Preparation and Aqueous Solution Properties of Thermoresponsive

Biomacromolecules 2009, 10, 1875–1887

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Preparation and Aqueous Solution Properties of Thermoresponsive Biocompatible AB Diblock Copolymers Jeppe Madsen* and Steven P. Armes* Dainton Building, Department of Chemistry, University of Sheffield, Brook Hill, Sheffield, Yorkshire, S3 7HF, United Kingdom

Karima Bertal and Sheila MacNeil The Kroto Research Institute, Department of Engineering Materials, University of Sheffield, Broad Lane, Sheffield, Yorkshire, S3 7HQ, United Kingdom

Andrew L. Lewis Biocompatibles UK Ltd., Chapman House, Farnham Business Park, Weydon Lane, Farnham, Surrey, GU9 8QL, United Kingdom Received March 11, 2009; Revised Manuscript Received May 14, 2009

The synthesis of a series of amphiphilic AB diblock copolymers in which the A block comprises poly(2(methacryloyloxy)ethyl phosphorylcholine) [PMPC] and the B block comprises poly(2-hydroxypropyl methacrylate) [PHPMA] by atom transfer radical polymerization (ATRP) is reported. The aqueous solution properties of these new diblock copolymers were examined using dynamic light scattering and variable temperature 1H NMR spectroscopy. Copolymers with shorter thermoresponsive PHPMA blocks formed relatively large aggregates, while copolymers with longer PHPMA blocks formed smaller aggregates. This apparently “anomalous” self-assembly behavior occurs because the PHPMA block becomes more hydrophobic as its degree of polymerization is increased. Therefore, shorter PHPMA blocks lead to the formation of loose highly hydrated aggregates, whereas longer blocks formed more compact dehydrated aggregates. In addition, these new PMPC-PHPMA diblock copolymers are highly biocompatible and can mediate the relatively rapid efficient uptake of a fluorescent dye by human dermal fibroblast cells. Interestingly, dye uptake kinetics appear to depend on the hydrophobic/hydrophilic balance of the copolymer. This not only bodes well for in vitro imaging of live cells for biomedical applications but also highlights the importance of copolymer design to ensure efficient drug delivery.

Introduction The aggregation behavior of amphiphilic AB diblock copolymers in solvents that are selective for one of the blocks has been of considerable interest for several decades.1-5 A wide range of aggregate morphologies have been identified, including spherical micelles, cylindrical micelles, and vesicles.6-9 If the selective solvent is water, such self-assembled nanostructures have potential biomedical and pharmaceutical applications for controlled drug release.3,10,11 Probably the most extensively investigated system comprises copolymers of water-soluble poly(ethylene oxide) [PEO], and thermoresponsive poly(propylene oxide) [PPO].1 Many other examples of PEO-based diblock copolymers, where the second block comprises either a permanently hydrophobic or a stimulus-responsive block, have been reported over the past decade or so.1,3,4 One alternative to PEO is poly(2-(methacryloyloxy)ethyl phosphorylcholine) [PMPC], which can be readily prepared via atom transfer radical polymerization (ATRP).12,13 The MPC repeat units are biomimetic, thus MPC-based copolymers confer clinically proven biocompatibility on a range of surfaces, including coronary stents, ear grommets, soft contact lenses, and artificial hip joints.14-16 We have recently reported examples of pHresponsive PMPC-based diblock copolymers, where the second * To whom correspondence should be addressed. E-mail: p.madsen@ sheffield.ac.uk (J.M.); [email protected] (S.P.A.).

block comprises poly(2-(diisopropylamino)ethyl methacrylate) [PDPA].17,18 PDPA is highly hydrophobic in its neutral form at physiological pH, thus these PMPC-PDPA diblock copolymers form micellar or vesicular aggregates, depending on the relative block lengths. Below pH 6.3, the PDPA blocks become protonated, causing molecular dissolution of the copolymer chains. These vesicular aggregates can be used to deliver DNA efficiently to cell nuclei, with high transfection efficiencies being achieved.10,11 In addition, we have recently reported a series of ABA triblock gelators, where the B blocks comprise PMPC and the A blocks comprise various stimulus-responsive blocks such as PDPA,19 poly(N-isopropylacrylamide) [PNIPAM],20,21 or poly(2-hydroxypropyl methacrylate) [PHPMA].22,23 The PDPAbased triblocks proved to be efficient pH-responsive gelators.19 On the other hand, both the PNIPAM- and PHPMA-based triblocks were found to be thermoresponsive gelators.20-23 Copolymer gelators based on PNIPAM exhibited a critical gelation temperature close to the lower critical solution temperature (LCST) reported for PNIPAM homopolymer,20 whereas the gelation properties of the PHPMA-PMPC-PHPMA triblock copolymers were highly dependent on the copolymer composition and concentration.22,23 In addition, these latter copolymer gels did not exhibit any cytotoxicity, making them potential candidates for biomedical application such as wound dressings. The thermoresponsive behavior of the PHPMA-PMPCPHPMA triblock copolymers was initially unexpected because

10.1021/bm9002915 CCC: $40.75  2009 American Chemical Society Published on Web 06/05/2009

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PHPMA homopolymer is known to be water-insoluble.24,25 In the present study we synthesized a series of analogous PMPCPHPMA diblock copolymers. In contrast to the earlier triblock copolymers, these diblocks do not form interconnected gel networks. This fundamental difference simplifies their aqueous solution behavior, which was studied by means of variable temperature dynamic light scattering and variable temperature 1 H NMR spectroscopy. In particular, our aim was to examine the effect of varying the mean degree of polymerization of the PHPMA block at a fixed PMPC block length. Finally, we assessed the efficiency of intracellular uptake of an amphiphilic fluorescent dye mediated by selected PMPC-PHPMA diblock copolymers. Such studies should inform the future design of PMPC-based copolymers for biomedical applications.

Experimental Section Materials. 2-(Methacryloyloxy)ethyl phosphorylcholine monomer (MPC, 99.9% purity) was donated by Biocompatibles Ltd., U.K. 2-Hydroxypropyl methacrylate (HPMA) was donated by Cognis Performance Chemicals (Hythe, U.K.). Basic alumina (Brockmann I, standard grade, ∼150 mesh, 58 Å), anhydrous methanol (MeOH 99.8%), copper(I) bromide (Cu(I)Br, 99.999%), 4-(dimethylamino)pyridine (DMAP, 99%), rhodamine B octadecyl ester perchlorate, 4-(2hydroxyethyl)morpholine (99%), 2,2′-bipyridine (bpy, 99%), and Sepharose 4B (40-165 µm beads diameter) were all purchased from Sigma-Aldrich U.K. and used as received. The silica gel 60 (0.063 0.200 µm) used to remove the spent ATRP catalyst was purchased from E. Merck (Darmstadt, Germany) and was also used as received. 2-Phenoxyethanol (99%) was from Acros Organics and used as received. Magnesium sulfate, sodium hydrogen carbonate, sodium chloride, sodium sulfate, and triethylamine were laboratory reagent grade from Fisher Scientific (Loughborough, U.K.) and used as received. Dichloromethane, chloroform, methanol, and tetrahydrofuran were all HPLC-grade solvents obtained from Fisher Scientific (Loughborough, U.K.) and used as received. Phosphate-buffered saline (PBS) was prepared from tablets obtained from Oxoid (Basingstoke, U.K.). Regenerated cellulose dialysis membrane (1000 MWCO) was purchased from Spectra/Por. Disposable UV-grade cuvettes were obtained from Fisher Scientific (Loughborough, U.K.). Dubelco’s Modified Medium (DMEM), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), and EDTA were all purchased from Sigma-Aldrich (Poole, Dorset, U.K.). Foetal calf serum (FCS) was purchased from Labtech (East Sussex, U.K.). Glutamine, amphotericin B, penicillin, and streptomycin were purchased from Gibco (Paisley, U.K.). Trypsin was purchased from Difco Laboratories (Detroit, MI). Collagenase A was purchased from Boehringer-Mannheim (East Sussex, U.K.). Synthesis of the 2-Phenoxyethyl 2-Bromoisobutyrate Initiator, PhOBr. 2-Phenoxyethanol (5.013 g, 0.0363 mol) was dissolved in dichloromethane (20 mL). DMAP (0.3299 g, 0.0027 mol) and triethylamine (3.63 g, 5.0 mL, 0.0359 mol) were added, and the resulting solution was cooled on ice and kept under a nitrogen atmosphere. 2-Bromoisobutyryl bromide (11.5 g, 6.2 mL, 0.050 mol) was dissolved in dichloromethane (20 mL) and added dropwise over 40 min to this solution, which was then stirred overnight at room temperature (∼20 °C). The reaction mixture was filtered and the precipitate was washed with additional dichloromethane (50 mL). The combined organic fractions were washed with water (2 × 20 mL), saturated NaHCO3 (3 × 30 mL), water (3 × 30 mL), and saturated NaCl (50 mL). The organic phase was dried over MgSO4, filtered and passed through basic alumina using dichloromethane as eluent and evaporated at 50 °C followed by thorough drying under reduced pressure. Yield: 4.84 g (46%). Elemental microanalyses gave C ) 50.24% (theory 50.19%), H ) 5.52% (theory 5.27%), and Br ) 27.46% (theory 27.83%), which suggested that the initiator purity exceeded 98% (based on Br). 1H NMR (400 MHz, CDCl3) δ 7.19 (m, 2H, Ar), 6.83 (m, 3H, Ar), 4.41 (t, 2H, J ) 4.9 Hz, -CH2-O-CdO), 4.09 (t, 2H, J ) 4.9 Hz, -CH2-O-Ar), 1.83 (s, 6H,

Madsen et al. (CH3)2C) ppm. 13C NMR δ (400 MHz, CDCl3) δ 171.6 (CH2-O-C)O), 158.5 (Ar), 129.6 (Ar), 121.3 (Ar), 114.8 (Ar), 65.6 (CH2-O-CdO), 64.3 (CH2-O-Ar), 55.7 (Br-C-), 30.8 (Br-C-(CH3)2) ppm. MS (EI+): m/z 286 (M+), 288 (M+). Copolymer Synthesis and Purification. One-pot ATRP syntheses of the PMPC-PHPMA diblock copolymers were conducted using sequential monomer addition without purification of the intermediate PMPC macroinitiator, as reported earlier.23 A typical synthesis was conducted as follows: to MPC (5.0027 g, 16.94 mmol, 25 equiv) under nitrogen was added a solution of PhOBr (0.1945 g, 0.6773 mmol, 1 equiv) in anhydrous methanol (3.0 mL) via cannula. The flask was washed with anhydrous methanol (3.0 mL), which was added to the MPC solution. After purging this solution with nitrogen for 25 min, a mixture of Cu(I)Br (97.1 mg, 0.677 mmol, 1 equiv) and bpy (212.0 mg, 1.357 mmol, 2 equiv) was added. After 30 min, an aliquot was analyzed by 1H NMR and GPC to determine the monomer conversion and molecular weight of the PMPC block. HPMA monomer (8.7880 g, 60.96 mmol, 90 equiv), which had been purged with nitrogen for 3.5 h prior to its addition, was added via cannula immediately after removing this aliquot. After 44 h, 1H NMR confirmed the disappearance of the vinyl signals, and the reaction solution was diluted with methanol and exposed to aerial oxygen to quench the polymerization. The resulting green copolymer solution was passed through a silica column to remove the spent copper catalyst. The residual solution was dialyzed first against methanol for three days, and then against a 3:1 chloroform/ methanol mixture for three days, with daily changes of solvent. Solvent was removed under reduced pressure, 50 mL water was added, and this aqueous solution/dispersion was also evaporated under reduced pressure at 50 °C. Water (50 mL) was again added and removed under reduced pressure at 50 °C. Finally, water (50 mL) was added for a third time and the aqueous solution/dispersion was freeze-dried overnight. The dry copolymer was placed in a vacuum oven at 80 °C for 48 h and then subjected to further heating a 90 °C for 3 h. Overall yield: 10.0 g (73%). This somewhat time-consuming purification protocol was previously found to be necessary to removes traces of methanol from PHPMA-PMPC-PHPMA triblock copolymers in order to ensure excellent biocompatibility with various cell types.23 1 H NMR Spectroscopy. 1H NMR spectra were recorded in CD3OD to determine block compositions and mean degrees of polymerization. Copolymer spectra were also recorded in D2O using either a 400 MHz Bruker AV1-400 or a 500 MHz Bruker DRX-500 spectrometer. For the variable temperature studies in D2O, the integrated peak intensity due to the pendent methyl groups in the PHPMA chains at 1.3 ppm was compared to that due to the pendent azamethylene groups of the PMPC chains at 3.7 ppm. This numerical value was normalized with respect to the actual diblock copolymer composition, as determined by 1H NMR in CD3OD, which is a good solvent for both the PHPMA and the PMPC blocks. Thus, the apparent relative PHPMA content of each diblock copolymer in D2O could be estimated at any given temperature. Molecular Weight Determination. Chromatograms were assessed using a Hewlett-Packard HP1090 Liquid Chromatograph pump unit and two Polymer Laboratories PL Gel 5 µm Mixed-C (7.5 × 300 mm) columns in series with a guard column at 40 °C connected to a Gilson Model 131 refractive index detector. The eluent was a 3:1 v/v % chloroform/methanol mixture containing 2 mM LiBr at a flow rate of 1.0 mL min-1. A series of near-monodisperse poly(methyl methacrylate) [PMMA] samples were used as calibration standards. Toluene (2 µL) was added to all samples as a flow rate marker. Data analyses were conducted using CirrusTM GPC Software supplied by Polymer Laboratories. Dynamic Light Scattering. Copolymer solutions/dispersions for light scattering studies were prepared as either 1.0 or 5.0 w/v % stock solutions/dispersions in PBS at pH 7.2. The initial mixtures were then equilibrated for 24 h at 4 °C to ensure complete homogeneity. These stock solutions/dispersions were diluted to the desired concentration and filtered through a 0.43 µm Nylon filter immediately before the

Thermoresponsive Biocompatible AB Diblock Copolymers measurements. Dynamic light scattering experiments were performed using a Zetasizer Nano-ZS instrument (Malvern Instruments, U.K.) at a scattering angle of 173°. Dispersion Technology Software version 4.20 supplied by the manufacturer was used for cumulants analysis according to ISO 13321:1996. Polymer Film Formation and Entrapment of Rhodamine. Diblock copolymer (20 mg) was codissolved with rhodamine B octadecyl ester (25 µg) in methanol (5 mL). A thin copolymer/dye film was subsequently formed on the walls of the glass vials by solvent evaporation using a vacuum oven at 25 °C. This copolymer/dye film was resuspended in water (5 mL), which was subsequently evaporated using the vacuum oven at 50 °C to ensure complete removal of methanol. The film was rehydrated in 10 mL PBS and then passed through a Sepharose 4B column to obtain a uniform aggregate size and remove any non-entrapped rhodamine. The collected solution/ dispersion was then diluted 10-fold in fibroblast medium to give a solution/dispersion with 0.2 mg/mL of copolymer and 0.25 µg/mL rhodamine dye. This solution/dispersion was exposed to primary human fibroblast cells. Fibroblasts cells were also exposed to the rhodamine B octadecyl ester solution in the absence of any copolymer. Dye uptake by the fibroblast cells was not affected by passing the rhodamine dye solution through the Sepharose 4B column. Cell Culture Studies. Skin was obtained from patients undergoing breast reductions and abdominoplasty elective surgical procedures. Patients gave informed consent for skin not required for their treatment to be used for experimental purposes under a protocol approved by the Ethical Committee of the Northern General Hospital Trust (NHS), Sheffield, U.K. Further details concerning the isolation and culture of fibroblasts can be found in the literature.23,26 Effect of Copolymers on the Viability of Cell Monolayers. Fibroblasts were cultured for 24 h in a humidified atmosphere of 5% CO2 at 37 °C at a density of 40000 cells per well in 24-well plates. The cells were then exposed to the copolymer/dye mixture (0.2 mg/ mL copolymer plus 0.25 µg/mL rhodamine B octadecyl ester dye) in fibroblast media for 24 h before assessing cell viability. Assessment of Cell Viability Using the MTT-Eluted Stain Assay. Viable cell density on 2D cell monolayers was assessed using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay. Further details on the experimental method and mode of action of this MTT assay can be found in the literature.21,23 Kinetics of Intracellular Delivery. Primary human dermal fibroblasts were cultured in 24-well plates at a density of 30000 cells/well for 48 h. The cells were then exposed to the copolymer/rhodamine dye mixture (0.2 mg/mL copolymer plus 0.25 µg/mL rhodamine dye) for various time frames before being thoroughly washed with PBS to remove any free rhodamine dye. Fluorescence-activated cell sorting (FACS) analysis was then performed to determine the rate of dye uptake using a commercial FACS instrument (Guava Express, CytoSoft 2.1.4, Guava Technologies Inc.). Confocal Microscopy. Fibroblasts were cultured for 24 h in a humidified atmosphere of 5% CO2 at 37 °C at a density of 40000 cells per well in 24-well plates. The cells were then exposed to the copolymer/rhodamine dye mixture (0.2 mg/mL copolymer plus 0.25 µg/mL rhodamine dye) in fibroblast media for 24 h before imaging. Micrographs of living cells were taken at λex ) 543 nm/λem ) 602 nm for detection of the rhodamine dye. This analysis was performed using a ZEISS LSM 510 M instrument.

Results and Discussion Initiators. The PhOBr initiator was prepared according to a previously reported protocol using the commercially available 2-phenoxyethanol instead of bis(2-hydroxyethyl)disulfide.23 1H and 13C NMR spectroscopy, mass spectroscopy, and elemental microanalyses were consistent with the target compound being isolated in high purity (>98%). This aromatic initiator was selected to aid determination of mean degrees of polymerization


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Scheme 1. Synthesis of PMPCm-PHPMAn Diblock Copolymers via ATRP Using Sequential Monomer Additiona

a

MPC monomer polymerized first.

from 1H NMR spectra. In addition, its initiator efficiency was close to 100% and the aromatic ester group appeared to be hydrolytically stable during workup. Copolymer Synthesis. The PMPC-PHPMA diblock copolymers were synthesized by ATRP in a one-pot protocol according to Scheme 1 using sequential monomer addition following a previously published protocol.23 In our previous study, PMPCrich triblock copolymers were easily purified by precipitation into excess THF.23 However, the PHPMA blocks are highly soluble in THF (and most other common organic solvents), thus, purification of these PHPMA-rich diblock copolymers required nonaqueous dialysis to remove residual catalyst and unreacted monomer. Characterization data for the purified PMPC-PHPMA diblock copolymers are summarized in Table 1. The block compositions determined by 1H NMR utilized the aromatic endgroup signals originating from the PhOBr initiator. This approach yielded copolymer compositions that corresponded well with the target compositions, indicating high initiator efficiencies. All GPC traces proved to be unimodal and polydispersities for these diblock copolymers were generally below 1.30, indicating well-controlled polymerizations (Figure 1). PMPC25PHPMA120 has a small high molecular weight shoulder. This is possibly due to a very low degree of branching, because HPMA monomer contains a small amount of dimethacrylate impurity due to its slow transesterification during storage.25 An alternative plausible reason for this high molecular weight shoulder may be radical recombination of the active chain ends. This alternative explanation is perhaps less likely, because a similar shoulder was not observed for copolymers with shorter PHPMA blocks, although the overall comonomer conversions were equally high (>99%) in all cases, as judged by 1H NMR (data not shown). In addition, this copolymer has a low molecular weight shoulder, which is most likely due to a small amount of unreacted PMPC25 macroinitiator. Nevertheless, the polydispersity of this copolymer is 1.29 (Table 1), indicating that the polymerization is reasonably well controlled. Temperature-Dependent Dynamic Light Scattering Studies. The temperature dependence of the count rate and hydrodynamic diameter observed for 1.0 w/v % aqueous solutions/ dispersions of the PMPC-PHPMA diblock copolymers are shown in Figure 2. The observed behavior is highly dependent on the copolymer composition. For example, neither the scattering intensity nor the hydrodynamic radius of PMPC23PHPMA24 is significantly affected by the temperature. Its hydrodynamic radius is approximately 4 nm, which is consistent with a molecularly dissolved copolymer. In contrast, the scattered light intensity obtained for PMPC25-PHPMA39 in-

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Table 1. Summary of Block Compositions and Molecular Weight Data Obtained from 1H NMR and GPC Studies of the Diblock Copolymersa entry

target composition

1 H NMR composition

Mn 1H NMR

Mn GPC

Mw/Mn

wt % HPMA

1 2 3 4 5 6 7 8

PMPC25-PHPMA25 PMPC25-PHPMA40 PMPC25-PHPMA60 PMPC25-PHPMA90 PMPC25-PHPMA120 PMPC50-PHPMA30 PMPC50-PHPMA50 PMPC50-PHPMA70

PMPC23-PHPMA24 PMPC25-PHPMA39 PMPC25-PHPMA58 PMPC25-PHPMA90 PMPC25-PHPMA120 PMPC49-PHPMA26 PMPC49-PHPMA49 PMPC49-PHPMA67

10500 13000 16000 20600 25000 18400 21500 24400

18300 22400 23000 29700 34600 25700 25900 28300

1.18 1.21 1.24 1.28 1.29 1.33 1.25 1.27

34 43 53 64 70 21 33 40

a 1 H NMR spectra were recorded at 400 MHz. GPC data were obtained using a 3:1 v/v chloroform/methanol eluent and a series of PMMA calibration standards.

Figure 1. Gel permeation chromatograms of the PMPC-PHPMA diblock copolymers obtained using a 3:1 chloroform/methanol eluent and a series of near-monodisperse poly(methyl methacrylate) calibration standards.

creases by almost two orders of magnitude between 4 and 15 °C. The corresponding hydrodynamic radii increase from 70 to 130 nm between 4 and 7 °C, followed by a reduction to 110 nm between 10 and 15 °C. These relatively large aggregates suggest that at least some fraction of this diblock copolymer may not be molecularly dissolved, even at low temperature. Visual inspection of this aqueous copolymer dispersion revealed that it had significant turbidity at all temperatures. In addition, cumulants analyses indicated that some degree of aggregation occurred even at the lowest temperature examined (see Figure S1). The scattered light intensity for PMPC25-PHPMA58 increased by approximately a factor of two from 4 to 12 °C, with no further change at higher temperatures. However, the corresponding hydrodynamic radii of 60 nm are almost constant over the entire temperature range. The scattered light intensities obtained for PMPC25-PHPMA90 and PMPC25-PHPMA120 diblock copolymers both increase

between 4 and 10 °C, with no further changes occurring up to 50 °C. The hydrodynamic radii are almost constant for these two copolymer dispersions over the entire temperature range, with the PMPC25-PHPMA90 forming slightly smaller aggregates. At first sight, it may seem surprising that the hydrodynamic radii are not affected by the temperature for all these copolymer dispersions, since the greater scattering intensity suggests either a larger aggregation number or a higher concentration of copolymer aggregates. However, similar behavior has been reported for thermoresponsive diblock copolymers based on poly(ethylene oxide) (PEO) and poly(propylene oxide) (PPO).27 In this case, the reduced solvation of the PEO block on raising the solution/dispersion temperature is compensated by a higher aggregation number. A similar mechanism may well operate for the current copolymer system. Raising the temperature causes progressive dehydration of the PHPMA chains, which leads to gradual contraction of the aggregates. However, for copolymers where aggregates and individual copolymer chains (unimers) coexist, the greater hydrophobic character at higher temperature may lead to additional aggregation of unimers. This would lead to a larger hydrodynamic diameter, which offsets the effect of dehydrating the PHPMA chains. The net effect is that there is very little change in the hydrodynamic diameter. Although the PMPC23-PHPMA24 diblock copolymer did not undergo detectable aggregation over the entire temperature range studied (4-50 °C), the four copolymers with longer PHPMA blocks formed aggregates at all temperatures. Somewhat surprisingly, the hydrodynamic radii of these aggregates at 22 °C follow the order: PMPC25-PHPMA39 (106 nm) > PMPC25PHPMA58 (58 nm) > PMPC25-PHPMA120 (35 nm) > PMPC25PHPMA90 (27 nm). Thus, the copolymer with the shortest hydrophobic PHPMA block forms the largest aggregates. Moreover, extending this hydrophobic block leads to a reduction in the hydrodynamic radius, which is in contrast to most of the literature data reported for amphiphilic block copolymers. In the vast majority of cases, the hydrodynamic size increases as the mean degree of polymerization of the hydrophobic block is increased, which is usually interpreted in terms of a higher aggregation number.27,28 The behavior of the series of three diblock copolymers with a fixed PMPC DP of 49 and a variable PHPMA DP (see Figure 2b,d) is slightly different from the series comprising a shorter fixed PMPC block (DP ∼ 25). At 4 °C, 1.0 w/v % solutions of PMPC49-PHPMA26 and PMPC49-PHPMA49 have hydrodynamic radii below 10 nm, indicating molecular dissolution. Increasing the temperature leads to thermoresponsive behavior, with large increases in both scattering intensity and hydrodynamic radius. For PMPC49-PHPMA49, enhanced scattering begins at 12 °C. The hydrodynamic radius increases up to 100 nm at 25 °C, followed by a reduction to around 80 nm at 50 °C. A 1.0 w/v

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Figure 2. (a,b) Scattering intensity vs temperature plots for 1.0 w/v % PMPC-PHPMA diblock copolymers in PBS (pH 7.2). (c,d) Hydrodynamic radius vs temperature plots for the same aqueous diblock copolymer solutions/dispersions.

% solution/dispersion of PMPC49-PHPMA26 behaves similarly, although additional scattering ensues at 20 °C and a hydrodynamic radius of more than 200 nm is attained at 34 °C. PMPC49PHPMA67 does not exhibit any significant thermoresponsive behavior: its hydrodynamic radius is reduced from ∼90 nm at 4 °C to ∼70 nm at 50 °C. It is also noteworthy that the high temperature behavior of these three copolymers follows the same anomalous behavior observed for the diblock copolymers containing shorter PMPC blocks, that is, the copolymer with the shortest PHPMA block forms the largest aggregates. One possible explanation for this anomalous behavior is that the degree of hydration of the PHPMA block is strongly dependent on its degree of polymerization. Cloud point data obtained for poly(propylene oxide),29 poly(2-(dimethylamino)ethyl methacrylate),30 poly(2-(N-morpholino)ethyl methacrylate),31 and poly(2-hydroxyethyl methacrylate)32 also shows this trend and is in accordance with both classical Flory-Huggins theory33 and also the observation that HPMA monomer is watermiscible up to 13%. In contrast, cloud point data obtained for PNIPAM34,35 indicates somewhat weaker dependence on the mean degree of polymerization, while the LCST values of statistical copolymers of 2-(2-methoxyethoxy)ethyl methacrylate and oligo(ethylene glycol) methacrylate recently reported by Lutz et al. exhibit little or no dependence on copolymer molecular weight.35 It is noteworthy that, if shorter PHPMA blocks aggregate but have some affinity for water, the formation of relatively large, hydrated colloidal aggregates is likely. There are a few literature examples of other diblock copolymers that form similarly large aggregates.24,36,37 Diblock copolymers comprising poly(methyl methacrylate) and poly(sulfonated glycidyl methacrylate), [PMMA-b-PSGMA] reported by Gohy et al.36 form large colloidal aggregates in water, apparently due to slow dissolution kinetics caused by the glassy PMMA cores. There are several literature reports describing systems in which the hydrophobic cores are partially hydrated.24,37 For example, Save and co-workers24 reported that diblock copolymers comprising PPO and poly(glycerol monomethacrylate) [PGMA] formed spherical aggregates of 150-200 nm diameter in aqueous medium, as judged by DLS. Similarly, Ikerni et al.37

studied an ABA triblock copolymer where A ) poly(2hydroxyethyl methacrylate) (PHEMA, DP ∼ 13) and B ) PEO (DP ∼ 165) by fluorescence spectroscopy, static light scattering, and small-angle X-ray spectroscopy. These copolymer aggregates had hydrophobic PHEMA cores and PEO coronas. However, the hydrodynamic radius was almost 100 nm and there was apparently a distinct boundary between the hydrophobic and hydrophilic domains. It is perhaps noteworthy that, based on the PHEMA DP alone, these copolymers should be fully water-soluble.32 It is likely that hydrogen bonding between the PHEMA and the PEO chains plays a significant role in this particular case. Concentration-Dependent Dynamic Light Scattering. Our Malvern DLS instrument detects scattered light at 173°, which allows meaningful measurements to be made on significantly more concentrated dispersions than those used in conventional DLS experiments. Figure 3 shows the hydrodynamic radius as a function of copolymer concentration at both 4 and 37 °C. The hydrodynamic radius of PMPC23-PHPMA24 at 4 °C is less than 5 nm, regardless of the copolymer concentration, suggesting that this copolymer is molecularly dissolved. However, raising the temperature to 37 °C leads to the formation of large aggregates for copolymer concentrations exceeding 1.0 w/v % (see Figure 3b). The temperature dependence of the hydrodynamic radius for three PMPC23-PHPMA24 concentrations is shown in Figure 3e. As already shown in Figure 2, this copolymer remains molecularly dissolved at a concentration of 1.0 w/v % at all temperatures. Large colloidal aggregates are formed above 30 °C at a concentration of 2.0 w/v %. At higher concentrations aggregation occurs at around 15 °C. However, the size of these aggregates is 500-900 nm, which is clearly far too large to be simple “core-shell” micelles. Visual inspection confirmed gradual precipitation of the 5.0 w/v % copolymer dispersion on standing at 22 °C. This precipitate redissolved on cooling to 4 °C. These observations suggest that the colloidal aggregates formed in more concentrated aqueous solution are at best metastable structures. This is in contrast to the temperature-responsive PHPMA-PMPC-PHPMA triblock copolymer gelators described earlier, which form transparent

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Figure 3. Concentration dependence of the apparent hydrodynamic radius of solutions/dispersions of (a) PMPC∼25-PHPMAn diblock copolymers in PBS, pH 7.2 at 4 °C; (b) PMPC∼25-PHPMAn diblock copolymers in PBS, pH 7.2 at 37 °C; (c) PMPC49-PHPMAn diblock copolymers in PBS, pH 7.2 at 4 °C; (d) PMPC49-PHPMAn diblock copolymers in PBS, pH 7.2 at 37 °C. (e) Hydrodynamic radius as a function of temperature for 1.0, 2.0, and 5.0 w/v % solutions/dispersions of PMPC23-PHPMA24. Dotted lines indicate aggregation/precipitation. (f) Hydrodynamic radius as a function of temperature for 1.0, 2.0, and 5.0 w/v % solutions/dispersions of PMPC49-PHPMA49. Dotted lines indicate aggregation/precipitation.

dispersions or gels with no signs of precipitation up to 30 w/v %.23 However, it is noteworthy that these triblocks are PMPCrich, with PHPMA contents of only 10-20 wt %. In contrast, the diblock copolymers described in the present work have PHPMA contents ranging from 20 to 70 wt %. The hydrodynamic radii of the remaining four PMPCPHPMA diblock copolymers with a fixed PMPC DP of 25 all increase at higher copolymer concentrations. Similar observations have been made for other copolymers where aggregates and unimers coexist in equilibrium and this phenomenon is attributed to enhanced incorporation of unimers within aggregates.27 In the present study, this appears to be strongly dependent on the DP of the PHPMA block. At 4 °C, the mean hydrodynamic radius of the PMPC25-PHPMA39 aggregates increases from 10 nm at 0.1 w/v % to 500 nm at 5.0 w/v % (Figure 3a). Clearly, the copolymer is only weakly aggregated at low concentration at this temperature because its radius is close to that expected for individual copolymer chains (∼5 nm). At higher copolymer concentrations, interchain interactions such as hydrogen bonding may well contribute to the formation of

very large aggregates. Copolymer aggregates with a mean radius of ∼35 nm at a concentration of 0.1 w/v % are formed at 37 °C, with this radius increasing up to 400 nm for copolymer concentrations of 5.0 w/v %. Thus, the copolymer’s propensity toward aggregation at low concentration is greater at 37 °C than at 4 °C, probably due to progressive dehydration of the PHPMA chains. At higher copolymer concentrations, smaller aggregates are formed at 37 °C than at 4 °C due to greater dehydration at the higher temperature, leading to more compact aggregates. This implies that, at some critical copolymer concentration where the two curves shown in Figure 3a,b for PMPC25PHPMA39 intersect, the dehydration-driven contraction eventually outweighs the formation of larger aggregates due to unimer incorporation. This crossover concentration seems to lie between 1.0 and 2.0 w/v % for PMPC25-PHPMA39. The hydrodynamic radii of copolymers with longer PHPMA blocks also increase with copolymer concentration. However, the relative increase is much smaller than for PMPC25-PHPMA39 (see Figure 3a,b). For example, in the case of PMPC25-PHPMA58 at 4 °C, the aggregate radius increases from approximately 40


nm at 0.1 w/v % up to 90 nm at 5.0 w/v %. At 37 °C, the radii are typically 5-10 nm smaller than at 4 °C (except at 0.10 w/v %, where the radius is larger by almost 20 nm). This behavior is reminiscent of that observed for the PMPC25-PHPMA39 copolymer, albeit with the critical copolymer concentration shifted to a much lower value. This is to be expected, because the hydrophobicity of the PHPMA block should be greater for higher degrees of polymerization. Unfortunately, the excess scattering intensity at such dilutions is very low, which adds significant uncertainty to the data. Therefore the exact position of this crossover concentration is rather hard to determine reliably. Increasing the concentration of either PMPC25PHPMA90 or PMPC25-PHPMA120 from 0.1 to 5.0 w/v % leads to an increase of a few nm in aggregate size, indicating a shift in the unimer-aggregate equilibrium. The concentration dependence of the hydrodynamic radius of the series of diblock copolymers with a fixed PMPC DP of 50 is more complex (see Figure 3c,d). At 4 °C, PMPC49-PHPMA49 has a hydrodynamic radius of approximately 5 nm up to a concentration of 3.0 w/v %, indicating molecular dissolution. A further increase in the copolymer concentration leads to the abrupt formation of aggregates with a hydrodynamic radius of approximately 100 nm at 5.0 w/v %. In contrast, the hydrodynamic radius of PMPC49-PHPMA26 aggregates increases almost exponentially from 6 nm at 0.5 w/v % up to 200 nm at 5.0 w/v % (Figure 3c). This is unexpected because a more hydrated, shorter PHPMA block should favor molecular dissolution. This behavior may be due to the higher polydispersity of this copolymer. Indeed, cumulants analyses of the light scattering data indicate at least two populations for this copolymer at 4 °C, which correspond to molecularly dissolved chains and colloidal aggregates (Figure S3). This aggregation from a small but measurable fraction of the copolymer leads to a larger calculated hydrodynamic radius. The hydrodynamic radius of PMPC49PHPMA67 increases between 0.1 and 1.0 w/v %, after which an almost constant value of approximately 200 nm is attained. This indicates that the equilibrium is shifted toward aggregates, which is consistent with the cumulants analysis (see Figure S3). Increasing the temperature also leads to aggregation of PMPC49PHPMA26 and PMPC49-PHPMA49 between 0.5 and 1.0 w/v %. In both cases, increasing the copolymer concentration apparently leads to a modest reduction in the hydrodynamic radius, which may be due to either formation of more compact aggregates or a lower aggregation number.38 In general, the DLS data indicates the presence of large nonmicellar aggregates whose size is largely independent of the copolymer concentration above 1.0 w/v %. However, PMPC49-PHPMA67 exhibits qualitatively different behavior, because its apparent hydrodynamic radius increases monotonically with increasing copolymer concentration. This is somewhat surprising because the longer PHPMA block should favor aggregate formation, in accordance with the behavior observed for the copolymer series with a fixed PMPC DP of 25. Inspection of the cumulants analyses for this copolymer (Figure S3) indicates that it forms two types of aggregate: one with a hydrodynamic radius of around 20 nm and the other with a radius in excess of 200 nm. Increasing the copolymer concentration shifts the equilibrium toward the larger species, as expected. Figure 3f shows how the hydrodynamic radius varies with temperature for three PMPC49-PHPMA49 concentrations. Below 15 °C, the hydrodynamic radius observed for concentrations of 1.0 and 2.0 w/v % is less than 5 nm, which indicates molecular dissolution. Increasing the temperature leads to formation of


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Figure 4. 500 MHz1H NMR spectra of PMPC25-PHPMA39 recorded in CD3OD at 21 °C and in D2O at 4.6 °C, 25 and 37 °C. All spectra are normalized relative to peak “a”. The arrows indicate those PHPMA signals that are significantly attenuated at elevated temperature.

aggregates of 100 nm radius at both concentrations. At 5.0 w/v %, the same copolymer has a hydrodynamic radius of around 10 nm below 12 °C. This is around twice as large as the molecularly dissolved unimers observed at lower concentrations, indicating that the 5.0 w/v % copolymer is weakly aggregated even at low temperature, although the effect of a change in refractive index cannot be excluded (we assumed a refractive index for pure water of 1.330, which is a valid approximation to within 0.5% over the temperature interval studied39). However, the change in solution/dispersion refractive index due to dissolved solids may have an influence especially at the relatively high concentrations used. This effect was not investigated. Above 12 °C, aggregates are formed with radii of 150-200 nm. Thus, the aqueous dispersion behavior of PMPC49PHPMA49 is somewhat different from that of PMPC23PHPMA24, even though the PHPMA content is almost identical. Essentially, the critical aggregation temperature is much less concentration-dependent for the larger copolymer. In addition, the aggregate size is much smaller for the larger copolymer and macroscopic precipitation is not observed. Hence it appears that longer PHPMA blocks leads to the formation of more compact, smaller aggregates. In addition, the longer PMPC block is more efficient in forming colloidally stable aggregates in solution. Temperature-Dependent 1H NMR Studies. The degree of solvation of these PMPC-PHPMA copolymers was examined by temperature-dependent 1H NMR spectroscopy in D2O. Figure 4 shows 1H NMR spectra recorded for 1.0 w/v % solutions of PMPC25-PHPMA39 in (i) CD3OD at 22 °C and (ii) in D2O at three different temperatures. Because CD3OD is a good solvent for both blocks, this solvent was used to determine the true block composition. D2O is a good solvent for the PMPC blocks at all temperatures and therefore the PMPC signals were used as internal standards. The 1H NMR spectrum of the molecularly dissolved copolymer in CD3OD is quite complex, with several overlapping peaks. Moreover, the HPMA repeat unit is actually a 75:25 mixture of two isomers.37 This is why the signals labeled h and f comprise more than one peak. The block composition was assessed in CD3OD by the integral of signal g (1H; assigned to the major HPMA isomer). Multiplying this integral by 4/3 and comparing it to an appropriate PMPC signal (a, 2H) allows calculation of the block composition. This approach produced

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Figure 5. Temperature dependence of the apparent PHPMA content of 1.0 w/v % aqueous solutions/dispersions of various PMPC-PHPMA diblock copolymers in D2O normalized with respect to their actual block compositions (as determined in CD3OD). The monotonic reduction in apparent PHPMA content on increasing the temperature indicates progressively poorer solvation and lower mobility for this block; this is consistent with the onset of micellar self-assembly. (a) Data set obtained for PMPC-PHPMA diblock copolymers with a fixed PMPC DP of ∼25; (b) data set obtained for PMPC-PHPMA diblock copolymers with a fixed PMPC DP of ∼49. Lines are guides for the eye, rather than fits to the data.

results that were consistent with the target block compositions (see Table 1). Alternatively, if the peak integrals for a (2H) and f (3H) were compared, similar compositions were obtained (within a few percent) despite the overlap between f and the backbone signals. Increasing the temperature of the diblock copolymer solutions in D2O leads to gradual attenuation of the PHPMA signals (Figure 4). This attenuation is due to the reduced mobility of these chains and a reduction in the number of molecularly dissolved polymer chains due to their incorporation into aggregates.40 Inspecting the 1H NMR spectra recorded in D2O (Figure 4), only the signal from the side-chain methyl groups (f) of the PHPMA chains is readily detectable in aqueous solution, with signals g and h being either almost completely attenuated or obscured by overlapping PMPC signals. Therefore the integrals of signals a and f were compared to calculate apparent block compositions for each of the diblock copolymers at a given temperature. These compositions were then normalized with respect to the actual block composition obtained from the spectrum recorded in CD3OD to produce an “apparent” PHPMA content. The results are shown in Figure 5. The apparent PHPMA content of a 1.0 w/v % solution of PMPC23-

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PHPMA24 in D2O at 5 °C is identical to the true content within experimental error, indicating that this copolymer is molecularly dissolved. Increasing the temperature leads to an apparent reduction in the PHPMA content, but even at 37 °C this block has a degree of solvation of more than 85%. Thus, these NMR results are consistent with the DLS data obtained for this copolymer, which indicated molecular dissolution up to 50 °C (Figure 2). Increasing the DP of the PHPMA block leads to a reduction in the apparent PHPMA content regardless of the temperature. This is because longer PHPMA blocks are more hydrophobic and hence more prone to aggregation. For both PMPC25PHPMA39 and PMPC25-PHPMA58, the apparent PHPMA content is around 70% of the true value at 5 °C. This value is progressively reduced to 20% on heating to 37 °C. For these two copolymers, the light scattering intensity increased significantly with temperature, whereas their hydrodynamic radii were relatively large but did not change significantly (Figure 2). In addition, concentration-dependent light scattering indicated that unimers and aggregates coexist in such solutions (Figure 3). Thus, attenuation of the NMR signals due to PHPMA at higher temperature is consistent with the incorporation of unimers into aggregates, although there may also be some contribution due to a reduction in segmental motion within the aggregates. PHPMA blocks with higher DP values are significantly less solvated: above 15 °C, the apparent PHPMA content is less than 10% and remains relatively constant for both PMPC25PHPMA90 and PMPC25-PHPMA120. At 5 °C, the apparent PHPMA content is 20-40%, with PMPC25-PHPMA90 being more solvated as expected. For these two copolymers, slightly more intense light scattering was observed between 4 and 15 °C, whereas the hydrodynamic radius did not change significantly. In addition, there was almost no increase in the hydrodynamic radius with concentration, suggesting that few, if any, copolymer chains are present as unimers (Figure 3). Thus, the PHPMA chains are highly dehydrated for these two copolymers and the additional attenuation observed on raising the temperature from 5 to 15 °C is probably mainly due to further dehydration of the aggregates. Figure 5b shows the apparent PHPMA content as a function of temperature for three copolymers with a fixed mean PMPC DP of 49. Their behavior is similar to that observed for the series of copolymers comprising shorter PMPC chains. Increasing the temperature leads to reduced PHPMA signal intensities and this attenuation is highly dependent on the mean DP of the PHPMA chains. It is noteworthy that, for all copolymers, these variable temperature 1H NMR experiments do not indicate a well-defined critical aggregation temperature but rather continuous dehydration and a progressive shift in the unimer/aggregate equilibrium. This is in contrast to the behavior observed for other thermoresponsive polymers such as PPO41 or PNIPAM,20,42 where a critical aggregation temperature or LCST could be inferred from the attenuated 1H NMR signals. Figure 6a shows the variation in the apparent PHPMA content with the DP of this block at 5 and 37 °C for both series of PMPC-PHPMA diblock copolymers. At 5 °C, the apparent PHPMA content decreases continuously from 100 to 17% on increasing the DP of the PHPMA block from 24 to 120. At 37 °C, the apparent PHPMA content decreases from 100 to 4% on increasing the DP of the PHPMA block from 24 to 120. Intermediate apparent block compositions were observed at 22 °C (data not shown). Hence, the degree of solvation of the PHPMA chains is very sensitive to their mean degree of polymerization, whereas the influence of the PMPC block is


Figure 6. (a) Apparent PHPMA content measured by 1H NMR spectroscopy for 1.0 w/v % solutions/dispersions in D2O of PMPC25PHPMAn (triangles) and PMPC50-PHPMAn (circles) diblock copolymers as a function of the actual degree of polymerization of the PHPMA block at 5 °C (open symbols) and 37 °C (closed symbols). (b) DLS hydrodynamic radius obtained for 1.0 w/v % solutions/ dispersions in PBS at pH 7.2 containing PMPC25-PHPMAn (triangles) and PMPC50-PHPMAn (circles) diblock copolymers as a function of the actual degree of polymerization of the PHPMA block at 4 °C (open symbols) and 37 °C (closed symbols). Lines are guides to the eye rather than data fits.

relatively weak. In addition, the reduced apparent PHPMA content at elevated temperature is also highly dependent on the DP of this block. Figure 6b shows the effect of varying the PHPMA DP on the hydrodynamic radius at 4 and 37 °C. Copolymers containing relatively short PHPMA blocks may be either molecularly dissolved or thermoresponsive. Increasing the PHPMA DP leads to aggregation, with no discernible difference in aggregate dimensions observed at 4 and 37 °C. For those copolymers that do aggregate, the hydrodynamic radius is reduced as the PHPMA DP is increased up to 90. In view of our 1H NMR data, this indicates that the aggregation behavior is highly dependent on the hydration of the PHPMA blocks. Preliminary variable angle light scattering studies only indicate a weak dependence of the diffusion coefficient on the scattering angle in most cases, indicating approximately spherical aggregates (Figure S4). Aggregation Mechanism. In general, the thermoresponsive behavior of nonionic water-soluble polymers is due to a significant reduction in hydrogen bonding interactions between the polymer chains and water molecules occurring at a particular temperature. For example, temperature-dependent IR studies of PPO in water confirmed weaker hydrogen bonding between water and the ether oxygens on the polymer backbone,43 as well as partial dehydration of the pendent methyl groups.44 Similar


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studies of aqueous solutions of PNIPAM provided spectroscopic evidence for hydrogen bonding between the N-H protons and the carbonyl oxygens in the collapsed state above the LCST but not for the soluble chains below the LCST.45 The aggregation of PMPC-PHPMA diblock copolymers in aqueous solution strongly depends on both the DP of the PHPMA block and the solution temperature. This behavior is summarized in Figure 7. Copolymers with sufficiently short PHPMA blocks remain molecularly dissolved at all temperatures in dilute solution. Increasing the DP of the PHPMA block or the copolymer concentration eventually leads to aggregation, although a sufficiently long PMPC block may suppress this significantly. The aggregates formed by diblock copolymers with relatively short PHPMA blocks are very large, presumably due to their highly hydrated nature. These aggregates exist in equilibrium with molecularly dissolved copolymer chains at low concentrations. Increasing the solution temperature causes progressive dehydration of the PHPMA blocks, which leads to incorporation of unimers into aggregates as well as to more compact aggregates (presumably due to expulsion of water). Increasing the PHPMA DP has a similar effect; the equilibrium between unimers and aggregates shifts toward aggregates and these aggregates are significantly less hydrated and therefore smaller. At a PHPMA DP of 90 or above, there are essentially no unimers present. There are two main differences between these PHPMA-based thermoresponsive diblock copolymers and those based on other thermoresponsive blocks: First, these block copolymers form relatively large, water-rich aggregates if the PHPMA block has a relatively low DP. Perhaps counterintuitively, relatively short copolymers form larger aggregates than those with longer PHPMA blocks. Similar behavior has been reported by Soga et al. for three diblock copolymers of PEO and poly(N-(2hydroxypropyl) methacrylamide lactate46). In this case, a minimum in the aggregate size was also found for the copolymer with an intermediate degree of polymerization for the poly(N(2-hydroxypropyl) methacrylamide lactate) block. However, these copolymers are harder to compare directly because their polydispersities vary considerably.46 Second, whether a given diblock copolymer forms large aggregates or becomes molecularly dissolved seems to depend on the DP of the water-soluble PMPC block, with longer “buoy” blocks favoring unimers. For those copolymers that do exhibit thermoresponsive behavior, this transition is relatively ill-defined, typically occurring over a temperature range of 10-15 °C. Similar broad transitions have been observed for thermoresponsive polymers based on PPO27 and poly(2-hydroxyethyl methacrylate),32 whereas thermoresponsive polymers based on PNIPAM33,34 and poly(2-(2-methoxyethoxy)ethyl methacrylate47 typically exhibit sharper transitions. It is conceivable that the isomeric nature of the PHPMA block may be important in dictating its aqueous phase behavior. Another possibility may be a polydispersity effect; the diblock copolymers described in the present work typically have polydispersities ranging from 1.2 to 1.3 (Table 1). If the aqueous solubility of the PHPMA block at a given temperature is sensitive to its degree of polymerization, such polydispersities may well “smear out” any thermal transition. In this context, Sugihara48 recently studied thermoresponsive diblock copolymers based on poly(2-(2-ethoxy)ethoxyethyl vinyl ether) [EOEOVE] and poly(2-methoxyethyl vinyl ether) [MOVE], where phase separation of PEOEOVE200-PMOVE400 with a polydispersity of 1.10 occurred over less than 5 °C. In contrast, an ad-mixture comprising three near-monodis-

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Figure 7. Schematic representation of the effect of raising the temperature and increasing the mean degree of polymerization of the PHPMA block on the colloidal aggregates produced by self-assembly.

perse diblock copolymers (PEOEOVE100-PMOVE200, PEOEOVE200-PMOVE400 and PEOEOVE300-PMOVE600) had an overall polydispersity of 1.81 and only underwent partial phase-separation over a temperature range of approximately 20 °C.48 Apart from this work, there seem to be few, if any, detailed studies on the influence of the polydispersity on the aqueous phase behavior of stimulus-responsive diblock copolymers. Intracellular Delivery. Preparation of Dye-Loaded Copolymer Structures. The PMPC-PHPMA diblock copolymers did not exhibit any significant cytotoxicity toward human dermal fibroblasts (see Supporting Information, Figure S5). Therefore, they were examined as potential drug delivery vehicles using a model compound, rhodamine B octadecyl ester. The dye-loaded copolymer structures were prepared by dissolving a copolymer/ dye film in PBS, followed by column chromatography to remove excess dye. In the case of diblock copolymers with a PHPMA DP of 58 or higher, the final copolymer solutions were highly colored and no dye was left on the column. However, use of the PMPC23-PHPMA24 copolymer (or, in control experiments, the dye alone) led to a significant fraction of the dye remaining on the column. This is in accordance with this copolymer being molecularly dissolved at low concentration; thus, it has little or no capacity for dye solubilization. It should be noted that, even in the absence of any diblock copolymer, solutions that had been passed through the column were faintly colored. This may be due to formation of aggregates of the amphiphilic dye. Images Using Confocal Microscopy. Confocal microscopy was used to visualize fibroblast cells after incubation with various copolymer/dye solutions. As a control, cells were also incubated with rhodamine B octadecyl ester in the absence of any copolymer. The results are shown in Figure 8. All cells became fluorescent within 24 h. However, in the absence of any copolymer or in the presence of the copolymer with the shortest PHPMA block (PMPC23-PHPMA24) the fluorescence is relatively weak (see Figure 8a and b, respectively). The uptake of macromolecules by mammalian cells is mainly mediated via endocytosis.49 This is a broad term encompassing various internalization mechanisms. Endocytosis involves the formation of membrane-bound vesicles and includes the fol-

lowing subcategories: clathrin-dependent endocytosis, macropinocytosis (also known as phagocytosis), caveolae-mediated internalization, and clathrin/caveolae independent pathways.50 The intracellular domain is divided into various compartments to allow the trafficking of various different materials such as proteins, lipids, and other solutes.50 Once inside the cells, both the mode of entry of the macromolecule and its physicochemical properties will determine the extent of its intracellular localization.51 Microtubules are protein filaments found inside mammalian cells; they play an important role in mediating the intracellular transport of caveolae vesicles and organelles such as mitochondria between different compartments of the cell.52 It has been shown that mitochondria organelles are coaligned with microtubule filaments.53 Specific staining of these organelles with rhodamine 12354 produced images that are remarkably similar to those obtained in the present study using either rhodamine B octadecyl ester alone or in conjunction with PMPC23-PHPMA24 (see Figure 8a,b). Moreover, similar images were also obtained when microtubules were specifically stained.55 Thus, using either the dye alone or in conjunction with the most hydrophilic copolymer, specific staining of either microtubules or mitochondria can be achieved. Due to the colocalization of the organelle along the filaments, the precise identification of either component is difficult. In contrast, using copolymers with high dye-loading capacities (e.g., PMPC25PHPMA58 and PMPC25-PHPMA120, see Figure 8c and d, respectively) leads to highly fluorescent cells with a strong perinuclear fluorescence, indicating either endosomal escape or internalization via an alternative mechanism. This suggests that the copolymer aggregates enable much more efficient intracellular delivery of this model drug. It is not apparent from our images whether the dye remains within such copolymer aggregates or whether release has occurred. However, because the aggregates exist in equilibrium with unimers, it is quite feasible that release is simply mediated by gradual dilution. Kinetics of Intracellular DeliVery of Rhodamine B Octadecyl Ester. The cellular uptake kinetics of a fluorescent model drug, rhodamine B octadecyl ester mediated by PMPC-PHPMA diblock copolymers were also investigated (see Figure 9).



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Figure 8. Confocal microscopy images of human dermal fibroblasts exposed to a solution of rhodamine B octadecyl ester in PBS containing (a) no copolymer, (b) PMPC23-PHPMA24, (c) PMPC25-PHPMA58, and (d) PMPC25-PHPMA120. The scale bar corresponds to 20 µm.

According to Rejman et al., who examined the rate of internalization of fluorescent latex beads by murine melanoma cells, larger objects should be taken up more slowly.56 Thus, it is anticipated that intracellular uptake of the larger copolymer aggregates should be retarded, provided that the precise mechanism of internalization remains unchanged. The intracellular delivery of the rhodamine dye using various diblock copolymers with the same fixed PMPC block but different PHPMA blocks indicate that its uptake kinetics differ significantly depending on the block composition (see Figure 9). More specifically, a positive correlation between the rate of intracellular rhodamine uptake and PHPMA block length was observed. Rhodamine B octadecyl ester exhibited both statistically similar kinetics and mean fluorescence intensity per cell (P g 0.3) whether in the absence of any copolymer or mediated by PMPC24-PHPMA23. In both cases, exposure to cells for 4 h led to a maximum dye uptake of 50% of the total cell population, while the mean fluorescence intensity per cell was only around 10 au. It was not possible to distinguish between the cellular uptake kinetics obtained using dye solutions prepared in the absence of any copolymer (after passage through a size exclusion column) from nonseparated dye solutions (data not shown). This suggests that cellular uptake is mediated by the formation of dye aggregates, rather than molecularly dissolved dye molecules. On the other hand, when PMPC24-PHPMA58 or PMPC24PHPMA120 copolymers were used to deliver the same dye, 80-90% of the cells became fluorescent after 4 h incubation, with mean fluorescence intensities per cell exceeding 150 (see Figure 9). Interestingly, a higher fluorescence intensity per cell was obtained for PMPC24-PHPMA120 compared to PMPC24PHPMA58, with the former copolymer producing approximately twice the mean fluorescence intensity (Figure 9b). However, this difference was statistically significant for the first hour only

(P ) 0.006). These results are in good agreement with previous drug delivery studies using amphiphilic copolymers. For example, molecularly dissolved PEO-PPO-PEO block copolymers modified the biological activity of compounds with respect to multiple drug-resistant tumors by inhibition of glycoprotein P-mediated drug efflux, which is responsible for pumping therapeutic compounds out of resistant cells.57 This effect depended on the hydrophilic/hydrophobic balance of the block copolymers. Copolymers with intermediate degrees of polymerization for the PPO block and relatively low degrees of polymerization for the PEO block led to enhanced inhibition of glycoprotein P and subsequently enhanced cytotoxicity for the anticancer drug (doxorubicin). These results were explained by stronger interaction of the more hydrophobic copolymers with cellular membranes.57 Although this study only examined the effect of molecularly dissolved copolymers rather than aggregates, a similar principle may well apply to the aggregateforming PMPC-PHPMA diblock copolymers. Thus, diblock copolymers with longer PHPMA blocks may exhibit increased interaction with cellular membranes and hence trigger faster, more efficient endocytosis than the more weakly aggregated copolymers comprising shorter PHPMA blocks. Another possible explanation for the data presented in Figure 9 is the higher colloidal stability of the aggregates obtained with longer PHPMA blocks. The thermodynamic stability of colloidal aggregates (including drug-loaded micelles) correlates well with the length of the hydrophobic block.58 A more stable aggregate is less likely to dissociate upon dilution and therefore it can deliver its drug load to cells more efficiently via endocytosis, rather than merely passive drug diffusion through the membrane.59 The rhodamine B octadecyl ester perchlorate used in these experiments has amphiphilic character and may well selfassemble to form weak aggregates in aqueous dye solution. Such

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Figure 9. Cellular uptake kinetics obtained for human dermal fibroblast cells using either rhodamine B octadecyl ester dye alone or the same dye in the presence of selected PMPC-PHPMA diblock copolymers: (a) fraction of fluorescent cells versus time; (b) mean fluorescence intensity per cell versus time.

aggregation should result in fluorescence quenching,60 compared to dye dispersions obtained using copolymers comprising longer PHPMA blocks. This suggests that such copolymers suppress dye aggregation and thereby reduce the fluorescence quenching. Although the relatively low fluorescence per cell observed in the absence of any copolymer or in the presence of PMPC23-PHPMA24 may be due to dye aggregation-induced quenching, the accompanying low level of fluorescently-active cells strongly suggests relatively inefficient dye delivery (Figure 9a,b).

Conclusions A novel class of amphiphilic PMPC-PHPMA diblock copolymers has been synthesized via ATRP. In general, these syntheses were well-controlled, affording copolymers with polydispersities between 1.20 and 1.30 and actual compositions that were close to the targeted compositions. These copolymers were readily dissolved or dispersed in cold aqueous solution and exhibited a range of phase behavior depending on the degree of polymerization of the PMPC and PHPMA blocks, the copolymer concentration and the solution temperature. Thus, a PMPC24-PHPMA23 diblock copolymer is molecularly dissolved at 4 °C in concentrations up to 5.0 w/v %. Increasing the temperature led to the formation of very large aggregates with dimensions of several hundred nanometers at concentrations above 2.0 w/v %, with a critical aggregation temperature that ranged from 30 °C at 2.0 w/v % to 15 °C at 5.0 w/v %.

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Increasing the PHPMA block length for a fixed PMPC block length of ∼25 led to concentration-dependent aggregation at all temperatures. PMPC49-PHPMA49 diblock copolymer is molecularly dissolved at 4 °C up to 3.0 w/v %, with large colloidal aggregates being formed at 5.0 w/v %. In this particular case, aggregation occurred at a temperature of approximately 10-12 °C almost independent of copolymer concentration. The solution behavior of PMPC49-PHPMA26 or PMPC49-PHPMA67 was more complicated due to the coexistence of several colloidal species, as revealed by cumulants analysis. In addition, 1H NMR analyses indicated that the PHPMA chains within these aggregates remained at least partially solvated, suggesting coexistence with unimers. Furthermore, longer PHPMA blocks led to the formation of smaller, less hydrated aggregates whose size was only weakly concentration-dependent. Cytotoxicity studies confirmed that these PMPC-PHPMA copolymers were biocompatible, as expected. Selected copolymers were evaluated for the intracellular delivery of a fluorescent dye (rhodamine B octadecyl ester). Only those copolymers that formed colloidal aggregates efficiently solubilized this amphiphilic dye: molecularly dissolved copolymers exhibited little or no dye uptake. This behavior was also reflected in cell uptake studies: delivery of the rhodamine B dye into human dermal fibroblasts proved to be particularly effective and rapid for copolymers containing longer PHPMA blocks that formed colloidal aggregates in aqueous solution. In contrast, intracellular dye uptake was slower and less effective for copolymers that contained shorter (less hydrophobic) PHPMA blocks or for the dye alone. Although these results were obtained using an amphiphilic model compound, they are likely to be also applicable to hydrophobic drugs. Thus, careful copolymer design should allow enhanced drug solubilization, as well as efficient intracellular delivery. Moreover, because most of these copolymers exhibit relatively high critical aggregation concentrations, they may offer some potential for localized intracellular drug delivery. After delivering the drug, gradual dilution may lead to dissolution of the aggregates, allowing their excretion in the form of molecularly dissolved chains. Acknowledgment. The University of Sheffield is thanked for funding a Ph.D. studentship for J.M. The Algerian government is thanked for funding a Ph.D. studentship for K.B. Biocompatibles UK Ltd is thanked for funding CASE awards for J.M. and K.B. and for supplying the MPC monomer. S.P.A. is the recipient of a five-year Royal Society-Wolfson Research Merit Award. We thank the four reviewers of this manuscript for their constructive criticisms. Supporting Information Available. Cumulants analysis of the light scattering data versus temperature and concentration, angular dependence of the diffusion coefficient for selected copolymer solutions, and cytotoxicity data. This material is available free of charge via the Internet at http://pubs.acs.org.

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BM9002915

Preparation and Aqueous Solution Properties of Thermoresponsive

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