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Synthesis and characterization of a novel water-soluble cationic diblock copolymer with star conformation by ATRP
Materials Science and Engineering C 43 (2014) 350–358
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Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
Synthesis and characterization of a novel water-soluble cationic diblock copolymer with star conformation by ATRP Shuzhao Li a,b, Miaomiao Xiao a, Anna Zheng b, Huining Xiao a,⁎ a b
Department of Chemical Engineering, University of New Brunswick, Fredericton, NB E3B 5A3, Canada School of Materials Science and Engineering, East China University of Science and Technology, Shanghai 200237, China
a r t i c l e
i n f o
Article history: Received 9 October 2013 Received in revised form 14 April 2014 Accepted 30 June 2014 Available online 8 July 2014 Keywords: Star diblock copolymer ATRP β-Cyclodextrin Gene delivery
a b s t r a c t A water-soluble cationic diblock copolymer, CD-PAM-b-PMeDMA, was synthesized through atom transfer radical polymerization (ATRP) from a β-cyclodextrin (CD) macroinitiator with 10-active sites (10Br-β-CD). In order to reduce the cytotoxicity of the CD-PAM-b-PMeDMA, biocompatible polyacrylamide (PAM) was first introduced onto the surface of β-CD as a scaffold structure by ATRP using the 10Br-β-CD as a macroinitiator. The reaction conditions of AM were explored and optimized. The ATRP of [2-(methacryloyloxy)ethyl] trimethyl ammonium chloride (MeDMA) was also performed to synthesize the second cationic block using the resulting CD-PAM as a macroinitiator. The resulting diblock copolymer shows an increased hydrodynamic radius in aqueous solution with a pretty low concentration compared with β-CD. In addition, it appears a near-uniform coniform after being deposited on mica ascribed to the presence of an asymmetric 10-arm structure. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Gene therapy, which involves the insertion of a target gene into cells to prevent or treat a disease associated with a mutant or defective gene, is a promising and rapidly developing therapeutic technique. It enables the correction of inherited or acquired genetic defects or the ablation of diseased cells such as tumors in cancer therapy [1]. To design the gene delivery vectors with low cytotoxicity and high transfection efficiency is the most challenging task in gene therapy [2,3]. An ideal nonviral vector should deliver the nucleic acid with high gene expression in the target cells without any inflammatory response. It should be biodegradable, non-toxic, and stable against enzyme. Some factors such as molecular weight (Mw), polydispersity, charge density, cationic or anionic functionality, polymer structure, charge ratio and size of the polymer-nucleic acid complex, as well as the pH of the medium play crucial roles in efficacy and toxicity for in vitro and vivo applications [4–7]. Cationic water-soluble polymers have been widely investigated for various applications [8–11], and also considered as the major type of the non-viral gene delivery vectors [12–17]. To date, a number of cationic polymers have been reported to be able to affect gene transfection, including linear polymers such as polyethylenimine (PEI) [18] or star poly(2-(dimethylamino)ethyl methacrylate) (P(DMAEMA)) [19]. Compared with linear polymer, star polymer, with the same Mw, has a much smaller gyration radius according to the shrinking parameter theory [20], and meanwhile maintains most properties of the linear polymer ⁎ Corresponding author. Tel: +1 5064533532; fax: +1 506 453 3591. E-mail address: [email protected] (H. Xiao).
http://dx.doi.org/10.1016/j.msec.2014.06.031 0928-4931/© 2014 Elsevier B.V. All rights reserved.
with high Mw without the solution viscosity penalty [21,22]. There are generally two strategies to synthesize star polymers, namely the “arm-first” and “core-first” approaches. Star block copolymers have recently attracted much attention because of the tunable properties like solubility, topology, and temperature or pH sensitivity, which could be manipulated by the parameters such as the block composition, Mw, and arm number. Star block copolymers combine the properties of block copolymers and the unique properties of star-shaped polymers. In addition to reducing cytotoxicity, star block copolymer can further enhance the gene transfection efficiency. The studies of Xu et al. [19] showed that star polymers or star block copolymers exhibit much lower cytotoxicity and higher gene transfection efficiency than linear homopolymers. β-CD is one of the excellent candidates as core materials for the synthesis of star polymers due to its high biocompatibility and multi-reactive sites [23]. β-CD is a cyclic oligosaccharide consisting of seven glucose units linked by α-1,4-glucosidic bonds with seven primary and fourteen secondary hydroxyl groups, which can be further utilized for selective modification and surface grafting. Through the core-first approach, a variety of star polymers have been successfully synthesized by ATRP [24–28]. To identify some key parameters for the development of efficient carriers, controlled polymerization techniques are required. Compared with other polymerizations, ATRP is considered to be one of the most practical and facilitating approaches to synthesize star block copolymers; and a variety of functional star block polymers have been achieved via ATRP [19,29,30]. Lorenzo et al. [29] synthesized (PCL2)-b(PS2) 4-miktoarm star block copolymers by ATRP and ring opening polymerization, which behaved much differently from PCL-b-PS diblock copolymer in morphology, nucleation, and crystallization. Xu et al. [19]
S. Li et al. / Materials Science and Engineering C 43 (2014) 350–358
synthesized star-shaped cationic block copolymers by ATRP for nonviral gene delivery, and both the cytotoxicity and gene transfection efficiency were higher than liner homopolymers. Schramm et al. [30] prepared water-soluble star block copolymers, which could potentially be used as drug carriers through ATRP of PEGMA with hydrophobic starshaped polymeric core molecules. The generated polymers have high hydrophilicity, tunable hydrophobic/hydrophilic balance, and molar masses. The high cytotoxicity is mainly ascribed to the high concentration of amino groups [31–33]. It has been proved that the cytotoxicity will be lowered and gene transfection will be facilitated through introducing another biocompatible block without amino groups [19]. It has been shown that the conjugation of PEG to polyelectrolytes increases aqueous solubility and reduces cytotoxicity by masking the polymer's positive charge [4]. Therefore, in the present work, a novel 10-arm cationic star-shaped block polymer was synthesized, which consists of one slightly negative charged block, polyacrylamide (PAM), and one cationic block, poly[2-(methacryloyloxy)ethyl] trimethyl ammonium chloride (PMeDMA). The generated cationic star polymer could be potentially used as gene vector. Polymers of acrylamide and its derivatives are widely used in industry, agriculture, and medicine due to their remarkable properties including biocompatibility, lack of toxicity, water solubility, pH or temperature sensitivity, and so on. Among the applications of these polymers, the most important ones include dental fillers, tumor suppressants, hair spray components, shaving creams, and many others [34,35]. The incorporation of AM unit on the exterior or interior of the star diblock copolymer is proposed as a potential DNA vector. As far as we are aware, there have been no reports about the polymerization of star-shaped PAM by ATRP using β-CD as the core. Although some researchers synthesized linear PAM via ATRP by using small molecule initiators [36–39], the reaction conditions by using a macroinitiator, 10Br-β-CD, are much different from those by using small molecule initiators. In the current work, the optimum reaction conditions for ATRP of AM were identified. The star polymer, CD-PAM, was then utilized as a macroinitiator to induce the polymerization of MeDMA in an attempt to obtain the star-shaped cationic water-soluble block polymer, CDPAM-b-PMeDMA. The resulting star-shaped copolymer is expected to have lower cytotoxicity, better biocompatibility, and potentially higher transfection efficiency than quaternary ammonium-CD nanoparticles developed in our previous work [40]. Furthermore, with the aid of the AFM, the conformation of the block polymer, deposited on mica from aqueous solution, was evidently revealed for the first time. 2. Experimental
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added to the solution as deacid reagent and catalyst, respectively, before the mixture was cooled to 0 °C. BiBB (50 mmol) dissolved in anhydrous NMP (25 mL) at 0 °C was drop by drop added into the β-CD solution, with magnetic stirring under N2 atmosphere. Afterwards, the reaction temperature was maintained at 0 °C for 2 h and then at room temperature for 24 h. The final mixture was filtered to remove the triethylammonium bromide. The residual brown solution was precipitated in a large amount of water to get rid of NMP. The dried pale yellow precipitate was dissolved in dichloromethane (50 mL) and then crystallized in cold n-hexane twice to produce a white product (12.23 g, yield 93%). The structure of 10Br-β-CD was confirmed using 1 H NMR (CDCl3, 300 MHz). The degree of substitution of the hydroxyl groups on the outside surface of β-CD is determined by the area ratio of the methyl protons at δ1.82 ppm and methylidyne and methylene protons at the region of δ3.7–4.6 ppm [19]. 2.3. ATRP of AM using 10Br-β-CD as an initiator An example of ATRP of AM using the 10Br-β-CD/CuCl/PMDETA catalyst system is as follows. 10Br-β-CD (0.3 g, 0.114 mmol) was dissolved in 50 mL DMF/water mixture (4:1 v/v) placed in the flask, and then CuCl2 (30.7 mg, 0.229 mmol), PMDETA (476.23 mg 2.75 mmol), and AM (1.62 g, 22.8 mmol) were added sequentially under room temperature. The pH was adjusted to 7 with dilute hydrochloric acid (1 N HCl) to ensure the narrow molecular weight distribution (MWD) [41]. The mixture was deoxygenated by three vacuum-nitrogen purge cycles and subsequently purged under a nitrogen atmosphere. After the Cu(I)Cl was quickly added into the flask, the reaction vessel was sealed and placed in an oil bath maintained at a temperature of 130 °C. After a 24-h reaction time, the reaction vessel was taken out of the oil bath, exposed to air to oxidize the Cu(I) catalyst, and then the product solution was passed through a short column of alumina to remove the catalyst residues. The polymer solution was concentrated by a rotary evaporator and then precipitated in acetone twice. The precipitated polymer, CDPAM, was separated by centrifuge and dried in vacuum oven at 40 °C for 48 h to conduct the 1H-NMR and GPC analysis. The monomer conversion was determined by comparing the peak integrals of the vinyl signals at δ5.5 ppm and δ6.0 ppm to those of the methacrylate backbone at δ0.5–1.1 ppm and δ1.5–2.0 ppm [26,42]. For kinetics experiments, samples were withdrawn from time to time with gas-tight syringes for the characterization of the monomer conversion and Mw after purified as above procedure. 2.4. Typical procedure for the ATRP of MeDMA using CD-PAM as the macroinitiator
2.1. Materials 2-Bromoisobutyryl bromide (BiBB), anhydrous 1-methyl-2pyrrolidione (NMP), triethylamine (TEA), 4-(N,N-dimethylamino)pyridine (DMAP), N,N,N′,N′,N″-pentamethyldiethylenetriamine (PMDETA), CuCl, CuCl2, and 2,2′-bipyridine (bpy) were all purchased from Aldrich. The chemicals were used without further purification except for PMDETA, which was distilled and degassed, and CuCl, which was treated with pure acidic acid and filtered to remove traces of Cu(II) compounds. β-CD was also purchased from Aldrich and then vacuum dried at 80 °C over phosphorus pentoxide overnight immediately before use. Acrylamide (AM), purchased from Sigma, was recrystallized for 3 times prior to use. [2-(methacryloyloxy)ethyl] trimethyl ammonium chloride (MeDMA) was obtained from Aldrich in 75% w/v aqueous solution and used without further purification. 2.2. Synthesis of the macroinitiator based on β-CD In this work, the arm number of the star-shaped polymer was designed to be 10. The starting material, β-CD (5 mmol), was dissolved in 90 mL anhydrous NMP. TEA (55 mmol) and DMAP (15 mmol) were
The ATRP of MeDMA was conducted using purified CD-PAM as the macroinitiator. The procedure of ATRP of MeDMA is similar to that of AM. The CD-PAM (100 mg, 0.01 mmol) was dissolved in 15 mL methanol/water mixture (3:1, v/v), and then MeDMA (620 mg, 3.0 mmol) monomer aqueous solution and bpy ligand (74.20 mg, 0.48 mmol) were added into the reaction vessel. Cu(I)Cl was quickly added into the flask after deoxygenated and sealed in the reaction vessel with a rubber septum. The reaction vessel was finally placed in an oil bath maintained at a temperature of 60 °C. After a 10-h reaction time, the reaction vessel was taken out of the oil bath, exposed to air to terminate the reaction. The product solution was purified with dialysis membrane (MWCO 1000) against water for 24 h to yield a colorless product. 2.5. Measurements 1
H NMR spectra were obtained using a Varian Unity 400 spectrometer operated at 300 MHz. 10Br-β-CD samples were dissolved in CDCl3, and CD-PAM and CD-PAM-b-PMeDMA polymers were dissolved in D2O. 1H NMR spectra of the samples in D2O were also employed to identify the monomer conversion in ATRP.
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Energy dispersive X-ray analysis (EDAX) was measured using a Hitachi SU-70 FE-SEM equipped with an Oxford-INCA (x-act LN2-free SDD Detector) energy dispersive X-ray analyzer (accelerating voltage 15 kV). The number-average Mw and MWD of polymers were measured on a GPCMax VE201 Gel Permeation Chromatography (GPC) (Viscotek) equipped with three columns PAA-202-204-205 and three detectors (viscometer, light scattering, and IR detector). The eluent was 0.05 N sodium nitrate aqueous solution and the flow rate was 1 mL/min at 30 °C. The calibration was based on poly(ethylene oxide) standards using multi-detector method. Apparent charge density of the anionic CD-PAM and cationic CDPAM-b-PMeDMA was determined via colloidal titration using a Particle Charge Detector Mütek PCD 03 (Herrsching) with standard cationic polyelectrolyte (poly-dimethyldially ammonim chloride (DADMAC) solution) (concentration = 1.099 mN) and standard anionic polyelectrolyte (potassium polyvinyl sulfate (PVSK) solution) (concentration = 0.189 mN), respectively. Three repeats were conducted to acquire an average value for each sample. Hydrodynamic radii of polymers were measured by Quasi Elastic Light Scattering (QELS), which was conducted with 90Plus/BI-MAS Multi Angle Particle Sizing Option (Brookhaven Instruments). The measurements were conducted at a scattering angle of 90° and different temperatures with low polymer concentrations in aqueous solution; and three repeats were performed for each sample. AFM measurements were performed using a Nanoscope IIIa from Veeco Instruments Inc., Santa Barbara, CA. The images were scanned in Multimode mode in air using a silicon tapping probe (NP-S20, Veeco Instruments) with a resonance frequency of about 273 kHz. For the AFM experiments of the samples, a drop of sample aqueous solution was deposited on the mica surface. After 5 min contact with the substrate the sample was dried by centrifugal force on a spin-coater.
3. Results and discussion
which led to a higher yield and purer product. During the synthesis, tertiary amine (TEA) was added to the solution as a deacid reagent and reacted with hydrogen bromide, a product of hydroxyl groups and BiBB in the reaction, to form triethylammonium bromide which cannot dissolve in NMP solvent, thus facilitating its removal by filtration after the reaction. The product, 10Br-β-CD, in the solution was readily obtained by precipitation in water. The reaction efficiency was as high as 93% by calculating the weight change, indicating that most of BiBB reacted with β-CD. EDAX was used to confirm the existence of bromine element on the surface of β-CD. In the EDAX spectrum of 10Br-β-CD (Fig. 1), the Br peak is observed around 1.48 keV, implying the formation of the 10Br-β-CD macromolecular initiator. A 10 active-sites initiator was designed to avoid the close proximity of reactive groups [44], and it was assumed that BiBB first reacted with primary hydroxyl protons because of its higher reaction activity than secondary hydroxyl protons on β-CD. Fig. 2(a) shows the 1H NMR spectrum of the 10Br-β-CD sample: the peak at δ1.82 ppm (a, 3H, \CH3) demonstrates the presence of 2-bromoisobutyryl groups; whereas the peaks at δ3.7–5.3 ppm correspond to the sugar residues [41]. In the region of δ3.7–5.4 ppm, the peaks at δ3.7–4.6 ppm are assigned to the inner methylidyne and methylene protons between the oxygen and carbon moieties (b, O\CH\C and O\CH2\C) on the glucose units of β-CD; the peak at δ4.8 ppm is attributed to the inner methylidyne protons between the oxygen moieties (b″, O\CH\O); and the peak located at δ5.3 ppm is associated with the hydroxyl protons adjacent to the methylidyne moieties (c′, CH\OH) of glucose units [19,45]. Furthermore, the relative peak area at δ5.3 ppm almost does not vary in comparison with the corresponding peak in 1H NMR of β-CD in Fig. 2(b), indicating that the hydroxyl protons on CH\OH do not react with BiBB. In addition, the signal of the hydroxyl protons adjacent to the methylene moieties (c, CH2\OH) disappears in Fig. 2(a), indicating that all the primary hydroxyl groups were substituted with 2-bromoisobutyryl groups. By calculating the area ratio of the methyl protons at δ1.82 ppm and methylidyne and methylene protons at the region of δ3.7–4.6 ppm, the degree of substitution of the hydroxyl groups on the outside surface of β-CD is determined to be approximately 10 [19].
3.1. Synthesis of 10Br-β-CD The synthetic approach of the CD-PAM-b-PMeDMA is shown in Scheme 1. 10Br-β-CD was synthesized according to the procedures reported by Kohji et al. [41] and Li et al. [26,43]. However, in the present work, the reaction and purification procedures were further optimized,
3.2. Synthesis of 10-arm star-shaped CD-PAM in DMF aqueous solution It is well known that (meth)acrylamides are not satisfactorily polymerized through ATRP as other notable monomers [39,46,47]. Although
Scheme 1. Synthesis route of star diblock polymers.
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Fig. 1. EDAX spectrum of 10Br-β-CD.
many researchers have investigated the ATRP of AM, however, most of them employed small molecule as an initiator to synthesize linear PAM [36–39]. In the synthesis of star-shaped PAM, the results from us
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were not as good as what they reported by simply duplicating their experimental conditions. In the present study, it was noticed that the macroinitiator, 10Br-β-CD, behaved differently because of the presence of steric-hindered effect among 10 active sites and the insolubility in water caused by the grafted BiBB. Overall, the 10Br-β-CD performed well since the system, i.e., CuCl/CuCl2/PMDETA catalyst and DMF/ water mixture solvent, allows the ATRP to proceed at 130 °С which is often unachievable for the linear polymer system. Fig. 2(c) shows the 1 H NMR spectrum of CD-PAM synthesized in this work. The chemical shifts, δ1.67–1.79 ppm and δ2.23–2.34 ppm, are mainly attributed to the \CH2\ (c) and \CH\ (d) groups of the PAM arms, respectively, indicating the presence of PAM chains. Table 1 summarizes the results of these polymerizations. Since CuCl was employed as a catalyst, the majority of the polymer chains should be ended with Cl groups because of the halide exchange equilibrium and the strong carbon–halogen bonds [48–50]. Halide exchange contributes to the increase of the initiation rate relative to propagation. ATRP of AM has been successfully carried out in DMF [51] and glycerol aqueous solution [36]. In this work, the initiator, 10Br-β-CD, cannot dissolve in glycerol aqueous solution, but dissolves well in DMF. Therefore, initially, DMF was used as a solvent to conduct the ATRP of AM. From the results in Table 1, in the absence of any additives, the polymerization in DMF only reached a low monomer conversion (23.34%) with a high polydispersity index (PDI = 1.88). In ATRP, the presence of an X-CuII/ligand complex is necessary to reduce the concentration of radicals through a deactivation process in order to maintain
Fig. 2. 1H NMR spectra of 10Br-β-CD (a), β-CD (b), CD-PAM (c), and CD-PAM-b-PMeDMA (d).
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Table 1 Results of the ATRP of Acrylamide for 10Br-β-CD based initiating system (The molar ratio of acrylamide and 10Br-β-CD is 200:1, and the reaction was conducted at 130 °С for 24 h with PMDETA as the ligand ([PMDETA] / ([Cu(II)] + [Cu(I)]) = 2/1)). Entry
1 2 3 4 5 6 7
Additives CuIICl2 ([Cu(II)]/[Cu(I)])
LiCl (mol/L)
– 0.2 0.2 0.2 0.2 0.2 0.2
– – – – – 1 0.1
Solvent (V/V)
Conversion (%)
Mn (GPC)
Mn (theory)
PDI Mw/Mn
DMF DMF DMF/H2O DMF/H2O DMF/H2O DMF/H2O DMF/H2O
23.34 55.37 75.34 82.59 83.20 85.30 71.78
3150 6700 10,000 10,100 11,300 11,400 8200
5900 10,400 13,200 14,300 14,400 14,700 12,800
1.88 2.10 1.48 1.27 2.03 2.87 1.34
= = = = =
5/1 4/1 3/1 3/1 3/1
the activation–deactivation equilibrium for the controlled growth of the polymer chains with conversion [52]. To reduce the polydispersities, the addition of Cu(II) to the reaction mixture was employed in the earlier study [53]. From Table 1, it can be seen that, in the presence of CuCl2 as a deactivator, the monomer conversion was increased to 55.37%. However, a large amount of polymers precipitated out of the DMF solvent, leading to a high PDI, which is in accordance with Suresh's result [36]. In order to conduct the polymerization in homogeneous conditions during the designed reaction time, it is necessary to select suitable solvents. Since CD-PAM is water soluble, the introduction of small amount of water in the system may increase the solubility of the resulting products. Therefore, DMF and water mixture was used as the media in the following experiments for further optimizing ATRP conditions. With the addition of a small amount of water in DMF (DMF/H2O = 5/1(v/v)), the conversion was apparently increased to 75% with a narrow PDI (PDI = 1.48). However, there was still a trace amount of precipitate at the end of reaction. Compared with DMF/H2O = 5/1(v/v), when DMF/H2O = 4/1 was used, no precipitate appeared at the end of polymerization; and the relatively high monomer conversion (82.59%) and a low PDI (1.27) were obtained. However, with further increasing the content of water (DMF/H2O = 3/1(v/v)), the PDI became broader although the monomer conversion was slightly increased to 83.20%. In fact, Wang et al. [54] thought that the water leads to the formation of a more active Cu(I) catalyst, thus increasing the rate of ATRP in aqueous media. Also, it was suggested that, as the solvent polarity was increased, a higher concentration of mononuclear copper catalyst was produced; thus, both the radical concentration and the polymerization rate increased. As a result, the MWD broadened [53]. Therefore, in the present case, the PDI was broadened with the increase of the water in DMF, especially in the radical-confined reaction system in which the active chains were in close conjunction with β-CD. Jewrajka et al. [36] demonstrated that, in the presence of sufficient amount of LiCl, which ensured the high efficiency of CuCl2 as a deactivator in an aqueous medium, the linear kinetic plots for monomer disappearance was obtained. In this work, the addition of LiCl at a higher concentration was also attempted. However, when the concentration of LiCl was increased to 1 mol/L, the monomer conversion was only further increased to 85.30%; whereas the PDI became much broader (PDI = 2.87), and the precipitation of a large amount of polymer occurred. It might be because of the fact that the alkali halide concentration was so high that the solubility of polymer in the DMF aqueous solution was decreased. The polymer precipitation was enhanced by increasing ionic strength in the salt solution, thus leading to a broad MWD. In order to address this problem, the concentration of the LiCl was decreased to 0.1 mol/L, and there was no precipitate in the reaction system; consequently, the resulting PDI went down to 1.34. However, the monomer conversion decreased to 71.78%. Fig. 3 shows several aqueous GPC profiles of CD-PAM synthesized under different conditions listed in Table 1. Clearly, the sample synthesized under entry 4 conditions has compromised monomer conversions (82.59%) and PDI (1.27). Linear first-order kinetic plots of Ln[M]0/[M] against polymerization time can be only observed under entry 4 and entry 7 conditions
(Table 1) as shown in Fig. 4. The results indicate that, throughout the reaction, the concentration of propagating species is constant, and therefore the influence from chain transfer or other side reactions is negligible. Furthermore, the kinetic results further show that, in addition to the decrease of monomer conversion, the reaction rate of ATRP also decreases in the conditions of entry 7 in Table 1. The number average molecular weight (Mn) and PDI as a function of monomer conversion are plotted in Fig. 5(a) under the conditions of entry 4 and Fig. 5(b) under the conditions of entry 7 (Table 1), respectively. As expected, the Mn almost linearly increases with the monomer conversion. However, the observed Mws are slightly lower than the theoretical values calculated from the consumed monomer, which might be because of the fact that the GPC is calibrated using linear PEO narrow standards; however, their hydrodynamic volumes are different from the star polymers [55].
3.3. Synthesis of 10-arm star-shaped block copolymer CD-PAM-b-PMeDMA in methanol aqueous solution One of the ATRP advantages is that block copolymers can be generated from a macroinitiator synthesized by ATRP [56]. Using CD-PAM as a macroinitiator, the second cationic block was synthesized via ATRP in this work. It has been reported that the ATRP of methyl chloridequaternized 2-(dimethylamino)ethyl methacrylate (MeDMA) could be performed in an aqueous medium [26,42]. Li et al. [26] synthesized PMeDMA using a small molecule initiator in alcohol aqueous solution at room temperature with a very high monomer conversion. Using 21Br-β-CD, Cu(I)Br, and 2,2″-dipyridyl as an initiator, catalyst, and ligand, respectively, Li et al. [42] synthesized 21 arm star polymers. However, the synthesis was conducted in water solution. As previously described, the macroinitiator is insoluble in water, thus it was only dispersed in the
Fig. 3. Aqueous GPC elution curves of CD-PAM synthesized at 130 °C. (—) in presence CuCl2 ([Cu(II)]/[Cu(I)] = 0.2) and DMF/H2O = 4:1 v/v; (−) in the presence of CuCl2 ([Cu(II)]/[Cu(I)] = 0.2) and 0.1 mol/dm3 LiCl, DMF/H2O = 3:1 v/v; (…) in the presence of CuCl2 ([Cu(II)]/[Cu(I)] = 0.2) and 1 mol/dm3 LiCl, DMF/H2O = 3:1 v/v.
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Fig. 4. Ln [M0]/[M] versus t for ATRP of acrylamide with Cl-based initiating system at 130°С (◆) DMF/H2O = 4/1(v/v), (■) DMF/H2O = 3/1(v/v) and in the presence of 0.1 mol/dm3 LiCl.
aqueous solution in Li's work [42], implying that the polymerization was a heterogeneous reaction. In this work, CD-PAM is a water-soluble polymer; however, in pure water, the ATRP of MeDMA could not be properly controlled [26]. Therefore, in order to achieve a low PDI and ensure that the ligand, bpy, can dissolve in the solvent very well, two mixture solvents, CH3OH/water and DMF/water, were employed respectively. In addition, to ensure the star-shaped block copolymers soluble in the
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mixture solvent, the volume ratio of organic solvent (DMF or methanol) and water was maintained at 1:1. The results are summarized in Table 2. From the results in Table 2, it can be seen that monomer conversions were not high either in methanol aqueous solution or DMF aqueous solution at a low temperature (30 °C) or high temperature (60 °C) as using the higher-molecular-weight macroinitiator. This finding is much different from that reported by Li et al. [57], in which the monomer conversion could be very high (90% or above). This might result from the fact that the small molecular initiator is relatively easy to be initiated compared with the macroinitiator. In this work, the propagating radicals are connected with β-CD and close adjacent to each other, which in turn leads to some side reactions such as coupling reaction instead of polymer chain propagation. Therefore, the monomer conversion could be limited to some extent, although the reaction temperature was increased to 60 °C. It is also clear that methanol aqueous solution is better than DMF aqueous solution. This might be attributed to the higher polarity of DMF aqueous solution, which tends to increase the activation rate and leads to higher radical concentration [58,59], thus potentially enhancing the coupling reactions. Furthermore, DMF is a coordination solvent, which may displace ligands from the complex and may also saturate the coordination sphere around the Cu(I) species [60]. Therefore, in this study, synthesizing block copolymer was conducted in methanol aqueous (CH3OH/H2O = 1/1) at 60 °C. Noteworthily, when the CD-PAM with a lower Mw of 4200 g/mol was used as a macroinitiator, the conversion of MeDMA was increased to 79% in methanol aqueous (CH3OH/H2O = 1/1) at 60 °C. This could be attributed to that, with the decrease of Mw, the coupling reactions among different arms were reduced and more chlorin atoms could be access rather than buried in the PAM chains. In Fig. 2(c), the chemical structure of CD-PAM-b-PMeDMA synthesized in methanol aqueous solution was revealed by 1H NMR spectrum. There is an additional peak at δ = 3.3 ppm compared with spectrum (b), which is assigned to the methyl protons of -N(CH3)3+ [26]. 3.4. Confirmation of the hydrodynamic radius of the resulting CD-PAM-b-PMeDMA in aqueous solution In the test of the hydrodynamic radius of β-CD with a concentration of 1.3% (10 mM), a bimodal distribution was observed by filtrating through 0.2 μm, yielding two mean hydrodynamic radii of 0.8 nm and 114.7 nm, respectively [61]. The larger hydrodynamic radius is ascribed to the self-assembly of β-CD [62–65]. Furthermore, 3 mM sample is the less concentrated aqueous solution where aggregates are formed [66]. Herein, at a pretty low concentration of 3 × 10−6% (3 ppm), the native β-CD aqueous solution, filtered through 0.2 μm by using a syringe at 25 °C, was subjected to the QELS tests. As expected, it shows a single modal distribution at 0.75 nm in radius, which is in accordance with those of González-Gaitano [64]. Also, with the concentration of 3 × 10−6% at 25 °C, the CD-PAM-b-PMeDMA aqueous solution, filtered through 0.2 μm by using a syringe, of entry 3 in Table 2 shows a single modal Table 2 Results of the ATRP of MeDMA from CD-PAM synthesized under the conditions of entry 4 in Table 1.
Fig. 5. Dependence of molecular weight, Mn GPC, and molecular weight distribution, Mw/Mn, on monomer conversion for the solution polymerization of acrylamide at 130 °С (a) DMF/H2O = 4/1(v/v), (b) DMF/H2O = 3/1(v/v) and in the presence of 0.1 mol/dm3 LiCl.
Sample
Solvent (v/v)
Tep. (°C)
Conversion (%)
Mn (GPC)
Mn (theory)
Mw/Mn
1a 2a 3a 4a 5b
CH3OH/H2O = 1/1 DMF/H2O = 1/1 CH3OH/H2O = 1/1 DMF/H2O = 1/1 CH3OH/H2O = 1/1
30 30 60 60 60
35.32 30.92 49.45 34.50 79.60
19,300 17,000 25,600 18,300 32,900
25,900 23,100 34,700 23,500 49,400
1.28 1.27 1.22 1.24 1.26
a The reaction time is 24 h, and target DP is 30. The macroinitiator, CD-PAM was the sample of entry 4 in Table 1, synthesized using PMDETA as the ligand at 130 °C for 24 h. Mn = 10,100 g/mol, Mw/Mn = 1.27 (Table 1, entry 4). b The reaction time is 24 h, and target DP is 30. The macroinitiator, CD-PAM was withdrawn from the entry 4 reaction system in Table 1 at the fourth hour. Mn = 4200 g/mol, Mw/Mn = 1.18.
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smaller size corresponding to the monomeric sample (122.1 nm) and the larger one (452.7 nm) attributed to aggregates after the filtration as shown in Fig. 6.
3.5. Determination of the conformation of the star-shaped diblock copolymers
Fig. 6. DLS results of the aqueous solutions of CD-PAM-b-PMeDMA. 3 × 10−6% (3 ppm) at 25 °C (solid line), 3 × 10−6% (3 ppm) at 60 °C (dash line), 1 × 10−5% (10 ppm) at 25 °C (dot line).
distribution. However, in comparison with that of native β-CD aqueous solution, the hydrodynamic radius of the cationic copolymer increases to 130.0 nm as shown in Fig. 6. Furthermore, with the increase of the temperature to 60 °C, the radius distribution decreases because of the adequately stretching of the star-shaped copolymer in aqueous solution. The results are reasonable in light of the actual dimensions of the CDPAM-b-PMeDMA sample. However, when the concentration of CDPAM-b-PMeDMA aqueous solution increases to 1 × 10−5%, a bimodal distribution is detected even filtered through 0.2 μm by using a syringe, the
In order to further confirm the distribution and the morphology of the CD-PAM-b-PMeDMA sample, we performed AFM experiments on dried CD-PAM-b-PMeDMA molecules deposited on a mica surface from aqueous solutions with different concentrations, 3 × 10−6% (3 ppm) and 1 × 10−5% (10 ppm), respectively. The molecules deposited from the aqueous solution with a concentration of 3 × 10-6% are observed to form near-uniform coniform particles with a wide bottom about 36 nm (Fig. 7a). In the AFM images, the macromolecular chains of the star-shaped copolymer do not show the extended conformation like other reported cationic telechelic polyelectrolytes [67,68]. In fact, the charge density results indicate that the surface of CD-PAM is slightly negative-charged (−2.58 × 10−5 eq/g) as shown in Fig. 8. In contrast, the CD-PAM-b-PMeDMA bears a much larger amount of positive charges. Furthermore, from Fig. 8 it can be seen that the charge density of the cationic diblock copolymers with different Mws listed in Table 2 is increased with the increase of the Mws of the second block. Therefore, the two blocks of each arm may attract each other via electrostatic association, and thus leading to a compact structure. As described previously, there are ten arms on the star-shaped copolymer, which consists of seven arms locating at the narrow side and three arms at the other side of β-CD. We may speculate that the coniform structure arise from the asymmetry of ten arms schematically shown in Fig. 7b. In addition,
Fig. 7. Representative AFM topographic image of CD-PAM-b-PMeDMA deposited from aqueous solution with a concentration of 3 × 10−6% (a), 3D-AFM image (c), and schematic representation of the comformations in absorbed state of the copolymer on the mica surface (b, d) (red color indicates PAM chains). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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4. Conclusions
Fig. 8. Charge density of the cationic diblock copolymers with different Mws listed in Table 2.
A novel water-soluble cationic diblock polymer with star like shape, CD-PAM-PMeDMA, was successfully synthesized by ATRP. This appears to be the first report on an ATRP of acrylamide using modified β-CD as a macroinitiator. By the improved procedures, the synthesis of macroinitiator (10Br-β-CD) reached a very high yield. The polymerization of AM was conducted at 130 °C in a controlled manner using CuCl/ CuCl2/PMDETA catalytic system. The conversion of AM was increased to 82% in a controlled manner after the optimization of reaction conditions. The second cationic block was successfully initiated using CD-PAM to generate a diblock copolymer, CD-PAM-b-PMeDMA. The hydrodynamic radius of the resulting copolymer increases to 130 nm with a single model distribution in the aqueous solution with a lower concentration of 3 × 10−6%. Due to the electrostatic association of the diblocks, the CD-PAM-b-PMeDMA sample shows a near-uniform coniform conformation after deposited on mica due to the presence of the asymmetric 10-arms structure. Acknowledgments
once the copolymer molecules approach the mica surface, the PMeDMA arms are oriented toward the mica substrate due to strong attractions among the positively charged polyelectrolytes and the negatively charged surface (mica). Thus, the major PMeDMA constituent is located on the surface while PAM constituent is located on the top of PMeDMA arms because of the repulsion with the mica surface. However, after the rapid evaporation of water the molecular structure collapses in the Z-direction and forms a flat structure with a height around 6 nm as shown in Fig. 7(c). At the lower concentration of 3 × 10−6%, it can be seen that there are only few molecule superpositions in Fig. 7(a). Therefore, the result confirms the single model distribution of the hydrodynamic radius of CD-PAM-b-PMeDMA as shown in Fig. 6. However, with the increase of the concentration to 1 × 10−5%, aggregation phenomenon is detected by AFM as shown in Fig. 9. The result is in accordance with the bimodal distribution in Fig. 6 at a higher concentration.
Fig. 9. AFM topographic image of CD-PAM-b-PMeDMA deposited from aqueous solution with a concentration of 1 × 10−5%.
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Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
Synthesis and characterization of a novel water-soluble cationic diblock copolymer with star conformation by ATRP Shuzhao Li a,b, Miaomiao Xiao a, Anna Zheng b, Huining Xiao a,⁎ a b
Department of Chemical Engineering, University of New Brunswick, Fredericton, NB E3B 5A3, Canada School of Materials Science and Engineering, East China University of Science and Technology, Shanghai 200237, China
a r t i c l e
i n f o
Article history: Received 9 October 2013 Received in revised form 14 April 2014 Accepted 30 June 2014 Available online 8 July 2014 Keywords: Star diblock copolymer ATRP β-Cyclodextrin Gene delivery
a b s t r a c t A water-soluble cationic diblock copolymer, CD-PAM-b-PMeDMA, was synthesized through atom transfer radical polymerization (ATRP) from a β-cyclodextrin (CD) macroinitiator with 10-active sites (10Br-β-CD). In order to reduce the cytotoxicity of the CD-PAM-b-PMeDMA, biocompatible polyacrylamide (PAM) was first introduced onto the surface of β-CD as a scaffold structure by ATRP using the 10Br-β-CD as a macroinitiator. The reaction conditions of AM were explored and optimized. The ATRP of [2-(methacryloyloxy)ethyl] trimethyl ammonium chloride (MeDMA) was also performed to synthesize the second cationic block using the resulting CD-PAM as a macroinitiator. The resulting diblock copolymer shows an increased hydrodynamic radius in aqueous solution with a pretty low concentration compared with β-CD. In addition, it appears a near-uniform coniform after being deposited on mica ascribed to the presence of an asymmetric 10-arm structure. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Gene therapy, which involves the insertion of a target gene into cells to prevent or treat a disease associated with a mutant or defective gene, is a promising and rapidly developing therapeutic technique. It enables the correction of inherited or acquired genetic defects or the ablation of diseased cells such as tumors in cancer therapy [1]. To design the gene delivery vectors with low cytotoxicity and high transfection efficiency is the most challenging task in gene therapy [2,3]. An ideal nonviral vector should deliver the nucleic acid with high gene expression in the target cells without any inflammatory response. It should be biodegradable, non-toxic, and stable against enzyme. Some factors such as molecular weight (Mw), polydispersity, charge density, cationic or anionic functionality, polymer structure, charge ratio and size of the polymer-nucleic acid complex, as well as the pH of the medium play crucial roles in efficacy and toxicity for in vitro and vivo applications [4–7]. Cationic water-soluble polymers have been widely investigated for various applications [8–11], and also considered as the major type of the non-viral gene delivery vectors [12–17]. To date, a number of cationic polymers have been reported to be able to affect gene transfection, including linear polymers such as polyethylenimine (PEI) [18] or star poly(2-(dimethylamino)ethyl methacrylate) (P(DMAEMA)) [19]. Compared with linear polymer, star polymer, with the same Mw, has a much smaller gyration radius according to the shrinking parameter theory [20], and meanwhile maintains most properties of the linear polymer ⁎ Corresponding author. Tel: +1 5064533532; fax: +1 506 453 3591. E-mail address: [email protected] (H. Xiao).
http://dx.doi.org/10.1016/j.msec.2014.06.031 0928-4931/© 2014 Elsevier B.V. All rights reserved.
with high Mw without the solution viscosity penalty [21,22]. There are generally two strategies to synthesize star polymers, namely the “arm-first” and “core-first” approaches. Star block copolymers have recently attracted much attention because of the tunable properties like solubility, topology, and temperature or pH sensitivity, which could be manipulated by the parameters such as the block composition, Mw, and arm number. Star block copolymers combine the properties of block copolymers and the unique properties of star-shaped polymers. In addition to reducing cytotoxicity, star block copolymer can further enhance the gene transfection efficiency. The studies of Xu et al. [19] showed that star polymers or star block copolymers exhibit much lower cytotoxicity and higher gene transfection efficiency than linear homopolymers. β-CD is one of the excellent candidates as core materials for the synthesis of star polymers due to its high biocompatibility and multi-reactive sites [23]. β-CD is a cyclic oligosaccharide consisting of seven glucose units linked by α-1,4-glucosidic bonds with seven primary and fourteen secondary hydroxyl groups, which can be further utilized for selective modification and surface grafting. Through the core-first approach, a variety of star polymers have been successfully synthesized by ATRP [24–28]. To identify some key parameters for the development of efficient carriers, controlled polymerization techniques are required. Compared with other polymerizations, ATRP is considered to be one of the most practical and facilitating approaches to synthesize star block copolymers; and a variety of functional star block polymers have been achieved via ATRP [19,29,30]. Lorenzo et al. [29] synthesized (PCL2)-b(PS2) 4-miktoarm star block copolymers by ATRP and ring opening polymerization, which behaved much differently from PCL-b-PS diblock copolymer in morphology, nucleation, and crystallization. Xu et al. [19]
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synthesized star-shaped cationic block copolymers by ATRP for nonviral gene delivery, and both the cytotoxicity and gene transfection efficiency were higher than liner homopolymers. Schramm et al. [30] prepared water-soluble star block copolymers, which could potentially be used as drug carriers through ATRP of PEGMA with hydrophobic starshaped polymeric core molecules. The generated polymers have high hydrophilicity, tunable hydrophobic/hydrophilic balance, and molar masses. The high cytotoxicity is mainly ascribed to the high concentration of amino groups [31–33]. It has been proved that the cytotoxicity will be lowered and gene transfection will be facilitated through introducing another biocompatible block without amino groups [19]. It has been shown that the conjugation of PEG to polyelectrolytes increases aqueous solubility and reduces cytotoxicity by masking the polymer's positive charge [4]. Therefore, in the present work, a novel 10-arm cationic star-shaped block polymer was synthesized, which consists of one slightly negative charged block, polyacrylamide (PAM), and one cationic block, poly[2-(methacryloyloxy)ethyl] trimethyl ammonium chloride (PMeDMA). The generated cationic star polymer could be potentially used as gene vector. Polymers of acrylamide and its derivatives are widely used in industry, agriculture, and medicine due to their remarkable properties including biocompatibility, lack of toxicity, water solubility, pH or temperature sensitivity, and so on. Among the applications of these polymers, the most important ones include dental fillers, tumor suppressants, hair spray components, shaving creams, and many others [34,35]. The incorporation of AM unit on the exterior or interior of the star diblock copolymer is proposed as a potential DNA vector. As far as we are aware, there have been no reports about the polymerization of star-shaped PAM by ATRP using β-CD as the core. Although some researchers synthesized linear PAM via ATRP by using small molecule initiators [36–39], the reaction conditions by using a macroinitiator, 10Br-β-CD, are much different from those by using small molecule initiators. In the current work, the optimum reaction conditions for ATRP of AM were identified. The star polymer, CD-PAM, was then utilized as a macroinitiator to induce the polymerization of MeDMA in an attempt to obtain the star-shaped cationic water-soluble block polymer, CDPAM-b-PMeDMA. The resulting star-shaped copolymer is expected to have lower cytotoxicity, better biocompatibility, and potentially higher transfection efficiency than quaternary ammonium-CD nanoparticles developed in our previous work [40]. Furthermore, with the aid of the AFM, the conformation of the block polymer, deposited on mica from aqueous solution, was evidently revealed for the first time. 2. Experimental
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added to the solution as deacid reagent and catalyst, respectively, before the mixture was cooled to 0 °C. BiBB (50 mmol) dissolved in anhydrous NMP (25 mL) at 0 °C was drop by drop added into the β-CD solution, with magnetic stirring under N2 atmosphere. Afterwards, the reaction temperature was maintained at 0 °C for 2 h and then at room temperature for 24 h. The final mixture was filtered to remove the triethylammonium bromide. The residual brown solution was precipitated in a large amount of water to get rid of NMP. The dried pale yellow precipitate was dissolved in dichloromethane (50 mL) and then crystallized in cold n-hexane twice to produce a white product (12.23 g, yield 93%). The structure of 10Br-β-CD was confirmed using 1 H NMR (CDCl3, 300 MHz). The degree of substitution of the hydroxyl groups on the outside surface of β-CD is determined by the area ratio of the methyl protons at δ1.82 ppm and methylidyne and methylene protons at the region of δ3.7–4.6 ppm [19]. 2.3. ATRP of AM using 10Br-β-CD as an initiator An example of ATRP of AM using the 10Br-β-CD/CuCl/PMDETA catalyst system is as follows. 10Br-β-CD (0.3 g, 0.114 mmol) was dissolved in 50 mL DMF/water mixture (4:1 v/v) placed in the flask, and then CuCl2 (30.7 mg, 0.229 mmol), PMDETA (476.23 mg 2.75 mmol), and AM (1.62 g, 22.8 mmol) were added sequentially under room temperature. The pH was adjusted to 7 with dilute hydrochloric acid (1 N HCl) to ensure the narrow molecular weight distribution (MWD) [41]. The mixture was deoxygenated by three vacuum-nitrogen purge cycles and subsequently purged under a nitrogen atmosphere. After the Cu(I)Cl was quickly added into the flask, the reaction vessel was sealed and placed in an oil bath maintained at a temperature of 130 °C. After a 24-h reaction time, the reaction vessel was taken out of the oil bath, exposed to air to oxidize the Cu(I) catalyst, and then the product solution was passed through a short column of alumina to remove the catalyst residues. The polymer solution was concentrated by a rotary evaporator and then precipitated in acetone twice. The precipitated polymer, CDPAM, was separated by centrifuge and dried in vacuum oven at 40 °C for 48 h to conduct the 1H-NMR and GPC analysis. The monomer conversion was determined by comparing the peak integrals of the vinyl signals at δ5.5 ppm and δ6.0 ppm to those of the methacrylate backbone at δ0.5–1.1 ppm and δ1.5–2.0 ppm [26,42]. For kinetics experiments, samples were withdrawn from time to time with gas-tight syringes for the characterization of the monomer conversion and Mw after purified as above procedure. 2.4. Typical procedure for the ATRP of MeDMA using CD-PAM as the macroinitiator
2.1. Materials 2-Bromoisobutyryl bromide (BiBB), anhydrous 1-methyl-2pyrrolidione (NMP), triethylamine (TEA), 4-(N,N-dimethylamino)pyridine (DMAP), N,N,N′,N′,N″-pentamethyldiethylenetriamine (PMDETA), CuCl, CuCl2, and 2,2′-bipyridine (bpy) were all purchased from Aldrich. The chemicals were used without further purification except for PMDETA, which was distilled and degassed, and CuCl, which was treated with pure acidic acid and filtered to remove traces of Cu(II) compounds. β-CD was also purchased from Aldrich and then vacuum dried at 80 °C over phosphorus pentoxide overnight immediately before use. Acrylamide (AM), purchased from Sigma, was recrystallized for 3 times prior to use. [2-(methacryloyloxy)ethyl] trimethyl ammonium chloride (MeDMA) was obtained from Aldrich in 75% w/v aqueous solution and used without further purification. 2.2. Synthesis of the macroinitiator based on β-CD In this work, the arm number of the star-shaped polymer was designed to be 10. The starting material, β-CD (5 mmol), was dissolved in 90 mL anhydrous NMP. TEA (55 mmol) and DMAP (15 mmol) were
The ATRP of MeDMA was conducted using purified CD-PAM as the macroinitiator. The procedure of ATRP of MeDMA is similar to that of AM. The CD-PAM (100 mg, 0.01 mmol) was dissolved in 15 mL methanol/water mixture (3:1, v/v), and then MeDMA (620 mg, 3.0 mmol) monomer aqueous solution and bpy ligand (74.20 mg, 0.48 mmol) were added into the reaction vessel. Cu(I)Cl was quickly added into the flask after deoxygenated and sealed in the reaction vessel with a rubber septum. The reaction vessel was finally placed in an oil bath maintained at a temperature of 60 °C. After a 10-h reaction time, the reaction vessel was taken out of the oil bath, exposed to air to terminate the reaction. The product solution was purified with dialysis membrane (MWCO 1000) against water for 24 h to yield a colorless product. 2.5. Measurements 1
H NMR spectra were obtained using a Varian Unity 400 spectrometer operated at 300 MHz. 10Br-β-CD samples were dissolved in CDCl3, and CD-PAM and CD-PAM-b-PMeDMA polymers were dissolved in D2O. 1H NMR spectra of the samples in D2O were also employed to identify the monomer conversion in ATRP.
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Energy dispersive X-ray analysis (EDAX) was measured using a Hitachi SU-70 FE-SEM equipped with an Oxford-INCA (x-act LN2-free SDD Detector) energy dispersive X-ray analyzer (accelerating voltage 15 kV). The number-average Mw and MWD of polymers were measured on a GPCMax VE201 Gel Permeation Chromatography (GPC) (Viscotek) equipped with three columns PAA-202-204-205 and three detectors (viscometer, light scattering, and IR detector). The eluent was 0.05 N sodium nitrate aqueous solution and the flow rate was 1 mL/min at 30 °C. The calibration was based on poly(ethylene oxide) standards using multi-detector method. Apparent charge density of the anionic CD-PAM and cationic CDPAM-b-PMeDMA was determined via colloidal titration using a Particle Charge Detector Mütek PCD 03 (Herrsching) with standard cationic polyelectrolyte (poly-dimethyldially ammonim chloride (DADMAC) solution) (concentration = 1.099 mN) and standard anionic polyelectrolyte (potassium polyvinyl sulfate (PVSK) solution) (concentration = 0.189 mN), respectively. Three repeats were conducted to acquire an average value for each sample. Hydrodynamic radii of polymers were measured by Quasi Elastic Light Scattering (QELS), which was conducted with 90Plus/BI-MAS Multi Angle Particle Sizing Option (Brookhaven Instruments). The measurements were conducted at a scattering angle of 90° and different temperatures with low polymer concentrations in aqueous solution; and three repeats were performed for each sample. AFM measurements were performed using a Nanoscope IIIa from Veeco Instruments Inc., Santa Barbara, CA. The images were scanned in Multimode mode in air using a silicon tapping probe (NP-S20, Veeco Instruments) with a resonance frequency of about 273 kHz. For the AFM experiments of the samples, a drop of sample aqueous solution was deposited on the mica surface. After 5 min contact with the substrate the sample was dried by centrifugal force on a spin-coater.
3. Results and discussion
which led to a higher yield and purer product. During the synthesis, tertiary amine (TEA) was added to the solution as a deacid reagent and reacted with hydrogen bromide, a product of hydroxyl groups and BiBB in the reaction, to form triethylammonium bromide which cannot dissolve in NMP solvent, thus facilitating its removal by filtration after the reaction. The product, 10Br-β-CD, in the solution was readily obtained by precipitation in water. The reaction efficiency was as high as 93% by calculating the weight change, indicating that most of BiBB reacted with β-CD. EDAX was used to confirm the existence of bromine element on the surface of β-CD. In the EDAX spectrum of 10Br-β-CD (Fig. 1), the Br peak is observed around 1.48 keV, implying the formation of the 10Br-β-CD macromolecular initiator. A 10 active-sites initiator was designed to avoid the close proximity of reactive groups [44], and it was assumed that BiBB first reacted with primary hydroxyl protons because of its higher reaction activity than secondary hydroxyl protons on β-CD. Fig. 2(a) shows the 1H NMR spectrum of the 10Br-β-CD sample: the peak at δ1.82 ppm (a, 3H, \CH3) demonstrates the presence of 2-bromoisobutyryl groups; whereas the peaks at δ3.7–5.3 ppm correspond to the sugar residues [41]. In the region of δ3.7–5.4 ppm, the peaks at δ3.7–4.6 ppm are assigned to the inner methylidyne and methylene protons between the oxygen and carbon moieties (b, O\CH\C and O\CH2\C) on the glucose units of β-CD; the peak at δ4.8 ppm is attributed to the inner methylidyne protons between the oxygen moieties (b″, O\CH\O); and the peak located at δ5.3 ppm is associated with the hydroxyl protons adjacent to the methylidyne moieties (c′, CH\OH) of glucose units [19,45]. Furthermore, the relative peak area at δ5.3 ppm almost does not vary in comparison with the corresponding peak in 1H NMR of β-CD in Fig. 2(b), indicating that the hydroxyl protons on CH\OH do not react with BiBB. In addition, the signal of the hydroxyl protons adjacent to the methylene moieties (c, CH2\OH) disappears in Fig. 2(a), indicating that all the primary hydroxyl groups were substituted with 2-bromoisobutyryl groups. By calculating the area ratio of the methyl protons at δ1.82 ppm and methylidyne and methylene protons at the region of δ3.7–4.6 ppm, the degree of substitution of the hydroxyl groups on the outside surface of β-CD is determined to be approximately 10 [19].
3.1. Synthesis of 10Br-β-CD The synthetic approach of the CD-PAM-b-PMeDMA is shown in Scheme 1. 10Br-β-CD was synthesized according to the procedures reported by Kohji et al. [41] and Li et al. [26,43]. However, in the present work, the reaction and purification procedures were further optimized,
3.2. Synthesis of 10-arm star-shaped CD-PAM in DMF aqueous solution It is well known that (meth)acrylamides are not satisfactorily polymerized through ATRP as other notable monomers [39,46,47]. Although
Scheme 1. Synthesis route of star diblock polymers.
S. Li et al. / Materials Science and Engineering C 43 (2014) 350–358
Fig. 1. EDAX spectrum of 10Br-β-CD.
many researchers have investigated the ATRP of AM, however, most of them employed small molecule as an initiator to synthesize linear PAM [36–39]. In the synthesis of star-shaped PAM, the results from us
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were not as good as what they reported by simply duplicating their experimental conditions. In the present study, it was noticed that the macroinitiator, 10Br-β-CD, behaved differently because of the presence of steric-hindered effect among 10 active sites and the insolubility in water caused by the grafted BiBB. Overall, the 10Br-β-CD performed well since the system, i.e., CuCl/CuCl2/PMDETA catalyst and DMF/ water mixture solvent, allows the ATRP to proceed at 130 °С which is often unachievable for the linear polymer system. Fig. 2(c) shows the 1 H NMR spectrum of CD-PAM synthesized in this work. The chemical shifts, δ1.67–1.79 ppm and δ2.23–2.34 ppm, are mainly attributed to the \CH2\ (c) and \CH\ (d) groups of the PAM arms, respectively, indicating the presence of PAM chains. Table 1 summarizes the results of these polymerizations. Since CuCl was employed as a catalyst, the majority of the polymer chains should be ended with Cl groups because of the halide exchange equilibrium and the strong carbon–halogen bonds [48–50]. Halide exchange contributes to the increase of the initiation rate relative to propagation. ATRP of AM has been successfully carried out in DMF [51] and glycerol aqueous solution [36]. In this work, the initiator, 10Br-β-CD, cannot dissolve in glycerol aqueous solution, but dissolves well in DMF. Therefore, initially, DMF was used as a solvent to conduct the ATRP of AM. From the results in Table 1, in the absence of any additives, the polymerization in DMF only reached a low monomer conversion (23.34%) with a high polydispersity index (PDI = 1.88). In ATRP, the presence of an X-CuII/ligand complex is necessary to reduce the concentration of radicals through a deactivation process in order to maintain
Fig. 2. 1H NMR spectra of 10Br-β-CD (a), β-CD (b), CD-PAM (c), and CD-PAM-b-PMeDMA (d).
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Table 1 Results of the ATRP of Acrylamide for 10Br-β-CD based initiating system (The molar ratio of acrylamide and 10Br-β-CD is 200:1, and the reaction was conducted at 130 °С for 24 h with PMDETA as the ligand ([PMDETA] / ([Cu(II)] + [Cu(I)]) = 2/1)). Entry
1 2 3 4 5 6 7
Additives CuIICl2 ([Cu(II)]/[Cu(I)])
LiCl (mol/L)
– 0.2 0.2 0.2 0.2 0.2 0.2
– – – – – 1 0.1
Solvent (V/V)
Conversion (%)
Mn (GPC)
Mn (theory)
PDI Mw/Mn
DMF DMF DMF/H2O DMF/H2O DMF/H2O DMF/H2O DMF/H2O
23.34 55.37 75.34 82.59 83.20 85.30 71.78
3150 6700 10,000 10,100 11,300 11,400 8200
5900 10,400 13,200 14,300 14,400 14,700 12,800
1.88 2.10 1.48 1.27 2.03 2.87 1.34
= = = = =
5/1 4/1 3/1 3/1 3/1
the activation–deactivation equilibrium for the controlled growth of the polymer chains with conversion [52]. To reduce the polydispersities, the addition of Cu(II) to the reaction mixture was employed in the earlier study [53]. From Table 1, it can be seen that, in the presence of CuCl2 as a deactivator, the monomer conversion was increased to 55.37%. However, a large amount of polymers precipitated out of the DMF solvent, leading to a high PDI, which is in accordance with Suresh's result [36]. In order to conduct the polymerization in homogeneous conditions during the designed reaction time, it is necessary to select suitable solvents. Since CD-PAM is water soluble, the introduction of small amount of water in the system may increase the solubility of the resulting products. Therefore, DMF and water mixture was used as the media in the following experiments for further optimizing ATRP conditions. With the addition of a small amount of water in DMF (DMF/H2O = 5/1(v/v)), the conversion was apparently increased to 75% with a narrow PDI (PDI = 1.48). However, there was still a trace amount of precipitate at the end of reaction. Compared with DMF/H2O = 5/1(v/v), when DMF/H2O = 4/1 was used, no precipitate appeared at the end of polymerization; and the relatively high monomer conversion (82.59%) and a low PDI (1.27) were obtained. However, with further increasing the content of water (DMF/H2O = 3/1(v/v)), the PDI became broader although the monomer conversion was slightly increased to 83.20%. In fact, Wang et al. [54] thought that the water leads to the formation of a more active Cu(I) catalyst, thus increasing the rate of ATRP in aqueous media. Also, it was suggested that, as the solvent polarity was increased, a higher concentration of mononuclear copper catalyst was produced; thus, both the radical concentration and the polymerization rate increased. As a result, the MWD broadened [53]. Therefore, in the present case, the PDI was broadened with the increase of the water in DMF, especially in the radical-confined reaction system in which the active chains were in close conjunction with β-CD. Jewrajka et al. [36] demonstrated that, in the presence of sufficient amount of LiCl, which ensured the high efficiency of CuCl2 as a deactivator in an aqueous medium, the linear kinetic plots for monomer disappearance was obtained. In this work, the addition of LiCl at a higher concentration was also attempted. However, when the concentration of LiCl was increased to 1 mol/L, the monomer conversion was only further increased to 85.30%; whereas the PDI became much broader (PDI = 2.87), and the precipitation of a large amount of polymer occurred. It might be because of the fact that the alkali halide concentration was so high that the solubility of polymer in the DMF aqueous solution was decreased. The polymer precipitation was enhanced by increasing ionic strength in the salt solution, thus leading to a broad MWD. In order to address this problem, the concentration of the LiCl was decreased to 0.1 mol/L, and there was no precipitate in the reaction system; consequently, the resulting PDI went down to 1.34. However, the monomer conversion decreased to 71.78%. Fig. 3 shows several aqueous GPC profiles of CD-PAM synthesized under different conditions listed in Table 1. Clearly, the sample synthesized under entry 4 conditions has compromised monomer conversions (82.59%) and PDI (1.27). Linear first-order kinetic plots of Ln[M]0/[M] against polymerization time can be only observed under entry 4 and entry 7 conditions
(Table 1) as shown in Fig. 4. The results indicate that, throughout the reaction, the concentration of propagating species is constant, and therefore the influence from chain transfer or other side reactions is negligible. Furthermore, the kinetic results further show that, in addition to the decrease of monomer conversion, the reaction rate of ATRP also decreases in the conditions of entry 7 in Table 1. The number average molecular weight (Mn) and PDI as a function of monomer conversion are plotted in Fig. 5(a) under the conditions of entry 4 and Fig. 5(b) under the conditions of entry 7 (Table 1), respectively. As expected, the Mn almost linearly increases with the monomer conversion. However, the observed Mws are slightly lower than the theoretical values calculated from the consumed monomer, which might be because of the fact that the GPC is calibrated using linear PEO narrow standards; however, their hydrodynamic volumes are different from the star polymers [55].
3.3. Synthesis of 10-arm star-shaped block copolymer CD-PAM-b-PMeDMA in methanol aqueous solution One of the ATRP advantages is that block copolymers can be generated from a macroinitiator synthesized by ATRP [56]. Using CD-PAM as a macroinitiator, the second cationic block was synthesized via ATRP in this work. It has been reported that the ATRP of methyl chloridequaternized 2-(dimethylamino)ethyl methacrylate (MeDMA) could be performed in an aqueous medium [26,42]. Li et al. [26] synthesized PMeDMA using a small molecule initiator in alcohol aqueous solution at room temperature with a very high monomer conversion. Using 21Br-β-CD, Cu(I)Br, and 2,2″-dipyridyl as an initiator, catalyst, and ligand, respectively, Li et al. [42] synthesized 21 arm star polymers. However, the synthesis was conducted in water solution. As previously described, the macroinitiator is insoluble in water, thus it was only dispersed in the
Fig. 3. Aqueous GPC elution curves of CD-PAM synthesized at 130 °C. (—) in presence CuCl2 ([Cu(II)]/[Cu(I)] = 0.2) and DMF/H2O = 4:1 v/v; (−) in the presence of CuCl2 ([Cu(II)]/[Cu(I)] = 0.2) and 0.1 mol/dm3 LiCl, DMF/H2O = 3:1 v/v; (…) in the presence of CuCl2 ([Cu(II)]/[Cu(I)] = 0.2) and 1 mol/dm3 LiCl, DMF/H2O = 3:1 v/v.
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Fig. 4. Ln [M0]/[M] versus t for ATRP of acrylamide with Cl-based initiating system at 130°С (◆) DMF/H2O = 4/1(v/v), (■) DMF/H2O = 3/1(v/v) and in the presence of 0.1 mol/dm3 LiCl.
aqueous solution in Li's work [42], implying that the polymerization was a heterogeneous reaction. In this work, CD-PAM is a water-soluble polymer; however, in pure water, the ATRP of MeDMA could not be properly controlled [26]. Therefore, in order to achieve a low PDI and ensure that the ligand, bpy, can dissolve in the solvent very well, two mixture solvents, CH3OH/water and DMF/water, were employed respectively. In addition, to ensure the star-shaped block copolymers soluble in the
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mixture solvent, the volume ratio of organic solvent (DMF or methanol) and water was maintained at 1:1. The results are summarized in Table 2. From the results in Table 2, it can be seen that monomer conversions were not high either in methanol aqueous solution or DMF aqueous solution at a low temperature (30 °C) or high temperature (60 °C) as using the higher-molecular-weight macroinitiator. This finding is much different from that reported by Li et al. [57], in which the monomer conversion could be very high (90% or above). This might result from the fact that the small molecular initiator is relatively easy to be initiated compared with the macroinitiator. In this work, the propagating radicals are connected with β-CD and close adjacent to each other, which in turn leads to some side reactions such as coupling reaction instead of polymer chain propagation. Therefore, the monomer conversion could be limited to some extent, although the reaction temperature was increased to 60 °C. It is also clear that methanol aqueous solution is better than DMF aqueous solution. This might be attributed to the higher polarity of DMF aqueous solution, which tends to increase the activation rate and leads to higher radical concentration [58,59], thus potentially enhancing the coupling reactions. Furthermore, DMF is a coordination solvent, which may displace ligands from the complex and may also saturate the coordination sphere around the Cu(I) species [60]. Therefore, in this study, synthesizing block copolymer was conducted in methanol aqueous (CH3OH/H2O = 1/1) at 60 °C. Noteworthily, when the CD-PAM with a lower Mw of 4200 g/mol was used as a macroinitiator, the conversion of MeDMA was increased to 79% in methanol aqueous (CH3OH/H2O = 1/1) at 60 °C. This could be attributed to that, with the decrease of Mw, the coupling reactions among different arms were reduced and more chlorin atoms could be access rather than buried in the PAM chains. In Fig. 2(c), the chemical structure of CD-PAM-b-PMeDMA synthesized in methanol aqueous solution was revealed by 1H NMR spectrum. There is an additional peak at δ = 3.3 ppm compared with spectrum (b), which is assigned to the methyl protons of -N(CH3)3+ [26]. 3.4. Confirmation of the hydrodynamic radius of the resulting CD-PAM-b-PMeDMA in aqueous solution In the test of the hydrodynamic radius of β-CD with a concentration of 1.3% (10 mM), a bimodal distribution was observed by filtrating through 0.2 μm, yielding two mean hydrodynamic radii of 0.8 nm and 114.7 nm, respectively [61]. The larger hydrodynamic radius is ascribed to the self-assembly of β-CD [62–65]. Furthermore, 3 mM sample is the less concentrated aqueous solution where aggregates are formed [66]. Herein, at a pretty low concentration of 3 × 10−6% (3 ppm), the native β-CD aqueous solution, filtered through 0.2 μm by using a syringe at 25 °C, was subjected to the QELS tests. As expected, it shows a single modal distribution at 0.75 nm in radius, which is in accordance with those of González-Gaitano [64]. Also, with the concentration of 3 × 10−6% at 25 °C, the CD-PAM-b-PMeDMA aqueous solution, filtered through 0.2 μm by using a syringe, of entry 3 in Table 2 shows a single modal Table 2 Results of the ATRP of MeDMA from CD-PAM synthesized under the conditions of entry 4 in Table 1.
Fig. 5. Dependence of molecular weight, Mn GPC, and molecular weight distribution, Mw/Mn, on monomer conversion for the solution polymerization of acrylamide at 130 °С (a) DMF/H2O = 4/1(v/v), (b) DMF/H2O = 3/1(v/v) and in the presence of 0.1 mol/dm3 LiCl.
Sample
Solvent (v/v)
Tep. (°C)
Conversion (%)
Mn (GPC)
Mn (theory)
Mw/Mn
1a 2a 3a 4a 5b
CH3OH/H2O = 1/1 DMF/H2O = 1/1 CH3OH/H2O = 1/1 DMF/H2O = 1/1 CH3OH/H2O = 1/1
30 30 60 60 60
35.32 30.92 49.45 34.50 79.60
19,300 17,000 25,600 18,300 32,900
25,900 23,100 34,700 23,500 49,400
1.28 1.27 1.22 1.24 1.26
a The reaction time is 24 h, and target DP is 30. The macroinitiator, CD-PAM was the sample of entry 4 in Table 1, synthesized using PMDETA as the ligand at 130 °C for 24 h. Mn = 10,100 g/mol, Mw/Mn = 1.27 (Table 1, entry 4). b The reaction time is 24 h, and target DP is 30. The macroinitiator, CD-PAM was withdrawn from the entry 4 reaction system in Table 1 at the fourth hour. Mn = 4200 g/mol, Mw/Mn = 1.18.
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smaller size corresponding to the monomeric sample (122.1 nm) and the larger one (452.7 nm) attributed to aggregates after the filtration as shown in Fig. 6.
3.5. Determination of the conformation of the star-shaped diblock copolymers
Fig. 6. DLS results of the aqueous solutions of CD-PAM-b-PMeDMA. 3 × 10−6% (3 ppm) at 25 °C (solid line), 3 × 10−6% (3 ppm) at 60 °C (dash line), 1 × 10−5% (10 ppm) at 25 °C (dot line).
distribution. However, in comparison with that of native β-CD aqueous solution, the hydrodynamic radius of the cationic copolymer increases to 130.0 nm as shown in Fig. 6. Furthermore, with the increase of the temperature to 60 °C, the radius distribution decreases because of the adequately stretching of the star-shaped copolymer in aqueous solution. The results are reasonable in light of the actual dimensions of the CDPAM-b-PMeDMA sample. However, when the concentration of CDPAM-b-PMeDMA aqueous solution increases to 1 × 10−5%, a bimodal distribution is detected even filtered through 0.2 μm by using a syringe, the
In order to further confirm the distribution and the morphology of the CD-PAM-b-PMeDMA sample, we performed AFM experiments on dried CD-PAM-b-PMeDMA molecules deposited on a mica surface from aqueous solutions with different concentrations, 3 × 10−6% (3 ppm) and 1 × 10−5% (10 ppm), respectively. The molecules deposited from the aqueous solution with a concentration of 3 × 10-6% are observed to form near-uniform coniform particles with a wide bottom about 36 nm (Fig. 7a). In the AFM images, the macromolecular chains of the star-shaped copolymer do not show the extended conformation like other reported cationic telechelic polyelectrolytes [67,68]. In fact, the charge density results indicate that the surface of CD-PAM is slightly negative-charged (−2.58 × 10−5 eq/g) as shown in Fig. 8. In contrast, the CD-PAM-b-PMeDMA bears a much larger amount of positive charges. Furthermore, from Fig. 8 it can be seen that the charge density of the cationic diblock copolymers with different Mws listed in Table 2 is increased with the increase of the Mws of the second block. Therefore, the two blocks of each arm may attract each other via electrostatic association, and thus leading to a compact structure. As described previously, there are ten arms on the star-shaped copolymer, which consists of seven arms locating at the narrow side and three arms at the other side of β-CD. We may speculate that the coniform structure arise from the asymmetry of ten arms schematically shown in Fig. 7b. In addition,
Fig. 7. Representative AFM topographic image of CD-PAM-b-PMeDMA deposited from aqueous solution with a concentration of 3 × 10−6% (a), 3D-AFM image (c), and schematic representation of the comformations in absorbed state of the copolymer on the mica surface (b, d) (red color indicates PAM chains). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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4. Conclusions
Fig. 8. Charge density of the cationic diblock copolymers with different Mws listed in Table 2.
A novel water-soluble cationic diblock polymer with star like shape, CD-PAM-PMeDMA, was successfully synthesized by ATRP. This appears to be the first report on an ATRP of acrylamide using modified β-CD as a macroinitiator. By the improved procedures, the synthesis of macroinitiator (10Br-β-CD) reached a very high yield. The polymerization of AM was conducted at 130 °C in a controlled manner using CuCl/ CuCl2/PMDETA catalytic system. The conversion of AM was increased to 82% in a controlled manner after the optimization of reaction conditions. The second cationic block was successfully initiated using CD-PAM to generate a diblock copolymer, CD-PAM-b-PMeDMA. The hydrodynamic radius of the resulting copolymer increases to 130 nm with a single model distribution in the aqueous solution with a lower concentration of 3 × 10−6%. Due to the electrostatic association of the diblocks, the CD-PAM-b-PMeDMA sample shows a near-uniform coniform conformation after deposited on mica due to the presence of the asymmetric 10-arms structure. Acknowledgments
once the copolymer molecules approach the mica surface, the PMeDMA arms are oriented toward the mica substrate due to strong attractions among the positively charged polyelectrolytes and the negatively charged surface (mica). Thus, the major PMeDMA constituent is located on the surface while PAM constituent is located on the top of PMeDMA arms because of the repulsion with the mica surface. However, after the rapid evaporation of water the molecular structure collapses in the Z-direction and forms a flat structure with a height around 6 nm as shown in Fig. 7(c). At the lower concentration of 3 × 10−6%, it can be seen that there are only few molecule superpositions in Fig. 7(a). Therefore, the result confirms the single model distribution of the hydrodynamic radius of CD-PAM-b-PMeDMA as shown in Fig. 6. However, with the increase of the concentration to 1 × 10−5%, aggregation phenomenon is detected by AFM as shown in Fig. 9. The result is in accordance with the bimodal distribution in Fig. 6 at a higher concentration.
Fig. 9. AFM topographic image of CD-PAM-b-PMeDMA deposited from aqueous solution with a concentration of 1 × 10−5%.
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