Segmented Highly Branched Copolymers: Rationally Designed

Cheng Zhang , Shehzahdi Shebbrin Moonshi , Yanxiao Han , Simon Puttick , Hui Peng , Bryan John Abel Magoling , James C. Reid , Stefano Bernardi , Debr...
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Segmented Highly Branched Copolymers: Rationally Designed Macromolecules for Improved and Tunable 19F MRI Kewei Wang, Hui Peng, Kristofer J. Thurecht, Simon Puttick, and Andrew K. Whittaker* Australian Institute for Bioengineering and Nanotechnology; Centre for Advanced Imaging; ARC Centre of Excellence in Convergent Bio-Nano Science and Technology, The University of Queensland, St. Lucia, Queensland 4072, Australia S Supporting Information *

ABSTRACT: Highly branched polymers are a promising platform for the design of next-generation contrast agents for 19 F magnetic resonance imaging (MRI). A series of segmented highly branched polymers (SHBPs) consisting of fluoro- and PEG-based monomers were synthesized by self-condensing vinyl copolymerization (SCVP) using the reversible addition− fragmentation chain transfer (RAFT) technique. SHBPs having different compositions and degrees of branching were obtained by varying the monomer type and feed ratio of monomer to chain transfer agent (CTA). The chemical structures and physical properties of the branched polymers were thoroughly characterized in detail by NMR, SEC and DSC. The systematic variation in structural parameters allowed the relationships between molecular structure, sequence distribution, and imaging performance to be examined. The 19F NMR properties were strongly affected by the sequence distribution of the fluorinated monomers, the type of polymer backbone and the degree of branching. As a result, SHBPs consisting of statistical copolymeric segments of acrylate units were identified as excellent candidates for imaging due to a single 19F signal, long T2 relaxation times, and high fluorine contents. The SHBPs could be all imaged or selectively imaged by taking advantage of the differences in relaxation times, demonstrating tunable and selective imaging performance through tailoring the structure and composition of the SHBPs.

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reduced chemical shift imaging artifacts;10,11 (2) a short spin− lattice (T1) relaxation time for reduced scan time and long spin−spin (T2) relaxation time for increased image intensity in the case of the spin−echo pulse sequence.10 In recent years, polymeric CAs have attracted significant attention for 19F MRI due to the diverse functionalities and architectures possible with polymers.12,13 A variety of partly fluorinated polymeric CAs have been reported, including linear polymers,14−19 star polymers,20,21 dendritic polymers,22−25 hyperbranched polymers (HBPs),26−30 polymer nanogels,31−33 and so on. From among these classes of CAs, HBPs have been considered to be excellent candidates for 19F MRI, in particular, the HBPs synthesized via reversible addition−fragmentation chain transfer (RAFT) polymerization.26,27,30 Specifically, these novel HBPs display a number of attractive properties, including a high 19 F payload (up to 20 mol % fluorinated monomer) can be achieved while maintaining water solubility. Second, the strong tendency of fluorinated groups to aggregate in aqueous solution can be frustrated by positioning fluorine-containing segments within a 3D hyperbranched structure, leading to high 19F mobility and, hence, strong signal intensity. Third, the abundant functionalities (alkyne and trithiocarbonate groups) provided

INTRODUCTION Magnetic resonance imaging (MRI), which was developed in the early 1970s,1 has advanced rapidly in recent decades. Due to advantages of noninvasiveness, relatively high resolution, rich information content, and lack of radiation burden,2,3 MRI has become an indispensable diagnosis technique in both the clinic and in medical research. As a leading MRI modality, 1H MRI has been dominant in clinical scans because it can generate 3D anatomical images, particularly for soft tissues.4 However, limited sensitivity and contrast requires, in some instances, the use of a contrast agent, usually administered intravenously. These 1H contrast agents work by altering the relaxation times of protons in water molecules within surrounding tissue in a manner that can lead to contrast on use of an appropriate imaging sequence. Emerging only four years later than 1H MRI,5 19F MRI is recognized as a promising modality to complement 1H MRI. The most significant feature of 19F MRI is the physiological rarity of detectable 19F in the body, which minimizes confounding background signal during imaging.6,7 Therefore, 19F MRI is more advantageous in quantification and tracking, such as measurement of the partial pressure of oxygen (pO2), cell labeling, cell tracking, and so on.8,9 19 F MRI highly relies on the use of contrast agents (CAs), which introduce exogenous 19F to the body. A successful 19F MRI CA should fulfill the following two basic criteria: (1) a single and narrow 19F resonance for maximum sensitivity and © XXXX American Chemical Society

Received: June 16, 2015 Revised: July 26, 2015

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DOI: 10.1021/acs.biomac.5b00800 Biomacromolecules XXXX, XXX, XXX−XXX

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by the RAFT end groups can be utilized for conjugation reactions to attach targeting/drug molecules as well as other imaging moieties. Such RAFT-synthesized HBPs therefore represent a straightforward approach for the design of 19F MRI CAs. The conventional RAFT synthesis of HBPs requires the use of bifunctional monomers (or cross-linkers).34 Under optimized experimental conditions, well-defined HBPs with reasonably narrow molar mass dispersity can be obtained.35−38 Nevertheless, the structure of the resultant HBPs can be influenced by a number of factors related to the cross-linker (e.g., cross-linker type, cross-linker/chain transfer agent (CTA) ratio, cross-linker concentration), and sometimes unwanted macroscopic gelation occurs.39−41 An alternative approach to the synthesis of HBPs is self-condensing vinyl polymerization (SCVP), which utilizes initiator monomers (inimers).42−44 When the inimer is copolymerized with other conventional vinyl monomers, the degree of branching (DB) is dependent on the comonomer/inimer ratio (γ) and monomer conversion. In the past decade, RAFTmediated SCVP (RAFT SCVP) has been developed for the controlled synthesis of HBPs with a myriad of branched architectures and functionalities.45−56 It is also worth noting that RAFT SCVP is a robust strategy for the design of segmented branched polymers, which are composed of linear polymer chains and, thus, possess unique properties due to chain entanglement.57 As a result of the properties of the RAFT technique,58 HBPs synthesized by RAFT SCVP have controllable topologies, multifunctional groups, and relatively narrow molar mass dispersity. For example, by using a polymerizable trithiocarbonate chain transfer agent (CTA), Gao and co-workers synthesized segmented hyperbranched polymers (SHBPs) of poly(glycidyl methacrylate), bearing highly dense and heterofunctional groups, as a versatile platform for a collection of standard click chemistries.53 Zhao and co-workers explored RAFT SCVP for the synthesis of hyperbranched and star polymers via one or two steps.49 Methyl methacrylate (MMA), styrene (St), methyl acrylate (MA), and tert-butyl acrylate (tBA) were selected to copolymerize with a polymerizable CTA (S-(4-vinyl)benzyl S′propyltrithiocarbonate). The DB and branch length of HBPs were tunable, and the arm length of star polymers was controllable, confirming that RAFT SCVP is a robust approach to synthesize hyperbranched and star polymers with desired segmental and arm composition. Although a number of HBP-based CAs have been developed for 19F MRI, more detailed investigations are needed to understand how imaging properties are regulated by the specific hyperbranched structure. It would be especially challenging to examine HBPs synthesized using a cross-linking monomer because of the complexity of the structures formed. By comparison, partly fluorinated SHBPs synthesized by RAFT SCVP offer a simpler model for a systematic study due to the facile control over polymer structure as well as chain topology. To date, such polymers have not been reported for application as 19 F MRI agents. Therefore, in this work we report the synthesis of water-soluble, partly fluorinated SHBPs via RAFT SCVP using a polymerizable trithiocarbonate CTA. SHBPs with different degrees of branching and sequence distributions were prepared. A robust methodology was thus established for the design of SHBPs with targeted branched structure and composition. One particular aim is to determine how the hyperbranched structure, sequence distribution, and composition affect the 19F NMR properties. This understanding will allow the synthesis of partly fluorinated SHBPs with tailored structure and properties for selective and tunable 19F MRI.

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EXPERIMENTAL SECTION

Materials. All chemicals were purchased from Sigma-Aldrich unless otherwise stated. Poly(ethylene glycol) methyl ether methacrylate (PEGMA, MW = 475), poly(ethylene glycol) methyl ether acrylate (PEGA, MW = 480), 2,2,2-trifluoroethyl methacrylate (TFEMA), 2,2,2-trifluoroethyl acrylate (TFEA), and 2-hydroxyethyl methacrylate (HEMA) were passed through basic alumina columns to remove inhibitors prior to use. The initiator, 2,2′-azobis(2-methylpropionitrile) (AIBN), was recrystallized from ethanol twice before use. 4(Dimethylamino)pyridine (DMAP) and N,N′-dicyclohexylcarbodiimide (DCC, purchased from Alfa Aesar) were used as received. The trithiocarbonate RAFT agent, 4-cyano-4-(dodecylsulfanylthiocarbonyl)sulfanyl pentanoic acid was synthesized and purified according to a previously reported method.59 Milli-Q water with a resistivity of 18.4 MΩ cm−1 was used for the relevant experiments. Tetrahydrofuran (THF) and dichloromethane (DCM) were obtained from a solvent purification system (MB-SPS-800-Auto, Mbraun) and used directly. All other solvents were of analytical grade. Synthesis of the Polymerizable CTA, (S)-2-(Methacryloyloxy)ethyl 4-Cyano-4-(((propylthio)carbonothioyl)thio)pentanoate (MECP). 4-Cyano-4-(dodecylsulfanylthiocarbonyl)sulfanyl pentanoic acid (8.07 g, 0.02 mol), HEMA (5.21 g, 0.04 mol), and DMAP (0.489 g, 0.004 mol) were dissolved in 200 mL of DCM in a 500 mL roundbottom flask immersed in an ice bath. DCC (5.25 g, 0.04 mol) in 50 mL of DCM was added dropwise to the above solution. The resultant mixture was stirred at 0 °C for 1 h and then at room temperature for 24 h. After reaction, the mixture was filtered to remove the insoluble dicyclohexylurea byproduct. The filtrate was washed with 1 M HCl solution (200 mL × 3) and saturated NaHCO3 aqueous solution (200 mL × 3) and then dried over anhydrous MgSO4 overnight in a freezer. The solution was then filtered again and concentrated by rotary evaporation. The crude product was purified by flash column chromatography (silica gel, 230−400 mesh) using petroleum spirit (40−60)/ethyl acetate mixture (gradient from 9/1 to 7/3) as eluent. After removal of the solvents, a red oil was obtained and stored in a freezer away from light. Yield: 7.15 g, 69%. 1H NMR (500 MHz, CDCl3, ppm): 6.13 and 5.6 (s, 2H, CH2(CH3)C), 4.36 (m, 4H, COOCH2CH2), 3.32 (t, 2H, CH2CH2S), 2.36−2.56 (m, 4H, CH2CH2COO), 1.95 (s, 3H, CH2C(CH 3)COO), 1.87 (s, 3H, SCH 3C(CN)CH2), 1.69 (m, 2H, CH2CH2S), 1.26−1.40 (m, 18H, CH3(CH2)9CH2CH2S), 0.88 (t, 3H, CH3(CH2)9CH2CH2S). 13C NMR (125 MHz, CDCl3, ppm): 217.22, 176.61, 167.43, 136.22, 126.54, 119.30, 63.08, 62.54, 46.67, 37.43, 34.16, 32.26, 30.02, 29.96, 29.89, 29.76, 29.68, 29.41, 29.28, 28.02, 25.22, 23.03, 18.63. Synthesis of Segmented, Highly Branched Polymers via RAFT SCVP. A typical polymerization is described as follows. PEGMA (1.43 g, 3 mmol), TFEMA (0.336 g, 2 mmol), MECP (51.6 mg, 0.1 mmol), and AIBN (3.28 mg, 0.02 mmol) were dissolved in 1 mL of 1,4-dioxane in a 10 mL tube placed with a magnetic stir bar. The relative amounts of PEGMA/TFEMA/MECP/AIBN = 30/20/1/0.2, and the comonomer/MECP ratio (γ) = 50. The tube was sealed with a rubber septum and purged with argon for 15 min, followed by immersing in a 70 °C oil bath for 24 h. The polymerization was then quenched by immersing the flask in an ice bath and exposing to air. The conversions of PEGMA, TFEMA, and MECP were found to be all above 97% according to 1H NMR. The crude solution was diluted with THF and precipitated in cold hexane three times. A yellowish viscous oil was collected after drying at 35 °C under a vacuum for 24 h. Yield: 1.46 g, 80%. Following this procedure, a series of SHBPs was synthesized under the same conditions by varying the comonomer type and comonomer/ MECP ratio. The characteristics of the polymers are listed in Table 1. Synthesis of Linear Statistical Copolymer Poly(TFEA-coPEGA). PEGA (1.44 g, 3 mmol), TFEA (0.308 g, 2 mmol), 4-cyano-4(dodecylsulfanylthiocarbonyl)sulfanyl pentanoic acid (40.4 mg, 0.1 mmol), and AIBN (3.28 mg, 0.02 mmol) were dissolved in 1 mL of 1,4-dioxane in a 10 mL tube placed with a magnetic stir bar. After purging with argon for 15 min, the tube was placed in a 70 °C oil bath for 24 h. The polymerization was then quenched by immersing the flask in an ice bath and exposing to air. The crude sample was B

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Biomacromolecules Table 1. Molecular Characteristics of the SHBPs Synthesized by RAFT SCVP sample

PEG(M)A:TFE(M)A:CTAa

Mna

Mnb (kDa)

ĐM b

DBc

RBc

NCTAd

SHBP-1 SHBP-2 SHBP-3 SHBP-4 SHBP-5 SHBP-6 SHBP-7 SHBP-8 SHBP-9 SHBP-10 SHBP-11 SHBP-12 SHBP-13 SHBP-14 SHBP-15 LP-11

52:7:1 (PEGMA + TFEMA) 47:12:1 (PEGMA + TFEMA) 35:17:1 (PEGMA + TFEMA) 30:22:1 (PEGMA + TFEMA) 25:26:1 (PEGMA + TFEMA) 16:26:1 (PEGMA + TFEMA) 45:13:1 (PEGMA + TFEA) 48:15:1 (PEGA + TFEMA) 39:12:1 (PEGA + TFEA) 37:17:1 (PEGA + TFEA) 34:22:1 (PEGA + TFEA) 24:35:1 (PEGA + TFEA) 4:3:1 (PEGA + TFEA) 7:5:1 (PEGA + TFEA) 14:11:1 (PEGA + TFEA) 39:23:1 (PEGA + TFEA)

26400 24900 20000 18400 16800 12500 23900 26100 20600 20400 19700 16900 2900 4600 8900 22700

1440 690 1030 820 510 440 1030 430 370 230 210 130 140 160 270 13e

2.2 3.6 2.3 2.6 3.8 3.6 2.5 1.8 1.6 2.1 2.0 2.0 1.7 1.2 1.2 1.43e

0.033 0.033 0.037 0.037 0.038 0.045 0.033 0.036 0.038 0.036 0.034 0.033 0.219 0.142 0.074 0

30 30 27 27 26 22 30 28 26 28 29 30 5 7 14

55 28 51 45 30 35 43 16 18 11 11 8 49 35 31 1

a

Theoretical degree of polymerization (DP) was determined by 1H NMR. Mn (1H NMR) of a single polymer chain was calculated by using the equation Mn = MWPEG(M)A × DPPEG(M)A + MWTFE(M)A × DPTFE(M)A + MWMECP, assuming the polymerizations completely followed the RAFT process. bAbsolute MW and ĐM were measured by SEC MALLS. cDegree of branching (DB) was calculated by the equation introduced in the main text. Repeating unit per branch (RB) = 1/DB. dTheoretical number of trithiocarbonate functionality per SHBP (NCTA) = Mn (SEC MALLS)/Mn (1H NMR). eMeasured by SEC RI detector. All the SHBPs were synthesized in 1,4-dioxane at 70 °C for 24 h with [monomer] = 5 M and [MECP]/ [AIBN] = 5/1. diluted with THF and precipitated in cold hexane three times. The collected sample was dried at 35 °C in vacuum for 24 h. A yellowish viscous oil was obtained. Yield: 1.1 g, 62%.

Then A-b-M* can undergo two possible reactions through the addition of either inimer or comonomers at rate constants of KMA and KMM, respectively. Meanwhile, through similar monomer additions, A-b-A*-B* can be reacted with inimer and comonomers in six routes at its two activated sites A* and B*, resulting in the formation of the six depicted structures (Scheme 1) at rate constants of KAA, KAM, KBA, and KBM. As the polymerization proceeds, highly branched macromolecules composed of linear chains are gradually produced via these reactions. According to the nature of the RAFT process, the equilibrium between the active propagating radicals and dormant thiocarbonylthio compound is very fast. Hence, the chain growth progresses in a controlled fashion, yielding polymer segments with narrow molar mass distribution as well as the formation of branched structures.60 It should also be mentioned that in SCVP the degree of branching and degree of polymerization are dependent on [comonomer]/[inimer] ratio and monomer conversion, while the highly branched structure is affected by the monomer reactivity ratios.57 Therefore, by carefully choosing the experimental parameters, highly branched polymers with a targeted degree of branching, molecular weights, and structures can be synthesized. The detailed synthetic route is described in Scheme 2. 2,2,2Trifluoroethyl methacrylate (TFEMA) and 2,2,2-trifluoroethyl acrylate (TFEA) were selected as fluoro monomers, while poly(ethylene glycol) methyl ether methacrylate (PEGMA) and poly(ethylene glycol) methyl ether acrylate (PEGA) were chosen as PEG-based oligomers. Through RAFT SCVP using a polymerizable trithiocarbonate CTA (MECP), segmented highly branched p(TFE(M)A-co-PEG(M)A) were prepared. According to the mechanism, each SHBP macromolecule theoretically contains an unreacted vinyl group and numerous trithiocarbonate end groups. As described above, the SHBPs prepared here are intended for 19F MRI as CAs. A number of previous publications have pointed out that high 19F segmental mobility is of importance for achieving long spin−spin relaxation (T2) times, which are

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RESULTS AND DISCUSSION Polymerization Mechanism and Molecular Design. The aim of this work is to study the use of RAFT SCVP for the synthesis of segmented highly branched polymers (SHBPs) that are partly fluorinated and water-soluble for 19F MRI applications. A polymerizable RAFT agent, (S)-2-(methacryloyloxy)ethyl 4-cyano-4-(((propylthio)carbonothioyl)thio)pentanoate (MECP), was synthesized by the standard DCC/DMAP esterification between 4-cyano-4-(dodecylsulfanylthiocarbonyl)sulfanyl pentanoic acid and HEMA. As shown in Figure 1,

Figure 1. Chemical structure of the polymerizable CTA.

MECP is a typical AB*-type inimer for SCVP, in which B* is a trithiocarbonate function for the RAFT polymerization of the vinyl group A and the other comonomers. Therefore, MECP can be exploited for controlled polymerization via the RAFT process as well as the simultaneous formation of branching points through the A groups. As illustrated in Scheme 1, the polymerization process follows the typical reaction mechanism of a SCVP.57 In the scheme, the lower case letter, “b”, refers to the reacted trithiocarbonate. “M” and “m” stand for the unreacted and reacted monomer, respectively. Upon initiation through the decomposition of AIBN, A-b-M* is generated through the chain transfer of propagating radicals of comonomers M at a rate constant of KBM, while A-b-A*-B* is formed by the chain transfer of propagating radicals of inimer A-B* at a rate constant of KBA. C

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Biomacromolecules Scheme 1. Reaction Mechanism for RAFT SCVPa

A: vinyl group of MECP. B: trithiocarbonate function of MECP. M: comonomers TFE(M)A and PEG(M)A. Lowercase letters “a” and “b” stand for reacted A and B, respectively. Asterisks represent activated sites for the addition of monomers and inimer.

a

Scheme 2. Illustration of the synthesis of SHBPs via RAFT SCVPa

a Lowercase letters “a” and “b” stand for reacted A and B, respectively. “A” refers to unreacted vinyl groups in MECP, and “B*” denotes trithiocarbonate groups that have not engaged in the RAFT process.

preferred for 19F MRI.15,17 To achieve high 19F mobility in water, the fluorinated segments should be hydrated and separated from each other to avoid association. One strategy to

achieve this is to design statistical copolymers composed of units of fluoro monomers and hydrophilic PEG-based oligomers such as PEGMA and PEGA. Consequently, two issues are D

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formation of highly branched macromolecules via the polymerization of vinyl groups of MECP. Nevertheless, the conversion of the vinyl group of inimer could not be identified precisely by 1 H NMR because its characteristic peaks were significantly overlapped with those of PEGMA units. According to the SEC results, the RAFT SCVP exhibited hybrid chain-growth/stepgrowth behavior that could be described by two steps.57 In the first step (0−6 h), due to the much higher concentration of TFEMA and PEGMA compared with MECP, RAFT-mediated chain growth proceeded with rapidly increasing monomer conversions, yielding lightly branched polymers (macroinimers) with narrow molar mass distribution. In the second step (6−48 h), after the consumption of most comonomers, the macroinimers started to react with each other through the A group sites, resulting in highly branched polymers with dramatically increased MW, as evidenced by the bimodal SEC curves. In fact, these two processes occur simultaneously, but each step dominates at different stages.57 Similar observations have been reported by other workers.47,48 The effect of monomer concentration was investigated next. As displayed in Figure 3A, single SEC peaks with low MW and narrow ĐM were observed for the polymerization performed at low monomer concentration (0.25 and 0.5 M). When the monomer concentration was above 1 M, bimodal SEC curves were obtained, with a peak becoming more pronounced at MW of ∼105 g mol−1, suggesting that highly branched macromolecules were only produced at relatively high monomer concentrations instead of dilute conditions. We thus assume that a high monomer concentration increased the probability of condensation of macroinimers, facilitating the formation of polymers with highly branched structures. It was also found that an increase in the ratio [comonomer]/ [MECP] (γ) resulted in polymers with higher MW mainly due to the longer chain length between two branching points (Figure 3B). Based on these results, a series of SHBPs were synthesized via RAFT SCVP using a monomer concentration of 5 M. For an in-depth study, both methacrylates and acrylates were chosen to vary the polymer compositions. In addition, different [comonomer]/[MECP] ratios were selected for the synthesis of SHBPs with various degrees of branching. The details of the SHBPs are summarized in Table 1. One benefit of SCVP is that the degree of branching (DB) of the resultant HBPs can be predicted at given [comonomer]/[MECP]

to be addressed. First, the choice of monomers needs to be examined. As reported before,14,15 tapered or block copolymers could cause 19F aggregation in water. In addition to monomer placement, the type of monomer (acrylates or methacrylates) can affect the segmental motion. Thus, certain monomers with similar reactivity ratios are required for the synthesis of statistical copolymers that have highly flexible polymer backbones. A second requirement is that the fluorine content should be maximized. A high 19F payload is preferred for reducing scan time as well as improving image quality, but may lead to aggregation of fluorine-containing units (short T2) and decreased water solubility. Hence, the relationship of 19F payload and 19F mobility needs to be investigated. Following these findings, the next goal is to understand the influence of branched structure on the 19F NMR properties before 19F MRI evaluation. Synthesis of SHBPs via RAFT SCVP. The RAFT SCVP of TFEMA and PEGMA using MECP as CTA was carried out in 1,4-dioxane at 70 °C. The influence of reaction time was first studied by SEC and 1H NMR. As shown in Figure 2, from 2 to

Figure 2. SEC traces of the samples withdrawn at different time intervals during a RAFT SCVP. Inset gives the monomer conversions determined by 1H NMR. Polymerization conditions: [PEGMA]/ [TFEMA]/[MECP]/[AIBN] = 40/10/1/0.2, [monomer]0 = 1 M, 70 °C.

48 h, the SEC traces measured by RI detector evolved toward higher molecular weight with increasing monomer conversions, evidenced by 1H NMR. Meanwhile, owing to the RAFT process, the molar mass dispersity (ĐM, Mw/Mn) remained relatively low at 1.25−1.81. In addition, the monomer conversion reached ∼90% after 6 h with MW of ∼104 g mol−1, indicating a rapid polymerization rate. A shoulder peak in the SEC trace appeared after 6 h with MW of ∼105 g mol−1, suggesting the

Figure 3. SEC traces of RAFT SCVP at different monomer concentrations (A) and [comonomer]/[MECP] ratios. Condition for (A): [PEGMA]/ [TFEMA]/[MECP]/[AIBN] = 40/10/1/0.2, 70 °C, 24 h. Condition for (B): [PEGMA]/[TFEMA] = 4/1, [MECP]/[AIBN] = 5/1, [monomer]0 = 5 M, 70 °C, 24 h. E

DOI: 10.1021/acs.biomac.5b00800 Biomacromolecules XXXX, XXX, XXX−XXX

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Figure 4. NMR spectra of SHBP-4 in CDCl3. (A) 1H NMR spectrum, inset is the expanded area of 5.4−6.4 ppm. (B) 13C NMR spectrum, insets are expanded areas of 221.5−222 and 120−130 ppm.

175.3−177.4 (carbonyl groups), 126.0 and 107.9 (vinyl groups), 128.2 (CN groups), 121.0−125.7 ppm (CF3 groups), and so on. Both 1H and 13C NMR confirmed the successful incorporation of TFEMA, PEGMA, and MECP units into the SHBPs via RAFT SCVP. To get more detailed information, SHBP-4 was characterized by 1H−13C 2D NMR. 1H−13C HSQC can be used to confirm the direct attachment of 1H atoms to 13C atoms through displaying correlations between resonances of 1H and 13C nuclei with one-bond C−H couplings (1JCH). In Figure 5A, the

ratio and monomer conversion. In our study, the DBs of SBHPs can be calculated by the following equations.57 DB =

2(1 − e−(γ + 1)xM)⎡⎣xM − ⎡⎣ 1 − e−(γ + 1)xM / γ + 1 ⎤⎦

(

γxM + 1 − (1 − xM)(2 − e

)(

−(γ + 1)x M

)

) (1)

DB =

2(1 − e−(γ + 1))(γ + e−(γ + 1)) (γ + 1)2

(2)

In eqs 1 and 2, γ and xM refer to [comonomer]/[MECP] ratio and monomer conversion, respectively. Because nearly full monomer conversion was achieved, eq 1 can be simplified to eq 2. Accordingly, the degree of branching was calculated to be between 0.033 and 0.219 (Table 1). It should be stressed that the degree of branching is solely dependent on γ at fixed monomer conversions (e.g., 100%). Hence, it is feasible to obtain targeted DBs by simply using a stoichiometric [comonomer]/ [MECP] ratio. This feature is especially advantageous for the synthesis of SHBPs with tailored compositions and degrees of branching.57 As reported by Sumerlin and Vogt, highly branched poly(N-isopropylacrylamide) with different degrees of branching were prepared through RAFT SCVP by varying the ratio of monomer to CTA.51 Characterization of Structure and End Functionality of SHBPs. The chemical structure of the SHBPs was characterized by NMR spectroscopy. SHBP-4 was chosen as a typical sample for detailed NMR analysis. As displayed in Figure 4A, all the characteristic peaks of TFEMA, PEGMA were identified and assigned. Specifically, the peaks at 4.3 and 4.0 ppm are from CH2 adjacent to the ester bonds of TFEMA and PEGMA units, respectively. The dominant peak at ∼3.6 ppm can be ascribed to the CH2CH2O repeating units of PEGMA segments. Noticeably, the peaks of MECP were also identified, confirming the retention of CTA functionality. The presence of peaks from vinyl bonds is in accord with the aforementioned RAFT SCVP mechanism that each SHBP retains an unreacted vinyl group of MECP. According to 1H NMR, the measured ratio of PEGMA/ TFEMA/MECP in the SHBP-4 was calculated to be 30/22/1, which is very close to the feed ratio 30/20/1. Therefore, the Mn for a single polymer chain was calculated to be 18400 g mol−1 using the equation Mn = MWPEGMA × DPPEGMA + MWTFEMA × DPTFEMA + MWMECP. In addition, 13C NMR (Figure 4B) verified the chemical structure with all the relevant carbon peaks being observed, such as at 221.8 (trithiocarbonate groups),

Figure 5. 1H−13C HSQC spectrum (A) and 1H−13C HMBC spectrum (B) of SHBP-4 in CDCl3. The red circles highlight the correlations that are related to trithiocarbonate group and its neighboring CH2.

characteristic protonated carbons are labeled with numbers, and they can be assigned to the related cross-peaks. For example, the cross-peak 4 (δH = 4.3 ppm, δC = 60.9 ppm) arises from the correlation of resonances of CH2 connected to the ester bond F

DOI: 10.1021/acs.biomac.5b00800 Biomacromolecules XXXX, XXX, XXX−XXX

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Figure 6. 1H NMR spectra of 4 selected SHBPs with expanded region between 4−5 ppm. All spectra were recorded on samples in CDCl3, except that SHBP-7 was additionally characterized in D2O.

in TFEMA units, while cross-peak 5 (δH = 4.0 ppm, δC = 64.3 ppm) is from CH2 next to the ester bond in PEGMA units. It is worth mentioning that the CH2 adjacent to trithiocarbonate was also evidenced by two cross-peaks (red circle; see Supporting Information, Figure S1, for the enlarged spectrum). The spectrum also confirms retention of trithiocarbonate functionality in the SHBPs crucial for either further bioconjugation or chain extension. To be more specific, cross-peak 1 (δH = 3.2 ppm, δC = 37.1 ppm) is from the CH2 next to the reacted trithiocarbonate groups that engaged in the RAFT process, while cross-peak 1′ (δH = 3.3 ppm, δC = 37.2 ppm) can be attributed to the neighboring CH2 of unreacted trithiocarbonate groups that did not participate in the RAFT process. Hence, the coexistence of both reacted and unreacted RAFT species in the highly branched structure, a typical characteristic of RAFT SCVP, was elucidated by the 1H−13C HSQC spectrum. In addition, the correlations of CH2 in vinyl groups can also be confirmed by several cross-peaks (see Supporting Information, Figure S2), indicating different chemical environments of double bonds existing in the complicated highly branched structure. To further study the structure, SHBP-4 was also analyzed by 1 H−13C HMBC experiments, which can provide information about long distance connectivity between 1H and 13C atoms, in particular, the three-bond scalar coupling (3JCH), as well as occasional observation of 2JCH and 4JCH. As shown in Figure 5B, correlations between characteristic 1H−13C pairs were detected and labeled accordingly. The cross-peaks highlighted by a red circle are related to the correlations of 1H atoms in CH2 and 13 C atoms in the neighboring trithiocarbonate groups, providing further proof for the trithiocarbonate functionality in SHBP macromolecules. In addition to NMR analysis, the presence of trithiocarbonate was also confirmed by UV−vis spectroscopy and the UV detector of SEC at wavelength of ∼306 nm (see Supporting Information, Figure S3). Characterization of Chain Sequence Distribution. As stated in the Introduction, one design priority for 19F MRI polymeric CAs is to achieve a single 19F NMR resonance with strong signal intensity. Therefore, the choice of monomers

should be carefully considered as it directly determines the 19F NMR properties of the resulting polymers. In this study, hydrophobic TFEA and TFEMA were selected as fluoro-monomers to provide the 19F NMR signal. Both of these monomers have moderate fluorine contents that can potentially generate strong 19F signals while avoiding association of the fluorinated segments in water. In addition, both monomers have only one fluorine-containing chemical structure (CF3) and so is expected to produce a single 19F NMR resonance, a desirable characteristic for 19F MRI. PEG-based hydrophilic oligomers, PEGA and PEGMA, were copolymerized with TFE(M)A to obtain water-soluble polymers. SHBPs consisting of a range of compositions were synthesized, including TFEA/PEGA, TFEA/ PEGMA, TFEMA/PEGA, and TFEMA/PEGMA pairs (see Table 1 for details). 1H NMR was used for the investigation of the chain composition and sequence. In Figure 6, 1H NMR spectra of SHBP-2, -7, -8, and -9 having relatively close TFE(M)A/PEG(M)A ratios are compared particularly within the chemical shift of 4−5 ppm to highlight several differences. For the sample containing TFEA and PEGMA, two peaks “a” and “b” (in solid box) were observed from 4.25 to 4.60 ppm, which could be assigned to the resonances of protons in CH2 adjacent to the ester bond in TFEA, as further evidenced by the 1H−13C HSQC spectrum (see Supporting Information, Figure S4). In addition, a shoulder peak c (in dash box) was also found on the peak (4.07 ppm) for the CH2 next to the ester bond in PEGMA. These results imply that both TFEA and PEGMA segments exist in different chemical environments. To gain further insights, SHBP-7 was also analyzed in D2O using the diffusion NMR technique (1D double-stimulated echo experiment) that suppressed the water signal at 4.7 ppm. Compared with the spectrum in CDCl3, only one peak of CH2 in TFEA was found and it was shifted to 4.6 ppm, while a single peak due to the CH2 adjacent to the ester bond in PEGMA appeared at 4.1 ppm without the previous shoulder peak. The 1 H NMR spectrum in D2O indicates that the peaks “b” and “c” could be attributed to the hydrophobic segments in the polymer. In our previous paper, the reactivity ratios of PEGMA and TFEA were determined to be 2.46 and 0.22, respectively, G

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As reported in our previous study,15 the fluorinated units connected to acrylate backbones have higher local mobility compared to those in methacrylate backbones. Therefore, acrylate monomers are favored for achieving high 19F NMR signal intensity. The ratios of peak integral to fluorine concentration were calculated based on 19F NMR spectra. In Figure 9B, the samples consisting of acrylate monomers exhibit higher ratios of peak integral to fluorine concentration, indicating that they have higher 19F signal intensity at a given 19F concentration. It is noteworthy that SHBP-7 shows a considerably low intensity because it contained 40% of “19F NMR-invisible” 19F, as revealed by 1H NMR spectrum. In the normalized spectra (see Supporting Information, Figure S5), SHBP-7 displays an asymmetrical peak, which was caused by multiple 19F chemical environments in the gradient copolymers. In contrast, the statistical copolymer chains in SHBP-2 and -9 resulted in relatively symmetrical peaks. This is in good agreement with the 1H NMR results shown in Figure 6. Spin−lattice (T1) and spin−spin (T2) relaxation times, which are two important parameters for 19F MRI, were measured by 19 F NMR. Generally, highly mobile fluorine nuclei experience reduced dipolar couplings and chemical shift interactions, which results in narrow 19F peaks and long T2 relaxation times. As depicted in Figure 10, it is clear that samples composed of acrylates have much longer T2 relaxation times than their methacrylate counterparts, providing further evidence that fluorine atoms in acrylate backbones are more mobile. In addition, the sample with TFEA/PEGMA (SHBP-7) units exhibits a slightly longer T2 than the polymer consisting of TFEMA/ PEGA (SHBP-8). Considering the gradient in sequence of the chains in SHBP-7, we assume that the 19F NMR-detectable nuclei were mainly from its PEGMA-rich sequences, where the 19 F units were loosely dispersed, affording comparably higher mobility and longer T2 relaxation times than SHBP-8. Furthermore, when the 19F content increases, T2 decreases dramatically. For example, the T2 of samples with TFEA/PEGA dropped by 76% when the fluorine content was increased from 3.3 wt % to 11.8 wt %. This tendency confirms that the 19F mobility is becoming restricted at higher 19F contents due to the significantly increased association of the fluorinated segments in an aqueous environment. Compared with T2, the T1 values stayed relatively short at ∼300 ms, implying that the spectral density of motions in the range of 700 MHz was not significantly affected by the change in polymer conformation. Similar trends were observed in previous reports.20,21 The glass transition temperature (Tg) of the SHBPs is also a measure of the molecular mobility of the main chains and can be related to the NMR properties. The Tg of the homopolymers PTFEA and PTFEMA were reported to be 2 and 60 °C,62,63 respectively, whereas the Tg of PPEGA and PPEGMA were in the range of −70 ∼ −65 °C.64−66 As listed in Table 2, for the SHBPs consisting of TFEMA and PEGMA (from SHBP-1 to -6), Tg increases gradually with the mole fraction of TFEMA from −65.8 to −51.6 °C. The same trend was also noted for SHBPs composed of TFEA and PEGA (from SHBP-9 to -12), as evidenced by the stack of DSC curves (see Supporting Information, Figure S6). In addition, the Tg values were predicted using the Fox equation based on the composition determined by 1H NMR. In Supporting Information, Figure S7, the experimental Tg values are generally in agreement with the values determined using the Fox equation. It is expected that chain segmental flexibility at ambient temperature is more restricted for samples with higher glass temperatures, and therefore it is not

suggesting a tendency of forming gradient copolymers.61 Based on these observations, we propose that SHBP-7 was constructed of poly(TFEA-co-PEGMA) gradient copolymer chains, which had TFEA-rich sequences (hydrophobic) and PEGMArich sequences (hydrophilic). Therefore, the gradient incorporation of TFEA and PEGMA results in different chemical environments of TFEA and PEGMA units, as reflected by the changes in the 1H NMR spectra. On the basis of the 1H NMR results, the water-insoluble TFEA segments in SHBP-7 account for 40% of TFEA units, estimated by comparing the integration of peaks “b” and peak “a”, indicating a considerable loss of effective 19F loading. This is likely to have implications for 19F MRI performance. The 1H NMR spectrum of SHBP-8 (24 mol % of TFEMA and 76 mol % of PEGA) was quite similar to that of SHBP-7. This may be due to the large differences in reactivity ratios of TFEMA and PEGA. However, for SHBP-9 and -2, a single peak for CH2 was found for both TFEA and PEGMA, illustrating that near-ideal copolymers were prepared owing to the close reactivity ratios of TFEA/PEGA and TFEMA/PEGMA. We suggest that statistical copolymers (poly(TFEA-co-PEGA) or poly(TFEMA-co-PEGMA) are more preferable than gradient copolymers (poly(TFEA-co-PEGMA) or poly(TFEMA-coPEGA) (Figure 7) for 19F MRI, because the latter would not

Figure 7. Illustration of the structures of copolymers with different sequence distributions.

only reduce the effective of 19F loading, but also potentially give rise to multiple peaks in the 19F NMR spectrum. 19 F NMR Studies. In order to evaluate their potential as 19F MRI CAs, the SHBPs were examined by 19F NMR. Samples were dissolved in PBS/D2O (90/10, v/v) at a concentration of 20 mg mL−1. The SHBPs could form nanoparticles with diameters of 4−8 nm in PBS by direct dissolution (see Table 2). It should be mentioned that all the SHBPs were water-soluble, even with incorporation of fluorinated units up to 60 mol % due to the excellent hydrophilicity of the PEG-based oligomers. As can be seen in Figure 8A, all the 19F NMR spectra exhibited a single peak at −73.1 ∼ −72.6 ppm. The small triplet at −76.6 ppm could be ascribed to residual fluorinated compounds, including monomer. Clearly, the peaks become broader when the 19F content increases, indicating the decreasing signal intensity caused by the elevated chance of 19F association at higher 19F content, and consequently decrease in modulation of residual dipolar and chemical shift interactions. The relevant values of peak half widths are listed in Table 2. To study the effect of copolymer composition and sequence on the 19F NMR spectra, SHBP-2, -7, -8, and -9 were selected for comparison as these samples have similar fluorine contents of approximately ∼3.0 wt %. From Figure 9A, one can see that SHBP-9 has the highest 19F NMR signal intensity, while SHBP7 has the lowest intensity. At a given fluorine content, the signal intensity is dependent on mobility of the fluorinated segments, which can be affected by the flexibility of the polymer chains. H

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Biomacromolecules Table 2. Physical and 19F NMR Properties of the SHBPs sample SHBP-1 SHBP-2 SHBP-3 SHBP-4 SHBP-5 SHBP-6 SHBP-7 SHBP-8 SHBP-9 SHBP-10 SHBP-11 SHBP-12 SHBP-13 SHBP-14 SHBP-15 LP-11

Dha

F%b (wt %)

T2c (ms)

T1c (ms)

half-widthc (Hz)

Tg (°C)

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

1.5 2.8 4.8 6.8 8.8 11.9 3.1 3.3 3.3 4.8 6.4 11.8 5.9 6.1 6.7 5.8

17.9 11.3 6.5 4.7 3.6 3.0 44.0 33.9 87.1 51.7 35.3 20.3 17.6 24.4 33.1 43.9

291.4 288.0 282.4 282.4 284.4 291.4 308.5 302.3 345.4 330.0 317.7 308.5 307.4 309.1 318.1 319.3

119.4 161.7 288.9 310.8 783.7 1115.8 200.6 248.0 223.9 320.5 373.4 422.2 349.7 333.7 355.6 371.2

−65.8 −60.7 −59.3 −57.9 −53.3 −51.6 −60.9 −62.1 −61.2 −59.3 −57.7 −48.7

7.90 7.52 6.24 7.42 5.89 8.12 6.45 6.29 6.31 6.07 5.62 5.56 5.12 4.09 4.52 3.95

0.65 1.1 0.51 0.79 0.50 0.42 0.22 0.24 0.36 0.31 0.08 0.06 0.02 0.06 0.21 0.15

F MRI intensityd

19

1.33 0.65 0.07 0.01 0.0009 0.0001 10.62 8.59 17.59 18.69 17.34 13.61 4.50 9.88 16.80 19.72

Hydrodynamic diameters were measured in PBS (1 mg mL−1) by DLS at 25 °C, and number-averaged diameters are reported here. b19F payloads were calculated based on 1H NMR results. c19F NMR spectra and measurements of relaxation times were performed by 19F NMR at 659 MHz in PBS/D2O (90/10, v/v) at 25 °C. dHalf-width is the peak width at half height. a

Figure 8. Stacked 19F NMR spectra of the SHBPs in PBS/D2O (90/10, v/v) at 20 mg mL−1 at 25 °C. (A) Samples consisted of P(TFEMA-coPEGMA) copolymer chains. (B) Samples composed of P(TFEA-co-PEGA) copolymer chains. Note: the peak intensities have been normalized to the same value (1.0).

surprising that longer T2 relaxation times were observed for the samples with lower Tg values because of the enhanced averaging of the dipole−dipole and chemical shift interactions.15 Although the DSC measurements were conducted in solid state, the results were in good agreement with the findings revealed by solution-state NMR. Therefore, PEGbased oligomers are ideal monomers for the copolymerization with fluoro monomers to yield copolymers with low Tg and good water solubility. As discussed before, the degree of branching (DB) of the SHBPs can be adjusted by varying the [comonomer]/[MECP] ratio. In this work, SHBPs with different DBs and similar [TFEA]/[PEGA] ratios were prepared to examine the relationship between DB and relaxation times. A linear analogue of SHBP-11 (LP-11, DB = 0) was also prepared for comparison. As displayed in Figure 11, a remarkable decrease of T2 from 43.9 to 17.6 ms was observed with an increase of DB, implying poor 19F mobility for samples with high degrees of branching.

This result suggests that samples with high DBs are more compact, leading to the increased chain rigidity, restricted macromolecular motion, and reduced segmental flexibility. The change in macromolecular compactness was also confirmed by SEC MALLS, which revealed that samples with higher DBs showed larger absolute MWs at the same retention time (see Supporting Information, Figure S8). However, the spin−lattice relaxation time T1 was relatively constant at ∼310 ms, again indicating minimal effect of DB on chain motion at a frequency of 700 MHz. 19 F MRI Studies. The ultimate aim of this work was to examine the SHBPs as potential contrast agents for 19F MRI. Following 19F NMR characterization, the imaging performance was evaluated by in vitro 19F MRI. For comparison, SHBP-2, -7, -8, and -9 were selected for imaging as they possess different compositions but similar 19F content (∼3 wt %). Samples were dissolved in pure water at a concentration of 100 mg mL−1, and the solutions were loaded in 30 × 8 mm clear glass vials, I

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Figure 9. 19F NMR results of four SHBPs. (A) Stacked 19F NMR spectra of SHBP-2, -7, -8, and -9. (B) The ratios of peak integral to fluorine concentration. Samples were dissolved in PBS/D2O (90/10, v/v) at 20 mg mL−1 at 25 °C.

Figure 10. T2 relaxation times of the SHBPs. Samples were dissolved in PBS/D2O (90/10, v/v) at 20 mg mL−1 at 25 °C.

of which the cross sections were shown in phantom images. The vials in the resonator were localized using the 1H RARE sequence, then the samples were imaged using 19F FLASH, which is a T1-weighted gradient echo sequence with a short scan time.67 As depicted in Figure 12, all four samples were effectively visualized using 19F FLASH (repetition time TR = 30 ms) within 47 min. The signal-to-noise ratios (SNRs) for the images are displayed in Figure 12 (bottom right). Since the samples have different spin−spin relaxation times, T2-weighted imaging was then conducted using the RARE spin−echo pulse sequence. As shown in Figure 12, using the RARE sequence with an echo time TE of 41 ms, SHBP-7, -8, and -9 that have T2s above 30 ms were all well imaged, while SHBP-2 having a short T2 of 11.3 ms exhibited a poor SNR. Notably, SHBP-9 with the longest T2 showed the brightest image. When the TE was increased to 157 ms, only SHBP-9 could be imaged, while SHBP-7 and -8 were detected with fairly low SNRs. SHBP-2 was found to be hardly visualized owing to its very short T2, affording a dark field image. The 19F MRI results demonstrate that the SHBPs can be selectively imaged by making use of the differences in T2 values and selection of the appropriate pulse sequences.

Figure 11. 19F T1 and T2 relaxation times of SHBPs with different degrees of branching and similar 19F contents of ∼3 wt %. DB from low to high: LP-11, SHBP-11, SHBP-13, SHBP-14, and SHBP-15.

For 19F MRI using a spin−echo pulse sequence, the imaging intensity can be described as the following equation.68 ⎛ − TE ⎞⎡ ⎛ − (TR − (TE/2)) ⎞ ⎛ − TR ⎞⎤ I ≈ N (F )exp⎜ ⎟⎢1 − 2 exp⎜ ⎟ + exp⎜ ⎟⎥ T1 ⎝ T2 ⎠⎢⎣ ⎝ ⎠ ⎝ T1 ⎠⎥⎦

(3)

In eq 3, I is the imaging intensity, N(F) is a measure of the number of fluorine nuclei in the sensitive volume of the imaging coil, and TR and TE are the pulse sequence repetition time and echo delay times, respectively. According to this equation, high fluorine content, long T2 and short T1 are required for the design of efficient contrast agents. The 19F MRI intensity J

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Figure 12. 19F MRI results for four selected SHBPs. Top: phantom images of the samples using different sequences. Note that 1H RARE was performed to localize the sample vials. Bottom left: concentrations and 19F NMR profiles of the samples. Bottom right: signal-to-noise ratios for the imaging experiments.

chain flexibility was also revealed by thermal analysis, and longer T2 relaxation times were observed for the samples with lower glass temperatures, Tg. The imaging performance of the SHBPs was confirmed by 19F MRI. All the samples could be well imaged by T1-weighted imaging, and they could be selectively visualized using a T2-weighted sequence using different echo times. In summary, we have demonstrated therefore a robust concept for the controlled design of polymeric 19F MRI contrast agents. By tailoring the branched structure and composition, the imaging properties of the agents can be effectively controlled, enabling the realization of selective and tunable 19F MRI agents. Furthermore, this work provides useful guidance for the future design of polymeric CAs with high 19F content, long T2, and short T1 for effective 19F MRI.

values were estimated using eq 3 using the parameters for the RARE-8 pulse sequence (TE = 41 ms, TR = 1 s, sample concentration =100 mg mL−1). As listed in Table 2, the calculated imaging intensity generally decreases with the T2 relaxation time. It is not surprising that the SHBPs consisting of P(TFEA-coPEGA) exhibit considerably higher imaging intensities compared to those containing P(TFEMA-co-PEGMA). In addition, the calculated imaging intensity decreases with the increase of DB, providing further evidence that SHBPs with low DBs are preferred for 19F MRI. Furthermore, the fluorine content plays an important role in imaging intensity. Compared with SHBP-9 (3.3 wt %), SHBP-10 (4.8 wt %) has a shorter T2 relaxation time but a slightly higher imaging intensity due to the increased fluorine content. These results suggest that SHBPs consisting of P(TFEA-coPEGA) with low DB are excellent candidates as 19F MRI contrast agents.

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ASSOCIATED CONTENT

* Supporting Information S

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Characterization techniques, 1H−13C HSQC and 19F NMR spectra, UV−vis spectra, SEC trace, DSC curves, plot of Fox equation, and absolute Mn versus retention time given by SEC MALLS. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/ acs.biomac.5b00800. (PDF)

CONCLUSIONS Segmented and highly branched polymers were successfully synthesized by RAFT SCVP of TFE(M)A, PEG(M)A, and a polymerizable trithiocarbonate CTA. By varying monomer type and [comonomer]/[MECP] ratio, SHBPs with desired compositions and degree of branching were prepared. The structure of the SHBPs was thoroughly characterized by a range of different analytical techniques. Statistical copolymers were obtained using TFEA/PEGA or TFEMA/PEGMA, while gradient copolymers were produced with monomers having largely different reactivity ratios. The 19F NMR properties were affected by both chain composition and monomer sequence. Higher 19F mobility (longer T2) was achieved in statistical copolymers consisting of acrylate units due to greater segmental flexibility. In addition, the 19 F mobility was greatly restricted for the samples with higher 19F content due to increased association of the fluorinated segments. A shortened T2 was also observed with an increase in degree of branching, which caused increased compactness, rigidity, and restricted segmental motion. For gradient copolymers, the 19F signal intensity was significantly reduced by the loss of solubilized fluorinated units in the chains. The relationship between T2 and

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AUTHOR INFORMATION

Corresponding Author

*Tel: +61-7-33463885. Fax: +61-7-33463973. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors would like to acknowledge the Australian Research Council (CE140100036, DP0987407, DP110104299, LE0775684, LE0668517, and LE0882357) for funding of this research. The Australian National Fabrication Facility, Queensland Node is also acknowledged for access to some items of K

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equipment. H.P. thanks the University of Queensland for her UQ Postdoctoral Research Fellowship for Women.

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DOI: 10.1021/acs.biomac.5b00800 Biomacromolecules XXXX, XXX, XXX−XXX