Cationic Amphiphilic Star and Linear Block Copolymers: Synthesis

ARTICLE pubs.acs.org/Biomac

Cationic Amphiphilic Star and Linear Block Copolymers: Synthesis, Self-Assembly, and in Vitro Gene Transfection Anu M. Alhoranta,† Julia K. Lehtinen,‡,§ Arto O. Urtti,‡ Sarah J. Butcher,|| Vladimir O. Aseyev,† and Heikki J. Tenhu*,† Department of Chemistry, Laboratory of Polymer Chemistry, ‡Centre for Drug Research and §Division of Biopharmacy and Pharmacokinetics, Faculty of Pharmacy, and Institute of Biotechnology, 00014 University of Helsinki, Finland )

†

bS Supporting Information ABSTRACT: A series of amphiphilic star and linear block copolymers were synthesized using ATRP. The core consisted of either polystyrene (PS) or poly(n-butyl acrylate) (PBuA), having different glass-transition (Tg) values. These polymers were used as macroinitiators in the polymerization of the cationic 2-(dimethylamino)ethyl methacrylate (DMAEMA). The polymers were used to study the effects of polymer architecture and flexibility on the selfassembling properties, DNA complexation, and transfection. All polymers formed core
shell micelles in aqueous solutions and condensed plasmid DNA. Linear PDMAEMA-PBuA-PDMAEMA has transfection efficiency comparable to PEI25K in ARPE19 cell line. Glassy state of the micellar core and star-shaped architecture decreased the DNA transfection compared with the rubbery and linear polymer structures. The polymers showed low cellular toxicity at low nitrogen/phosphate (n/p) ratios.

1. INTRODUCTION Block copolymers are intriguing polymeric materials because of their ability to self-assemble both in the solid state and in selective solvents.1,2 In bulk, block copolymers self-organize to form characteristic morphologies such as spheres, cylinders, and lamellae, whereas in selective solvents they form micellelike aggregates with various structures. The most common structure is the multimolecular spherical micelle, consisting of a core formed by the insoluble block and a corona of the soluble block. These spherical assemblies have diameters of tens to hundreds of nanometers and have numerous applications in many fields, such as biomedicine and nanotechnology. Polymeric micelles show many advantages in biomedical applications because they are highly stable in aqueous solutions as a result of their low critical micelle concentration (cmc).3
5 A low cmc prevents micelle dissociation upon sudden dilution, for example, in the bloodstream. The architecture and composition of the copolymer are essential in controlling the morphologies of the forming aggregates.6,7 Amphiphilic star block copolymers have smaller hydrodynamic dimensions and lower solution and melt viscosities and thus may form different self-assemblies compared with their linear counterparts. Starlike polymers have an extremely low cmc, and they can even exist as unimolecular core
shell micelles.8 Unimolecular core
shell architectures have attracted considerable attention because of their high stability in solution. Unimolecular micelles in solution can be utilized, for example, to r 2011 American Chemical Society

encapsulate a large variety of small-molecular-weight guest molecules.9,10 Cationic polymers are known to bind and condense nucleic acids and thus have promising characteristics as nonviral gene delivery systems.11,12 Cationic polymers interact electrostatically with the phosphate groups of DNA to form stable nanoparticles, called polyplexes. Poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA) is a biocompatible water-soluble polymer having a pKa value around 7.4 to 7.5, depending on the molecular weight.13,14 This polymer can be endocytosed into cells and thus be used as a nonviral DNA vector. To achieve transfection, a plasmid needs to be delivered into the nucleus in transcriptionally active form. This requires cellular uptake of the polyplex, endosomal escape, dissociation of the polyplex, and transport of the plasmid to the nucleus. The great efficiency of PDMAEMA compared with other methacrylated polymers is due to its ability to destabilize endosomes owing to its tertiary amines acting as a proton sponge and the easy dissociation of the polyplex once present in the nucleus.15,16 The molecular size, shape, DNA binding affinity, and buffering capacity of the polymer in endosomes are important factors in gene transfer.17 The pK values are particularly important because they seem to determine the endosomal buffering properties and Received: May 20, 2011 Revised: July 15, 2011 Published: July 15, 2011 3213

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units into the PDMAEMA polymer significantly expanded the effective range of polymer/DNA weight ratios. Herein we report the synthesis of two analogous star block copolymers and linear triblock copolymers using controlled/living polymerization (ATRP23) (Figure 1). The polymers have either a glassy or a rubbery core-forming block. The shell-forming block in all cases is composed of water-soluble cationic PDMAEMA. The aim was to investigate the effects of polymer architecture and flexibility on the self-assembling properties. The DNA complex formation, in vitro transfection efficiency, and toxicity of these block copolymer micelles were also evaluated. We show that the shape and rigidity of the neutral part of the copolymers has an impact on self-assembly, DNA condensation, and transfection.

2. MATERIALS AND METHODS

Figure 1. Structure scheme of the synthesized block copolymers.

the release of the polymer/DNA complexes from the endosomes. Optimal pK values are in the range of 5
7.18,19 M€annist€ o and coworkers examined the DNA binding, condensation, cell uptake, and transfection efficiency of linear, grafted, dendritic, and branched poly(L-lysines) (PLLs).17 They suggest that the architecture of polylysines affects the condensation of DNA, with linear polymers being more efficient than dendritic ones. They found that the dendritic shape of PLL seems to be unfavorable for DNA binding, and thus dendritic PLLs have a poor transfection efficiency and cell uptake in in vitro studies. They reasoned that the shape and orientation of amines in dendrimers are not optimal for DNA binding. Georgiou et al. aimed to combine the optimal polymer architecture with the optimal range of pK values and synthesized PDMAEMA star homopolymers to be used as a transfection agent for the first time.18 Star polymers mimic the structure of dendrimers but are more flexible and are easier to synthesize. Georgiou et al. found that star PDMAEMA homopolymers are powerful transfection agents compared to the commercial PAMAM dendrimer-based transfection reagent. Xu et al. also discerned that the star-shaped architecture of PDMAEMA enhanced gene transfection efficiency and exhibited much lower cytotoxicity compared with a linear PDMAEMA homopolymer and PEI25K.20 They additionally observed that incorporation of biocompatible PPEGEEMA (poly[poly(ethylene glycol)ethyl ether methacrylate]) end blocks into the PDMAEMA arms reduces cytotoxicity and can further enhance the transfection efficiency. A series of PDMAEMA copolymers with methyl methacrylate (MMA), ethoxytriethylene glycol methacrylate (triEGMA), or N-vinyl-pyrrolidone (NVP) was synthesized by van de Wetering and coworkers.21 A PDMAEMA copolymer having a 20 mol % hydrophobic content of MMA exhibited reduced transfection efficiency and increased cytotoxicity compared with the analogous homopolymer. A copolymer of triEGMA (48 mol %) showed both reduced transfection efficiency and cytotoxicity, whereas a copolymer of NVP (54 mol %) showed increased transfection efficiency and decreased cytotoxicity. Contrary to van de Wetering’s result, Kurisawa et al. discovered that a PDMAEMA copolymer having a hydrophobic butylmethacrylate (BMA) block established increased transfection efficiency compared with the PDMAEMA homopolymer.22 Moreover, incorporation of hydrophobic monomer

2.1. Materials. 4-tert-Butylcalix[6]arene (95%), 2-bromoisobutyrylbromide (98%), copper(I) bromide (CuBr, 99.999%), copper(I) chloride (CuCl, 99%), 2,20 -bipyridyl (99%), 1,1,4,7,10,10-hexamethyltriethylenetetramine (HMTETA, 97%), and dimethyl 2,6-dibromoheptanedioate (97%) (all from Aldrich) were used as received. Triethylamine (99%, Aldrich), styrene (Merck), and n-butyl acrylate (99%, Aldrich), N,N,N0 ,N00 , N00 -pentamethyldiethylenetriamine (PDMETA, 99%, Aldrich), and tetrahydrofuran (THF, Lab-Scan) were dried on molecular sieves and distilled prior to use. 2-(Dimethylamino)ethyl methacrylate (DMAEMA, 98% Aldrich), dimethylformamide (DMF, Lab-Scan), and anisole (99% Aldrich) were filtered through a basic alumina column before use. Deuterated chloroform (Euriso-Top) and dichloromethane-d2 (99.9% Aldrich) were used as received. For dialysis-regenerated cellulose membranes, MWCO 3500 and 6000
8000 (Cellu Sep) were used. Polyethylene imine 25 kDa (PEI25K), 2-(N-morpholino)ethanesulfonic acid sodium salt (MES), 4-(2hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES), ethidium bromide, ο-nitrophenyl-β-galactosidase (ONPG), and β-galactosidase standard were purchased from Sigma Aldrich. Phosphate-buffered saline (PBS) and Alamar Blue were from Invitrogen, agarose was from Promega, and Triton X-100 was from Fluka. 2.2. Molecular Characterization. To estimate the purity and mass of the derivatized calix[6]arene initiator, we performed elemental analysis using an Elementar VarioMICRO cube (CHNS) instrument and MALDI-TOF mass spectrometry with Bruker Daltonics Microflex MS 9 (2,5-acid (DHB) as a matrix and NaI as a cationizing agent. The conversions of the polymerizations and the compositions of the synthesized polymers were determined with a Varian Gemini 2000 operating at 200 MHz for 1H NMR and 50.3 MHz for 13C NMR. The NMR spectra of the synthesized products were measured at ambient temperature using deuterated chloroform or dichloromethane-d2 as a solvent. The chemical shifts are presented in parts per million downfield from the internal TMS standard. The FTIR spectra were recorded from solid samples on a Perkin-Elmer spectrum one Fourier transform infrared spectrometer. The apparent number-average molar masses (Mn) and molar mass distributions (PDI, Mw/Mn) of the synthesized polymers were determined using a Waters size exclusion chromatography (SEC) equipment with Styragel columns, a Waters 2410 refractive index (RI) detector, and a Waters 2487 UV detector. Either pure THF or THF with tetrabutylammonium bromide (TBAB 1 g/L) was used as an eluent (flow rate 0.8 mL/min). Monodisperse polystyrene was used as a calibration standard (Scientific Polymer Products). Samples were filtered using 0.45 μm glass fiber filters (Life Sciences). The absolute number-average molar masses of the macroinitiators were determined with Gonotec Osmomat 090 membrane osmometry with a regenerated cellulose membrane (Millipore PLGC, NMWL: 10 000) using THF as solvent. The measuring cell was kept at 30 °C. 3214

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Biomacromolecules 2.3. Light Scattering. Methodological aspects of dynamic and static light scattering (DLS, SLS) can be found elsewhere,24,25 and the detailed aspects of the data analysis used in this Article are presented in the Supporting Information. Light scattering experiments were conducted using an LS setup composed of a Brookhaven Instruments BI-200SM goniometer, a BIC-TurboCorr digital pseudo-cross-correlator, and a BI-CrossCorr detector, including two BIC-DS1 detectors; pseudocross-correlation functions of the scattered light intensity were collected with the self-beating method,26 a Sapphire 488-100 CDRH laser from Coherent GmbH operating at λo = 488 nm. All LS studies were performed at 20 °C, and solutions were filtered directly into the glass sample cuvette through Acrodisc CR PTFE (0.45 μm) filters. The solution concentrations were 0.05 to 0.5 mg/mL for aqueous solutions (0.05 M NaCl) and 0.25
5.0 mg/mL for THF. The samples were allowed to equilibrate for 15 min before the measurement. Scattered light was collected in the angular range between 30 and 150°. Correlation functions of the light intensity scattered at an angle θ, G2(t), were recorded simultaneously with the integral time average intensities, I(θ) t I(q), where q (= (4πno/λo) sin(θ/2)) and no are the scattering vector and the RI of the medium, respectively. Intensities measured in counts of photons per second (cps) were normalized with respect to the Rayleigh ratio of toluene. The Zimm double extrapolation method was used to determine the weight-average molar mass (Mw) of the scattering objects and the radius of gyration, Rg. 2.4. Refractive Index Increment Measurements. The specific RI increments (dn/dc) were determined with an Optilab rEX differential refractometer (λo = 632.8 nm) both in aqueous solutions (0.05 M NaCl) and in THF. The solution concentrations were the same as those for light scattering measurements. Values for dn/dc were measured separately for each polymer sample, after which the data were gathered together using data analysis. The average values for dn/dc were used in all calculations. 2.5. Cryoelectron Microscopy. For cryoelectron microscopy imaging, the micellar samples (1 mg/mL in 0,05 M NaCl, filtered through 0.45 μm Acrodisc CR PTFE filters) were vitrified in liquid ethane on holey carbon grids (QUANTIFOIL R 2/2) using a Leica EM GP vitrification robot at 22 °C and 70% humidity. The samples were investigated using a Gatan 626 cryoholder maintained at
180 °C in a FEI Tecnai F20 microscope operated at 200 kV. The images were recorded with a Gatan US4000 CCD camera at a magnification of 68 000. 2.6. Preparation of the Polymer/DNA Complexes. Plasmid DNA encoding β-galactosidase under control of a cytomegalovirus promoter (pCMVβ) was amplified and purified as previously reported.27 Plasmid DNA was used as 0.1 mg/mL solution. PDMAEMA block copolymer/DNA complexes were prepared in 50 mM MES-50 mM HEPES-75 mM NaCl buffer (pH 7.2) at different PDMAEMA nitrogen/DNA phosphate ratios (n/p) of 0.5
32. Polyethylene imine 25 kDa (PEI25K)/DNA complex was prepared at n/p ratio 8 and was used as a positive control. 2.7. Gel Electrophoresis. DNA binding by PDMAEMA copolymers was confirmed by gel electrophoresis. PDMAEMA/DNA complexes (0.4 μg of DNA) were prepared at n/p ratios 0.5
32 and loaded with bromophenol blue in glycerol onto a 1% agarose gel in Tris-acetateEDTA (TAE) buffer (pH 8.0) containing 0.5 μg/mL ethidium bromide. The gel was electrophoresed at 80 V, for 40 min and then transluminated and imaged (Syngene Gene Genius Bio Imaging System, Synoptics) to visualize the DNA. 2.8. Transfection and Cytotoxicity Assays. Retinal pigment epithelial cells (ARPE 19) and monkey kidney fibroblasts (CV1-P) were cultured in growth medium (DMEM, 10% fetal bovine serum, 100 units/mL penicillin, 100 μg/mL streptomycin (Invitrogen)) and seeded onto 96-well plates (Sarstedt) 1 day before transfection at a density of 20 000 and 15 000 cells per well, respectively. Polymer/DNA

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complexes were prepared at n/p ratios of 0.5
32 for PDMAEMA copolymers and 8 for PEI. The cells were incubated with the complexes (0.6 μg of DNA/well) in the serum-free medium for 4 h at 37 °C, after which the cells were washed with PBS, and the growth medium was added for 43 h. For analysis of transfection efficiency, the cells were washed twice with PBS and lysed with 2% Triton-X 100 in 250 mM Tris-HCl (pH 8.0). β-Galactosidase activity was measured spectrophotometrically (Varioskan, Wallac, Turku, Finland) using ο-nitrophenol galactosidase as a substrate. Purified β-galactosidase was used as a standard. The cytotoxicity of the polyplexes was determined with the same procedure as the transfection followed by the AlamarBlue assay that is based on the metabolic activity of the cells. A solution of 10% AlamarBlue in growth medium was added to the cells and incubated for 1 (ARPE19) or 3 h (CV1-P), after which fluorescence at ex 560/em 590 was measured (Varioskan). The percentage of viability was calculated by comparing treated cells with blank cells representing 100% viability.

3. SYNTHESIS 3.1. Synthesis of Hexafunctional Initiator 4-tert-Butyl-(2bromoisobutyryloxy)calix[6]arene. The modification of 4-tert-

butylcalix[6]arene was conducted using 2-bromoisobutyrylbromide following the procedure described by Angot et al.28 In a 250 mL three-necked flask equipped with a magnetic stirrer, 4-tert-butylcalix[6]arene (10 g, 0.010 mol) was dissolved in 100 mL of dry THF, followed by 14.6 mL of freshly distilled triethylamine. The solution was cooled to 0 °C, and a solution of 2-bromoisobutyrylbromide (21 g, 0.092 mol) dissolved in 100 mL of dry THF was added dropwise over 1 h with vigorous stirring and under nitrogen flow. The solution was then stirred at 0 °C for 1 h and at room temperature for 48 h. The solution was concentrated with a rotary evaporator without further heating. The concentrated product was precipitated in cold water and redissolved in dichloromethane. The solution was washed several times with 0.1 M aqueous K2CO3 solution and finally with pure water and dried over MgSO4. The solution was concentrated, and the product was precipitated twice in a mixture of methanol and water (9/1 v/v) and dried in vacuo at room temperature. 4-tert-Butyl-(2-bromoisobutyryloxy)calix[6]arene exists as a solid white powder. Yield: 54%. 1H NMR (200 MHz, CD2Cl2, δ): 7.2 (2H, Ar protons), 3.8 (2H,
CH2
), 2.1 (3H,
CH3), 1.1 (9H, tert-butyl). 13C NMR (50.3 MHz, CD2Cl2, δ): 171 (carbonyl), 150, 146, 132, 130 (Ar carbons), 55 (CH3
C
Br), 34 (C(CH3)3), 31.8, (
CH3 of tert-butyl), 31.6, (CH3
C
Br), 30 (Ar
CH2
Ar). Elemental analysis for C90H114Br6O12: Calculated: C, 57.9%; H, 6.2%; Br, 25.7%; O, 10.3%. Found: C, 57.9%; H, 6.6%. Mass spectrum (MALDI-TOF): 1836 [M + Na]+. 3.2. Polymerizations Using Hexafunctional 4-tert-Butyl-(2bromoisobutyryloxy)calix[6]arene. Hexafunctional 4-tert-butyl(2-bromoisobutyryloxy)calix[6]arene initiator was used in the polymerization of styrene and n-butyl acrylate. The typical procedure in polymerization of styrene using hexafunctional initiator is as follows: The polymerization was carried out in an oven-dried flask equipped with magnetic stirrer. The flask was charged with hexafunctional initiator (75 mg, 0.04  10
3 mol), monomer (5 g, 0.05 mol), and 2,20 -bipyridyl (113 mg, 0.72  10
3 mol). The solution was purged with nitrogen for 15 min before CuBr catalyst (34 mg, 0.24  10
3 mol) was added under nitrogen counter flow. The reaction mixture was purged with nitrogen for 30 min before being placing in an oil bath thermostatted at 100 °C. After the reaction, the solution was 3215

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Biomacromolecules cooled by dipping in liquid nitrogen. The solution was diluted with THF and passed through a column of neutral alumina to remove copper salts. The polymer was precipitated in methanol and dried in vacuo at room temperature. The polymer was purified by reprecipitation. The polymerization of n-butyl acrylate was conducted as above. 3.3. Polymerizations Using Bifunctional Dimethyl 2,6Dibromoheptanedioate. The typical procedure in polymerization of styrene using bifunctional initiator is as follows: The polymerization was carried out in an oven-dried flask equipped with magnetic stirrer. The flask was charged with dimethyl 2,6dibromoheptanedioate (42 mg, 0.12  10
3 mol), monomer (5 g, 0.05 mol), and PMDETA (83 mg, 0.48  10
3 mol). The solution was purged with nitrogen for 15 min before a CuBr catalyst (69 mg, 0.48  10
3 mol) was added under nitrogen counter flow. The reaction mixture was purged with nitrogen for 30 min before placing in an oil bath thermostatted at 70 °C. After the reaction, the solution was cooled by dipping in liquid nitrogen. The solution was diluted with THF and passed through a column of neutral alumina to remove copper salts. The polymer was precipitated in methanol and dried in vacuo at room temperature. The polymer was purified by reprecipitation. The polymerization of n-butyl acrylate as a monomer was conducted as above. 3.4. Block Copolymerizations of 2-(dimethylamino)ethyl Methacrylate (DMAEMA). Block copolymerizations were conducted using purified starlike and linear macroinitiators. The general polymerization procedure is as follows: A dry roundbottomed flask, equipped with a magnetic stirrer, was charged with polystyrene macroinitiator (0.67 g, 0.03  10
3 mol, with molar mass 24 600 g/mol) dissolved in 5 mL of dry DMF. DMAEMA monomer (2.5 g, 0.03 mol) and HMTETA (37 mg, 0.16  10
3 mol) were added, and the solution was purged with nitrogen for 15 min. A CuCl catalyst (16 mg, 0.16  10
3 mol) was added under nitrogen counter flow. The reaction mixture was purged with nitrogen for 30 min before being placed in an oil bath thermostatted at 80 °C. After the reaction, the solution was cooled to room temperature, dissolved in DMF, and passed through a column of neutral alumina. The solution was dialyzed against pure water (Cellu Sep T2MWCO: 6000
8000) to ensure that all copper salts have been removed. The aqueous polymer solution was freeze-dried and redissolved before the polymer was precipitated in cold n-hexane. The product was dried in vacuo at room temperature. The polymer was purified by reprecipitation in n-hexane. The polymerizations using linear macroinitiators were conducted as above except that anisole (7 mL) was used as solvent. 3.5. Hydrolysis of Polystyrene Star. Alkaline hydrolysis of polystyrene star was utilized to detach the arms from the calixarene core using the alkaline conditions.29 Hexafunctional polystyrene star (0.15 g) was dissolved in 10 mL of dry THF in a double-necked flask equipped with a condenser and nitrogen inlet. The solution was degassed for 30 min before 5 mL of KOH (1 M solution in ethanol) was added. The solution was refluxed for 72 h, after which the solution was evaporated to dryness, redissolved to THF, and precipitated to cold methanol. The hydrolyzed star arms were purified by reprecipitation. The functionality (f) of the polystyrene star was calculated as follows: f = Mn(star)/Mn(arm), and the functionality was found to be f = 6 as expected. 3.6. Micelle Formation. Micelles were made using a water addition method to obtain equilibrium structures. The general

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procedure is as follows:30 Block copolymers were dissolved in DMF (5 mg/mL), which is a common solvent for both blocks. Pure water (ultra-high quality, UHQ) was added dropwise, making the solvent gradually poorer for the hydrophobic block. This causes aggregation, which is seen as turbidity of the solutions. The resulting slightly opaque solutions were placed in dialysis bags (Cellu Sep T1, nominal MWCO: 3500) and dialyzed against pure water to remove DMF. Finally, the micellar solutions were dialyzed against saline water (0.05 M NaCl), yielding solutions of 1 mg/mL. A series of concentrations for light scattering studies were obtained by dilution using the aqueous dialysis solution (0.05 M NaCl). Micellar solutions (0.05 M NaCl) are from now on referred to as aqueous solutions.

4. RESULTS AND DISCUSSION 4.1. Synthesis and Characterization of Polystyrene and Poly(n-butyl acrylate) Macroinitiators. The synthesis of poly-

styrene and poly(n-butyl acrylate) star polymers having precisely six arms by core-first technique was possible using an initiator possessing six initiating sites. The proper ATRP initiator was synthesized by modifying commercially available 4-tert-butylcalix[6]arene by a simple esterification reaction. The obtained 4-tertbutyl(2-bromoisobutyryloxy)calix[6]arene was characterized by 1H and 13C NMR, IR, elemental analysis, and mass spectrometry (MALDI-TOF). The 1H and 13C NMR spectra showed no signals from the chemical impurities or phenolic protons, indicating full esterification of the phenolic functions. When the phenolic functions are fully converted to isobutyryl groups, the structure of the calixarene macrocycle becomes less flexible. This is seen in broadening of the proton signals in the NMR spectrum (Figures 2 and 3). 4-tert-Butyl-(2-bromoisobutyryloxy)calix[6]arene was utilized as an hexafunctional ATRP initiator for the bulk polymerization of styrene and n-butyl acrylate in the presence of CuBr/2,20 bipyridyl catalyst at elevated temperatures. The polymerizations of styrene and n-butyl acrylate were conducted under the same conditions and using the same monomer to initiator ratios to achieve similar degrees of polymerization (DP) for both monomers. The conversions were kept low (∼20%) to as much as possible to avoid star
star coupling reactions. The conversions were determined by 1H NMR spectroscopy and were used to calculate the theoretical molar masses of the polymers (Table 1). The number-average molar masses (Mn) of the macroinitiators were determined by SEC using RI and UV detectors, linear polystyrene standards, and THF as an eluent. The SEC eluograms were narrow and monomodal, and the number-average molar masses were in good agreement with the theoretical ones, indicating controlled polymerization and quantitative efficiency of the hexafunctional initiator (Table 1). The polydispersities were rather low, and no clear shoulder was observed at the high molar mass region, indicating no star
star coupling reactions. Because of the lower hydrodynamic volumes of star polymers compared with their linear analogs, number-average molar masses determined by SEC are usually underestimated; therefore, SEC gives only apparent values. Therefore, the absolute number-average molar masses of the star polymers were determined by membrane osmometry, also in THF. The obtained values are comparable, although slightly smaller, than the ones obtained by SEC and calculated from the conversion. This suggests that molar masses determined by NMR, SEC, and 3216

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Figure 2. 1H NMR (200 MHz) spectra of 4-tert-butyl calix[6]arene in CDCl3.

Figure 3. 1H NMR (200 MHz) spectra of 4-tert-butyl-(2-bromoisobutyryloxy)calix[6]arene in CD2Cl2.

osmometry are comparable. Molar masses obtained by osmometry are used in further calculations. To analyze further the molar masses of the polystyrene and poly(n-butyl acrylate) (both star and linear), SLS experiments were performed in THF. (See the Supporting Information.) Measured values of the RI increments (dn/dc) were 0.1914 mL/g for polystyrene and 0.0484 mL/g for poly(n-butyl acrylate). The weight-average molar masses (M w ) obtained by SLS were somewhat larger than the M n

values obtained by SEC, as expected (Table 1). In the case of poly(n-butyl acrylate) star polymer, SLS gave a weightaverage molar mass of 88 400 g/mol, whereas the M w determined by SEC was only 40 800 g/mol. This large difference in molar masses does not come from the calibration of SEC by linear polystyrene standards but shows that some unavoidable coupling has taken place. However, the SEC eluograms are nearly symmetrical, indicating a very low fraction of coupled stars. 3217

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Table 1. Synthesized Polymers and Block Copolymers GPC conversion

Mn(theor)

Mn(NMR)

(%)

(g/mol)

(g/mol)c

(PS)6 macroinitiator

20.8

28 000

a

(PBuA)6 macroinitiator

20.2

33 400a

(PS-PDMAEMA)6

54.5

126 000b

(PBuA-PDMAEMA)6

61.9

153 500b

Br-PS-Br macroinitiator

76.3

Br-PBuA-Br macroinitiator

sample: polymers

PDMAEMA-PS-PDMAEMA PDMAEMA-PBuA-PDMAEMA

Mw

Mn

Mw(SLS)

dn/dc

(g/mol)f

(g/mol)g

(mL/g)h

(g/mol)

(g/mol)

d

28 200

32 500

1.15

26 400

33 200

0.191

32 000d

40 800

1.27

29 300

88 400

0.048

101 000

134 000e

195 000

1.46

147 600

0.117

103 800

127 700e

209 800

1.64

981 400

0.075

27 300a

37 300d

43 500

1.17

36 200

43 400

0.191

65.0

33 900a

35 400d

37 700

1.07

38 700

56 500

0.048

74.7 75.8

b

93 900e 107 300e

141 300 132 900

1.50 1.23

348 000 197 000

0.117 0.075

87 600 106 400b

90 600 104 700

PDI

Mn(osm)

a Mn(theor) = [M]/[I]*conv*M(monomer) + M(initiator), where “conv” is for conversion calculated using 1H NMR. b Mn(theor) = [M]/[I]*conv*M(monomer) + Mn(osm). c Number-average molecular weight (Mn) estimated by 1H NMR analysis using the block ratio and Mn(osm) of macroinitiator. d Apparent Mn determined by size exclusion chromatography (SEC) in THF using PS standards. e Apparent Mn determined by SEC in THF + TBAB (1 g/L) (tetrabutylammonium bromide) using PS standards. f Experimental Mn determined by membrane osmometry in THF. g Weightaverage molecular weight (Mw) determined by static light scattering (SLS) in THF (20 °C, 488 nm). h Refractive index increment (dn/dc) determined by refractometer in THF (25 °C, 633 nm).

The functionality of the star polymers and thus the functionality of the initiator can be determined by comparing the molar mass of cleaved star arms to the molar mass of the whole star polymer. Because polystyrene arms are linked to the calixarene core by ester functions, they can be cleaved off by alkaline hydrolysis without damaging the yielding linear polystyrene chains. The molar mass of the individual polystyrene arms corresponds to the theoretical one. The SEC eluogram was monomodal, and the polydispersity was only slightly higher (1.31) than that for star polystyrene (1.15), which is in agreement with values obtained by other groups. (See the Supporting Information.)31,29 The functionality of the star polystyrene was found to be six (f = 6), confirming that the polystyrene star has on average six arms of similar lengths. This result thereby reveals that the functionality of the initiator is also six, which makes it possible to expect the functionality of poly(n-butyl acrylate) star to be the same as that for polystyrene. Linear polystyrene and poly(n-butyl acrylate) were synthesized using bifunctional dimethyl 2,6-dibromoheptanedioate as an initiator. Linear macroinitiators were synthesized using the same conditions and technique as in the case of starlike macroinitiators. The monomer-to-initiator ratio was adjusted so that the molar masses would become similar to that of the star polymers (Table 1). 4.2. Synthesis and Characterization of Diblock Copolymers. Carefully purified starlike and linear polystyrenes and poly(n-butyl acrylate)s were further used as macroinitiators in the polymerization of the second monomer, DMAEMA. The aim was to have a hydrophobic/hydrophilic block ratio over 1:2 to have water-soluble polymers. The polymerization of DMAEMA was conducted by ATRP reaction using CuCl/HMTETA as a catalyst complex. Halogen exchange was utilized to provide high initiating efficiency and better control over the molecular weight during the reaction.32 HMTETA was used as a ligand instead of 2,2-bipyridyl because of the higher activity of the CuCl/HMTETA complex owing to the lower redox potential.33 The conversions of the polymerizations and block ratios were characterized with 1H NMR, either in CDCl3 or in CD2Cl2. The hydrophobic/hydrophilic block ratios were 1:3 and 1:2 for star

block copolymers and linear block copolymers respectively. The DP of PDMAEMA were 80 per star arm and 200 per block of linear polymers. Size exclusion measurements for the block copolymers were performed in THF with 1 g/L TBAB to minimize the adsorption of the amino-containing polymers on the column. Numberaverage molar masses were in good agreement with the values estimated theoretically, which indicates that the initiation efficiency of the macroinitiators is close to one (Table 1). The SEC eluograms of the block copolymers were monomodal and symmetrical, and no shoulder was found at the high-molecularweight region (Figure 4). Molar masses obtained by 1H NMR are somewhat more reliable, especially in the case of star polymers, than molar masses obtained using SEC and thus are used in further calculations. The SLS experiments were also performed for block copolymers in THF. The measured RI increments (dn/dc) were 0.1165 mL/g for polystyrene-PDMEAMA and 0.0751 mL/g for poly(n-butyl acrylate)-PDMAEMA block copolymers. Mw values obtained by SLS for block copolymers were somewhat larger than values obtained by SEC, especially in the case of linear PDMAEMA-PS-PDMAEMA and starlike (PDMAEMA-PBuA)6 block copolymers (Table 1). These polymers also exhibited angular dependence, and thus the molar mass was calculated by extrapolating the LS intensity to zero angle and to zero concentration. (See the Supporting Information.) Light scattering emphasizes large scatterers in solution, such as coupled polymers. Like in the case of macroinitiators, SEC reveals that the fraction of coupled polymers must be minor because the eluograms are symmetrical. 4.3. Micellization. In block selective solvents, amphiphilic polymers tend to self-assemble into aggregates of various morphologies depending on the structure of the polymer and solvent conditions. Here, both starlike and linear block copolymers were used to prepare polymer micelles in aqueous solutions (0.05 M NaCl). Micelles were made in a water-addition method to achieve equilibrium structures.34 In the case of styrene-containing polymers, the proper term would be micelle-like aggregate because T < Tg(core).30 However, the term micelle is extensively used in the literature. DLS was used to estimate the hydrodynamic sizes 3218

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Figure 4. SEC traces of star like poly(n-butyl acrylate) macroinitiator and corresponding (PBuA-PDMAEMA)6 block copolymer using THF with 1 g/L TBAB as an eluent (0.8 mL/min flow rate).

Table 2. Micelles Formed by Water-Addition Method in Aqueous Solution (0.05 M NaCl) dn/dc (mL/g)a

Mw (g/mol)b

Rh (nm)c

Rg (nm)d

Rh/Rg

Nagge

0.1605 0.1605

4 200 000 7 200 000

64.9 80.9

48.1 49.0

0.74 0.61

22 34

PDMAEMA-PS-PDMAEMA

0.2026

37 400 000

89.8

56.9

0.63

265

PDMAEMA-PBuA-PDMAEMA

0.1605

11 700 000

57.0

40.0

0.70

88

sample: micelles (PS-PDMAEMA)6 (PBuA-PDMAEMA)6

dn/dc determined by refractometer (25 °C, 633 nm). b Mw determined by dynamic light scattering (DLS)(20 °C, 488 nm). c Hydrodynamic radius determined by DLS obtained by extrapolating to q = 0 and c = 0. d Radius of gyration determined by DLS obtained by extrapolating to q = 0 and c = 0. e Aggregation number determined by the ratio of Mw(micelles)/Mw(polymers, SEC). a

and molar masses of the micelles. Cryoelectron microscopy was used to confirm the morphology of the micelles. RI increments (dn/dc) were also determined for the micellar solutions. All micellar solutions exhibited the dn/dc value of 0.1605 mL/g, except linear PDMAEMA-PS-PDMEAMA block copolymer micelle (0.2026 mL/g). The reason for this kind of behavior of linear PDMAEMA-PS-PDMEAMA is hard to interpret. RI measurements may give some estimation of the structure of polymer micelles. Because the dn/dc value of 0.1605 mL/g is close the dn/dc value of PDMEMA star homopolymer (0.139 mL/g in acetone,35 0.149 mL/g in THF36), it most probably indicates that the micelles are core
shell micelles having PDMAEMA blocks as the outer shell. Therefore the difference in dn/dc value of linear PDMAEMA-PS-PDMEAMA in water might be due to different architecture. Light scattering was used to estimate the molar masses of the micelles and to determine the hydrodynamic sizes of the particles. Molar masses of micellar solutions were on the order of millions, with linear PDMAEMA-PS-PDMEAMA having the largest molar mass (37 000 000 g/mol) (Table 2). Aggregation numbers (Nagg) for the micelles in aqueous solutions were also calculated. Linear PDMAEMA-PS-PDMEAMA is having the largest (265) aggregation number. This suggests that linear PDMAEMA-PS-PDMEAMA

forms larger assemblies in aqueous solutions than the linear PDMAEMA-PBuA-PDMEAMA of the same molecular weight or the corresponding star polymers. This is because of the restricted chain topology of the molecule, which disables the close packing of the polymer chains. Using LS, we may roughly estimate the structure of the polymer micelles by comparing the radius of gyration (Rg) with the hydrodynamic radius (Rh). Ratios of these radii (F = Rg/Rh) for all micellar solutions are in the range of 0.6 to 0.7, which suggests core
shell structures. (See the Supporting Information.) The hydrodynamic radius for a star (PS-PDMAEMA)6 micelle decreased with increasing the polymer concentration. The second virial coefficient (A2) is ∼0 or slightly negative for that system within the experimental error. This can be due to either intermolecular interactions or the particle growth. The latter contradicts with DLS data, and thus the decrease in Rh with increasing concentration most likely owes to the hydrodynamic interactions rather than actual decrease in the micellar size. Furthermore, the glassy core of the (PS-PDMAEMA)6 aggregates restricts the chain exchange between the particles. 4.4. Cryoelectron Microscopy. Filtered micelle samples (1 mg/mL) were imaged using cryoelectron microscopy to support the light scattering results. Electron micrographs clearly confirm the presence of spherical micelle-like aggregates. 3219

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Figure 5. Cryoelectron micrograph of PDMAEMA-PS-PMAEMA in 1 mg/mL (0.05 M NaCl) with size distribution figure obtained with DLS (0.5 mg/mL, 65°).

Figure 6. Cryoelectron micrograph of (PBuA-PDMAEMA)6 in 1 mg/ mL (0.05 M NaCl) with size distribution figure obtained with DLS (0.5 mg/mL, 65°).

Small spherical micelle-like aggregates with visible arms pointing from the dense cores can be observed (Figures 5 and 6). This is most prominent for the large micelle-like aggregates formed by linear PDMAEMA-PS-PDMAEMA, which also has the largest molar mass (Mn = 37 400 000 g/mol). For other micelle-solutions the dense cores are clearly visible with less contrast for the arms. The cryoelectron microscopy shows areas of high contrast, such as the core of the particle, rather than the low contrast periphery, and thus the scale bar does not give a proper estimate about the size of the micelles. DLS gives the true estimation of the sizes of these micelles (Table 2). 4.5. DNA Condensation by PDMAEMA Block Copolymers. The ability of PDMAEMA block copolymers to condense plasmid DNA was confirmed and evaluated by an electrophoresis retardation assay. Block copolymers were complexed with DNA at different nitrogen/phosphate (n/p) ratios ranging from 0.5 to 32. PEI25K at n/p ratio 8 condenses DNA totally and is thus used as a positive control. For all block copolymers, the intensity of

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migrating free DNA bands was decreased gradually with increasing charge ratio until DNA was completely retarded (Figure 7). For all polymers, at n/p ratio 2 or higher, the free DNA was completely retarded, and only bound DNA could be seen. At n/p ratio 8 or higher, all copolymers except (PS-PDMAEMA)6 can condense DNA completely. Starlike (PS-PDMAEMA)6 forms rather loose complexes with DNA because ethidium bromide has access to DNA at all charge ratios. This is due to the stiff and glassy polystyrene core, which restricts the DNA from wrapping around the polymer. Linear polymers seem to condense DNA more effectively. Flexible linear chains of the polymer make the structure more favorable for DNA condensation than more restricted starlike architecture. In long linear chains, the amines are more accessible and thus reach the phosphate groups of DNA more readily. 4.6. Cytotoxicity Evaluation and in Vitro Transfection Activity. The cytotoxicity and transfection efficacy of polymer
DNA complexes were evaluated using two different cell lines, ARPE19 and CV1-P. ARPE19 is the most representative cell model for retinal pigment epithelium and thus is an interesting target for gene therapy studies. CV1-P is the generally used cell line in studies of transfection efficacy. Cationic polymers are known to be potentially cytotoxic as they damage cell membranes by electrostatic interactions.37 With both cell lines, all polymer micelles showed >80% cell viability at n/p ratios 0.5
4. (See the Supporting Information.) With increasing n/p ratio, the cell viability decreased gradually, and at n/p ratio 32, the viability was almost 0%. This is most probably due to the high cationic content. Starlike (PS-PDMAEMA)6 showed the highest cell viabilities at all n/p ratios. Otherwise, the architecture of the polymers did not seem to affect the cell viability. The ability of polymer micelles to transfect ARPE19 and CV1-P cells was evaluated with varying n/p ratios. Gene transfection efficiency was evaluated as β-galactosidase expression ability. With ARPE19 cell line, reasonable β-galactosidase expression was observed only for poly(n-butyl acrylate)-based polymers at n/p ratios of 2 and 4 (Figure 8). For linear PDMAEMA-PBuAPDMAEMA, the expression level at n/p 2 was similar to the positive control PEI25K. Polystyrene-based polymers were almost totally unable to transfect the ARPE19 cells. The lack of transfection at high n/p ratios arises from the cytotoxicity and at low n/p ratios from the inefficient complex formation with DNA. Notably, no cytotoxicity was observed at n/p region 2
4. With the CV1-P cell line, all polymers, except (PSPDMAEMA)6, exhibited enhanced expression at n/p ratios 2 to 8, with linear PDMAEMA-PBuA-PDMAEMA having again the best transfection efficiency (Figure 9). In the case of the CV1-P cell line, the positive control PEI25K (n/p 8) exhibited almost 10-fold higher β-galactosidase activity than linear PDMAEMA-PBuA-PDMAEMA at n/p ratio 4. None of the PDMAEMA-based polymer micelles have cytotoxic character at low n/p ratios (0.5
4). Therefore, the enhanced ability of linear PDMAEMA-PBuA-PDMAEMA to transfect cells compared with other polymer arises from its composition and architecture. The rubbery core-forming poly(n-butyl acrylate) block and linear chain architecture allows it to wrap DNA most effectively and provide sufficient transfection compared to commercial PEI25K. Linear architecture provides a positive effect also for polystyrene-based polymers because PDMAEMA-PS-PDMAEMA has enhanced transfection efficiency with CV1-P cell line compared with (PS-PDMAEMA)6. Star (PS-PDMAEMA)6 was unable to transfect cells most likely 3220

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Figure 7. Agarose gel electrophoresis of PDMAEMA block copolymer/DNA complexes based on (a) (PS-PDMAEMA)6, (b) (PBuA-PDMAEMA)6, (c) PDMAEMA-PS-PDMAEMA, and (d) PDMAEMA-PBuA-PDMAEMA at varying n/p ratios. Above the figures, mw represents molecular marker, lane 0 represents free DNA, lanes 0.5
32 represent PDMAEMA block copolymer/DNA complexes at n/p ratios 0.5
32, respectively, and PEI corresponds to PEI/DNA complex at n/p ratio 8.

Figure 8. Transfection efficiency of DNA polyplexes based on PDMAEMA block copolymers in ARPE19 cells at n/p ratios 0.5
32. PEI/DNA n/p 8 was used as a positive control. The data are given as milliunits of β-galactosidase/well ( sem (n = 3 to 4).

Figure 9. Transfection efficiency of DNA polyplexes based on PDMAEMA block copolymers in CV1-P cells at n/p ratios 0.5
32. PEI/DNA n/p 8 was used as a positive control. The data are given as milliunits of β-galactosidase/well ( sem (n = 3 to 4).

because of its poor DNA condensation ability at low n/p ratios, which in turn is a consequence of its glassy and restricted core.

Star and linear amphiphilic polymers formed spherical core
shell micelles of ∼80 nm in aqueous environment. The aggregation numbers of the micelles varied from 22 to 265. More dense and restricted star polymers had lower aggregation numbers than linear polymers. All polymers complexed DNA completely at large nitrogen/ phosphate (n/p) ratios, except (PS-PDMAEMA)6, which formed loose DNA complexes. All polymers showed >80% cell viability at low n/p ratios. Gene transfection efficiency was highest for linear PDMAEMA-PBuA-PDMAEMA (at n/p 2), comparable to that of PEI25K. A glassy polystyrene core hinders the DNA condensation and cellular DNA transfection; only the linear form was able to mediate transfection. The architecture and the composition of the block copolymers have a clear effect on DNA transfection. A rubbery core-forming block of the polymer as well as the linear architecture of the

5. CONCLUSIONS In conclusion, tert-butylcalix[6]arene was modified by esterification to obtain a six-functional ATRP-initiator with alkyl halide groups. Using modified calix[6]arene as a core molecule, novel amphiphilic star block copolymers, with narrow size distribution, were synthesized using the core-first method and ATRP. Two different star block copolymers with different coreforming blocks were synthesized. Hydrophobic core consisted of either glassy polystyrene or rubbery poly(n-butylacrylate). The outer block consisted of cationic PDMAEMA. Corresponding triblock copolymers were also synthesized. Synthesized polymers were well characterized with various methods.

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Biomacromolecules polymer chain enhances the ability of polymer to wrap around the DNA and thus permit good expression levels.

’ ASSOCIATED CONTENT

bS

Supporting Information. Detailed aspects of the data analysis of the light scattering, additional SLS and DLS data, as well as size exclusion data for hydrolysis of polystyrene star. This material is available free of charge via the Internet at http://pubs. acs.org

’ AUTHOR INFORMATION Corresponding Author

*E-mail: heikki.tenhu@helsinki.fi.

’ ACKNOWLEDGMENT We would like to thank Pasi Laurinm€aki and Eevakaisa Vesanen for excellent technical assistance. We thank the Biocenter Finland National Cryoelectron Microscopy Unit, Institute of Biotechnology, Helsinki University for providing facilities. ’ REFERENCES (1) Smart, T.; Lomas, H.; Massignani, M.; Flores-Merino, M., V.; Ruiz Perez, L.; Battaglia, G. Nanotoday 2008, 3, 38–46. (2) Harada, A.; Kataoka, K. Prog. Polym. Sci. 2006, 31, 949–982. (3) Croy, S. R.; Kwon, G. S. Curr. Pharm. Des. 2006, 12, 4669–4684. (4) Gaucher, G.; Dufresne, M.; Sant, V. P.; Kang, N.; Maysinger, D.; Leroux, J. J. Controlled Release 2005, 109, 169–188. (5) Miyata, K.; Christie, J., R.; Kataoka, K. React. Funct. Polym. 2011, 71, 227–234. (6) Pispas, S.; Hadjichristidis, N.; Potemkin, I.; Khokhlov, A. Macromolecules 2000, 33, 1741–1746. (7) Lin, C.-M.; Chen, Y.-Z.; Sheng, Y.-J.; Tsao, H.-K. React. Funct. Polym. 2009, 69, 539–545. (8) Yoo, M.; Heise, A.; Hedrick, J. L.; Miller, R. D.; Frank, C. W. Macromolecules 2003, 36, 268–271. (9) Meier, M. A. R.; Schubert, U. S. J. Comb. Chem. 2005, 7, 356–359. (10) Kul, D.; Van Renterghem, L., M.; Meier, M. A. R.; Strandman, S.; Tenhu, H.; Yilmaz, S. S.; Schubert, U. S.; Du Prez, F. E. J. Polym. Sci., Part A: Polym. Chem. 2008, 46, 650–660. (11) Tiera, M. J.; Winnik, F. M.; Fernandes, J. C. Curr. Gene Ther. 2006, 6, 59–71. (12) Massignani, M.; LoPresti, C.; Blanazs, A.; Madsen, J.; Armes, S. P.; Lewis, A. L.; Battaglia, G. Small 2009, 5, 2424–2432. (13) van de Wetering, P.; Zuidam, N. J.; van Steenbergen, M. J.; van der Houwen, O. A. G. J.; Underberg, W. J. M.; Hennink, W. E. Macromolecules 1998, 31, 8063–8068. (14) Bougard, F.; Jeusette, M.; Mespouille, L.; Dubois, P.; Lazzaroni, R. Langmuir 2007, 23, 2339–2345. (15) Arigita, C.; Zuidam, N. J.; Crommelin, D. J. A.; Hennink, W. E. Pharm. Res. 1999, 16, 1534–1541. (16) Boussif, O.; Lezoualc’h, F.; Zanta, M. A.; Mergny, M. D.; Scherman, D.; Demeneix, B.; Behr, J. Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 7297–7301. (17) M€annist€o, M.; Vanderkerken, S.; Toncheva, V.; Elomaa, M.; Ruponen, M.; Schacht, E.; Urtti, A. J. Controlled Release 2002, 83, 169–182. (18) Georgiou, T. K.; Vamvakaki, M.; Patrickios, C. S.; Yamasaki, E. N.; Phylactou, L. A. Biomacromolecules 2004, 5, 2221–2229. (19) Merdan, T.; Kopecek, J.; Kissel, T. Adv. Drug Delivery Rev. 2002, 54, 715–758. (20) Xu, F. J.; Zhang, Z. X.; Ping, Y.; Li, J.; Kang, E. T.; Neoh, K. G. Biomacromolecules 2009, 10, 285–293.

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