Importance of Thermally Induced Aggregation on 19F Magnetic

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Importance of Thermally-Induced Aggregation on 19F Magnetic Resonance Imaging of Perfluoropolyether-Based Comb-Shaped Poly(2-oxazoline)s Cheng Zhang, Ronny Javier Pibaque Sanchez, Changkui Fu, Ryan Clayden-Zabik, Hui Peng, Kristian Kempe, and Andrew K. Whittaker Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.8b01549 • Publication Date (Web): 29 Nov 2018 Downloaded from http://pubs.acs.org on December 2, 2018

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Biomacromolecules

Importance of Thermally-Induced Aggregation on 19F

Magnetic Resonance Imaging of

Perfluoropolyether-Based Comb-Shaped Poly(2oxazoline)s Cheng Zhang,1,2 Ronny Javier Pibaque Sanchez,1,2 Changkui Fu,1,2 Ryan Clayden-Zabik,1 Hui Peng,1,2 Kristian Kempe*3,4 and Andrew K. Whittaker*1,2 1Australian

Institute for Bioengineering and Nanotechnology and 2ARC Centre of Excellence

in Convergent Bio-Nano Science and Technology, The University of Queensland, Brisbane, Qld 4072, Australia; E-mail: [email protected]. 3Drug

Delivery Disposition and Dynamics and 4ARC Centre of Excellence in Convergent Bio-

Nano Science and Technology, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, VIC 3052, Australia; E-mail: [email protected].

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ABSTRACT An understanding of thermally-induced aggregation, and consequent 19F magnetic resonance imaging (MRI) performance is essential for improved design of thermoresponsive

19F

MRI

contrast agents. Herein we describe a series of novel thermoresponsive perfluoropolyether (PFPE)-based comb-shaped poly(2-oxazoline)s (POxs) with different side-chain structures (2methyl- (MeOx), 2-ethyl- (EtOx) and 2-(n-propyl)-2-oxazoline (nPrOx)). The comb polymers were

prepared

through

reversible

addition-fragmentation

chain

transfer

(RAFT)

polymerization of the respective oligo(2-oxazoline)acrylates using a perfluoropolyether macroRAFT agent. The fluoropolyether chain end drives aggregation of the polymers, with small aggregates forming at 300 K for both poly(OMeOx5A)9-PFPE and poly(OEtOx4A)9-PFPE. The aggregates decrease in size and display increases in 19F MRI intensity with temperature, and at 350 K the MeOx polymers are in the form of unimers in solution, similar to the oligoethylene glycol (OEG)-based PFPE polymer. Above the TCP of poly(OEtOx4A)9-PFPE, the polymer forms large aggregates, and the

19F

MR imaging performance is degraded. Likewise,

poly(OnPrOx4A)-PFPE is above the LCST at all temperature studied (300-350 K), and so weak imaging intensity is obtained. This report of novel thermoresponsive POx-based PFPE polymers highlights the importance of understanding self-association of polymers in solution, and provides important insights for the development of ‘smart’ thermoresponsive

19F

MRI

contrast agents. INTRODUCTION Thermoresponsive partly-fluorinated polymers that exhibit a cloud point temperature (TCP) have received significant attention from researchers, particularly in the biomedical field.1, 2 Such fluorinated polymers are soluble in water at temperatures below the TCP, however above the TCP they phase separate into polymer-rich and polymer-depleted phases. These interesting properties have prompted investigations into many applications, for example the application of 2 ACS Paragon Plus Environment

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thermoresponsive fluorinated polymers as 19F magnetic resonance imaging (MRI) agents for disease detection. 1-6 Over the last years, a large number of thermoresponsive fluorinated polymers have been designed and characterized. The reported studies have mainly focused on polymers incorporating poly(oligo(ethylene glycol) (meth)acrylates) (POEG(M)As) or poly(Nisopropylacrylamide) (PNIPAAm) as the thermoresponsive segments.7-11 These two classes of thermoresponsive polymers have been reported to be highly biocompatible and nontoxic.12-14 However, a number of drawbacks for biomedical applications of these polymers have been identified, for example their unwanted interactions with various immunological entities and the tendency to accumulate in the body.15 For example, Garay et al. reported that an intravenous injection of PEG-based materials caused a second dose, injected a few days later, to have a reduced blood circulation time. This phenomenon is referred to the accelerated blood clearance and has been observed with PEGylated proteins, liposomes, micelles and nanocarriers.16-19 The preparation of thermoresponsive fluorinated polymers with alternative thermoresponsive segments is required to overcome the above limitations and to achieve truly biocompatible fluorinated materials. In recent years, significant efforts have been devoted to developing poly(2-oxazoline)s (POxs) as an alternative to PEG and PNIPAAm.

20, 21

POxs provide high

stability, tunability and functionalization, while retaining the requisite features of biocompatibility, stealth behavior and low dispersity.22-25 Moreover, POx have been demonstrated to be highly suitable for the fabrication of micro- and nano-sized particles and diverse polymer architectures, such as comb polymers.22, 26-29 The thermoresponsive nature of POx allows for the design of ‘smart’ materials for various biomedical applications including MRI. The thermoresponsive properties of partly-fluorinated polymers have been widely studied.7, 8 In the majority of these studies, the thermal properties have been mainly characterized by 3 ACS Paragon Plus Environment


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measurements of properties when the temperature passes above the TCP, while the thermoresponsive behavior below the TCP is less investigated and understood. As such, a more complete and molecular-level understanding of the process responsible for the thermoresponsive changes at variable temperatures below the phase transition temperature is highly desired. Our group has previously described the use of high-resolution nuclear magnetic resonance for the analysis of polymer structure and dynamics of chain segments as a function of temperature.7, 30-34 In particular, two-dimensional diffusion-ordered spectroscopy (DOSY) and nuclear Overhauser effect spectroscopy (NOESY) can provide rich information on aggregation behavior, and interactions between specific segments over short distances in space.8,

9, 35, 36

Our extensive previous studies have demonstrated the strong link between

polymer conformation, NMR and MR imaging performance.31-33, 37-39 This is not unexpected because of the well-understood relationship between polymer structure/conformation and the motions responsible for longitudinal (T1) and transverse (T2) relaxation. Therefore, NMR together with MRI techniques are applicable to a number of different thermoresponsive polymer classes and can provide rich information for understanding their thermoresponsive properties at the molecular level. In this study, we prepared thermoresponsive fluorinated comb-shaped poly(2-oxazoline)s with a range of side-chain structures through a combination of cationic ring-opening polymerization (CROP) and reversible addition-fragmentation chain-transfer (RAFT) polymerization. The chemical structures, thermoresponsive properties and 19F MRI performance were examined in detail. By conducting 1H 2D DOSY and NOESY experiments as a function of temperature, we observed changes in aggregation states (small aggregates to unimers or to large aggregates). Such transitions were found to be highly dependent on the structure of the side chain of the POx. Moreover, the

19F

MRI performance of the fluorinated comb-shaped POx was

investigated as a function of temperature. This study provides a detailed understanding of the 4 ACS Paragon Plus Environment

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self-association and 19F MR imaging of the fluorinated POx as a function of temperature, in particular below the TCP. Such understanding provides a useful guide for the design of thermoresponsive 19F MRI contrast agents. EXPERIMENTAL SECTION Materials. All chemicals were purchased from Sigma-Aldrich unless otherwise stated. Oligo(ethylene glycol) methyl ether acrylate (OEGA, MW = 480 g/mol) was passed through basic alumina columns to remove inhibitors prior to use. 2-Methyl-2-oxazoline (MeOx) and 2ethyl-2-oxazoline (EtOx) were distilled to dryness over barium oxide (BaO) and stored in a nitrogen atmosphere. The monohydroxy perfluoropolyether (PFPE-OH, MW ~1450 g/mol) was supplied by Apollo Scientific Ltd, UK. 2,2′-Azobis(2-methylpropionitrile) (AIBN) was recrystallized

twice

from

methanol

before

use.

The

RAFT

agent

2-

(butylthiocarbonothioylthio)propionic acid (PABTC) was synthesized according to a previously reported procedure.40 2-(n-propyl)-2-oxazoline (nPrOx) was synthesized according to a literature procedure.41 Milli-Q water with a resistivity of 18.4 MΩ/cm was used for the relevant experiments. The dialysis tubing with molecular weight cut-off (MWCO) of 3.5 kDa was purchased from Thermo Fisher Scientific Inc. and Spectrum Laboratories Inc., respectively. Synthesis of POx macromonomers. POx macromonomers were prepared following a modified literature protocol using acrylic acid as the terminating agent.42 Oligo(2-methyl-2oxazoline) (OMeOxA): methyl tosylate (6.98 mmol) and MeOx (35.3 mmol) were dissolved in acetonitrile (90.1 mmol) under nitrogen, yielding a total monomer concentration of 4 M and a [MeOx]:[I] ratio of 5:1. The sealed Schlenk flask was heated in an oil bath at 80 °C for 50 min and subsequently the polymerization was quenched by the addition of acrylic acid (8.4 mmol) and triethylamine (9.1 mmol). The polymer solution was stirred at 50 °C overnight and subsequently 0.6 g potassium carbonate were added and stirring was continued for another 24 5 ACS Paragon Plus Environment


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h. The precipitate was filtered off and the polymer was precipitated in ice-cold diethyl ether. Oligo(2-ethyl-2-oxazoline)acrylate (OEtOxA) and oligo(2-(n-propyl)-2-oxazoline)acrylate (OnPrOxA): under nitrogen, methyl tosylate (6.55 mmol) and EtOx/nPrOx (26.5 mmol) were dissolved in acetonitrile (48.7 mmol) yielding a total monomer concentration of 4.1 M and [Ox]:[I] ratio of 4:1. The sealed Schlenk flask was heated in an oil bath at 80 °C for 45 min and subsequently the polymerization was quenched by the addition of acrylic acid (8.5 mmol) and triethylamine (9.8 mmol). The polymer solution was stirred at 50 °C overnight and purification was performed by washing with sodium bicarbonate and brine. The combined organic phases were dried over magnesium sulfate, filtered and the solvent was removed under reduced pressure. 1H NMR analysis of the crude products revealed quantitative conversion of all monomers. Synthesis of POx-based PFPE polymers through RAFT polymerization. In a typical experiment for the synthesis of poly(OEtOx4A)-PFPE, PABTC-PFPE macro-RAFT agent (187 mg, 0.11 mmol), OEtOx4A (550 mg, 1.1 mmol) and AIBN (3.28 mg, 0.02 mmol) were dissolved in trifluorotoluene/methanol (2 mL) and sealed in a 5 mL flask fitted with a magnetic stirrer bar. The solution was then deoxygenated by purging thoroughly with argon for 15 min, heated to 70 °C in an oil bath, and allowed to react for 12 h. Upon completing the reaction, the crude sample was collected to run 1H NMR to determine the conversion of monomer to polymer (Figure S1). The remaining solution was precipitated into hexane and redissolved in THF three times. The precipitate was then dissolved in water and purified by dialysis for two days (molecular weight cut-off of 3500 Da), yielding a yellow viscous solid after freezer drying. Polymers with different side chains (2-methyl- and 2-(n-propyl-)) were prepared under identical conditions. The detailed structural characteristics of the polymers are summarized in Table 1.


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Characterization methods. NMR spectroscopy. 1H NMR spectra were obtained in CDCl3 using a Bruker Avance 400 MHz (9.4 T) spectrometer to analyze the conversion of monomer to polymer and the structure of the polymers. Solution spectra were measured under the following measurements conditions: 90o pulse width 14 µs, relaxation delay 1 s, acquisition time 4.1 s and 32 scans. Chemical shifts are reported relative to TMS using the residual solvent peak as reference. 19F

NMR spectra were acquired using a Bruker Avance 400 MHz spectrometer with either

CDCl3 or PBS/D2O (90/10, v/v) as solvent. The spectra were measured under the following measurements conditions: 90o pulse width 15 µs, relaxation delay 2 s, acquisition time 0.73 s and 128 scans. 19F

spin-spin relaxation times (T2) were measured using the Carr-Purcell-Meiboom-Gill

(CPMG) pulse sequence at different temperatures (300 to 350 K) in PBS/D2O (90/10, v/v) at a concentration of 20 mg/mL. The relaxation delay was 1 s and the number of scans was 16. The relaxation times are reported for the major 19F NMR peaks. 19F

spin-lattice (T1) relaxation times were measured using the standard inversion-recovery

pulse sequence at different temperatures (300 to 350 K) in PBS/D2O (90/10, v/v) at a concentration of 20 mg/mL. For each measurement, the relaxation delay was 2 s and the number of scans was 16. The relaxation times are reported for the major 19F NMR peaks. The 2D DOSY NMR spectra were obtained on a Bruker Avance 400 MHz instrument in CDCl3 or PBS/D2O (90/10, v/v), with a 2 s relaxation delay and 16 scans acquired. The 90 o pulse width was 14 µs and the spectral width was 4801 Hz. The data size of F1 and F2 was 16 and 16 k points without linear prediction. The spectra were analysed using the Bruker Topspin software to obtain the diffusion coefficients of species giving rise to each peak. The StokesEinstein equation was used to calculate the particle size.



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The 2D NOESY spectra were acquired with the standard pulse program (90o-t1-90o-tm-90oacquisition). The NOESY program was run with a 14 µs 90 o pulse, a 2 s relaxation delay, and a 4807 Hz spectral window. The data size was 128 and 2048 for F1 and F2, respectively. F1 was linear predicted to 1024 points. The data was treated by shifted sine bell (SSB=2) weighting functions in both dimensions. Magnetic resonance imaging (MRI) of polymer solutions. Images of polymer solutions were acquired on a Bruker BioSpec 94/30 USR 9.4 T small animal MRI scanner. Polymer solutions (20 mg/mL in PBS) were loaded in 5 mm NMR tubes, which were placed in a 1H/19F dual resonator 40 mm volume coil. 1H were acquired for localization of the samples using a rapid acquisition with relaxation enhancement (RARE) sequence (rare factor = 16, TE = 88 ms, TR = 1500 ms, FOV = 40 × 40 mm, matrix = 128 × 128). 19F MRI images were acquired in the same stereotactic space as the 1H image using RARE sequence (rare factor = 32, TE = 11 ms, TR = 1500 ms, number of averages = 128, FOV = 40 × 40 mm, matrix = 64 × 64, measurement time = 25 minutes 36 seconds). The 19F MRI signal-to-noise ratio (SNR) is defined as the ratio of the average signal intensity to the standard deviation of the background. RESULTS AND DISCUSSION The aim of this study is to examine the potential of novel partly-fluorinated thermoresponsive comb polymers as 19F MRI agents, and to understand their aggregation behavior as a function of temperature. The polymers are well-defined fluorinated comb-shaped poly(2-oxazoline)s prepared through a combination of cationic ring-opening polymerization and reversible addition-fragmentation chain transfer polymerization.43 To this end, a series of POx macromonomers with acrylate end groups was prepared by CROP and subsequently “grafting through” RAFT polymerization in the presence of a perfluoropolyether (PFPE) macro-RAFT agent was performed. Detailed high-resolution 1D, 2D NMR and MRI studies of polymer structure, solution dynamics, aggregation states and imaging properties as a function of 8 ACS Paragon Plus Environment

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temperature were conducted. Oligoethylene glycol (OEG)-based PFPE polymers were prepared as a comparison to the POx polymers (Scheme 1). Synthesis of poly(2-oxazoline)s-based perfluoropolyether polymers (POx-PFPE). Three different POx macromonomers were synthesized according to the previous report by Weber et al. via living CROP of 2-methyl- (MeOx), 2-ethyl- (EtOx) or 2-(n-propyl)-2-oxazoline (nPrOx) with methyl tosylate as the initiator at 80 °C in acetonitrile. 27, 44 The reaction was terminated by a mixture of acrylic acid (AA) and triethylamine creating the termination agent, a acrylate anion, in situ to yield acrylate end-functionalized POx (OMeOxA, OEtOxA and OnPrOxA, Scheme 1a). The chemical structures of the three macromonomers were confirmed by the 1H NMR spectra (Figures 1b and S1). Comparison of the peak integrals of the terminal methyl groups (group R in Scheme 1a) with the acrylic group allowed determination of the side-chain length of the macromonomers. Taking OEtOxmA as an example (Figure 1b), the ratio of the integrals of peaks due to the terminal methyl group (M7, three protons) to the acrylic group (M1+M2, three protons) is approximate 4:1, indicating the successful synthesis of the OEtOx4A macromonomer. Similarly, the side-chain length of OMeOxA and OnPrOxA was calculated to be five and four, respectively. Thus, three POx macromonomers of similar molecular weight but different side groups and hence hydrophilicity were prepared. Note that the molecular weights of the three POxA macromonomers are approximately the same as the OEGA monomer. The perfluoropolyether-modified RAFT agent was prepared according to our previous reports through standard EDCI/DMAP esterification between 2-propanoic acid butyl trithiocarbonate (PABTC) and monohydroxy PFPE of a molecular weight of ∼1450 g/mol (PABTC-PFPE macro-CTA) (Scheme 1b and Figure 1a).32



(a) n N

O

methyl tosylate, acetonitrile, 80 oC

O

R N

(b) S

CF3 OH

+ HO

S

R

O

N

N

O

n

O

R

O S

O

AA, NEt3, 50 oC, overnight

n-1

R

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CF F O C C O F2 8 CF3

F2 C

C F2

CF3

O

EDCI/DMAP, TFT, 0 oC

S

S

O

S O

TFT/Methanol, AIBN, 70 oC

CF3 F2 CF CF3 F O C C C C O F2 F2 8 CF3

R

O

N

O

n

O S

S m

S

O

O

O

CF3 F2 CF CF3 F O C C O C C F2 F2 8 CF3

O n OEG-based PFPE Polymer

O S

S

O

m

S

O

O

R N n O

CF3 F2 CF3 CF F O C C C C O F2 F2 8 CF3

R = CH3, CH2CH3, (CH2)2CH3

POx-based PFPE Polymer

Scheme 1. Synthetic scheme of POx-based PFPE polymers. (a) Synthesis of poly(2oxazoline)acrylate macromonomers by CROP with methyl tosylate as the initiator and terminated by acrylic acid/triethylamine. (b) Synthesis of PFPE macro-RAFT agent and RAFT polymerization of poly(2-oxazoline)acrylate macromonomers. The structure of the OEG-based PFPE polymer is also illustrated. Homopolymers of OMeOx5A, OEtOx4A and OnPrOx4A with PFPE as terminal unit were prepared through RAFT polymerization. The extent of conversion of the OROxA macromonomer to polymer during the polymerization can be determined from the peak integrals in the 1H NMR spectra of the crude samples (Figure S2 and Table 1). A typical 1H spectrum of the poly(OEtOx4A)-PFPE polymer in CDCl3 after purification and the assignments to the spectrum is displayed in Figure 1c, indicating the successful synthesis of the poly(OEtOx4A)-PFPE polymer through RAFT polymerization. All polymers have low molar mass dispersity (Đ ≈ 1.2, Figure S3). In addition, the 1H and

19F

NMR spectra of

poly(OMeOx5A)-PFPE and poly(OnPrOx4A)-PFPE after purification are provided in Figure S4 and S5. The detailed structural characteristics of all polymers are summarized in Table 1.


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Figure 1. 1H NMR spectra of the (a) PFPE macro-RAFT agent, (b) OEtOx4A macromonomer and (c) poly(OEtOx4A)-PFPE polymer in CDCl3 at 9.4 T. The assignments to the peaks were included in the figure. Table 1. Detailed structural characteristics and 19F NMR and MRI properties of the POx- and OEG-based PFPE polymers. conv. (%)

a

poly(OMeOx5A)8PFPE

93.0

Fluorine content (wt %)a 20.9

poly(OEtOx4A)9PFPE

91.6

poly(OnPrOx4A)9PFPE poly(OEGA)9PFPE

Mn,GPC Mn,NMR (g/mol)b (g/mol)c

Ðb

Dh (nm)d

19F

3600

5900

1.24

8.7

NMR T1/T2 (ms)e 402.5/50.0

TCP (K)f

19.6

4200

6300

1.22

11.5

388.7/52.1

338

93.5

18.6

4400

6660

1.21

>1000

N/A

< r.t.

89.0

17.0

3410

6600

1.20

9.3

404.7/97.4

>350

>350

the weight percentage of fluorine in the samples. b Mn,GPC and Ð were acquired by SEC in

dimethylacetamide (DMAc) (Figure S3).

c

The Mn, NMR for the polymers was calculated by 11

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considering the integrals of the peaks due to protons P2 (2H) and the RAFT agent protons P8 (3H) as shown in Figure 1c. d Dh was obtained by DLS in PBS and these are number-average values which are similar compared to the sizes obtained from the diffusion-ordered spectroscopy (DOSY) NMR. The concentration was 20 mg/mL. e The

19F

NMR T1/T2 were

measured in PBS/D2O (90/10, v/v) at 310 K at 9.4 T. The 19F NMR relaxation times and MR image SNR of polymers were acquired for the peak F1 (Figure S5). f TCP of the PFPE polymers (20 mg/mL) was determined from the turbidity measurements at a heating rate of 1 oC/min. Transition from small aggregates to unimers or large aggregates. The temperature dependence of the aggregation behavior of the newly developed POx-based PFPE polymers in solution was examined initially by high-resolution NMR spectroscopy. Note that the aqueous solutions of poly(OnPrOx4A)9-PFPE were cloudy, indicative of the presence of large aggregates at room temperature and a low cloud point temperature (TCP) (Table 1 and Figure S6). Consequently the following studies focused on poly(OMeOx5A)8-PFPE, poly(OEtOx4A)9PFPE and poly(OEGA)9-PFPE. The hydrodynamic diameters (Dh) of the polymers were determined using 1H diffusion ordered spectroscopy (DOSY) NMR using either PBS or chloroform (a good organic solvent for the PFPE polymers) as solvent. Figure 2a shows that the hydrodynamic diameters of poly(OMeOx5A)8-PFPE and poly(OEGA)9-PFPE decreased with increasing temperature (300 to 340 K), similar to the behavior of poly(N-isopropylacrylamide) below the TCP.45 The decrease in hydrodynamic diameter with increasing temperature may be ascribed to dehydration of the OROxA/OEGA side chains and partial disassociation of the multi-chain aggregates at high temperature.46 In addition, the values of Dh determined for poly(OMeOx5A)8-PFPE and poly(OEGA)9-PFPE in water are much larger than those measured in CHCl3 (green dash line, Figure 2a), indicating that these two polymers formed multi-chain aggregates within this temperature range (Figure 2c). Similar observations were 12 ACS Paragon Plus Environment

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obtained in our previous report, where multi-chain aggregates of poly(OEGA)9-PFPE with approximate four chains per aggregate were observed in water at 300 K.32 The values of Dh for both polymers in PBS were further decreased to ~ 1 nm (~ 0.91 nm in CHCl3) as the temperature was increased to 350 K, indicating disassembly of the multi-chain aggregates to unimers (~ 100 % unimers was confirmed by DOSY NMR, Figure S7). On the other hand, poly(OEtOx4A)9-PFPE displayed evidence of a transition from small to large aggregates above 340 K. As with the other polymers, the value of Dh decreased as the temperature increased from 300 to 330 K. However, a significant size increase was noted at 340 K, indicating the formation of large aggregates (~ 66%) confirmed by the NMR DOSY experiment, Figure S7 and S8. The change in aggregation behavior is clearly observed at the TCP of poly(OEtOx4A)9PFPE (TCP = 338 K, see Table 1 and Figure S6). NMR spin-spin (T2) relaxation times are sensitive to segmental motions of polymer chains, and are thus expected to reflect the aggregation state of the macromolecules (Figure 2b).47 The 19F NMR T2 relaxation times of poly(OMeOx5A)8-PFPE and poly(OEGA)9-PFPE increased continuously across the whole temperature range 300 to 350 K due to the increase in segmental mobility with increased thermal energy, and likely the reduction in aggregation number. In sharp contrast, a drastic decrease in T2 of poly(OEtOx4A)9-PFPE was observed at 340 K, consistent with the formation of large aggregates above TCP. The values of 19F T2 for the OEGbased PFPE polymers are significantly longer than for the POx-based polymer across the whole temperature range. This observation indicates the importance of chain flexibility of attached polymer units on the relaxation properties of the PFPE chain ends. The chain flexibility is reflected in the glass transition temperatures, being 47, 32 and -62 oC for poly(OMeOx5A)8PFPE, poly(OEtOx4A)9-PFPE and poly(OEGA)9-PFPE, respectively, Figure S9. It is clear that the greater stiffness of the POx chains leads to formation of more rigid assemblies, and hence shortened

19F

T2 compared with the OEGA-based polymer, despite both systems forming 13 ACS Paragon Plus Environment


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assemblies of similar size at room temperature, and having similar monomeric molecular weight. In the absence of inhomogeneous local magnetic fields, the NMR line width is expected to be inversely related to the T2 relaxation time. The

19F

NMR T2 relaxation times of

poly(OMeOx5A)8-PFPE measured here increased from 31.0 to 136.1 ms with increasing temperature from 300 to 350 K (Figure 2b). Despite this, the 19F NMR line widths remained constant at ~250 Hz across the same temperature range. The full widths at half maximum of the major peak labelled F1 were 268 and 237 Hz at 300 and 350 K, respectively, Figure S10. These line widths correspond to a T2* almost two orders of magnitude smaller than the T2 measured with the CPMG pulse sequence which is able to compensate for the effects of inhomogeneous magnetic fields. We conclude therefore that the structure of the assemblies, across the whole temperature range examined, leads to strong local field inhomogenities experienced by the fluorine nuclei. Experimental measurements of magnetic susceptibilities of fluorocarbons have not be often reported. For example, Beran and Kevan reported calculations of diamagnetic susceptibilities of a range of halogenated organic compounds, and showed that substitution of a linear alkane with fluorine led to significant increases in susceptibility.48 Magnetic field effects in dense polymers melts have been reported by us and others.49-51 We conclude that the large difference in magnetic susceptibility between the assembly of fluoroether chain ends and the surrounding polar media gives rise to strong distortions of the local magnetic fields and hence broader NMR lines.


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Figure 2. (a) Hydrodynamic diameters (Dh) and (b)

19F

NMR T2 relaxation times of

poly(OMeOx5A)8-PFPE, poly(OEtOx4A)9-PFPE and poly(OEGA)9-PFPE as a function of temperature in PBS. The green dashed line shows the Dh of these PFPE polymers in CHCl3. (c) Illustration of the chemical structure and transition from multi-chain aggregates to unimers of poly(OMeOx5A)8-PFPE. The LCST of poly(OEtOx4A)9-PFPE was determined by the turbidity measurements (20 mg/mL; 338 K, yellow dash line). No gross phase separation of poly(OEGA)9-PFPE and poly(OMeOx5A)8-PFPE was observed in the tested temperature range (300 to 350 K, Figure S6). For the NMR measurements the temperature was maintained at the specified temperature for 20 min prior to measurement. Data in (a) are mean ± standard deviation (SD). In order to further understand the changes in aggregation states and

19F

T2 as a function of

temperature, 2D nuclear Overhauser effect spectroscopy (NOESY) was employed. NOESY NMR provides rich information on the spatial displacement of different nuclear spins, typically over distances smaller than 5 Å.52 The mixing time and other parameters used for the NOESY 15 ACS Paragon Plus Environment


NMR experiments were carefully chosen based on our previous studies.8,

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9, 35

The relative

cross-peak intensities between different nuclei were calculated and compared across the temperature range examined. Figure 3 and S11 displayed the results of the NOESY measurements of poly(OMeOx5A)8-PFPE, poly(OEtOx4A)9-PFPE and poly(OEGA)9-PFPE at different temperatures. It demonstrates that the cross-peak intensities between specific protons from the side chain of all three polymers were continuously decreased with increasing temperature from 300 to 330 K. The expected increases in cross-peak intensities on dehydration were not observed in these NOESY experiments. 8, 9, 35 The magnitude of the NOE and hence the cross peak intensity depends not only on the distance between the specific nuclei spins but also on the motional correlation time τc. In order to clarify the different contributions from the above two effects to the NOE signal, the cross-peak intensity between the ethyl CH3 and CH2 protons of OEtOx units of poly(OEtOx4A)9-PFPE (P5 to P7) was calculated and found to be markedly changed with increasing temperature (Figure S12). Obviously, the distance between these protons in the ethyl group cannot change with temperature and therefore the τc, which is not determined by proton distances, was the dominant factor leading to the decrease in crosspeak intensity at high temperature.52 The cross-peak intensities between protons P2 and P4 of poly(OMeOx5A)8-PFPE and poly(OEtOx4A)9-PFPE decreased by 48 and 45% when the temperature was increased from 300 to 330 K, respectively. These numbers are significantly smaller than observed for poly(OEGA)9-PFPE (~ 70%, Figure S13). This observation again illustrates that the effect of temperature on the conformations and segmental motions of the OEG-based polymer was more pronounced compared to the POx-based polymers, due to the higher Tg and lower flexibility of the side chain of the POx polymers. Notably, a significant increase in cross-peak intensities was observed for poly(OEtOx4A)9-PFPE as the temperature was increased to 340 K (Figure


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3b), indicative of large aggregation of the poly(OEtOx4A)9-PFPE chain above its TCP (338 K) which further leads to a decrease in 19F T2.

Figure 3. Chemical structures and inter-nuclear correlations in (a) poly(OMeOx5A)8-PFPE and (b) poly(OEtOx4A)9-PFPE. Calculated cross peak intensities between different proton pairs were measured by the NOESY NMR technique as a function of temperature. For the NOESY measurements the temperature was maintained at the specified temperature for 20 min prior to the measurement. 19F

MRI performance of the POx-based PFPE polymers. The POx-based PFPE polymers

have a high fluorine content at ~ 20 wt %, higher than previously reported polymeric 19F MRI agents. 53-55 A high fluorine content is important for achieving high 19F MR imaging sensitivity. In addition to the fluorine content, segmental motion of the fluorinated segments are important considerations for the development of 19F MRI contrast agents with high imaging sensitivity.56, 57

Chain segmental motion is expected to be restricted in large aggregates. 17 ACS Paragon Plus Environment


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The hydrodynamic diameters determined for poly(OMeOx5A)8-PFPE, poly(OEtOx4A)9-PFPE and poly(OEGA)9-PFPE in water at 300 K are all around 10 nm (Table 1 and Figure 2a). Large aggregates are not formed, ensuring high mobility of fluorinated segments and hence long T2 and sharp and intense 19F NMR signals (Figure S14). The 19F NMR T1 relaxation times of all three polymers examined are approximately 400 ms at 300 K, indicating similar motional characteristics in the high megahertz frequency regime. The

19F

T2 relaxation times of

poly(OMeOx5A)8-PFPE, poly(OEtOx4A)9-PFPE and poly(OEGA)9-PFPE in PBS at 300 K are 50, 52 and 97 ms, respectively, indicating again that motional averaging of the dipolar interactions in the PFPE segments of the fluorinated POx is less efficient compared to the OEGbased polymer. In contrast, poly(OnPrOx4A)-PFPE is above the LCST at 300 K, and so a cloudy solution containing large aggregates was formed with weak

19F

NMR signal in PBS

(Figure S14). 19F

MRI was performed on solutions of the polymers at different concentrations ranging from

2 to 40 mg/mL (~ 25 to 400 mM of 19F) at 300 K. The 19F MR images and signal-to-noise ratio (SNR) as a function of polymer concentration are plotted in Figure 4. Higher concentration polymer solutions showed brighter

19F

MRI images, and the SNR increased linearly with

increasing fluorine concentration in solution (Figure 4b). Note that the Dh (~ 10 nm) and 19F NMR T2 (~ 50 ms) of poly(OMeOx5A)8-PFPE and poly(OEtOx4A)9-PFPE did not significantly change with increasing concentration. This suggests that the POx-based PFPE polymers are a promising tracer for quantitative

19F

MRI. In addition, the in vitro cytotoxicity of the POx-

based PFPE polymers were tested against MCF-7 breast cancer cells through the MTS cell viability assay. It can be concluded that the POx-based polymers are not toxic to the cells in the concentration range from 1 to 20 mg/ml at a 48 h incubation time (Figure 4c).


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Figure 4. (a)

19F

MR images at 9.4 T and 300 K of poly(OMeOx5A)8-PFPE and

poly(OEtOx4A)9-PFPE in PBS at different concentrations ranging from 2 to 40 mg/mL (~ 25 to 400 mM of 19F) in PBS. (b) Plot of 19F MRI signal-to-noise ratio (SNR) as a function of fluorine concentration in solution. (c) Cell viability of MCF-7 cancer cells after incubation with the POx-based PFPE polymers at different concentrations for 48 h. The results are the average of three replicates ± standard deviation. Temperature dependent 19F MRI properties. 19F MR images of the PFPE polymers in PBS at a fluorine concentration of ~ 400 mM at 300 K, shown in Figure 5a, clearly indicating the higher imaging intensity for the solution of the OEG-based polymer compared to the POxbased polymers at that temperature. Our small animal MRI systems is not capable of controlling the temperature over an extended range. Therefore the expected relative 19F MRI intensities were calculated using the equation described in equation 1 at five temperatures from 300 K to 350 K (Table S1). From equation 1, the MRI intensity is directly related to the fluorine content within the imaging volume, as well as the 19F T1 and T2 relaxation times.30 The calculations 19 ACS Paragon Plus Environment


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made using experimental imaging parameters and measured relaxation times, show that the MRI intensities of poly(OMeOx5A)8-PFPE and poly(OEGA)9-PFPE should increase continuously with temperature (Figure 5b). The image intensity for solutions of poly(OEGA)9PFPE will be larger for poly(OMeOx5A)8-PFPE in the temperature range of 300 to 340 K, however, a similar intensity is expected when the temperature was further increased to 350 K. At 350 K both poly(OEGA)9-PFPE and poly(OMeOx5A)8-PFPE are in the form of unimers and have similar relaxation properties. In summary, the OEG-based PFPE polymers should show superior imaging performance compared to OMeOx-based PFPE polymer when the latter forms small aggregates (300-340 K), while at 350 K when unimers are present, the imaging properties of these two polymers should be similar (Figure 5b).

[

𝐼 = 𝑁(F) 1 ― 2exp

(

(

― 𝑇R ― 𝑇1

𝑇E 2

)

)

+ exp

]

( ) exp ( ―𝑇R 𝑇1

―𝑇E 𝑇2

)

(1)

In equation 1, I is the image intensity, N(F) is a measure of the fluorine content in the volume element of the image, TR and TE are the pulse sequence repetition and echo times, respectively, and T1 and T2 are spin-lattice and spin-spin relaxation times, respectively. The TR and TE used in experimental studies and calculations using equation 1 were 1500 ms and 11 ms, respectively. The predicted image intensity for poly(OEtOx4A)9-PFPE is similar to poly(OMeOx5A)8-PFPE over the temperature range of 300 to 330 K. A dramatic drop in image intensity is expected for poly(OEtOx4A)9-PFPE as the temperature is further increased to 340 K, due to the formation of large aggregates. As discussed above, such aggregation can result in strong homo- and heteronuclear dipolar interactions and cause severe attenuation of the 19F NMR and MRI signal (Figure 5b).1


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Figure 5. (a) 19F MRI of the PFPE polymers in PBS at 300 K. The concentration of fluorine in solution was ~ 400 mM. (b) The calculated 19F MR imaging intensities of the PFPE polymers at different temperatures. Values of TR and TE used in the experimental studies and calculations were 1500 ms and 11 ms, respectively. CONCLUSIONS In summary, we have successfully prepared a series of novel thermoresponsive perfluoropolyether (PFPE)-based comb-shaped poly(2-oxazoline)s (POxs) with different side chain structures (2-methyl-, 2-ethyl- and 2-n-propyl-2-oxazoline). The side chains of POxs control the hydrophilicity of the polymer and thus have a strong effect on the chain aggregation behavior. This in turn directly influences the

19F

magnetic resonance properties, revealed

through studies using DLS, 1H 2D NOESY, 19F NMR relaxation times and MRI performance as a function of temperature. At the lowest temperature examined, poly(OMeOx5A)8-PFPE and poly(OEGA)9-PFPE form small aggregates, however smaller aggregates and eventually unimers are formed with increasing temperature. This leads to a continuous increase in 19F MRI signal intensity with temperature. Above the LCST of poly(OEtOx4A)9-PFPE (338 K), the polymer formed large aggregates which resulted in a decrease in the 19F MRI intensity. The LCST of poly(OnPrOx4A)9-PFPE was below room temperature and a low

19F

NMR signal

intensity was observed. Comparisons were made with poly(OEGA)9-PFPE, which possesses a 21 ACS Paragon Plus Environment


Page 22 of 30

more flexible acrylate backbone and a lower Tg in the bulk state than the POxs. As a consequence of the more extensive motional averaging of dipolar couplings, the OEGA-based polymer had superior imaging performance at 300 - 340 K. However, at 350 K when both poly(OEGA)9-PFPE and the poly(OMeOx5A)8-PFPE polymer are in the form of unimers, comparable imaging intensity is expected. In conclusion, this study demonstrates the power of NMR and MRI methods for understanding the aggregation of thermoresponsive partlyfluorinated polymers. More significantly, the results reveal insights about the importance of solution aggregation of macromolecules for achieving superior 19F MR imaging performance. It is expected the results reported here will assist the design of advanced MRI agents. ASSOCIATED CONTENT Supporting Information. This material is available free of charge via the Internet at http://pubs.acs.org. Additional experimental and characterization details, NMR, GPC and UV-vis spectra as well as the relaxation times at different temperatures. AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] *E-mail: [email protected] ORCID Cheng Zhang: 0000-0002-2722-7497 Kristian Kempe: 0000-0002-0136-9403 Andrew K. Whittaker: 0000-0002-1948-8355


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ACKNOWLEDGMENT The authors acknowledge the Australian Research Council (CE140100036, DP0987407, DP110104299, LE0775684, LE0668517, LE0882357, LE140100087 and LE160100168) and the National Health and Medical Research Council (APP1021759) for funding of this research. The Australian National Fabrication Facility, Queensland Node, is also acknowledged for the access to some items of equipment. C.Z. acknowledges the University of Queensland for his Early Career Researcher Grant (UQECR1720289). K. K. gratefully acknowledges the award of a NHMRC-ARC Dementia Research Development Fellowship (APP1109945). REFERENCES 1.

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Importance of Thermally Induced Aggregation on 19F Magnetic

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