Highly Efficient and Versatile Formation of Biocompatible Star

Nov 13, 2014 - However, PNIPAM CCS made with the lowest [CL]/[MI] of 8:1 shows ...... Abrol , S.; Kambouris , P. A.; Looney , M. G.; Solomon , D. H. M...
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Highly Efficient and Versatile Formation of Biocompatible Star Polymers in Pure Water and Their Stimuli-Responsive Self-Assembly Thomas G. McKenzie, Edgar H. H. Wong, Qiang Fu, Shu Jie Lam, Dave E. Dunstan, and Greg G. Qiao* Polymer Science Group, Department of Chemical and Biomolecular Engineering, The University of Melbourne, Parkville, VIC 3010, Australia S Supporting Information *

ABSTRACT: This study demonstrates the rapid and efficient formation of functional core cross-linked star polymers via copper-mediated reversibledeactivation radical polymerization (RDRP) in pure water using fully soluble monomers and cross-linkers. This high throughput “arm-first” methodology allows the generation of complex nanoarchitectures with tailored core, shell, or periphery- functionalities and is potentially well-suited for biomedical applications given that the macromolecular synthesis is performed entirely in water. To exemplify this approach, different homo- and miktoarm star polymers composed of either poly(N-isopropylacrylamide) (PNIPAM), poly(2-hydroxyethyl acrylate) (PHEA), and poly(ethylene glycol) (PEG) as the polymeric arms are formed. The star products are generated in high yield (88−96%) in one-pot and require short reaction times (1−3 h) and minimal purification steps (dialysis and lyophilization). In addition, the thermal responsivity of PNIPAM-based miktoarm star polymers leading to reversible supramolecular self-assembly is confirmed by DLS and 2D-NOESY NMR analysis. Furthermore, cytotoxicity studies using human embryonic kidney (HEK239T) cells as the model mammalian cells revealed that the star polymers are nontoxic even up to high polymer concentrations (2 mg mL−1). The simplistic product formation and isolation, combined with the use of water as the polymerization medium, mean that this procedure is highly attractive as a low-cost pathway toward functional, biocompatible organic nanoparticles for commercial applications.

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INTRODUCTION Over the past few decades, the development of organic nanoparticles (ONPs) has been a major research focus across various scientific disciplines.1,2 These materials, typically made from organic macromolecules such as polymers or dendrimers, have potential in a wide range of applications such as clean energy generation,3 biomedical imaging, drug delivery,4 and catalysis.5,6 Typical pathways toward ONPs include the self-assembly of polymeric “unimers” to form higher-order controlled aggregates of specific morphology,7−9 through the intramolecular selffolding of individual polymer chains,6,10−12 or by the design of complex polymer architectures (e.g., star, brush) achieved via controlled polymerization techniques.13−15 Star polymers belong to a unique class of ONPs that are made possible by the advent of controlled polymerization.13,16−21 This includes the core cross-linked star (CCS) polymers (generally 10−50 nm in diameter) synthesized via an “arm-first” approach, where linear macroinitiators (MIs) are reacted with a crosslinking moiety to generate star-shaped macromolecule architectures with linear polymeric “arms” radiating from a central cross-linked core. The preparation of CCS polymers with core− shell structures is most readily achieved via controlled radical polymerization (now referred to as reversible deactivation radical polymerization (RDRP)22), with high yielding synthetic methodologies only recently becoming available.23−26 CCS © XXXX American Chemical Society

polymers are structurally similar to micelles prepared via solution self-assembly of amphiphilic diblock copolymers. However, CCS polymers have several distinct advantages: (i) their inherently cross-linked nature means they have no lower critical concentration (such as the critical micelle concentration (CMC) present in self-assembled systems) and are thus stable up to infinite dilution; (ii) lesser synthetic steps involved (i.e., the absence of the assembly process); and (iii) access to a wider range in the number of radiating “arms”. These advantages are of crucial importance when considering the design of ONPs for use in commercial applications. One aspect that is highly desirable for RDRP techniques is the use of water as a polymerization medium, replacing traditionally used organic solvents. Water as a polymerization medium is attractive not just due to the lower associated costs but also due to its “green” nature, which makes water particularly well-suited to be used as a polymerization medium for synthesizing biomacromolecules.27 However, there have been many challenges associated with using water as a polymerization medium for RDRPsmainly, the hydrolysis of propagating end-groups. The use of water as a solvent for the generation of CCS polymers Received: September 28, 2014 Revised: October 27, 2014

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esterification of 1,2-ethanediol with α-bromoisobutyryl bromide (98%, Aldrich), purified by flash column chromatography, and characterized by 1H and 13C NMR. Distilled water from an RO water purification system was used directly in all cases. Copper(I) bromide (CuBr, >98%, Aldrich) was washed sequentially with glacial acetic acid and ethanol, dried under vacuum, and then stored under argon. Tris[2(diethylamino)ethyl]amine (Me6TREN) was synthesized according to the procedure reported in the literature.36 Poly(ethylene glycol) methyl ether (PEG-OH, Mn ∼ 5000 Da, Aldrich) was converted into the macroinitiator PEG-Br via esterification with α-bromoisobutyryl bromide. The PEG-Br product was then washed sequentially with aqueous solutions of saturated NaCl, 0.5 M HCl, and saturated NaHCO3, followed by precipitation in cold diethyl ether and dried under vacuum.21 Dialysis membrane (Spectrum Laboratories, Inc., Spectra/Por Dialysis (MWCO 50 kDa)) was rinsed with RO water prior to use. Dulbecco’s Modified Eagle Medium (DMEM, GIBCO Cat. No. 11995), fetal bovine serum (FBS, GIBCO Cat. No. 10099), GlutaMAX supplement (100×, GIBCO Cat. No. 35050), Dulbecco’s Phosphate Buffered Saline (DPBS, GIBCO 14190), and 0.05% trypsin-EDTA (1×, GIBCO Cat. No. 25300) were purchased from Invitrogen and used as received. Penicillin-stretomycin was purchased from Aldrich and used as received. CellTiter 96 Aqueous One Solution Cell Proliferation Assay (MTS) (Promega) was used to perform cell viability assays. 96-well cell culture plates and T75 cell culture flasks (Corning) were used for cell culture. Characterizations. Gel Permeation Chromatography (GPC). Molecular weight characterizations were carried out via GPC using DMF as the mobile phase unless otherwise stated. The GPC analysis was conducted on a Shimadzu liquid chromatography system equipped with a PostNova PN3621 MALLS detector (λ = 532 nm), Shimadzu RID-10 refractometer (λ = 633 nm), and Shimadzu SPD-20A UV−vis detector using three identical Jordi columns (5 μm bead size, Jordi Gel Fluorinated DVB Mixed Bed) in series operating at 70 °C. DMF (>99%, Aldrich) with 0.05 mol L−1 LiBr was employed as the mobile phase at a flow rate of 1 mL min−1. NovaMALLS software (PostNova Analytics) was used to determine the molecular weight characteristics using literature dn/dc values. When aqueous-phase GPC was employed, a separate Shimadzu liquid chromatography system was utilized, equipped with a Shimadzu RID-10 refractometer (λ = 633 nm), using three Waters Ultrahydrogel columns in series ((i) 250 Å porosity, 6 μm diameter bead size; (ii) and (iii) linear, 10 μm diameter bead size), operating at room temperature. The eluent was Milli-Q water containing 20% v/v acetonitrile and 0.1% w/v TFA at a flow rate of 1 mL min−1. The molecular weight characteristics of the analyte were determined by comparison to narrow molecular weight distribution poly(ethylene glycol) standards. All samples were filtered through 0.45 μm nylon filters prior to injection. Nuclear Magnetic Resonance (NMR) Spectroscopy. 1H NMR spectroscopy was conducted on a Varian Unity 400 MHz spectrometer operating at 400 MHz, using the solvent deuterium oxide (D2O) (Cambridge Isotope Laboratories) as reference and sample concentrations of approximately 10 mg mL−1. Dynamic Light Scattering (DLS). DLS measurements and analysis were performed on a Wyatt DynaoPro NanoStar DLS/SLS instrument with a GaAs laser (658 nm) at an angle of 90 °C and a temperature of 25 ± 0.1 °C. Spectra were obtained at concentrations of 1 mg mL−1 in water. Calculation of Number of Arms, Narm. The calculation of the average number of radiating arms for each star polymer was carried out using light scattering data obtained via the GPC systems listed above and processed using a series of equations reported in the literature.37 Procedures. Synthesis of Homoarm CCS Polymers. Ligand (Me6TREN, 0.035 mmol, 0.4 equiv) in water (1 mL) was degassed via bubbling with N2 for 20 min, after which CuBr (0.070 mmol, 0.8 equiv) was added, and the mixture was bubbled with N2 for a further 20 min and then cooled to 0 °C in an ice/water bath. Meanwhile, a mixture of monomer (e.g., NIPAM, 7.0 mmol, 80 equiv) and initiator (HEBriB, 0.088 mmol, 1 equiv) in 3.5 mL of water was degassed via bubbling with N2 for 20 min. The monomer/initiator mixture was then added to the copper solution to mark the start of the polymerization. Samples were

also poses similar challenges, in that any side-reactions or loss of chain-end fidelity to hydrolysis during CCS formation will render the dead polymer chains unable to participate further in the cross-linking step, thus lowering the yield of the star product. A few innovative works have tried to circumvent these problems by the use of complex heterogeneous systems designed to isolate the propagating chain ends in a hydrophobic domain so as to minimize the likelihood of hydrolysis. An example of this is the work by Matyjaszewski et al. in which an amphiphilic MI was selfassembled in water prior to the addition of a hydrophobic crosslinking reagent to form CCS polymers.28 Similar studies have been shown to work for other polymerization systems using cross-linkable monomers that are only partially soluble in the reaction medium, hence enabling the MIs to self-assemble in situ into a micelle-like structure during the cross-linking step.26,29,30 However, such approaches limit the range of monomers and cross-linkers that can be used to form CCS polymers. Recently, Haddleton et al. have reported the rapid and efficient synthesis of well-defined linear polymers in pure water (without relying on the isolation of propagating groups in hydrophobic domains) using metal catalysts that are generated via the predisproportionation of copper(I) bromide (CuBr).31 They have illustrated that control over the polymerization is best afforded via disproportionation of a CuBr/ligand complex prior to the addition of the initiator and monomer. Rapid formation of linear homo- and multiblock acrylate and acrylamide polymers with narrow molecular weight distributions and good chain-end fidelity were demonstrated even at high monomer conversions, representing a significant advancement in the field of aqueousbased RDRP.32,33 There remains however some debate in the literature as to the activation mechanism in such aqueous systems,34 with similar results achievable under conditions considered analogous to atom transfer radical polymerization (ATRP) reactions.35 Regardless, encouraged by the efficiency and simplicity of the polymerization system of Haddleton et al., we aim at employing the method of copper predisproportionation to synthesize CCS polymers in pure water and in a “one-pot” fashion, providing an efficient synthetic route to generate highyielding ONPs in water using fully soluble monomers and crosslinkers. In demonstrating the efficiency of our approach, linear MIs of polyacrylamide and polyacrylate are formed and taken to (near) full monomer conversion followed by the sequential addition of a water-soluble cross-linker to generate homo- and miktoarm CCS polymers, wherein the thermoresponsive miktoarm CCS polymers are subsequently shown to self-assemble into larger nanostructures at elevated temperatures. The synthesized materials are carefully characterized using GPC, DLS, and NMR analysis. In addition, the cytotoxicity of the prepared ONPs toward a model mammalian cell line (human embryonic kidney (HEK239T) cells) was also evaluated. We envisage that the current approach described herein will provide a highly promising platform for building complex ONPs in a highthroughput, low-cost manner that is particularly suited for polymer-based biomedical applications.

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EXPERIMENTAL SECTION

Materials. N-Isopropylacrylamide (NIPAM, >97%, Aldrich) was recrystallized twice from hexane to remove inhibitors. 2-Hydroxyethyl acrylate (HEA, 96%, Aldrich) was purified via distillation prior to use. N,N′-Methylenebis(acrylamide) (MBA, 99%, Aldrich) was recrystallized twice from hexane/toluene (1:1) to remove inhibitors prior to use. 2-Hydroxyethyl α-bromoisobutyrate (HEBriB) was synthesized via the B

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extracted via degassed syringe to monitor monomer conversion via 1H NMR. Upon (near) full monomer conversion, a degassed solution of cross-linker (MBA, 0.70−1.05 mmol, 8−12 equiv) was added to the reaction flask via degassed syringe, and the flask was kept at 0 °C for the duration of the reaction. Samples were extracted periodically to determine star conversion via GPC and 1H NMR. After high star conversions had been reached, the reaction mixture was exposed to air and transferred to a dialysis membrane (MWCO 50 kDa). The crude product was then dialyzed against pure water (200 mL) for 6−8 h, changing the water twice during this period. The mixture was then transferred to a vial and lyophilized overnight. The star product was collected as a white powder. Synthesis of Miktoarm CCS Polymers. Two separate catalyst/ligand suspensions were prepared in the same fashion as above. Into each was added the degassed monomer/initiator solution for monomer 1 (e.g., NIPAM) and monomer 2 (e.g., HEA). Once (near) full monomer conversion had been reached for both concurrent reactions, the reaction mixtures were combined into a single flask by transferring the contents from one flask into another using a wide-bore syringe needle (to allow the transfer of any copper solids), followed by the immediate addition of the degassed cross-linker solution. The reaction flask was kept at 0 °C, and samples were withdrawn periodically to monitor star conversion via GPC and 1H NMR. After high star conversions had been reached, the products were isolated in the same manner as the homoarm CCS polymers. Self-Assembly of Miktoarm Polymers. The isolated CCS polymers of interest were dissolved directly in water to a polymer concentration of 1 mg mL−1. This solution was then used for DLS measurements, where the sample was heated and cooled inside the DLS cuvette. The DLS scans were run after 1−2 min incubation time at the set temperature. The 2D-NOESY NMR measurements were obtained by dissolution of the isolated CCS products directly into D2O at a concentration of 10 mg mL−1. Cytotoxicity Studies. Cell Culture. Human embryonic kidney (HEK293T) cells were cultivated in DMEM medium (supplemented with 10% FBS, 1× GlutaMAX, and 1× penicillin-streptomycin) in a humidified atmosphere containing 5% CO2 at 37 °C. Cells were seeded in a T75 flask (ca. 3 × 106 cells mL−1) and passaged twice a week prior to the performance of the subsequent cell viability studies. Cell Viability Studies. Adherent HEK293T cells were grown to 80% confluence and trypsinized prior to assay. The cells were counted on a cell counter (Coulter Particle Counter Z series, Beckman Coulter), diluted with “complete” DMEM medium and seeded in a 96-well plate at a density of 10 000 cells per well. The cells were incubated at 37 °C in 5% CO2 for 24 h. Spent medium was removed. Varying concentrations of isolated CCS polymers were prepared in “complete” DMEM medium (100 μL) and incubated with cells at 37 °C in 5% CO2 for a further 48 h. After 48 h, 20 μL of MTS solution was added to each well. Plates were further incubated at 37 °C in 5% CO2 for 2 h. The absorbance at 490 nm was measured with a plate reader (PerkinElmer 1420 Multilabel Counter VICTOR). Two independent runs of the assay were conducted, and two replicates were used in each run for each polymer concentration. Cells that were untreated were used as positive growth control. Percentage viability of cells was calculated using the formula

% viability =

Scheme 1. One-Pot Synthesis of PNIPAM and PHEA-Derived CCS Polymers in Pure Water

Figure 1. (a) GPC DRI chromatograms of the PNIPAM macroinitiator and corresponding CCS products made with three different ratios of [CL]/[MI]. (b) DLS mass distributions of PNIPAM CCS polymers (1 mg mL−1 in water).

out to generate highly active, heterogeneous Cu0 particles as the “activator” as well as equimolar amounts of CuBr2/Me6TREN complexes as the “deactivating” species.31 This suspension was cooled to 0 °C in an ice/water bath before the addition of a degassed aqueous solution of monomer and initiator. The initial monomer-to-initiator ratio was set at 80 for all cases, generating linear polymers with number-averaged molecular weights (Mn) of 8600−9800 Da. The rapid polymerization of the water-soluble monomers allowed for the preparation of “living” MIs with high end-group fidelity at (near) full monomer conversion within 30− 90 min, as confirmed by 1H NMR analysis. Once high monomer conversions (generally >99%) had been reached for the linear MI, a degassed aqueous solution of the cross-linker N,N′methylenebis(acrylamide) was added via degassed syringe to initiate the cross-linking and hence star formation step. Progress of star formation was monitored closely by both 1H NMR spectroscopy and GPC analysis. It is important to note that throughout both the MI synthesis and the star forming step, the reaction flask was kept at 0 °C. This sub-ambient reaction temperature is employed to reduce the rate of bromine chain-end hydrolysis.31 Minimizing this loss of active polymer chain-end is essential in the current work, as it helps to maximize the star conversion by allowing a higher percentage of linear MIs to participate in the cross-linking reaction. For PNIPAM CCS polymers, high star conversions (88−96%) were attained within 1−3 h after the addition of the cross-linker. Specifically, the effect of cross-linker-to-macroinitiator ratio ([CL]/[MI]) was investigated. The molecular weight of the CCS polymers generally increased with increasing [CL]/[MI] as expected (Figure 1a and Table 1).23 However, at the lowest [CL]/[MI] ratio investigated (8:1), only 88% star conversion is

A490 test sample − A490 background × 100 A490 cells alone − A490 background

where A490 refers to the absorbance value at a wavelength of 490 nm.

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RESULTS AND DISCUSSION Homoarm CCS Polymers. The synthetic pathway for the generation of CCS polymers in water is illustrated in Scheme 1. As shown in Scheme 1, two model polymers, namely poly(Nisopropylacrylamide) (PNIPAM) and poly(2-hydroxyethyl acrylate) (PHEA), were employed to demonstrate the efficacy of our one-pot two-step CCS formation approach. Initially, predisproportionation of CuBr/Me6TREN complex was carried C

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Table 1. Summary of GPC and DLS Data of All CCS Polymers Synthesized in This Study code

MI

Mn,MIa (Da)

CL/MIb

star convc (%)

Mn,stard (kDa)

Đstard

Narme

Dhf

H1 H2 H3 H4 H5 M6 M7

PNIPAM PNIPAM PNIPAM PHEA PEG PNIPAM/PHEA PNIPAM/PEG

9700 10300 9650 8300 7000 10400/12000 9800/7000

8 10 12 10 10 12 12

88 96 94 92 89 88 90

134 188 272 450 243 701 271

1.12 1.10 1.11 1.31 1.11 1.35 1.15

14 20 29 46 44 63 29

13.6 15.0 14.4 20.0 16.3 19.4 13.8

a

Calculated from GPC using narrow molecular weight standards. bMolar ratio of cross-linker (CL) to macroinitiator (MI). cDetermined based on the ratio of integral area of star and arm peaks in GPC DRI chromatograms. dCalculated based on the GPC light scattering data. eCalculated using equations described in the Experimental Section. fNumber-averaged hydrodynamic diameter (Dh) measured using DLS.

Figure 2. 1H NMR (D2O) of (a) crude PNIPAM macroinitiator after 30 min and (b) the in situ formed CCS polymer after a further 3 h reaction time.

obtained compared to ≥94% when higher ratios of [CL]/[MI] were used. The correlation between low star conversion efficiency with low [CL]/[MI] is due to the higher propensity of propagating chains to undergo termination events over propagation/cross-linking reactions. This results in a higher population of “dead” linear species that are unable to partake in the star formation process. A [CL]/[MI] ratio of 10 was shown to be sufficient for near complete incorporation of MIs into the star structure (96%). Increasing [CL]/[MI] to 12 gave the same star conversion within experimental error, indicating that 10:1 is sufficient in generating high yielding stars using the present approach. Additionally, the average number of arms (Narm) per star molecule can be tailored depending on the [CL]/[MI]

(Table 1). In general, the dispersity (Đ) values of the PNIPAM MI and star products are low (Đ ≤ 1.12) (Table 1, H1−H3), while the symmetrical GPC differential refractive index (DRI) chromatograms of the star species indicate the absence of star− star coupling or aggregation and hence the well-controlled nature of the polymerizations (Figure 1a). The sizes of the CCS products were also investigated via DLS using 0.1 wt % solutions of the isolated products prepared directly in pure water. Changing the [CL]/[MI] had little effect on the measured hydrodynamic diameter (Dh) in solution (Figure 1b). However, PNIPAM CCS made with the lowest [CL]/[MI] of 8:1 shows a slightly smaller Dh than those made with higher [CL]/[MI] (13.6 D

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taken to near full monomer conversion. The spectrum obtained after star formation is almost identical to that of linear PNIPAM MI (Figure 2b). This is not surprising given that the cross-linked moieties in the core are not only highly shielded by the radiating “arms”, but also have very low chain mobility in solution, and therefore the corresponding spectroscopic signals of the core protons are greatly reduced in intensity. Most importantly, the 1 H NMR spectrum of the CCS polymer showed the complete absence of peaks corresponding to vinyl protons, which indicates that the cross-linker has been fully incorporated into the CCS polymer structure, along with any remaining monomer species. A model water-soluble acrylate monomer, 2-hydroxyethyl acrylate (HEA), was also employed to generate PHEA-based CCS polymers in a similar fashion. The PHEA MI proceeded with good star conversion efficiency (92%) to generate polyacrylate CCS polymers in high yields with low dispersity (Đ ∼ 1.31) (Figure 3a). DLS revealed that the size of the PHEA CCS polymer in solution (Dh = 20.0 nm) to be slightly larger than the PNIPAM-based products (Figure 3b and Table 1). This is due to the higher number of arms per star (Narm ∼ 46) in the PHEA CCS product and hence the larger overall dimensions in solution. We also investigated the possibility of using a premade, isolated MI to form CCS polymers via our approach. In demonstrating this, a commercially available poly(ethylene glycol) monomethyl ether (PEG) (Mn ∼ 5000 Da) was functionalized with an α-bromoester initiating group and subsequently employed in the star formation process using the above-mentioned protocols, which include the key copper predisproportionation step. The cross-linking reaction was carried out at 0 °C, and star formation was monitored via 1H NMR spectroscopy and GPC analysis. As with the polyacrylate and polyacrylamide MIs, the PEG MI was shown to achieve high star conversions (89%) within 1 h (Figure 3c). The Dh of the PEG CCS product is 16.3 nm as measured by DLS (Figure 3d), with a calculated Narm of 44. Closer inspection of Table 1 revealed that the star polymers made with different types of MI (i.e., H2, H4, and H5) possess different Dh and Narm even though the same [CL]/[MI] of 10 was used. The PEG-based polymer may be expected to have a high Narm due to the smaller size of the MI species (7 kDa, compared to 8−10 kDa for the polyacrylate/ acrylamide MIs). However, it is thought that the difference in random coil configuration adopted by each arm type in solution will influence the ability of MIs to incorporate into a growing star polymer, creating different levels of steric barrier for each MI to overcome during star formation and hence ultimately influencing the value of Narm.38 Miktoarm CCS Polymers. For increasing the structural complexity of the generated ONPs, we have combined two different types of MIs in a single cross-linking step to generate miktoarm star polymers. This can lead to the design of nanostructures with multiple functionalities incorporated into a single particle, providing an efficient pathway toward functional ONPs for targeted applications. Tailoring of functional group density (on the peripheries) can also be readily achieved by simply adjusting the molar ratio between the different linear MIs used or the [CL]/[MI]total to form stars with controllable Narm. The formation of miktoarm CCS structures was shown to proceed with high yields (>88%) using the current polymerization technique, with high consumption of both MI species. Two different approaches in forming miktoarm CCS polymers have been demonstrated. In the first approach, two different linear MIs were prepared simultaneously in separate flasks via the

Figure 3. (a) GPC DRI traces of PHEA MI (dashed) and PHEA CCS product (solid). (b) DLS of PHEA CCS (1 mg mL−1 in water). (c) GPC DRI traces of PEG macroinitiator (dashed) and PEG CCS product (solid). (d) DLS of PEG CCS (1 mg mL−1 in water).

Figure 4. (a) GPC DRI chromatograms of PNIPAM MI (red), PHEA MI (blue), and PNIPAM0.5-PHEA0.5 miktoarm CCS polymer (black) after 3 h. (b) DLS mass distribution of PNIPAM0.5-PHEA0.5 miktoarm CCS polymer (1 mg mL−1 in water). (c) GPC DRI chromatograms of PNIPAM MI (red), PEG MI (blue), and PNIPAM 0.66-PEG0.33 miktoarm CCS polymer (black) after 3 h. (d) DLS mass distribution of PNIPAM0.66-PEG0.33 miktoarm CCS polymer (1 mg mL−1 in water).

vs 15.0 nm), because of the fewer Narm per star molecule and smaller core size (Table 1). A typical 1H NMR spectrum of the reaction mixture immediately prior to the addition of the cross-linker (at [CL]/ [MI] = 10) during the synthesis of PNIPAM-based CCS polymers is shown in Figure 2a. Vinyl resonances corresponding to unreacted monomer (at δH = 5.0−6.5 ppm) make up less than 1% of the observed NIPAM signal as determined by peak integration, indicating that the synthesis of PNIPAM MI was E

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Figure 5. 2D-NOESY 1H NMR contour plots for 1 wt % solution of PNIPAM0.5-PHEA0.5 miktoarm CCS polymer in D2O at (a) 28 °C and (b) 35 °C. PNIPAM (red) and PHEA (blue) peaks of interest are circled, with arrows indicating potential cross-peaks when the two chains are in close proximity.

above-mentioned procedures. The reaction mixtures were then combined into a single flask along with the cross-linker to initiate the cross-linking and hence star-forming step. For example,

PNIPAM and PHEA linear MIs were combined in a 1:1 molar ratio, with a 12:1 ratio of [CL]/[MI]total (Table 1, M6). GPC analysis indicates high star conversion (88%) (Figure 4a), and F

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Figure 7. Percentage of living HEK239T cells after exposure to different concentrations of various CCS polymers.

Indirect evidence that the star structures are in fact mikto (or hetero) arm in nature, and not simply a mixture of homoarm stars, was determined using 2D-NOESY NMR spectroscopy. Because of the thermoresponsive behavior of PNIPAM (having lower critical solution temperature (LCST) of around 32 °C in water), we would expect the PNIPAM chains to phase-separate above this temperature, even when bound to the confines of the star structure. However, given that the star also contains an equimolar amount of PHEA (or PEG) covalently bounded to the same central core, we may predict some internal phase segregation within each individual star. Thus, this would lead to anisotropic, asymmetrical ONPs with possible Janus-type morphology. Through-space homonuclear proton coupling has been used in other studies to indicate phase segregation of polymer chains within the corona of micelle-like structures.39,40 In a similar fashion, we performed 2D-NOESY NMR spectroscopy experiments to determine if phase segregation within the star structure was actually occurring above the LCST, which would indicate that the star is in fact miktoarm in nature. In analyzing the 2D-NOESY NMR spectrum of CCS polymer M6 at 28 °C, a cross-peak representing some degree of interchain mixing was observed below the LCST of PNIPAM, indicating that the PNIPAM and PHEA chains are freely interacting with one another without any signs of phase segregation (Figure 5a). When the temperature was raised to above its LCST at 35 °C, the cross-peak which was present at 28 °C disappeared, strongly indicating the occurrence of phase segregation within the star. Noteworthy, the spectroscopic conditions for each temperature are identical (i.e., number of scans, relaxation time, sample, etc.), and so direct comparison between the two spectra can be made. This result reinforces the proposed internal phase segregation model39,40 and also provides indirect evidence that the stars are heteroarm in nature. Stimuli-Responsive Self-Assembly of Miktoarm CCS Polymer. Anisotropic (i.e., asymmetrical) structures are well-known to aggregate in a controlled manner in order to minimize unfavorable interactions to lower the overall enthalpy of the system. The most common example of such behavior is perhaps the solution self-assembly of a surfactant, or diblock copolymer, into micelle or vesicular structures.41 However, more recently, examples on the assembly of anisotropic colloidal particles to generate assemblies that occupy much larger size domains have

Figure 6. (a) Proposed internal phase segregation and pathway for star self-assembly (note: final supramolecular structure is a schematic depiction only; work is currently being undertaken to elucidate exact morphology). (b) Temperature-responsive reversible aggregation of PNIPAM0.5-PHEA0.5 miktoarm CCS polymers in water (1 mg mL−1) at temperatures above and below the LCST of PNIPAM (32 °C)three cycles shown. (c) Visualization of rapid reversible aggregation: (i) sample at room temperature; (ii) application of heat (from body temperature) for 1 min; (iii) localized aggregation observed by change in solution transmittance; (iv) sample reverts back to its initial state after 15 s.

the purified/lyophilized PNIPAM0.5-PHEA0.5 miktoarm CCS polymer has a Dh of 19.4 nm as measured by DLS (Figure 4b). The size of the PNIPAM0.5-PHEA0.5 CCS polymer is much closer to that of the homoarm PHEA stars rather than the PNIPAM because of the high Narm. In the second approach, one linear MI (PNIPAM) was synthesized according to the standard procedure, followed by the addition of a degassed solution containing presynthesized MI (PEG) and cross-linker to initiate miktoarm star formation. For this reaction, a 2:1 molar ratio of PNIPAM to PEG was used, with a 12:1 ratio of [Cl]/[MI]total (Table 1, M7). The PNIPAM0.66PEG0.33 miktoarm stars were formed in high yield (90%), with high consumption of both MI species (Figure 4c). The Dh of the isolated product is 13.8 nm as measured by DLS (Figure 4d), while the Narm is 29. Regardless of the approaches used, the synthesized miktoarm CCS polymers have relatively low Đ (80%) even at high polymer concentrations of up to 2 mg mL−1 (Figure 7). These findings indicate that the presented methodology provides a facile route to complex, biocompatible ONPs.

wide variety of complex functional ONPs can be developed. Simple purification and isolation protocols combined with the use of water as polymerization medium make this approach a potentially promising technique for commercial bioapplications.

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ASSOCIATED CONTENT

* Supporting Information S

Experimental details; Figures S1−S4; calculation of number of arms. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected] (G.G.Q.). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors acknowledge financial support from the Australian Research Council via the Future Fellowship (FT110100411, G.G.Q.) scheme. T.M. is the recipient of an Australian Postgraduate Award (APA). S.J.L. acknowledges the Australian Government for providing an International Postgraduate Research Scholarship (IPRS) and an Australian Postgraduate Award (APAInt).

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REFERENCES

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CONCLUSION A facile, versatile, and highly efficient procedure for the production of well-defined PNIPAM, PHEA, and PEG CCS polymers/ONPs in pure water via copper-mediated reversibledeactivation radical polymerization using fully water-soluble monomers and cross-linkers is demonstrated. In addition, miktoarm stars composed of PNIPAM arms undergo reversible thermally activated supramolecular assembly, as confirmed by 2D-NOESY NMR and DLS analysis. Cell viability tests performed on a wide range of synthesized ONPs show that they are relatively nontoxic, even up to high polymer concentrations (2 mg mL−1). The ease with which the functionality of the product can be tuned, for example, by simply controlling the molar ratio of [CL]/[MI] or between MIs of different species, demonstrates a strong platform from which a H

dx.doi.org/10.1021/ma502008j | Macromolecules XXXX, XXX, XXX−XXX

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dx.doi.org/10.1021/ma502008j | Macromolecules XXXX, XXX, XXX−XXX