Synthesis, Characterization, and DNA Adsorption Studies of

574

Biomacromolecules 2008, 9, 574–582

Synthesis, Characterization, and DNA Adsorption Studies of Ampholytic Model Conetworks Based on Cross-Linked Star Copolymers Theoni K. Georgiou† and Costas S. Patrickios* Department of Chemistry, University of Cyprus, P.O. Box 20537, 1678 Nicosia, Cyprus Received October 9, 2007; Revised Manuscript Received November 11, 2007

Five model conetworks based on cross-linked star ampholytic copolymers were synthesized by group transfer polymerization. The ampholytic copolymers were based on two hydrophilic monomers: the positively ionizable 2-(dimethylamino)ethyl methacrylate (DMAEMA) and the negatively ionizable methacrylic acid (MAA). Ethylene glycol dimethacrylate was used as the cross-linker. These five ampholytic model conetworks were isomers based on equimolar DMAEMA-MAA copolymer stars of different architectures: heteroarm (two), star block (two), and statistical. The two networks based on the homopolymer stars were also synthesized. The MAA units were introduced via the polymerization of tetrahydropyranyl methacrylate and the acid hydrolysis of the latter after network formation. All the precursors to the (co)networks were characterized in terms of their molecular weights using gel permeation chromatography (GPC). The mass of the extractables from the (co)networks was measured and characterized in terms of molecular weight and composition using GPC and proton nuclear magnetic resonance (1H NMR) spectroscopy, respectively. The degrees of swelling (DS) of all the ampholytic conetworks were measured as a function of pH and were found to present a minimum at a pH value which was taken as the isoelectric point, pI. The DS and the pI values did not present a dependence on conetwork architecture. Finally, DNA adsorption studies onto the ampholyte conetworks indicated that DNA binding was governed by electrostatics.

Introduction Polyampholytes are highly functional polymers comprising weakly acidic and weakly basic units.1–4 This constitution leads to their rich pH responsiveness, with high solubilities and great chain extensions at extreme pH values and a minimum solubility and chain collapse at an intermediate pH range around the isoelectric point, pI, the pH of zero net change. Proteins represent the most abundant class of polyampholytes. In these biological polymers, the ionizable groups come from the second carboxylic acid and second amine groups of amino acids, as well as from the protein carboxy and amino termini. In addition to naturally occurring polyampholytes, synthetic polyampholytes exist since the early 1950s when they were first prepared from the free-radical copolymerization of acidic and basic vinyl monomers.5 In contrast to the well-defined structure of many proteins (a precisely determined amino acid sequence, known as the primary structure), these primitive synthetic polyampholytes comprised random sequences of weakly acidic and weakly basic monomer repeating units. Synthetic polyampholytes with a diblock copolymer structure, comprising one polyacid segment and one polybase segment connected together at a single point, were prepared only 2 decades later using sequential anionic polymerization.6 With further developments in anionic polymerization chemistry and the advent of new “living”/controlled polymerization methods in the 1980s,7 new synthetic polyampholytes with controlled structure were prepared, including linear diblocks,8 linear ABA9 and ABC10 triblocks, stars,11 and shell-cross-linked micelles.12 Polyampholyte conetworks of controlled structure

(model13 conetworks) attracted much less attention, with only one example reported in the literature to date.14 In this example, ampholytic ABA triblock copolymers of well-defined molecular weight (MW) and composition were end-linked to produce model polyampholyte conetworks. It is noteworthy that the literature abounds with reports on the synthesis and characterization of random polyampholyte conetworks (gels).15 These materials are obtained by the simultaneous terpolymerization of the two monomers (acidic and basic) and the cross-linker and lack a well-defined structure since the chains between the cross-link points have broad MW and composition distributions. In contrast, model polyampholyte conetworks with chains between cross-link points of precisely known MW and composition are highly desirable materials that can allow the derivation of accurate structure–property relationships. The aim of this investigation is to present the preparation and study of model polyampholyte conetworks of a controlled but more complicated structure than those previously reported. In particular, this structure is based on interconnected star polyampholytes. The synthetic procedure was performed using a “living” polymerization technique, group transfer polymerization (GTP),16 and was accomplished in (up to) six stages in one pot. One-half of the arms of the produced star polymers were used for their interconnection to a conetwork, while the other half remained as dangling conetwork chains. These materials were thoroughly characterized in terms of their aqueous swelling as a function of pH and were also evaluated as biomaterials for the storage and delivery of DNA.

Experimental Section * Author to whom correspondence should be addressed: e-mail: [email protected]. † Present address: Surfactant & Colloid Group, Department of Chemistry, The University of Hull, Hull, HU6 7RX, United Kingdom.

Network Synthesis. Materials. 2-(Dimethylamino)ethyl methacrylate (DMAEMA) (98%, hydrophilic, cationic monomer, weak base), ethylene glycol dimethacrylate (EGDMA, 98%, cross-linker), 1-methoxy-

10.1021/bm701123s CCC: $40.75  2008 American Chemical Society Published on Web 12/29/2007

Cross-Linked Star Model Ampholytic Conetworks

Biomacromolecules, Vol. 9, No. 2, 2008 575

Figure 1. Chemical structures and names of the main reagents used for the (co)network synthesis.

1-(trimethylsiloxy)-2-methyl propene (MTS, 95%, GTP initiator), methacrylic acid (MAA, 99%, anionic monomer, weak acid), 3,4dihydro-2H-pyran (DHP, 97%), tetrabutylammonium hydroxide (40% in water), benzoic acid, calcium hydride (CaH2, 90–95%), 2,2-diphenyl1-picrylhydrazyl hydrate (DPPH, 95%, free radical inhibitor), basic alumina, and potassium metal (98%) were all purchased from Aldrich, Germany. Figure 1 shows the chemical structures and names of the monomers, the cross-linker, and the initiator. Sodium metal was purchased from Fluka, Germany. Tetrahydrofuran (THF) was purchased from Labscan, Ireland, and was used both as the polymerization solvent (reagent grade) and as the mobile phase in chromatography (HPLC grade). Methods. The methods used in this investigation were the same as those employed for typical GTP syntheses. The polymerization solvent, THF, was dried by refluxing it over a potassium/sodium alloy for 3 days prior to use. The tetrahydropyranyl methacrylate (THPMA) monomer was in-house synthesized by the catalytic esterification of MAA with 100% excess DHP at 55 °C17a using a modification of the procedure reported by Hertler.17b The monomers and the cross-linker were passed twice through basic alumina columns to remove inhibitors and protic impurities. They were subsequently stirred over calcium hydride in the presence of a free-radical inhibitor, DPPH, and stored at 4-8 °C. All monomers and the cross-linker were freshly distilled under vacuum and kept under a dry nitrogen atmosphere until use. The initiator was distilled once prior to the polymerization. The dried tetrabutylammonium bibenzoate (TBABB) catalyst powder was inhouse synthesized by the method of Dicker et al.16c and was stored in a round-bottom flask under vacuum until use. All glassware was dried overnight at 120 °C and assembled hot under dynamic vacuum prior to use. Polymerizations. The synthetic procedure for network preparation was similar to that reported previously for the preparation of crosslinked star polymer (co)networks based on homopolymers18a–c and amphiphilic,18d,e double-hydrophilic,18f and double-hydrophobic18g copolymers. The reactions were carried out in 250 mL round-bottom flasks at ambient temperature (20 °C) without thermostating the polymerization reactor. The polymerization exotherm was monitored by a digital thermometer which was used to follow the progress of the reaction. The polymerization procedure followed for the synthesis of one ampholytic conetwork based on cross-linked stars composed of arms comprising linear diblock copolymers with 10 DMAEMA units and 10 THPMA units is detailed below and is illustrated in Figure 2. Freshly distilled THF (60 mL) and MTS initiator (0.5 mL, 0.43 g, 2.46 mmol) were syringed to a 250 mL round-bottom flask containing a small amount (∼10 mg) of TBABB. DMAEMA (4.1 mL, 3.9 g, 24.6 mmol) was slowly added under stirring. The polymerization exotherm (23.2–30.5 °C) abated within 5 min, a sample was extracted, and THPMA (4.2 mL, 4.2 g, 24.6 mmol) was added, slowly giving an exotherm (27.1–33.9 °C). After sampling, EGDMA (1.86 mL, 1.95 g, 9.84 mmol) was added which produced an exotherm (28.9–33.6 °C). A sample was withdrawn again before THPMA (4.2 mL, 4.2 g, 24.6 mmol) was added for a second time, giving an exotherm from 28.9 to 34.7 °C. A sample

Figure 2. Schematic representation of the synthetic procedure followed for the preparation of the block copolymer-based network 3: (DMAEMA10-b-THPMA10)-star-(THPMA10-b-DMAEMA10)-network. The dark red and light blue colors indicate THPMA and DMAEMA segments, respectively, while the asterisks denote active polymerization sites.

was withdrawn once more, followed by the addition of DMAEMA (4.1 mL, 3.9 g, 24.6 mmol) with an exotherm from 29.0 to 33.3 °C. In the final stage, EGDMA (1.86 mL, 1.95 g, 9.84 mmol) was added which promoted gelation within seconds. Characterization of the Network Precursors. Gel Permeation Chromatography. The molecular weights (MWs) and the molecular weight distributions (MWDs) of the linear and the star precursors to the cross-linked star (co)polymer model (co)networks were determined by gel permeation chromatography (GPC) using a single high MW range Polymer Laboratories PL-Mixed “D” column. The mobile phase was THF, delivered at a flow rate of 1 mL min-1 using a Polymer Laboratories PL-LC1120 isocratic pump. The refractive index signal was measured using an ERC-7515A refractive index detector also supplied by Polymer Laboratories. The calibration curve was based on eight narrow MW (630, 2600, 4250, 1300, 22650, 50000, 128000 and 260000 g mol-1) linear polyMMA standards, which provided rather good estimations for the MWs of the linear polymer precursors but only rough estimations for the MWs of the star polymer precursors to the (co)networks. GPC was also used to deduce copolymer composition. The complete absence of signals from the monomer or the cross-linker in all the GPC traces implied their full consumption and, therefore, coincidence of the copolymer composition with the monomer feed composition. 1 H NMR Spectroscopy. The compositions of the extractables of the conetworks were determined by proton nuclear magnetic resonance (1H NMR) spectroscopy using a 300 MHz Avance Bruker NMR spectrometer equipped with an Ultrashield magnet. The solvent was CDCl3 containing traces of tetramethylsilane (TMS) which was used as an internal reference. Characterization of the (Co)networks. Determination of the Sol Fraction. The prepared (co)networks were taken out of the polymerization flasks and were washed in 200 mL of THF for 1 week to remove the sol fraction. Next, the THF solution was recovered by filtration.

576 Biomacromolecules, Vol. 9, No. 2, 2008

Georgiou and Patrickios

Figure 3. Synthetic sequences employed for the preparation of the various cross-linked star (co)network architectures of this study.

The extraction procedure was repeated once more after 1 week, and the solvent from the combined extracts was evaporated using a rotary evaporator. The recovered polymer was further dried for 24 h in a vacuum oven at room temperature. The sol fraction was calculated as the ratio of the dried mass of the extracted polymer divided by the theoretical mass of the polymer in the (co)network. The latter was calculated from the polymerization stoichiometry as the sum of the masses of the monomers, the cross-linker, and the initiator. The dried extractables were subsequently characterized in terms of their MW and composition by GPC and 1H NMR spectroscopy, respectively. Hydrolysis of the THPMA Units. Samples from all the THPMAcontaining (co)networks were transferred to 1 L glass jars. To each jar, 200 mL of THF and 34 mL of a 2 M HCl aqueous solution (the volume of the HCl solution was 68 mL in the case of the THPMA homopolymer network) were transferred. The number of moles of HCl was more than twice the number of moles of THPMA plus DMAEMA (DMAEMA does not get hydrolyzed but captures HCl to get ionized) equivalents in each (co)network sample. The system was allowed to hydrolyze for 3 weeks, followed by washing with distilled water for another 2 weeks to remove the THF and the excess of HCl. The water was changed twice a day. Characterization of the Degree of Swelling. The washed (co)networks were cut into small cubes of size 5–10 mm. The mass of the THF swollen cubes was measured gravimetrically before placing all samples in a vacuum oven for drying for 72 h at room temperature. The dry network mass was determined, followed by the transfer of the (co)networks in THF or water. For the samples in water, the pH was adjusted within the range of 2-12 by the addition of the appropriate number of drops of 0.5 M HCl or 0.5 M NaOH standard solutions. The samples were allowed to equilibrate for 2 weeks, and the swollen (co)network masses were measured. The swollen (co)network masses were measured at least five times for each sample, and the average was calculated for each sample. The degrees of swelling (DS) were calculated as the ratio of the average swollen (co)network mass divided by the dry (co)network mass. Calculation of Degree of Ionization, the pK Values, and the pI Values. The degrees of ionization (DI) of the two homopolymer networks were calculated as the number of HCl or NaOH equivalents added divided by the number of DMAEMA or MAA unit equivalents present in the sample. The hydrogen ion titration curves were obtained by plotting the calculated DI against the measured solution pH. The

effective pK values of the DMAEMA and the MAA units in each of the two homopolymer networks were estimated from the hydrogen ion titration curves as the pH (of the supernatant solution) at 50% ionization. The pI of the polyampholyte conetworks was taken as the pH where the conetwork presented the minimum DS. DNA Adsorption Studies. The synthesized ampholytic conetworks were used for adsorption studies of herring’s sperm DNA. First, DNA solutions that span a range of concentrations (1 × 10-7 to 1 × 10-4 g mL-1) were prepared and their absorbance was measured at 260 nm using a Lambda 10 Perkin-Elmer UV/vis spectrometer to construct a calibration curve (Α ) f(C)). Subsequently, choosing one of the more concentrated DNA solutions, and, in particular, the one with a concentration of 4 × 10-5g mL-1 which presented an absorbance value at 260 nm close to 1, the absorbance was measured as a function of pH and a calibration curve with respect to pH (Α ) f(pH)) was constructed. The pH values were adjusted by the addition of the appropriate volume (in number of drops) of HCl and NaOH 0.5 M standard solutions into the DNA solution, taking into account the protonation of the DMAEMA units in the (co)network samples that would subsequently equilibrate with the DNA solution, to achieve acidic and alkaline pH values, respectively. Next, three or four THF-swollen cubic samples of size ∼10 mm from each (co)network were placed in a vacuum oven and were dried at room temperature for 48 h, and they were finally weighed. Six milliliters of the dilute acidic or alkaline solution of DNA was transferred to each vial. Given that DNA precipitates at pH e4, no solutions were prepared in this lower pH range. The samples were allowed to equilibrate for 3 weeks, and the absorbance at 260 nm of the supernatant solutions was measured using the UV/vis spectrometer. The DNA concentration in the supernatant was calculated using the relevant calibration curve. The amount of adsorbed DNA was calculated from the amount initially added and its final concentration in the supernatant. The percentage of adsorption was calculated as the ratio of the amount of the adsorbed DNA divided by the amount of DNA initially loaded. Finally, the pH of the supernatant and the DS of each (co)network were measured.

Results and Discussion Synthesis and Structure of the Ampholytic Conetworks. The synthetic sequences followed for the preparation of the (co)networks are summarized in Figure 3, while schematic



Figure 5. Gel permeation chromatograms of the five precursors to and the extractables from network 3: (DMAEMA10-b-THPMA10)-star(THPMA10-b-DMAEMA10)-network. In the curve labeling, D, T and s are (further) abbreviations for DMAEMA, THPMA, and star, respectively, while the polymer nomenclature follows that introduced in Figure 3.

Figure 4. Schematic representation of the structures of the model (co)networks of this study. The DMAEMA units are depicted light blue, while the THPMA (MAA) units are colored dark red.

representations of the structure of all the (co)networks prepared are shown in Figure 4. The GTP synthesis comprised a successful multi- (4- or 6-) step sequential addition in a onepot preparation. The linear “living” (co)polymers produced upon the addition of methacrylate monomer(s) to a solution containing the monofunctional GTP initiator and the GTP catalyst were converted to “arm-first” star polymers with “living” cores upon the addition of the dimethacrylate cross-linker. These “armfirst” star polymers were further grown from the core outward, upon the addition of more monomer(s) to yield the “in-out” star polymers. Finally, the dimethacrylate cross-linker was added a second time to interconnect these stars to a (co)network. Cross-linked heteroarm stars were prepared when only one type of monomer was added between cross-links and the monomer added second was different from that used first (networks 6 and 7). If the monomer added second were the same as that added first, a homopolymer cross-linked star was produced (networks 1 and 2). The simultaneous addition of the two different monomers before each addition of the EGDMA cross-linker resulted in the preparation of arms of statistical architecture and it ultimately led to the formation of cross-linked statistical copolymer stars (network 5). The preparation of cross-linked block copolymer stars required the sequential addition of the two different monomers before the EGDMA additions (networks 3 and 4). All seven (co)networks have theoretical degrees of polymerization of the primary and secondary arms constant and equal to 20. Two of them are homopolymer networks and five are isomeric (co)networks with equimolar DMAEMA-THPMA (MAA) compositions. Molecular Weights and Compositions. Figure 5 displays the GPC traces of the five precursors to the N3 block copolymer conetwork [(DMAEMA10-b-THPMA10)-star-(THPMA10-bDMAEMA10)]-network and of the sol fraction extracted from this conetwork. The MWDs of the linear DMAEMA homopolymer and the DMAEMA-THPMA diblock copolymer were narrow and unimodal, as expected. The MWD of the main peak corresponding to the “arm-first” star was also narrow, but the distribution was bimodal, containing a small amount of unattached linear chains in addition to the stars. A fraction of these chains had originated from accidental deactivation during synthesis and thus it could not grow upon further addition of

monomer, while a second part had not participated in the reaction with EGDMA due to steric hindrance and was still “living” and, therefore, it could grow upon further addition of monomer. The chromatograms of the “in-out” stars were also bimodal for the same reasons as above, with a narrow MWD of the main peak corresponding to the star polymers. The MW of the main peak increased from the initial homopolymer to the final “in-out” star polymer, as expected, indicating the growth of the structure as a whole. Similar results were obtained with the precursors to all (co)networks synthesized in this study as well as in our previous studies on (co)networks based on cross-linked stars.18 Table 1 lists the structures of the precursors to the crosslinked star polymers prepared in this study and their GPC characterization data. These include the apparent numberaverage MWs, Mn, the polydispersity indices (PDIs, Mw/Mn), and the apparent peak MWs, Mp. The PDIs of all linear (co)polymers were sufficiently low (e1.22), whereas their Mn were higher compared to the theoretical value due to partial initiator deactivation. For the “arm-first” star polymers, the PDIs were higher than those corresponding to the linear polymers but still relatively low (e1.27). Similarly, low PDIs were recorded for the “in-out” star polymers, whose Mn values were always higher than those of the respective “arm-first” stars, as expected. It is noteworthy that after each reagent addition, both the Mn and the Mp grew without exception, indicating that the “livingness” of the polymerization was preserved throughout the syntheses. It is pointed out, however, that the reported Mn and Mp for the star polymers as determined by GPC are only apparent values and lower than the true values, due to the compact nature of the star polymers compared with the linear polyMMA MW calibration standards. Sol Fractions. Table 2 presents the percentage, the apparent Mp values, the apparent Mn values, and the PDIs of the main peak, and the composition of the extractables for each (co)network as measured by gravimetry, GPC, and 1H NMR. In all cases, the sol fraction was relatively low, from 5% to 14%. These sol fractions were similarly low to sol fractions measured previously for other (co)networks based on cross-linked star (co)polymers.18 The Mp values of the main peaks of the extractables were comparable to those of the corresponding linear polymer precursors that were not attached to the star. It is noteworthy that the sol fractions determined in the present study were comparable to the percentage of unattached linear polymer in the “arm-first” stars (see GPC traces in Figure 5) of ∼15%, similar to that determined for other “arm-first” star copolymers prepared using both “living” anionic and “living”



Table 1. Molecular Weight Characteristics of the Cross-Linked Star (Co)network Precursors GPC results net. no.

theoretical structure

theoretical MW

Mn

Mw/Mn

Mp

N1

D20 D20-star D20-star-D20 T20 T20-star T20-star-T20 D10 D10-b-T10 D10-b-T10-star D10-b-T10-star-T10 D10-b-T10-star-T10-b-D10 T10 T10-b-D10 T10-b-D10-star T10-b-D10-star-D10 T10-b-D10-star-D10-b-T10 D10-co-T10 D10-co-T10-star D10-co-T10-star-D10-co-T10 D20 D20-star D20-star-T20 T20 T20-star T20-star-D20

3240 * * 3500 * * 1670 3370 * * * 1800 3370 * * * 3370 * * 3240 * * 3500 * *

4460 41900 68100 4620 52000 88900 2480 4570 47800 61700 75700 2180 4360 42400 55100 70500 3910 38600 63600 3940 33864 59000 4960 62000 95200

1.12 1.25 1.20 1.13 1.25 1.28 1.18 1.13 1.25 1.23 1.22 1.22 1.16 1.27 1.20 1.18 1.15 1.25 1.19 1.15 1.26 1.17 1.14 1.26 1.29

5381 56700 73700 5520 66400 90900 3360 5530 63000 69900 81900 3020 5520 58200 66400 77700 4850 52500 71820 4980 48500 68200 5970 73700 95800

N2 N3

N4

N5 N6 N7

a

a

D and T are (further) abbreviations for DMAEMA and THPMA, respectively.

Table 2. Mass Percentage, Molecular Weights and Compositions of the Sol Fraction Extracted from the Cross-Linked Star (Co)Networks, as measured by gravimetry, GPC, and 1H NMR GPC results a

% mol DMAEMA

network no.

theoretical stucture

extract, w/w %

Mn

Mw/Mn

Mp

Theor. of net.

1 2 3 4 5 6 7

(D20-star-D20)-network (T20-star-T20)-network [(D10-b-T10)-star-(T10-b-D10)]-network [(T10-b-D10)-star-(D10-b-T10)]-network [(D10-co-T10)-star-(D10-co-T10)]-network (D20-star-T20)-network (T20-star-D20)-network

5.4 13.8 9.6 8.9 8.0 7.3 14.0

5290 4410 4370 2900 4070 5250 2580

1.30 1.13 1.19 1.30 1.12 1.32 1.50

5970 5500 5380 4180 4850 5250 4800

100 0 50 50 50 50 50

a

1

H NMR extract 100 0 83 44 53 82 20

D and T are (further) abbreviations for DMAEMA and THPMA, respectively.

cationic polymerizations.19 The highest percentage of extractables was measured for (co)networks where the first addition of EGDMA to form the “arm-first” star (co)polymer was after the addition of the THPMA monomer. This may be attributed to a higher cross-reactivity for the DMAEMA-EGDMA pair compared to the THPMA-EGDMA pair. The low amounts of extractables indicate satisfactory control over the (co)network structure during synthesis. From the 1H NMR data, also shown in the table, it was observed that the compositions of the extractables were, in most cases, different from those of the conetworks (coinciding with those of the comonomer feed due to complete monomer consumption, as confirmed by GPC). In particular, the extractables from conetworks N3 and N6 were richer in DMAEMA compared to the conetwork composition, whereas those from conetworks N4 and N7 were richer in THPMA. Thus, the extractables were richer in the component polymerized first, which would be available for deactivation from the beginning of the polymerization reaction. The composition of the extractables from conetwork N5, based on equimolar statistical star copolymers, was more balanced, reflecting the simultaneous copolymerization of the two monomers in this case. Degrees of Swelling of the (Co)networks. In Figure 6, the experimentally measured DS of all the (co)networks are plotted against the pH of the supernatant solution. The figure shows

that the pH profoundly affected the DS values of all the (co)networks. In particular, for the DMAEMA and the MAA homopolymer networks, as the pH decreased, the DS increased and decreased, respectively. This was due to the weakly basic and weakly acidic nature of the DMAEMA and the MAA units, respectively. The DMAEMA units are uncharged at high pH but become positively charged at low pH,20 while the MAA units are uncharged at low pH but become negatively charged at high pH.21 The electrostatic repulsion created by the network charges and the osmotic pressure due to the counterions both promoted network swelling.22 The DS of all the polyampholyte conetworks presented a characteristic minimum in their DS at intermediate pH values, while they increased again at acidic and basic pH. This behavior is typical of polyampholytes, due to the existence of the isoelectric point, pI, the pH of zero net charge. At and near the pI, the van der Waals and the hydrophobic attractive forces contribute significantly to the polyampholyte collapse. Moreover, in this pH range, all counterions to the charged groups are “dialyzed out” of the conetwork,1 while Coulombic attractions replace Coulombic repulsions. Therefore, the favorable contribution of electrostatic forces to swelling at the pI is not only annihilated but also reversed and becomes unfavorable due to the Coulombic attractions between the equal in number positively and nega-



Figure 6. pH dependence of the aqueous degrees of swelling and the degrees of ionization of the cross-linked star (co)networks. D and M are further abbreviations for DMAEMA and MAA.

tively charged units, thus further contributing to the polyampholyte collapse. Aqueous DS at Low and High pH and at the pI or the pK. Figure 7 shows the aqueous DS at low (∼3) and high (∼11) pH for all the (co)networks, and the DS at the pK and the pI for the homopolymer networks and the polyampholyte conetworks, respectively. The DS values of the cross-linked star copolymers at high (∼11) and low (∼3) pH were always between the values of the DS of the two homopolymer networks at the same pH. In particular, the DS of all the polyampholyte conetworks at low pH were lower than the DS of the DMAEMA homopolymer

network at low pH and higher than that of the MAA homopolymer network at the same pH, since the polyampholytes at that pH had only 50% of their units ionized (the DMAEMA units), while the DMAEMA and MAA homopolymer networks were 100% and 0% ionized, respectively. Similarly, at high pH, the DS of all the polyampholyte conetworks were lower than that of the MAA homopolymer network which was fully ionized and higher than that of the DMAEMA homopolymer network which was uncharged. It is noteworthy that the DS of each polyampholyte conetwork at high and low pH were almost equal, since the conetwork was in both cases 50% ionized. This is in agreement with the


Figure 7. Degrees of swelling of the cross-linked star (co)networks in low pH (∼3) water, in high pH (∼11) water, and at pH ) pK (homopolymer networks) or pH ) pI (polyampholyte conetworks). D, M, and s are (further) abbreviations for DMAEMA, MAA, and star, respectively.

previous report on equimolar linear DMAEMA-MAA polyampholyte-based model conetworks, that presented the same DS at high and low pH.14 At their pI, the DS of the all polyampholyte conetworks were lower than the DS of the two homopolymer networks at their corresponding pK, as expected, since the homopolymer networks were 50% ionized at their pK, while the polyampholytes conetworks had a zero net charge at their pI. Moreover, at their pI, the polyampholyte conetworks had the same DS values as the DS of the two homopolymer networks at their neutral state (high pH for the DMAEMA and low pH for the MAA homopolymer networks), since all the (co)networks at these pH were completely uncharged. It is noteworthy that the DS of the polyampholyte conetworks did not present an architecture dependence at any pH. The DS of the polyampholyte conetworks depended only on the DI of their units. This is in agreement with the previous polyampholyte model conetwork investigation, where the effect of pH on the DS was also thoroughly investigated.14 The DS in this previous study were higher at both high and low pH than those of the present study, due to the higher degrees of polymerization of the elastic chains, which were up to 40 as compared to 20 for these cross-linked star polyampholytes. Effective pK and pI. Table 3 lists the effective pK of the DMAEMA and MAA units in the homopolymer networks and the pI of the polyampholyte conetworks. The pK of the DMAEMA units and the MAA units in the two homopolymer networks were 6.0 and 7.8, respectively, while the pI of the polyampholyte conetworks ranged from 6.8 to 7.2, intermediate between the two pK values. These values are in good agreement with the experimentally determined pI of the previous study with cross-linked linear polyampholyte conetworks of the same DMAEMA-MAA composition,14 as well as with theoretical predictions.23 It is noteworthy that the experimentally determined pI of the previous as well as of the present study were not dependent on polyampholyte conetwork architecture. This is also in agreement with previous studies on the pK of cross-linked


star copolymer conetworks where only a conetwork-composition, but not a conetwork-architecture, dependence of the pK values was observed.18d,e DNA Adsorption Studies. There are many literature reports on the binding of DNA to polyampholytes.24 Most of them concern linear polyampholytes,24a–d but there are also reports on branched systems, including graft polyampholytes24e and polyampholyte gels.24f Figure 8 presents the results for the adsorption of DNA onto the cross-linked star (co)networks of this study. The figure displays the % DNA adsorption vs pH profiles for the seven (co)networks. The DS vs pH profiles of these (co)networks, measured in the presence of adsorbed DNA, are plotted on the second (right) y axis of each graph. These DS values were close to those presented in Figure 6 and measured in the absence of DNA. The data in Figure 8 suggested that the DNA adsorption was driven by the electrostatic attractions between the negatively charged DNA (the negative charge arising from the phosphate groups in DNA) and the positively charged (co)networks (due to the presence of the DMAEMA units) for pH below ∼8. This is supported by the fact that there was no DNA adsorption onto the MAA homopolymer network at pH >6 and onto the other six networks at alkaline pH (pH >10) where the DMAEMA units were uncharged, while adsorption onto the DMAEMAcontaining (co)networks was almost 100% at pH 5.5, where the DMAEMA units were fully charged. For the DMAEMA homopolymer network, DNA adsorption was high even when the DMAEMA units were slightly charged (pH 7). This was due to the very high ratio of the DMAEMA positive charges to the DNA negative charges arising from the very low concentration of the DNA solution added to the network. It is noteworthy that all five polyampholyte conetworks presented qualitatively similar DNA adsorption, independent of their structure. This supports once again the explanation given above that DNA adsorption onto these ampholytic conetworks was electrostatic, as previously observed for the adsorption of DNA onto linear polyampholytes in solution,24c,d for the adsorption of DNA onto cationic amphiphilic cross-linked star conetworks,18e and for the adsorption of negatively charged proteins onto cationic double-hydrophilic conetworks.20g Similar behavior has also been observed for the adsorption of anionic drugs onto polyampholyte cationic conetworks,15n proteins on methacrylic acid/acrylic microgels, and multivalent metal cations on (anionic) sodium acrylate networks.25 The present data suggest that these cross-linked star polyampholyte conetworks can be used as DNA reservoirs where DNA can be stored in a friendly aqueous environment. Subsequently, the stored DNA can be conveniently released by straightforward pH manipulations and be delivered at the appropriate location.

Conclusions The successful GTP synthesis of two homopolymer networks and five polyampholytic conetworks based on crosslinked “in-out” star (co)polymers of various structures (homopolymer, heteroarm, block and statistical) was accomplished using a monofunctional initiator in a four- or sixstep synthetic procedure. It is noteworthy that GTP was “living” even after six monomer/cross-linker additions, allowing the synthesis of (co)networks with such complicated structures. The aqueous DS of all the (co)networks synthesized were measured in aqueous solutions of different pH.



Table 3. Effective pK and pI Values of the Cross-Linked Star (Co)networks

a

pK.

b

network no.

theoretical structure

pK or pI

1 2 3 4 5 6 7

(DMAEMA20-star-DMAEMA20)-network (MAA20-star-MAA20)-network [(DMAEMA10-b-MAA10)-star-(MAA10-b-DMAEMA10)]-network [(MAA10-b-DMAEMA10)-star-(DMAEMA10-b-MAA10)]-network [(DMAEMA10-co-MAA10)-star-(DMAEMA10-co-MAA10)]-network (DMAEMA20-star-MAA20)-network (MAA20-star-DMAEMA20)-network

6.0a 7.9a 7.2b 7.0b 7.0b 6.8b 7.1b

pI.

Figure 8. Percentage of adsorbed DNA onto and aqueous degrees of swelling of the cross-linked star (co)networks as a function of pH. D and M are further abbreviations for DMAEMA and MAA.

The DS of the polyampholytic conetworks presented a minimum around the isoelectric point where the net charge was zero, while the DS of the MAA and DMAEMA homopolymer networks increased at alkaline and acidic pH, due to the ionization of the weak acid and weak base monomer repeat units, respectively. It was found that the DS were not affected by the conetwork architecture. DNA adsorption occurred for the DMAEMA homopolymer and the

polyampholytic conetworks when they were positively ionized under acidic conditions, while no significant adsorption was measured with the MAA homopolymer network. Acknowledgment. The University of Cyprus Research Committee (grant 2000–2003) is thanked for the financial support for this work. The A. G. Leventis Foundation is also thanked



for a generous donation that enabled the purchase of the NMR spectrometer at the University of Cyprus.

References and Notes (1) Kudaibergenov, S. E. AdV. Polym. Sci. 1999, 144, 115–197. (2) Kudaibergenov, S. E. Polyampholytes: Synthesis, Characterization and Application; Plenum: New York, 2002. (3) Lowe, A. B.; McCormick, C. L. Chem. ReV. 2002, 102, 4177–4189. (4) Polyelectrolytes and Polyzwiterions: Synthesis, Properties and Applications; Lowe, A. B., McCormick, C. L., Eds.; ACS Symposium Series 937; American Chemical Society: Washington, DC, 2006. (5) (a) Alfrey, T.; Morawetz, H.; Fitzgerald, E. B.; Fuoss, R. M. J. Am. Chem. Soc. 1950, 72, 1864–1865. (b) Katchalsky, A.; Miller, I. R. J. Polym. Sci. 1954, 14, 57–68. (6) (a) Kamachi, M.; Kurihara, M.; Stille, J. K. Macromolecules 1972, 5, 161–167. (b) Kurihara, M.; Kamachi, M.; Stille, J. K. J. Polym. Sci., Polym. Chem. Ed. 1973, 11, 587–610. (c) Varoqui, R.; Tran, Q.; Pefferkorn, E. Macromolecules 1979, 12, 831–835. (7) Webster, O. W. Science 1991, 251, 887–893. (8) (a) Lowe, A. B.; Billingham, N. C.; Armes, S. P. Chem. Commun. 1997, 1035–1036. (b) Lowe, A. B.; Billingham, N. C.; Armes, S. P. Macromolecules 1998, 31, 5991–5998. (c) Creutz, S.; Teyssié, P.; Jérôme, R. Macromolecules 1997, 30, 6–9. (d) Creutz, S.; van Stam, J.; De Schryver, F. C.; Jérôme, R. Macromolecules 1998, 31, 681– 689. (e) Goloub, T.; de Keizer, A.; Cohen Stuart, M. A. Macromolecules 1999, 32, 8441–8446. (f) Hadjikallis, G.; Hadjiyannakou, S. C.; Vamvakaki, M.; Patrickios, C. S. Polymer 2002, 43, 7269–7273. (g) Liu, S.; Armes, S. P. Angew. Chem., Int. Ed. 2002, 41, 1413–1416. (h) Gotzamanis, G.; Tsitsilianis, C. Macromol. Rapid Commum. 2006, 27, 1757–1763. (9) (a) Sfika, V.; Tsitsilianis, C. Macromolecules 2003, 36, 4983–4988. (b) Bossard, F.; Sfika, V.; Tsitsilianis, C. Macromolecules 2004, 37, 3899–3904. (c) Bossard, F.; Tsitsilianis, C.; Yannopoulos, S. N.; Petekidis, G.; Sfika, V. Macromolecules 2005, 38, 2883–2888. (10) (a) Patrickios, C. S.; Hertler; W, R.; Abbott, N. L.; Hatton, T. A. Macromolecules 1994, 27, 930–937. (b) Patrickios, C. S.; Hertler, W. R.; Hatton, T. A. Biotechnol. Bioeng. 1994, 44, 1031–1039. (c) Patrickios, C. S.; Gadam, S. D.; Cramer, S. M.; Hertler, W. R.; Hatton, T. A. Biotechnol. Prog. 1995, 11, 33–38. (d) Chen, W.-Y.; Alexandridis, P.; Su, C.-K.; Patrickios, C. S.; Hertler, W. R.; Hatton, T. A. Macromolecules 1995, 28, 8604–8611. (e) Patrickios, C. S.; Hertler, W. R.; Hatton, T. A. Fluid Phase Equilib. 1995, 108, 243–254. (f) Patrickios, C. S.; Strittmatter, J. A.; Hertler, W. R.; Hatton, T. A. J. Colloid Interface Sci. 1996, 182, 326–329. (g) Patrickios, C. S.; Lowe, A. B.; Armes, S. P.; Billingham, N. C. J. Polym. Sci., Part A: Polym. Chem. 1998, 36, 617–631. (h) Patrickios, C. S.; Sharma, L. R.; Armes, S. P.; Billingham, N. C. Langmuir 1999, 15, 1613–1620. (i) Giebeler, E.; Stadler, R. Macromol. Chem. Phys. 1997, 198, 3815–3825. (j) Bieringer, R.; Abetz, V.; Müller, A. H. E. Eur. Phys. J. E 2001, 5, 5–12. (k) Liu, F.; Eisenberg, A. J. Am. Chem. Soc. 2003, 125, 15059– 15064. (l) Sfika, V.; Tsitsilianis, C.; Kiriy, A.; Gorodyska, G.; Stamm, M. Macromolecules 2004, 37, 9551–9560. (m) Katsampas, I.; Roiter, Y.; Minko, S.; Tsitsilianis, C. Macromol. Rapid Commum. 2005, 26, 1371–1376. (11) Georgiou, T. K.; Phylactou, L. A.; Patrickios, C. S. Biomacromolecules 2006, 7, 3505–3512. (12) (a) Bütün, V.; Lowe, A. B.; Billingham, N. C.; Armes, S. P. J. Am. Chem. Soc. 1999, 121, 4288–4289. (b) Fujii, S.; Cai, Y.; Weaver, J. V. M.; Armes, S. P. J. Am. Chem. Soc. 2005, 127, 7304–7305. (13) Hild, G. Prog. Polym. Sci. 1998, 23, 1019–1149. (14) Demosthenous, E.; Hadjiyannakou, S. C.; Vamvakaki, M.; Patrickios, C. S. Macromolecules 2002, 35, 2252–2260. (15) (a) Karbarz, M.; Pulka, K.; Misicka, A.; Stojek, Z. Langmuir 2006, 22, 7843–7847. (b) Ogawa, Y.; Ogawa, K.; Kokufuta, E. Langmuir 2004, 20, 2546–2552. (c) Ogawa, K.; Nakayama, A.; Kokufuta, E. Langmuir 2003, 19, 3178–3184. (d) Ogawa, K.; Nakayama, A.; Kokufuta, E. J. Phys. Chem. B 2003, 107, 8223–8227. (e) Nisato, G.; Munch, J. P.; Candau, S. J. Langmuir 1999, 15, 4236–4244. (f) Kudaibergenov, S. E.; Sigitov, V. B. Langmuir 1999, 15, 4230–4235. (g) Guo, B.; Elgsaete, A.; Christensen, B. E.; Stokke, B. T. Polym. Gels Networks 1998, 6, 471–492. (h) Alvarez-Lorenzo, C.; Hiratani, H.; Tanaka, K.; Stancil, K.; Grosberg, A. Yu.; Tanaka, T. Langmuir 2001, 17, 3616–3622. (i) Baker, J. P.; Stephens, D. R.; Blanch, H. W.;

(16)

(17)

(18)

(19) (20)

(21)

(22)

(23) (24)

(25)

Prausnitz, J. M. Macromolecules 1992, 25, 1955–1958. (j) English, A. E.; Tanaka, T.; Edelman, E. R. Polymer 1998, 39, 5893–5897. (k) Annaka, M.; Tanaka, T. Nature 1992, 355, 430–432. (l) Casolaro, M.; Bottari, S.; Ito, Y. Biomacromolecules 2006, 7, 1439–1448. (m) Chen, L.; Tian, Z.; Du, Y. Biomaterials 2004, 25, 3725–3732. (n) Sutani, K.; Kaetsu, I.; Uchida, K.; Matsubara, Y. Radiat. Phys. Chem. 2002, 64, 331–336. (o) Than, L. T. M.; Makhaeva, E. E.; Khokhlov, A. R. Polym. Gels Networks 1997, 5, 357–367. (a) Webster, O. W.; Hertler, W. R.; Sogah, D. Y.; Farnham, W. B.; RajanBabu, T. V. J. Am. Chem. Soc. 1983, 105, 5706–5708. (b) Sogah, D. Y.; Hertler, W. R.; Webster, O. W.; Cohen, G. M. Macromolecules 1987, 20, 1473–1488. (c) Dicker, I. B.; Cohen, G. M.; Farnham, W. B.; Hertler, W. R.; Laganis, E. D.; Sogah, D. Y. Macromolecules 1990, 23, 4034–4041. (d) Webster, O. W. J. Polym. Sci., Part A: Polym. Chem. 2000, 38, 2855–2860. (e) Webster, O. W. AdV. Polym. Sci. 2004, 167, 1–34. (a) Kearns, J. E.; McLean, C. D.; Solomon, D. H. J. Macromol. Sci., Chem. 1974, A8, 673–685. (b) Hertler, W. R. US Patent 5,072,029, 1991. (a) Vamvakaki, M.; Hadjiyannakou, S. C.; Loizidou, E.; Patrickios, C. S.; Armes, S. P.; Billingham, N. C. Chem. Mater. 2001, 13, 4738– 4744. (b) Themistou, E.; Patrickios, C. S. Macromolecules 2004, 37, 6734–6743. (c) Kafouris, D.; Themistou, E.; Patrickios, C. S. Chem. Mater. 2006, 18, 85–93. (d) Vamvakaki, M.; Patrickios, C. S. Chem. Mater. 2002, 14, 1630–1638. (e) Achilleos, D. S.; Georgiou, T. K.; Patrickios, C. S. Biomacromolecules 2006, 7, 3396–3405. (f) Georgiades, S. N.; Vamvakaki, M.; Patrickios, C. S. Macromolecules 2002, 35, 4903–4911. (g) Georgiou, T. K.; Patrickios, C. S. Macromolecules 2006, 39, 1560–1568. (a) Kanaoka, S.; Omura, T.; Sawamoto, M.; Higashimura, T. Macromolecules 1992, 25, 6407–6413. (b) Tsitsilianis, C.; Voulgaris, D. Macromol. Chem. Phys. 1997, 198, 997–1007. (a) Siegel, R. A.; Firestone, B. A. Macromolecules 1988, 21, 3254– 3259. (b) Siegel, R. A. AdV. Polym. Sci. 1993, 109, 233–267. (c) Iván, B.; Feldthusen, J.; Müller, A. H. E. Macromol. Symp. 1996, 102, 81– 90. (d) Iván, B.; Haraszti, M.; Erdodi, G.; Scherble, J.; Thomann, R.; Mülhaupt, R. Macromol. Symp. 2005, 227, 265–274. (e) Triftaridou, A. I.; Hadjiyannakou, S. C.; Vamvakaki, M.; Patrickios, C. S. Macromolecules 2002, 35, 2506–2513. (f) Krasia, T. C.; Patrickios, C. S. Macromolecules 2006, 39, 2467–2473. (g) Loizou, E.; Triftaridou, A. I.; Georgiou, T. K.; Vamvakaki, M.; Patrickios, C. S. Biomacromolecules 2003, 4, 1150–1160. (h) Georgiou, T. K.; Patrickios, C. S.; Groh, P. W.; Iván, B. Macromolecules 2007, 40, 2335– 2343. (a) Kali, G.; Georgiou, T. K.; Iván, B.; Patrickios, C. S.; Loizou, E.; Thomann, Y.; Tiller, J. C. Macromolecules 2007, 40, 2192–2200. (b) Kali, G.; Georgiou, T. K.; Iván, B.; Patrickios, C. S.; Loizou, E.; Thomann, Y.; Tiller, J. C. Langmuir 2007, 23, 10746–10755. (c) Haraszti, M.; Tóth, E.; Iván, B. Chem. Mater. 2006, 18, 4952–4958. (d) Iván, B.; Haraszti, M.; Erdõdi, G.; Scherble, J.; Thomann, R.; Mülhaupt, R. Macromol. Symp. 2005, 227, 265–273. (a) Philippova, O. E.; Hourdet, D.; Audebert, R.; Khokhlov, A. R. Macromolecules 1997, 30, 8278–8285. (b) Vamvakaki, M.; Patrickios, C. S. J. Phys. Chem. B 2001, 105, 4979–4986. (c) Georgiou, T. K.; Vamvakaki, M.; Patrickios, C. S. Polymer 2004, 45, 7341–7355. (a) Patrickios, C. S. J. Colloid Interface Sci. 1995, 175, 256–260. (b) Patrickios, C. S.; Yamasaki, E. N. Anal. Biochem. 1995, 231, 82–91. (a) Wolff, J. A.; Hagstrom, J. E.; Budker, V. G.; Trubetskoy, V. S. US Patent 6,383,811, 2001. (b) Trubetskoy, V. S.; Hagstrom, J. E.; Budker, V. G.; Wolff, J. A.; Rozema, D. B.; Monahan, S. D. US Patent 6,919,091, 2002. (c) Izumrudov, V. A.; Domashenko, N. I.; Zhiryakova, M. V.; Rakhnyanskaya, A. A. Macromol. Rapid Commun. 2005, 26, 1060–1063. (d) Izumrudov, V. A.; Domashenko, N. I.; Zhiryakova, M. V.; Davydova, O. V. J. Phys. Chem. B 2005, 109, 17391–17399. (e) Asayama, S.; Nogawa, M.; Takei, Y.; Akaike, T.; Maruyama, A. Bioconjugate Chem. 1998, 9, 476–481. (f) Ohsugi, A.; Furukawa, H.; Kakugo, A.; Osada, Y.; Gong, J. P. Macromol. Rapid Commun. 2006, 27, 1242–1246. (a) Horkay, F.; Tasaki, I.; Basser, P. J. Biomacromolecules 2000, 1, 84–90. (b) Horkay, F.; Tasaki, I.; Basser, P. J. Biomacromolecules 2001, 2, 195–199.

BM701123S

Synthesis, Characterization, and DNA Adsorption Studies of

Recommend Documents