nitrogen (N)/phosphate (P) (or N/P) ratio, in comparison with those of the P(DMAEMA) homopolymer (Mn ) 1.89 × 104, about 120 DMAEMA repeat units)/pDNA and branched polyethylenimine (PEI, 25 kDa)/pDNA complexes. As expected, CDPD1, CDPD2, and CDPD3 show increasing capability to condense pDNA. They can condense pDNA effectively at N/P ratios of 2.5, 2.0, and 1.75, respectively, due to the increasing density of amino groups in the star polymers (Table 1). The migration of pDNA is completely retarded by

CDPD3PE at the N/P ratio of 2.5 (Figure 3d), indicating that the added P(PEGEEMA) end blocks do not significantly impede the condensation capability of the star polymer. The P(DMAEMA) and PEI control samples can inhibit the migration of pDNA at N/P ratios of 1.75 and 2.0, respectively. Thus, in comparison with the two well-known polymeric gene carriers,3-5 CDPDs and CDPD3PE have good condensation capability. The (a) particle size and (b) zeta potential of the cationic polymer (CDPD1, CDPD2, CDPD3, and CDPD3PE)/pDNA complexes are compared to those of the P(DMAEMA)/pDNA and PEI/pDNA complexes at various N/P ratios in Figure 4. All the cationic star polymers can efficiently compact pDNA into small nanoparticles. Generally, the hydrodynamic size of the complex decreases with increasing N/P ratio. After reaching the N/P ratio of 10, all the star polymers, as in the cases of P(DMAEMA) and PEI, can condense pDNA into nanoparticles of 100 to 200 nm in diameters. The polymer/pDNA complex within this size range can readily undergo endocytosis.4 In addition, there is no obvious difference between the particle sizes of CDPD3/pDNA and CDPD3PE/pDNA, as the P(PE-



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Figure 6. Cell viability assay in the HEK293 cell line with various concentrations of CDPD1, CDPD2, CDPD3, CDPD3PE, P(DMAEMA), and PEI. Cell viability was determined by the MTT assay and expressed as a percentage of the control cell culture.

Figure 5. STEM images of the (a) CDPD3/pDNA (at N/P ratio of 1), (b) CDPD3/pDNA (at N/P ratio of 20), and (c) CDPD3PE/pDNA (at N/P ratio of 20) complexes.

GEEMA) end block is relatively short. Zeta potential is an indicator of surface charges on the polymer/pDNA naoparticles. A positively charged surface allows electrostatic interaction with anionic cell surfaces and facilitates cellular uptake.29 For the pDNA complexes of CDPD1, CDPD2, CDPD3, CDPD3PE, P(DMAEMA), and PEI, the net surface charge increases dramatically as the N/P ratio increases from 1 to 5 and stabilizes at N/P ratios of 10 and above (Figure 4b). After reaching the N/P ratio of 10, the zeta potentials of the pDNA complexes of all the cationic polymers are strongly positive and vary within the narrow range of 25 to 40 mV, which will give rise to similar affinity for cell surfaces. In addition, at N/P ratios of 5 and above, the excess cationic polymers probably do not exert any significant effects on the particle size and zeta potential of the complexes (Figure 4). The results are consistent with those reported in the literature.30 Figure 5 shows the representative STEM images of the (a) CDPD3/pDNA (at a N/P ratio of 1), (b) CDPD3/pDNA (at a N/P ratio of 20), and (c) CDPD3PE/pDNA (at a N/P ratio of 20) complexes. The FESEM equipped with a STEM detector allows the visualization of polymer complexes in the TEM mode at a low voltage of 15 kV. In comparison with about 200 kV for conventional TEM, such a low voltage can prevent the

destruction of polymer/pDNA complexes. The STEM images clearly reveal the morphological difference between the polymer/ pDNA complexes at different N/P ratios. For the CDPD3/pDNA complex at an N/P ratio of 1, loose aggregates (over 400 nm), supercoiled plasmid pDNA, and compact nanoparticles (about 10 nm) are observed at the same time, indicating that pDNA has been partially condensed. When the N/P ratio reaches 20, pDNA can be packed tightly and condensed completely in all the complexes. The compacted complexes of pDNA with CDPD3 and CDPD3PE exist in the form of spherical nanoparticles of about 120 nm in size. 3.3. Cytotoxicity of the Star-Shaped Cationic Polymers. Cytotoxicity is one of the most important factors to be considered in selecting polymeric gene carriers. Figure 6 shows the in vitro MTT assay results of cytotoxicity of CDPD1, CDPD2, CDPD3, CDPD3PE, P(DMAEMA), and PEI in HEK293 cell cultures. All of the cationic polymers exhibit a dose-dependent cytotoxicity effect.30 Nevertheless, CDPD1, CDPD2, CDPD3, and CDPD3PE exhibit lower cytotoxicity than P(DMAEMA) and PEI (the controls). The slopes of the dosedependent cytotoxicity curves for P(DMAEMA) and PEI are much steeper than those for the copolymers. A high concentration of amino groups can lead to high cytotoxicity.23,31,32 The introduction of biocompatible CD cores (and P(PEGEEMA) end blocks in the case of CDPD3PE) not only lowers the relative concentration of amino groups, but has also imparted biocompatible characteristics to the cationic carriers. Because all the star polymers contain the same CD cores, CDPD1, CDPD2, and CDPD3 exhibit increasing cytotoxicity. The increase in cytotoxicity with the molecular weight is similar to that observed before by Georgiou et al.22 CDPD3PE is less toxic than CDPD2 and CDPD3 because of the presence of additional biocompatible P(PEGEEMA) segments. The above results are also supported by the calculated median inhibitory concentration (IC50). IC50 is defined as the concentration of a gene carrier at which the relative cell viability decreases to 50%.4 The IC50 values of P(DMAEMA) and PEI are 32 and 22 µg/mL, respectively, in comparison with those of CDPD1 (243 µg/mL), CDPD2 (93 µg/mL), CDPD3 (54 µg/mL), and CDPD3PE (130 µg/mL). 3.4. Gene Transfection Mediated by Star-Shaped Cationic Polymers. The in vitro gene transfection efficiency of the cationic polymers/pDNA complexes was assessed using luciferase as a gene reporter in HEK293 cell cultures. Figure 7 shows the gene transfection efficiency of CDPD1, CDPD2,

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Figure 7. In vitro gene transfection efficiency of the cationic polymers (CDPD1, CDPD2, CDPD3, CDPD3PE, and P(DMAEMA))/pDNA complexes in comparison with that of PEI (and naked DNA (ND)) at various N/P ratios in HEK293 cell cultures in the (a) absence and (b) presence of serum.

CDPD3, and CDPD3PE in comparison with those of P(DMAEMA) and PEI at various N/P ratios in the (a) absence and (b) presence of serum. In both cases, the transfection efficiency generally increases first and then decreases with the increase in N/P ratio from 5 to 30. The optimal N/P ratios of the cationic polymers correspond to their respective DNA condensation capability (Figure 3). At lower N/P ratios, pDNA cannot be condensed efficiently by the polymers, and the resultant loose polymer/pDNA complexes cannot enter the cell easily. At higher polymer/plasmid ratios, the transfection formulation contains also free polymers, besides the compact and positively charged polymer/pDNA complexes. Due to the presence of an increasing amount of free cationic polymers with the increase in N/P ratios, the corresponding increase in cytotoxicity may result in a reduction in transfection efficiency. In particular, the transfection efficiencies mediated by P(DMAEMA) and PEI decreases dramatically with the increase in N/P ratios, presumably due to the high cytotoxicity of the two polymers. At the highest N/P ratio of 30 in the serum-free case (Figure 7a), the respective transfection efficiencies mediated by CDPD1, CDPD2, CDPD3, and CDPD3PE are 13.1 (or 1.6), 61 (or 7.2), 20.2 (or 2.4), and 32.1 (or 3.8) times that of P(DMAEMA) (or PEI). A similar trend is also observed in the presence of serum (Figure 7b). The transfection results at the optimal N/P ratios indicate that, among the CDPDs examined, the increase in transfection capability generally follows the order CDPD1 < CDPD2 < CDPD3, with the more pronounced enhancements being observed in CDPD2 and CDPD3 (Figure 7). The observation indicates that the maximum transfection efficiency for CDPDs is dependent on the arm lengths of P(DMAEMA). However, Georgiou et al. evaluated a series of DMAEMA star homopolymers and found that their overall transfection efficiency decreased with the increase in the arm lengths of P(DMAEMA).22 A plausible explanation for the different observation is that the polymer investigated by Georgiou et al. has a

Xu et al.

much higher molecular weight and, thus, higher toxicity than the CDPDs used in this study. When the toxicity is too high, then the transfection efficiency is decreased. In this work, CDPDs possess lower toxicity than P(DMAEMA) homopolymer (Figure 6). The long P(DMAEMA) arms can increase the binding ability and complex stability, leading to a much higher transfection efficiency. The maximum transfection efficiency mediated by CDPD2 (and CDPD3) is comparable to (and higher than) that mediated by the control P(DMAEMA) homopolymer. The numbers of DMAEMA repeat units in CDPD2, CDPD3, and P(DMAEMA) are about 56, 99, and 120, respectively, indicating that the star cationic architecture containing a biocompatible CD core can enhance the gene transfection efficiency. One plausible explanation is that such unique architecture could increase the binding efficiency of the cationic P(DMAEMA) and enhance the interaction with pDNA or cellular membranes. As shown in Figure 7, CDPD3PE exhibits a higher transfection efficiency than CDPD3, both in the absence and presence of serum. The transfection efficiency mediated by CDPD3PE is also comparable to or higher than that mediated by PEI at most N/P ratios studied. The above results suggest that the external P(PEGEEMA) blocks can further enhance the gene transfection efficiency. Other studies have also shown that PEGylation can enhance the transfection efficiency.5,33,34 As shown in Figure 6, the introduction of P(PEGEEMA) reduced the cytotoxicity of CDPD3 significantly. The good biocompatibility of the P(PEGEEMA), as well as its shielding effect, probably have contributed to the enhanced transfection efficiency. Georgiou et al. reported different star copolymers consisting of P(DMAEMA) and polymethoxy hexa(ethylene glycol) methacrylate (P(HEGMA)) blocks.23 The star block copolymer having inner P(HEGMA) and outer P(DMAEMA) blocks exhibited a higher overall transfection efficiency than the P(DMAEMA) homopolymer. However, the prepared star block copolymer with inner P(DMAEMA) blocks exhibited lower transfection efficiency. In the present work, CDPD3PE consisting of P(DMAEMA) inner blocks and P(PEGEEMA) outer blocks exhibits a higher transfection efficiency than the P(DMAEMA) homopolymer. The difference can probably be attributed to the difference in the PEG-containing monomers. The HEGMA monomer used by Georgiou et al. has six EG units, while PEGEEMA used in the present study has an average of three EG units per monomer. The increased steric hindrance in HEGMA monomer probably had hindered the DMAEMA units from condensing pDNA and thus had decreased the transfection efficiency. In addition, PEGEEMA is more hydrophobic in comparison with HEGMA. The incorporated hydrophobic units at the end blocks are expected to increase the transfection efficiency by modulating the interactions of the polymer/DNA complex with cells, such as adsorption on cell surfaces and subsequent uptake by cells.35

4. Conclusions Well-defined star-shaped cationic CDPD, consisting of a β-cyclodextrin (β-CD) core and P(DMAEMA) arms, and CDPDPE, consisting of CDPD and P(PEGEEMA) end blocks, were prepared as gene carriers via ATRP from the bromoisobutyryl-terminated β-CD core. Both CDPD and CDPDPE can effectively bind pDNA, at N/P ratios of 10 and above, to form nanoparticle complexes of 100-200 nm in sizes and positive zeta potentials of 25-40 mV. The CDPD and CDPDPE exhibit much lower cytotoxicity than P(DMAEMA) and PEI. In comparison with P(DMAEMA), the unique architectures of the


star-shaped cationic polymers containing the CD cores can enhance the gene transfection efficiency. The incorporated P(PEGEEMA) end blocks on the P(DMAEMA) arms can reduce the cytotoxicity and further enhance the gene transfection efficiency. Thus, structural tailoring of functional star-shaped polymers via ATRP from CD cores provides a unique and versatile means for designing novel gene carriers for gene therapy applications.

References and Notes (1) De Smedt, S. C.; Demeester, J.; Hennink, W. E. Pharm. Res. 2000, 17, 113–126. (2) Yang, C.; Li, H.; Goh, S. H.; Li, J. Biomaterials 2007, 28, 3245– 3254. (3) Bisht, H. S.; Manickam, D. S.; You, Y.; Oupicky, D. Biomacromolecules 2006, 7, 1169–1178. (4) Wetering, P. V. D.; Moret, E. E.; Nieuwenbroek, N. M. E.; Steenbergen, M. J. V.; Hennink, W. E. Bioconjugate Chem. 1999, 10, 589– 597. (5) Xu, F. J.; Li, H. Z.; Li, J.; Zhang, Z. X.; Kang, E. T.; Neoh, K. G. Biomaterials 2008, 29, 3023–3033. (6) Agarwal, A.; Unfer, R.; Mallapragada, S. K. J. Controlled Release 2005, 103, 245–258. (7) Wagner, E.; Ogris, M.; Zauner, W. AdV. Drug DeliVery ReV. 1998, 30, 97–113. (8) Dimitrova, M.; Arntz, Y.; Lavalle, P.; Meyer, F.; Wolf, M.; Schuster, C.; Haı¨kel, Y.; Voegel, J. C.; Ogier, J. AdV. Funct. Mater. 2007, 17, 233–245. (9) Kukowska-Latallo, J. F.; Bielinska, A. U.; Johnson, J.; Spindler, R.; Tomalia, D. A.; Baker, J. R., Jr. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 4897–4902. (10) Roy, K.; Mao, H. Q.; Leong, K. W. Nat. Med. 1999, 5, 387–391. (11) Thanou, M.; Florea, B. I.; Geldof, M.; Junginger, H. E.; Borchard, G. Biomaterials 2002, 23, 153–159. (12) Ooya, T.; Choi, H. S.; Yamashita, A.; Yui, N.; Sugaya, Y.; Kano, A.; Maruyama, A.; Akita, H.; Ito, R.; Kogure, K.; Harashima, H. J. Am. Chem. Soc. 2006, 128, 3852–3853. (13) Gonzalez, H.; Hwang, S. J.; Davis, M. E. Bioconjugate Chem. 1999, 10, 1068–1074. (14) Popielarski, S. R.; Mishra, S.; Davis, M. E. Bioconjugate Chem. 2003, 14, 672–678. (15) Davis, M. E.; Brewster, M. E. Nat. ReV. Drug DiscoVery 2004, 3, 1023–35.


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(16) Arima, H.; Kihara, F.; Hirayama, F.; Uekama, K. Bioconjugate Chem. 2001, 12, 476–484. (17) Kihara, F.; Arima, H.; Tsutsumi, T.; Hirayama, F.; Uekama, K. Bioconjugate Chem. 2003, 14, 342–350. (18) Pun, S. H.; Bellocq, N. C.; Liu, A.; Jensen, G.; Machemer, T.; Quijano, E.; Schluep, T.; Wen, S.; Engler, H.; Heidel, J.; Davis, M. E. Bioconjugate Chem. 2004, 15, 831–840. (19) Forrest, M. L.; Gabrielson, N.; Pack, D. W. Biotechnol. Bioeng. 2005, 89, 416–423. (20) Huang, H.; Tang, G.; Wang, Q.; Li, D.; Shen, F.; Zhou, J.; Yu, H. Chem. Commun. 2006, 2383–2384. (21) Li, J.; Yang, C.; Li, H. Z.; Wang, X.; Goh, S. H.; Ding, J. L.; Wang, D. Y.; Leong, K. W. AdV. Mater. 2006, 18, 2969–2974. (22) Georgiou, T. K.; Vamvakaki, M.; Patrickios, C. S.; Yamasaki, E. N.; Phylactou, L. A. Biomacromolecules 2004, 5, 2221–2229. (23) Georgiou, T. K.; Vamvakaki, M.; Phylactou, L. A.; Patrickios, C. S. Biomacromolecules 2005, 6, 2990–2997. (24) Szejtli, J. Chem. ReV. 1998, 98, 1743–1753. (25) Li, J.; Xiao, H.; Kim, Y. S.; Lowe, T. L. J. Polym. Sci., Part A: Polym. Chem. 2005, 43, 6345–6354. (26) Ohno, K.; Wong, B.; Haddleton, D. M. J. Polym. Sci., Part A: Polym. Chem. 2001, 39, 2206–2214. (27) Xu, F. J.; Kang, E. T.; Neoh, K. G. Biomaterials 2006, 27, 2787– 2796. (28) Hudde, T.; Rayner, S. A.; Comer, R. M.; Weber, M.; Isaacs, J. D.; Waldmann, H.; Larkin, D. F. P.; George, A. J. T. Gene Ther. 1999, 6, 939–943. (29) Kunath, K.; von Harpe, A.; Fischer, D.; Peterson, H.; Bickel, U.; Voigt, K.; Kissel, T. J. Controlled Release 2003, 89, 113–125. (30) van de Wetering, P.; Cherng, J.-Y.; Talsma, H.; Hennink, W. E. J. Controlled Release 1997, 49, 59–69. (31) Van de Wetering, P.; Cherng, J.-Y.; Talsma, H.; Crommelin, D. J. A.; Hennink, W. E. J. Controlled Release 1998, 53, 145–153. (32) Van de Wetering, P.; Schuurmans-Nieuwenbroek, N. M. E.; van Steenbergen, M. J.; Crommelin, D. J. A.; Hennink, W. E. J. Controlled Release 2000, 64, 193–203. (33) Zhong, Z.; Feijen, J.; Lok, M. C.; Hennink, W. E.; Christensen, L. V.; Yockman, J. W.; Kim, Y. H.; Kim, S. W. Biomacromolecules 2005, 6, 3440–3448. (34) Banerjee, P.; Weissleder, R.; Bogdanov, A. J. Bioconjugate Chem. 2006, 17, 125–131. (35) Kurisawa, M.; Yokoyama, M.; Okano, T. J. Controlled Release 2000, 68, 1–8.

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Star-Shaped Cationic Polymers by Atom Transfer Radical

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