Fluorinated Glycopolymers as Reduction-responsive 19F MRI Agents

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Article

Fluorinated Glycopolymers as Reduction-responsive F MRI Agents for Targeted Imaging of Cancer 19

Changkui Fu, Joyce Tang, Aidan Pye, Tianqing Liu, Cheng Zhang, Xiao Tan, Felicity Han, Hui Peng, and Andrew K. Whittaker Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.9b00241 • Publication Date (Web): 17 Apr 2019 Downloaded from http://pubs.acs.org on April 17, 2019

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Fluorinated Glycopolymers as Reduction-responsive 19F

MRI Agents for Targeted Imaging of Cancer

Changkui Fu,1,2 Joyce Tang,1,2 Aidan Pye,1,2 Tianqing Liu,3 Cheng Zhang,1,2 Xiao Tan,1,2 Felicity Han,1,2 Hui Peng,1,2 Andrew K. Whittaker*1,2 1Australian

Institute for Bioengineering and Nanotechnology, The University of Queensland

Brisbane Qld 4072, Australia 2ARC

Centre of Excellence in Convergent Bio-Nano Science and Technology, The University of

Queensland Brisbane Qld 4072, Australia 3QIMR

Berghofer Medical Research Institute, PO Royal Brisbane Hospital, Brisbane, QLD 4029,

Australia

ACS Paragon Plus Environment

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ABSTRACT Imaging agents that can be targeted to specific diseases and respond to the microenvironment of the diseased tissue are of considerable interest due to their potential in diagnosing and managing diseases. Here we report a new class of branched fluorinated glycopolymers as 19F MRI contrast agents which respond to a reductive environment, for targeted imaging of cancer. The fluorinated glycopolymers can be readily prepared by a one-pot RAFT polymerization of glucose- and fluorine-containing monomers in the presence of a disulfide-containing crosslinking monomer. The incorporation of glucose units along the polymer chain enables these fluorinated glycopolymers to effectively target cancer cells due to interactions with the over-expressed sugar transporters present on the cell surface. In addition, the polymers exhibit an enhanced

19F

MRI

signal in response to a reductive environment, one of the unique hallmarks of many cancer cells, demonstrating their potential as promising candidates for targeted imaging of cancer.

INTRODUCTION Magnetic resonance imaging (MRI) have been widely used as a powerful medical imaging technique in clinical diagnostics to produce high resolution three dimensional detailed anatomical images in a non-invasive manner. To enhance the visibility of internal body structures, contrast agents are routinely used in MRI scans. The use of contrast agents provides improved sensitivity, enabling depiction of subtle changes in anatomy and function and facilitating diagnosis of many diseases.1 However, current contrast agents generally act in a passive manner, and induce image contrast regardless of variations in their local microenvironment. This makes it difficult to attribute


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image contrast to specific pathologies. On the other hand, responsive MRI contrast agents are able to alter their physiochemical properties when interacting with the intended biomarkers.2-4 These alterations in physiochemical properties lead to significant and measurable changes in the MRI signal, and thus may serve as specific indicators of pathologies.5-9 It follows that responsive MRI contrast agents have potential advantages for diagnosis and monitoring of diseases by providing useful information about changes in the physiological/pathological condition. Over the past two decades a number of MRI contrast agents have been developed able to respond to various stimuli such as pH,10,11 temperature,12,13 the presence of specific enzymes14,15 and reactive oxygen species.6,16 Most of these responsive agents are based on paramagnetic materials such as Fe(III),17 Mn(II),17,18 Gd(III)2 or nitroxide radicals.19 These 1H MRI contrast agents are not visualized directly in the MRI scan, but rather they alter the NMR relaxation properties of nearby water molecules to achieve improved image contrast. However, the MRI results mediated by these 1H

MRI contrast agents can be at times complicated by the high background signal due to the large

pool of water molecules in biological systems, as well as by the intrinsic sources of contrast in tissues. 19F MRI contrast agents can, on the other hand, be visualized directly by 19F MRI and used to generate so-called “hot spot” images.20 The lack of MRI-detectable endogenous fluorine in the body means that there is essentially no background signal in 19F MRI, and therefore 19F MRI has potentially a very high signal-to-noise ratio and unrivalled specificity.21-28 A number of responsive 19F

MRI contrast agents have been developed, allowing depiction of subtle changes in molecular

composition and function of biological systems by 19F MRI.6,7,29-34 Glycopolymers possessing a number of repeating carbohydrate units play an important role in biological recognition through carbohydrate-protein interactions. Glycopolymers have the ability to bind to a range of proteins, and so have been investigated as potent multivalent ligands to


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stimulate, mediate or inhibit various biological or pathological processes. Accordingly, glycopolymers have emerged as a prominent class of biocompatible polymeric materials for therapeutic applications.35,36 In these applications, glycopolymers not only display excellent water solubility but are widely used as a multivalent ligand able to facilitate receptor-mediated uptake through interactions with cell-surface carbohydrate-binding lectins.37 Cancer cells often exhibit many abnormal characteristics in comparison with healthy cells. For example, the concentration of glutathione (GSH) in many types of cancer cells has been reported to be at least four-fold higher than in their healthy cells.38 Moreover, cancer cells display a significantly increased uptake of glucose in order to maintain cellular homeostasis, growth and proliferation, as mediated by the overexpressed facilitative glucose transporters (GLUT) on their surface.39-43 Imaging agents that can recognise these abnormal characteristics and provide useful information about the physio/pathological variations will be of great value for effectively imaging and management of cancer. In this contribution we report a new class of macromolecular glycosylated 19F MRI contrast agents that can target cancer cells and respond to elevated levels of GSH. The contrast agents were readily prepared by RAFT polymerization of a newly-developed fluorinated monomer 2-((1,1,1,3,3,3hexafluoropropan-2-yl)oxy)ethyl acrylate (HFEA) and a D-glucose glycomonomer (GlcA) in the presence of a reductively cleavable crosslinking monomer N,N′-bis(acryloyl)cystamine (BAC) (Scheme 1). Enhanced cellular uptake of the contrast agents by cancer cells was observed owing to the presence of the glucose moiety. In vitro MRI studies revealed that the contrast agents are highly sensitive and provide intense 19F MRI signals. Moreover, the contrast agents can respond to a reducing environment, undergoing a change from a branched structure to a linear structure,


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which results in significant enhancement of the MRI signal. We anticipate that these fluorinated glycopolymers will find potential applications in targeted cancer imaging using 19F MRI. Scheme 1. Synthesis of branched fluorinated glycopolymer by a one-pot RAFT polymerization.

EXPERIMENTAL SECTION Materials. 2-hydroxyl ethyl acrylate (HEA, Sigma Aldrich, 96%), 1,1,1,3,3,3-hexafluoro-2propanol (HFP, Sigma Aldrich, 99%), diisopropyl azodicarboxylate (DIAD, Sigma Aldrich, 98%), triphenylphosphine (PPh3, Sigma Aldrich, 98%), N,N’-bis(acryloyl)cystamine (BAC, Sigma Aldrich, 98%), D-(+)-glucose (Sigma Aldrich, ≥99.5%), concanavalin A from Canavalia ensiformis (Jack bean) (Con A, type IV, Sigma Aldrich), 2,2’-azobis(2-methylpropionitrile) (AIBN,

Sigma

Aldrich,

98%),

1,4-dithiothreitol

(DTT,

Sigma

Aldrich,

97%)

2-

(((butylthio)carbonothioyl)thio)propanoic acid (BTPA) was prepared according to a previous report.44 D-glucose glycomonomer (GlcA) was prepared using a previously reported method.45 Synthesis of 2-((1,1,1,3,3,3-hexafluoropropan-2-yl)oxy)ethyl Acrylate (HFEA). A round bottom flask was dried using a flame. PPh3 (3.76 g, 14.4 mmol) was added into the flask and flushed with nitrogen. Subsequently, a solution of 2- hydroxyethyl acrylate (1.67 g, 14.4 mmol) in


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25 mL of diethyl ether was added to the PPh3 with a syringe. The reaction mixture was placed in an ice bath and DIAD (2.50 g, 14.4 mmol) was added dropwise. The mixture was stirred under dark for 30 min followed by addition of 1,1,1,3,3,3-hexafluoro-2-propanol (2.41 g, 14.4 mmol). The reaction was left to stir for 24 h under dark. Afterward, the solution was concentrated carefully using rotary evaporator under moderate reduced pressure. The product was purified by silica gel column with DCM/petroleum spirit (1:1, v/v) as the eluent. The product was obtained as a colourless liquid (Yield ~ 47%). 1H

NMR (CDCl3, 400 MHz): δ = 6.44 (1H, dd, J = 17.4, 1.4 Hz, CH2=CH), 6.15 (1H, dd, J = 17.4,

10.5 Hz, CH2=CH), 5.88 (1H, dd, J = 10.5, 1.4 Hz, CH2=CH), 4.36 (2H, t, J = 5.0 Hz, COOCH2), 4.14 (1H, sep, J = 4.0 Hz, OCH (CF3)2), 4.09 (2H, t, J = 5.0 Hz, CHOCH2) ppm. 13C

NMR (CDCl3, 100 MHz): δ = 166.7, 131.5, 127.8, 121.1, 76.4, 72.7, 62.9 ppm.

19F

NMR (CDCl3, 400 MHz): δ = -74.1 ppm.

ESI-MS m/z: calcd for C8H8F6O3 (M+Na+), 289.03; found: 289.01. Synthesis of Branched Fluorinated Glycopolymers. A typical procedure for the synthesis of hyperbranched glycosylated and fluorinated polymer is as follow: HFEA (71 mg, 0.26 mmol), GlcA (200 mg, 0.54 mmol), BAC (13.9 mg, 0.053 mmol), BTPA (6.36 mg, 0.026 mmol) and AIBN (1 mg, 0.005 mmol) were dissolved in 1 mL of DMF. The solution was deoxygenated with Ar for 20 min then immersed in preheated oil-bath at 70 oC for 48 h. The polymerization was stopped by cooling in ice-bath and exposed to the air. The polymer was purified by dialysis against water for 3 days. After lyophilisation, the polymer was obtained as pale yellow solid.


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Conjugation of Cy5.5 to Polymers. The polymer (100 mg) was dissolved in 2 mL of DMF followed by addition of n-butylamine (200 µL). The reaction proceeded under nitrogen for 2 h then the product subjected to dialysis in water for purification. The product obtained after lyophilisation was dissolved in 2 mL of DMSO followed by addition of 100 μL of triethylamine. Subsequently, 100 μL of maleimide-Cy5.5 solution (1 mg/mL in DMSO) was added to the polymer solution. The conjugation reaction proceeded for 24 h in the dark. Afterwards, the product was purified by dialysis against water (changed every 12 h) for 72 h. After lyophilisation, the product was obtained as a pale green solid. Size Exclusion Chromatography (SEC). SEC was conducted on a Polymer Laboratories GPC 50 Plus equipped with dual angle laser light scattering detector, viscometer, and differential refractive index detector. HPLC grade N,N-dimethylacetamide (DMAc, containing 0.03 wt % LiCl) was used as the eluent at a flow rate of 1.0 mL·min-1. Separations were achieved using two PLGel Mixed B (7.8 × 300 mm) SEC columns connected in series and held at a constant temperature of 50 °C. The system was calibrated using polystyrene standards with molecular weights ranging from 6.82 × 102 g/mol to 1.67 × 106 g/mol. The polymers were dissolved in DMAc, filtered through a PTFE membrane (0.45 μm pore size), and then subjected to injection. Turbidity Assay: Turbidity assays were performed on a Varian Cary 4000 UV-vis spectrophotometer. The light absorbance of the ConA solution (600 μL, 1.0 mg/mL) at 600 nm was initially set as 0. Then a solution of P1 or P2 (50 μL, 0.5 mg/mL) was added and the light absorbance of the solution monitored with time for 20 min.


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Nuclear Magnetic Resonance (NMR). NMR spectra were performed on a Bruker Avance 400 MHz spectrometer at 298 K using CDCl3, DMSO-d6 or D2O as solvents. All chemical shifts are reported in ppm (δ) relative to tetramethylsilane (TMS). Spin-spin Relaxation Times (T2). The T2 relaxation times of polymers were measured using the Carr-Purcell-Meiboom-Gill (CPMG) pulse sequence at 298 K. The relaxation delay was 1 s and the number of scans was 64. The samples were dissolved in a mixture of PBS/D2O (90/10, v/v) with a concentration of 10 mg/mL. For each measurement, the echo times were from 2 to 770 ms and 16 points were collected. The decay in amplitude of the spin echo could be described by a single exponential function, allowing the calculation of T2. Spin−lattice Relaxation Times (T1). The T1 relaxation times of polymers were measured using the standard inversion−recovery pulse sequence. The samples were dissolved in a mixture of PBS/D2O (90/10, v/v) with a concentration of 10 mg/mL. For each measurement, the relaxation delay was 2 s and the number of scans was 32. 19F

MR Imaging. Images of phantoms containing polymer solutions were acquired on a Bruker

BioSpec 94/30 USR 9.4 T small animal MRI scanner. Polymer solutions were loaded in 5 mm NMR tubes, which were placed in a 1H/19F dual resonator 40 mm volume coil. 1H MRI images were acquired for localization of the samples using a rapid acquisition with relaxation enhancement (RARE) sequence (TE = 15.4 ms, TR = 1500 ms, FOV = 60 × 60 mm, matrix = 256 × 256). 19F MRI images were acquired in the same stereotactic space as the 1H image using the RARE sequence (TE = 10.0 ms, TR = 1000 ms, FOV = 60 × 60 mm, matrix = 32 × 32, scan time = 34 minutes 8 seconds).


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Dynamic Light Scattering (DLS). DLS measurements were conducted using a Malvern Instrument Zetasizer nano series instrument equipped with a 4.0 mW He-Ne laser operating at 633 nm and a detection angle of 173°. The number-weighted hydrodynamic diameter was obtained from analysis of the autocorrelation functions using the method of cumulants. At least three measurements at 25 °C were made for each sample with an equilibrium time of 2 min before starting measurement. The concentration of polymers was 10 mg/mL in PBS. Cytotoxicity Assay. CHO cells were seeded at 10,000 cells per well in clear-bottom 96-well plates and left for 24 hours to adhere. They were then incubated with a range of concentrations (0.03125 mg/mL to 1.0 mg/mL) of polymer in media for 24 hours under appropriate growth conditions. Subsequently, MTS reagent (10 μL per well) was added and incubated for 2 hours at 37 °C. Absorbance readings were taken on a Tecan Infinite® 200 PRO plate reader at dual wavelengths of 490 nm and 650 nm (reference readings). The experiments were performed in quadruplets with 100 % viability determined from untreated cells. Flow Cytometry. MCF-7 cells were seeded at 100, 000 cells per well in 12-well plates. The cells were then treated with 0.5 mg/mL of polymers or with 0.5 mg/mL polymers and 10 mM glucose for three hours. Glucose-free Opti-MEM was used to prepare the polymer solutions. Excess polymer was removed by washing with cold PBS. The cells were harvested and the resulting single cell suspensions were fixed with 1 % paraformaldehyde (PFA) for analysis using CytoFLEX Flow Cytometer. Confocal Microscopy. MCF-7 cells were seeded onto coverslips at a density of 50 000 cells and incubated with 0.5 mg/mL of polymers, with or without 10 mM glucose at appropriate growth conditions for 3 hours. Excess polymers were removed and cells were fixed for 20 minutes in 4 %


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PFA at room temperature. MCF-7 cells treated without polymers was used as the control group. The fixed cells were rinsed with PBS and deionized water. The coverslips were then mounted with DAPI in Vectashield and sealed using nail polish. Confocal images were acquired using a Zeiss LSM 710 inverted confocal microscope and analysed using ZEN software (Zeiss). Excitation wavelengths for polymers and DAPI were 633 nm and 405 nm respectively. MCF-7 tumour spheroids culture and treatment. The 3D tumour spheroids were cultured using microwell devices as previously described.46 The growth of the spheroids was monitored using a light microscope. After the formation of MCF-7 tumour spheroids with the diameter above 500 μm, the tumour spheroids were treated with 0.5 mg/mL polymers for 24 h. The tumour spheroids were washed with PBS to remove excess polymers after the treatment and then fixed with 4% PFA for 2h. The cells were then stained with DAPI (1 μM/mL) in the dark at room temperature for 1h. The distribution of the polymers in the spheroids was imaged with Zeiss 780NLO Point Scanning Confocal microscope (Carl Zeiss, Oberkochen, Germany). RESULTS AND DISCUSSION In a number of previous studies, conventional fluorinated monomers such as 2,2,2-trifluoroethyl (meth)acrylate (TFE(M)A) were often employed for the preparation of fluorinated polymers as 19F MRI contrast agents. However, due to the strong electron-withdrawing effect of the -CF3 group, the ester bond of TFE(M)A is relatively instable and susceptible to hydrolysis in an aqueous environment. The potentially toxic molecule 2,2,2-trifluoroethanol is generated as a hydrolyzed byproduct,6 raising safety concerns in biological applications. To overcome this, a new fluorinated monomer 2-((1,1,1,3,3,3-hexafluoropropan-2-yl)oxy)ethyl acrylate (HFEA) is proposed in the current work. Compared with TFE(M)A, HFEA has an ester bond that is less affected by the


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electron-withdrawing –CF3 groups. Thus, HFEA is proposed to be more stable and resistant to hydrolysis in biological systems. Indeed no evidence of ester hydrolysis was seen in the extensive spectroscopic studies conducted here. The HFEA monomer was synthesized in a facile manner by reacting 2-hydroxyethyl acrylate (HEA) with hexafluoro-2-propanol (HFP) via the Mitsunobu reaction with an acceptable yield of ~ 47% (Scheme 2). HFEA was characterized by NMR (Figure S1-S3). Scheme 2. Synthesis of HFEA monomer by Mitsunobu reaction.

In the next step, branched fluorinated glycopolymers were prepared by a one-pot reversible addition−fragmentation chain-transfer (RAFT) copolymerization of HFEA and GlcA in the presence of a crosslinking monomer (BAC) with 2-(n-butyltrithiocarbonate) propionic acid (BTPA) as the RAFT agent and azobisisobutyronitrile (AIBN) as the initiator (Scheme 1). A polymerization reaction with a ratio of [HFEA]:[GlcA]:[BAC]:[CTA]:[AIBN] = 5:20:2:1:0.2 was initially conducted in DMF at 70 oC for 48 h. The overall conversion of the monomers was calculated to be 95% from integration of the NMR spectrum of the reaction mixture. Due to overlapping of characteristic peaks due to the three monomers, it is not possible to calculate the conversion of each monomer by NMR. However, a kinetic study of a copolymerization of GlcA and HFEA in the absence of crosslinking monomer was conducted, and the results revealed that the polymerization of GlcA proceeded slightly faster than HFEA (Figure S4), indicating the microstructure of the resultant polymer would present a slight gradient monomer composition. The polymer was purified by dialysis against water and subjected to lyophilization to yield a pale


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yellow powder as the product. NMR spectroscopy was used to characterize the final polymer. As shown in Figure 1a, the 1H NMR spectrum revealed all the characteristic peaks of the polymer. The proton due to the triazole ring of the glycomonomer GlcA was found at 8.10 ppm while the protons due to the glucose ring appear in the range of 5.42 ~ 3.16 ppm and 2.70 ~ 2.01 ppm. The proton signals (b, e and f) due to the fluorinated monomer HFEA are overlapped by the peaks of glucose monomer in the range between 4.50 ~ 4.00 ppm. However,

19F

NMR of the polymer

indicated an intense single peak, revealing the presence of fluorinated monomer HFEA within the polymer (Figure 1b). The number-average molecular weight (Mn, SEC) and dispersity (ÐM) of the polymer was measured by size exclusion chromatography (SEC) using a RI detector, and found to be 26,000 g/mol and 1.42 (Figure 2). To further characterize the structure of the polymer, SEC with a MALS detector was used and the absolute molecular weight (Mw, MALS) of the polymer was measured as 28,100 g/mol. End group analysis by NMR indicated a molecular weight (Ml, w, NMR) of 8,520 g/mol for the linear polymer components of the branched polymer. Therefore the branching ratio (BD) of the branched polymers can be calculated as the ratio of Ml, w, NMR to Mw, MALS

as 0.3. To investigate the effect of fluorine content on the NMR/MRI properties, a second

polymer with higher fluorine content was prepared using a molar ratio of HFEA:GlcA = 10:20 via a similar method. Table 1 summarizes the molecular characteristics of the fluorinated glycopolymers, which are denoted as P1 and P2.


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Figure 1. (a) 1H NMR and (b) 19F NMR spectrum of the branched fluorinated glycopolymer P1. The solvent was DMSO-d6 with one drop of D2O to exchange with the OH protons of the polymer. Table 1. Details of branched fluorinated glycopolymers prepared by RAFT copolymerization.

Polymer

Conversion HFEA:GlcA:BAC (%) a

19F

Mn, SEC

(wt%) b

g/mol

Mw, MALS

ÐM

BD c

g/mol

P1

5:20:2

95

6.0

26000

28100

1.42

0.30

P2

10:20:2

90

10.5

21080

38260

1.74

0.34

Note: a Total monomer conversion measured by NMR; b calculated based on the feed ratio of the monomers; c determined as Ml, w, NMR/Mw, MALS.


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Figure 2. SEC traces of branched fluorinated glycopolymers prepared by RAFT polymerization. The fluorinated polymers could be directly dispersed in water. Dynamic light scattering (DLS) was used to characterize the size of the polymers in PBS solution. As shown in Figure 3, P1 with a fluorine content of 6.0 wt% had a diameter of 3.9 nm, while P2 with a higher fluorine content of 10.5 wt% was slightly larger with a diameter of 7.8 nm. The spin-spin relaxation time T2 is an important NMR property closely related to the mobility of fluorinated chain segments in 19F MRI contrast agents in solution, and is an important parameter determining the resultant MRI performance. A longer T2 relaxation time is essential for generating intense

19F

MRI signals.

Therefore, the T2 relaxation times of P1 and P2 in aqueous solution were measured by NMR at a field strength of 9.4 T, and were 26 ms and 15 ms, respectively. In comparison with P1, P2 has a shorter T2 time indicative of relatively restricted segmental mobility of the fluorinated segments in aqueous solution, leading to enhanced dipolar coupling of the 19F spins at increased fluorine contents. Aqueous solutions of polymers with a series of concentrations from 10 mg/mL to 30 mg /mL were then imaged by 19F MRI to demonstrate their applicability as 19F MRI contrast agents. As shown in Figure 4a, 19F MR images of both polymers were successfully obtained. The MRI


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intensity (or signal to noise ratio, SNR) was found to be linearly dependent on the concentration of polymers (Figure 4b). This indicates that these imaging agents are suitable for quantitative MRI measurements.

Figure 3. DLS of branched fluorinated glycopolymers (10 mg/mL) in PBS solution.


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Figure 4. (a) 19F MR images of solutions of P1 and P2; (2) SNR of 19F MRI of solutions of P1 and P2 at a range of concentrations. As the branched fluorinated glycopolymers contain disulfide bonds within the crosslink points, the polymers are expected to be cleaved under reducing environment, changing from a branched to a linear structure (Figure 5a). This would lead to a significant decrease in the molecular size of the polymer, and is also expected to enhance the segmental mobility of the polymer segments and therefore elevate the 19F MRI signal intensity. Thus, the polymers were treated with 10 mM DTT at 37

oC

mimicking the reducing microenvironment of tumour cells. Diffusion-ordered

spectroscopy (DOSY) NMR was used to measure the diffusion coefficients of the polymers. Before the polymers were treated with DTT, the diffusion coefficients of P1 and P2 were determined as 7.6 × 10-11 m2/s and 4.4 × 10-11 m2/s, respectively. The hydrodynamic radii of the polymers can be calculated by the Stokes–Einstein equation as 3.22 nm and 5.57 nm. After the polymers were treated with DTT for 4 h, the diffusion coefficients of both P1 and P2 became larger at 8.5 × 10-11 m2/s and 5.6 × 10-11 m2/s, corresponding to hydrodynamic radii of 2.88 nm and 4.37, respectively. This was consistent with the SEC results in which the trace of the polymer shifted to the low molecular weight region due to degradation after treatment with DTT (Figure S5-S6). This change in molecule structure and size upon treatment with DTT was accompanied by an increase in T2 relaxation times of the polymers (Figure 5b). The T2 of P1 increased from 26 ms to 46 ms on reduction, while the T2 of P2 increased from 15 ms to 21 ms. As a result, an increase in 19F MRI signal intensity was observed after the polymers were exposed to the presence of DTT (Figure 5c and 5d). The enhanced

19F

MRI signal of these polymers in response to a reducing

microenvironment indicates the potential for improved image contrast within tumour tissue, enabling better discrimination of tumour tissue from normal tissue.


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Figure 5. (a) The polymer undergoes a change from a branched structure to a linear structure upon exposure to a high level of DTT; (b) T2 relaxation times of P1 and P2 before and after treatment with 10 mM DTT; (c) 19F MRI images of solutions of P1 and P2 before and after treatment with 10 mM DTT; (d) SNR of 19F MRI of solutions of P1 and P2 before and after treatment with 10 mM DTT. The cytotoxicity of the polymers was investigated on Chinese hamster ovary (CHO) cells by the standard MTS assay. As shown in Figure 6, no significant cytotoxicity was observed after incubating CHO cells in the presence of solutions of the polymers with a series of concentrations up to 1000 μg/mL.


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Figure 6. Cell viability of CHO cells in the presence of different concentrations of polymers. Glycopolymers have been demonstrated to strongly interact with carbohydrate recognition proteins (lectins) via a multivalent effect. The binding behaviour of P1 and P2 polymers toward a model lectin concanavalin A (ConA) was investigated via a turbid assay. The glycopolymer acts as a crosslinker resulting in clustering of the lectin molecules in solution and an increase in the optical turbidity of the solution. As shown in Figure 7, the optical absorbance of the ConA solution (600 μL, 1.0 mg/mL) at 600 nm increased on addition of a solution of P1 or P2 (50 μL, 0.5 mg/mL). The lectin binding assay demonstrates the potential of synthesized glycopolymers to recognise targeted cancer cells through carbohydrate-protein interactions. Such interactions may facilitate further cellular uptake of the polymers via sugar transporter-mediated endocytosis.

Figure 7. Turbidity assay of P1 and P2 with lectin monitored by UV-Vis spectroscopy.


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Targeting of glycosylated MRI contrast agents to cancer cells was further examined by measuring cellular uptake using MCF-7 breast cancer cells. The dye Cy5.5 was covalently conjugated to the fluorinated glycopolymers to facilitate measurement and visualisation of cellular uptake by confocal microscope. MCF-7 cells were incubated with a solution of 0.5 mg/mL polymer for three hours. Confocal images were acquired using excitation wavelengths for the dye attached to the polymer and for DAPI (for staining of the cell nuclei) of 633 nm and 405 nm, respectively. As shown in Figure 8a (top panels), red fluorescence due to Cy5.5 of polymers can be clearly observed, indicating the polymers have been successfully internalized by the cells. Glucose transporters (GLUT) especially GLUT1 are highly expressed in many malignant tumour cells, and have been often used as a tumour marker for targeted imaging of tumour cells and accurate drug delivery. To further understand whether GLUT1 mediates the internalization of the fluorinated glycopolymers, MCF-7 cells were incubated with polymers in the presence of a relatively high concentration of D-glucose (10 mM). The addition of D-glucose can block GLUT1 and thus inhibit GLUT1 transportation-mediated endocytosis.47 As shown in Figure 8a (bottom panel), much weaker fluorescence due to the polymers was observed on addition of excess glucose, indicating the expected significantly decreased cellular uptake of the fluorinated glycopolymers. Quantitative analysis using flow cytometry provide supporting results (Figure 8b).


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Figure 8. (a) Confocal images and (b) the flow cytometry results of MCF-7 cells incubated with P1 and P2 in the absence or presence of D-glucose. Cellular uptake of polymers P1 and P2 was also investigated in a 3D multicellular tumour spheroid model. The MCF-7 spheroids were initially prepared and then incubated with solutions of polymers for 24 h before washing with PBS buffer to remove excess polymers. As shown in Figure 9, both P1 and P2 can be successfully internalized by MCF-7 spheroids. Moreover, the polymers demonstrated good penetration into the 3D spheroids.

Figure 9. MCF-7 tumour spheroids incubated with polymers for 24 h. Blue: nucleus; red: polymers. CONCLUSIONS In summary, we have developed branched fluorinated glycopolymers as a new class of 19F MRI contrast agents. The polymers display a strong and unambiguous 19F MRI signal. Incorporation of disulfide bonds in the crosslinking monomer makes the polymers susceptible to cleavage upon exposure to a high level of DTT, leading to a change from a branched structure to a linear structure.


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Biomacromolecules

The change in polymer topology results in a more intense

19F

MRI signal, demonstrating the

potential for the molecule to respond to a reductive tumor microenvironment. Cellular uptake experiments showed that the glycosylated 19F MRI contrast agents can be successfully internalized, and that GLUT-mediated endocytosis plays a significant role through carbohydrate-protein interactions. In conclusion, the high sensitivity to a reductive environment, and the high affinity for cancer cells makes these fluorinated glycopolymers promising candidates for targeted imaging of cancer. ASSOCIATED CONTENT Supporting Information. NMR spectra of fluorinated monomer HFEA, polymerization kinetics and SEC traces of polymers after treatment of DTT. AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] ACKNOWLEDGEMENT A.W. acknowledges the financial support from Australian Research Council (CE140100036, DP0987407, DP110104299, DP180101221, LE0775684, LE0668517, and LE0882357) and the National Health and Medical Research Council (APP1021759). C.F. acknowledges the University of Queensland for a UQ Development Fellowship (UQFEL1831361). The Australian National Fabrication Facility, Queensland Node, is also acknowledged for access to some items of equipment.


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For Table of Contents Use Only Fluorinated Glycopolymers as Reduction-responsive 19F MRI Agents for Targeted Imaging of Cancer Changkui Fu, Joyce Tang, Aidan Pye, Tianqing Liu, Cheng Zhang, Xiao Tan, Felicity Han, Hui Peng, Andrew K. Whittaker


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Fluorinated Glycopolymers as Reduction-responsive 19F MRI Agents

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