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Article 19

Self-assembled Thermoresponsive Polymeric Nanogels for F MR Imaging Kristyna Kolouchova, Ondrej Sedlacek, Daniel Jirak, David Babuka, Jan Blahut, Jan Kotek, Martin Vít, Jiri Trousil, Rafal Konefal, Olga Janoušková, Bohumila Podhorska, Miroslav Slouf, and Martin Hruby Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.8b00812 • Publication Date (Web): 16 Jul 2018 Downloaded from http://pubs.acs.org on July 22, 2018

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Biomacromolecules

Self-assembled Thermoresponsive Polymeric Nanogels for 19F MR Imaging

Kristyna Kolouchova, 1 Ondrej Sedlacek,1,2 Daniel Jirak, 3,5 David Babuka, 1 Jan Blahut, 4 Jan Kotek, 4 Martin Vit, 3, 6 Jiri Trousil, 1,7 Rafał Konefał, 1 Olga Janouskova, 1 Bohumila Podhorska, 1

Miroslav Slouf,1 Martin Hruby 1*

1

Institute of Macromolecular Chemistry AS CR, v.v.i., Heyrovského sq. 2, Prague 6, 162 06,

Czech Republic 2

Department of Organic and Macromolecular Chemistry, Ghent University, Krijgslaan 281-S4,

9000 Ghent, Belgium. 3

Institute for Clinical and Experimental Medicine, Vídeňská 9, Prague 4, 140 21, Czech

Republic 4

Department of Inorganic Chemistry, Faculty of Science, Charles University, Hlavova 8, Prague

2, 128 00, Czech Republic 5

Institute of Biophysics and Informatics, 1st Medicine Faculty, Charles University, Salmovská 1,

Prague, 120 00, Czech Republic

ACS Paragon Plus Environment

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Biomacromolecules 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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TU Liberec, Faculty of mechatronics, informatics and interdisciplinary studies, Studentská

1402/2 Liberec 1,461 17, Czech Republic 7

Department of Analytical Chemistry, Charles University, Faculty of Science, Hlavova 8, 128 43

Prague 2, Czech Republic

*-corresponding author, e-mail: [email protected]

Keywords: Self-assembly, Thermoresponsive, Nanogel,

19

F MRI, Contrast agent,

Angiogenesis Graphical Abstract


2

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Biomacromolecules

Abstract Magnetic resonance imaging using fluorinated contrast agents (19F MRI) enables enhanced resolution due to the negligible fluorine background in living tissues. In this pilot study, we developed new biocompatible, temperature-responsive and easily synthetized polymeric nanogels containing a sufficient concentration of magnetically equivalent fluorine atoms for 19F MRI purposes. The structure of the nanogels is based on amphiphilic copolymers containing (PHPMA) or

two

blocks:

a

hydrophilic

poly(2-methyl-2-oxazoline)

poly[N(2,2difluoroethyl)acrylamide]

poly[N-(2-hydroxypropyl)methacrylamide]

(PMeOx)

(PDFEA)

block.

block, The

and

a

thermoresponsive

thermoresponsive

properties

of the PDFEA block allow us to control the process of nanogel self-assembly upon its heating in an aqueous solution. Particle size depends on the copolymer composition, and the most promising copolymers with longer thermoresponsive blocks form nanogels of suitable size for angiogenesis imaging or the labelling of pancreatic islets (approx. 120 nm). The in vitro


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F MRI experiments reveal good sensitivity of the copolymer contrast agents, while

the nanogels were proven to be non-cytotoxic for several cell lines.

Introduction Contrast agents for medical imaging methods such as magnetic resonance imaging (MRI), positron emission tomography (PET), computed tomography (CT), photon emission computed tomography (SPECT) or ultrasound techniques are crucial for the diagnosis of cancer and other diseases.1 The widely used

1

H MRI possesses advantages such as noninvasiveness,

high-resolution anatomical imaging and practically no limitation due to sample penetration. However, the main disadvantage of 1H MRI is a low capability to differentiate healthy tissue from diseased due to the small differences in relaxation times of protons within these tissue types. This disadvantage used to be at least partly resolved by using T1-contrast agents, including paramagnetic species, which contain paramagnetic metal (mostly GdIII) that accelerates the spin relaxation of surrounding water protons,2,3,4 or superparamagnetic iron oxide nanoparticles (SPIONs), serving as T2-contrast agents. Our approach is focused on 19

19

F MRI, which has similar benefits as 1H MRI. In addition,

F MRI using fluorinated contrast agents enables enhanced resolution (“hot-spot” imaging)

due to the very low (negligible) MR-active fluorine background in living tissues. 1H MRI and 19F MRI can be performed simultaneously, as fluorine atoms have similar resonance frequencies as protons,5,6,7 combining anatomical imaging (1H) with precise and selective positioning of the contrast agent (19F). Another benefit of fluorinated contrast agents over the use of standard paramagnetic species is their non-toxicity and stability with no risk of decomposition or release of toxic metal ions.8


4

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Biomacromolecules

Currently, numerous contrast agents suitable for

19

F MRI are available for applications

including cell targeting,9,10 plaque detection or suppression,11,12 pO2 sensitivity,13,14 monitoring of inflammation15 or antiproliferative drug delivery systems.16 The currently used contrast agents are based on various designs such as polyfluorinated nanoemulsions,17 drug delivery systems with fluorinated hydrocarbons as liposomal fluorinated particles,18 and fluorinated dendrimers, hyperbranched polymers19,20 or micelles.21 These systems suffer from various disadvantages, such as the distribution of signals due to multiple equivalent atoms of fluorine or the low solvation of fluorine atoms, either leading to low resolution or long processing time. The main principle of the imaging of angiogenesis is based on the Enhanced Permeability and Retention (EPR) effect.22 Due to the tumor-stimulated angiogenesis, the primary tumor and metastatic growth generate new blood vessels to better supply nutrients to tumor cells.23 The newly formed vessels are more permeable for larger particles (up to approx. 200 nm). In addition, these particles are retained in the tumor due to the lack of lymphatic vessels in the tumor mass. This effect is also observed in some inflammation states (where it helps the immune system to reach such places). Nanosized particles such as nanogels could be also used for labeling of pancreatic islets by microporation or endocytosis.24 Herein, we describe for the first time self-assembled thermoresponsive polymer nanogels suitable for 19F MRI with a high loading of magnetically equivalent fluorine atoms. The structure of the nanogels is based on amphiphilic copolymers containing two blocks: a hydrophilic biocompatible


(PHPMA)

or

poly(2-methyl-2-

oxazoline) (PMeOx) block, sterically stabilizing the system in aqueous media and protecting the thermoresponsive fluorinated core against unwanted nonspecific interaction with biological environments, and a thermoresponsive poly[N-(2,2-difluoroethyl)acrylamide] (PDFEA) block.


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According to our method of preparation, these nanogels are physically prepared via selfassembly, provided by non-covalent bonding, such as hydrophobic interactions and hydrogen bonding, instead of the more common chemical cross-linking. Moreover, these physically selfassembled particles show better biodegradability, due to reversibility of the phase transition, which is not the case for crosslinking by covalent bonds.25 The existence of one very sharp signal as the outcome from 19F MRI, due to the high content of magnetically equivalent fluorine atoms and their relatively high solvation compared to various systems with overly crystalline cores, makes these polymers very promising for further applications in MR imaging techniques. Another benefit of the improved solvation of the polymer chains in nanogels is presupposing physical degradability.26 The thermoresponsive properties of the PDFEA block allow us to control the process of nanoparticle self-assembly upon its heating in an aqueous solution. At laboratory temperatures, the system remains soluble as unimers, whereas the nanoparticles assemble upon heating to body temperature, which is the most controlled way of producing welldefined nanoparticles without using any nanoprecipitation method before injection.27,

28

The

molecular weight of the polymer chains (i.e., unimers in dynamic equilibrium with the assembled structures) forming nanogels is kept below the threshold of renal elimination to allow for the clearing of the polymer system after the completion of its task.

Experimental section Materials. (RS)-1-Amino-propan-2-ol, methacryloyl chloride (≥97%), tert-butanol (t-BuOH, anhydrous, ≥99.5%), 2,2’-azobis(2-methylbutyronitrile) (AIBN, ≥98%), dimethyl sulfoxide (DMSO, anhydrous, ≥99.9%), 2-methyl-2-oxazoline (MeOx, 98%), allyl alcohol (≥99%), 4-nitrobenzenesulfonyl chloride (97%), triethylamine (TEA, ≥99%), acetonitrile (ACN,


6

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anhydrous, 99,8%), 4-cyano-4-(phenylcarbonothioylthio)pentanoic acid 4-cyano-4-[(dodecylsulfanylthiocarbonyl)sulfanyl]pentanoic

acid

(CTA1,>97%),

(CTA2,

97%),

acryloyl

chloride (97,0%), N,N-dimethylformamide (anhydrous, 99,8%) and silica gel 60 were purchased from Sigma-Aldrich spol. s.r.o. (Czech Republic). 2,2-Difluoroethylamine (97%) was purchased from Fluorochem (Chempur Feinchemikalien GmbH, Germany). All other chemicals and solvents were of analytical grade. Solvents and liquid chemicals were dried and purified by conventional procedures or distillation before use. Synthesis of monomers and initiators. N-(2-Hydroxypropyl)methacrylamide (HPMA) was prepared by the reaction of methacryloyl chloride with 1-amino-propan-2-ol and sodium carbonate in dichloromethane according to the reference.29 N-(2,2-Difluoroethyl)acrylamide (DFEA) was synthesized by the reaction of acryloyl chloride with 2,2-difluoroethylamine in tetrahydrofuran (THF) in the presence of triethylamine (TEA), according to the reference.30 Allyl-4-nitrophenyl-sulfonate

was

synthesized

by

a

reaction

of

allyl

alcohol

with 4-nitrobenzenesulfonyl chloride, in the presence of triethylamine. 4-Nitrobenzenesulfonyl chloride (1.79 g, 8.09 mmol, 1.1 eq.) was dissolved in dry dichloromethane (40 mL) under an argon atmosphere and cooled in a dry ice/acetonitrile bath to -30 °C. Triethylamine (1.27 mL, 8.09 mmol, 1.1 eq) was added, followed by the dropwise addition of allyl alcohol (0.50 mL, 7.35 mmol) in dry dichloromethane (5 mL). The reaction mixture was allowed to warm to 0 °C using an ice bath and kept at that temperature for 2 h. Afterwards, the reaction products were washed

with

water,

dried

with

anhydrous

magnesium

sulfate

and

purified

by column chromatography on silica (mobile phase hexane:DCM 1:1 v/v).


7


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H NMR (CDCl3, 300 Hz): δ 4.66 (m, CH2), 5.34 (m, CH2), 5.83 (m, CH2), 8.12 (d, CH),

8.41 (d, CH) 13

C NMR (CDCl3, 300 Hz): δ 92.0 (CH2), 121.4 (CH2), 124.6 (CH), 129.4 (CH), 129.7 (CH),

142.3 (C), 150.1 (C) Synthesis of homopolymers. Poly[N-(2-hydroxypropyl)methacrylamide] (PHPMA) was prepared

by

consisted

of

RAFT

polymerization.

The

N-(2-hydroxypropyl)methacrylamide

polymerization (3

g,

mixture

20.97

mmol),

4-cyano-4-(phenylcarbonothioylthio)pentanoic acid (CTA1, 48 mg, 0.17 mmol) and AIBN (18 mg, 0.11 mmol) in tert-butanol (24 mL). The reaction mixture was flushed with argon in a dried Schlenk flask and polymerized for 6 h in an oil bath heated to 70 °C. Afterwards, the resulting polymer was precipitated in diethyl ether and purified by gel filtration using a Sephadex LH-20 column with methanol as the eluent. The polymer-containing fractions were evaporated under reduced pressure, and the polymer was isolated by freeze-drying, yielding 1.9 g (63 %) of macroCTA1. The concentration of CTA1 in macroCTA1 was determined by UV/VIS to be 0.04 mmol g−1 CTA1 in PHPMA (λ = 334 nm, ε = 3.71 × 103 mol−1 cm−1). Poly(2-methyl-2-oxazoline) (PMeOx) was prepared by ring-opening living cationic polymerization. 2-Methyl-2-oxazoline was purified by distillation from calcium hydride. The mixture of 2-methyl-2-oxazoline (4 mL, 47.01 mmol) with allyl-4-nitrophenyl-sulfonate (120 mg, 0.49 mmol) in dry acetonitrile (12 mL) was purged with argon in a dried Ace After

pressure

tube

polymerization,

and the

heated

to

reaction

120

°C

in

temperature

an was

oil

bath

for

lowered

to

30 70

min. °C

and cyano4[(dodecylsulfanylthiocarbonyl)sulfanyl]pentanoic acid (CTA2, 218 mg, 0.54 mmol, 1.1 eq.) and anhydrous triethylamine (0.17 mL, 1.18 mmol, 2.2 eq.) were added under an argon


8

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Biomacromolecules

atmosphere, and the mixture was stirred for an additional 2 h at room temperature. The resulting polymer was precipitated in diethyl ether and purified by gel filtration using a Sephadex LH-20 with methanol as the eluent. Methanol was evaporated under reduced pressure, and the polymer was

lyophilized

from

an

aqueous

solution

yielding

2.21

g

(55

%)

of macroCTA2. The concentration of CTA2 in macroCTA2 was determined by UV/VIS and was found to be 0.03 mmol g−1 CTA2 in PMeOx (λ = 308 nm, ε = 10.9 × 103 mol−1 cm−1). Synthesis of diblock copolymers. The three diblock copolymers of different block ratios of


(PHPMA)

were

prepared

by

RAFT

polymerization. The three reaction mixtures containing macroCTA1 (0.5 g), AIBN (3 mg, 0.02 mmol) and different amounts of N-(2,2-difluoroethyl)acrylamide (0.38 g, 0.75 g or 1.50 g, i.e., 2.81 mmol, 5.56 mmol and 11.11 mmol, respectively) in dry DMF (4.5 mL) were flushed with

argon

and

polymerized

overnight

in

an

oil

bath

heated

to

70

°C.

After polymerization, the resulting polymers were precipitated into diethyl ether and purified by gel filtration using a Sephadex LH-20 column with methanol. Methanol was evaporated under reduced pressure, and the polymers were lyophilized, yielding 0.42 g (48 %) of PHPMA-DFEA 2:1 (HF1), 0.71 g (56 %) of PHPMA-PDFEA 1:1 (HF2) and 1.10 g (55 %) of PHMA-PDFEA 1:2 (HF3). The three diblock copolymers with different block ratios of poly(2-methyl-2-oxazoline) (PMeOx) were also prepared by RAFT polymerization. The three reaction mixtures of macroCTA2 (0.5 g), AIBN (2.5 mg, 0.02 mmol) and different amounts of N-(2,2difluoroethyl)acrylamide (0.32 g, 0.64 g and 1.28 g, i.e., 2.37 mmol, 4.74 mmol and 9.48 mmol, respectively) in dry DMF (4.7 mL) were flushed with argon and polymerized overnight in an oil bath at 70 °C. After polymerization, the resulting polymers were precipitated in diethyl ether and


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purified by gel filtration using a Sephadex LH-20 column with methanol. Methanol was evaporated under reduced pressure, and the polymers were lyophilized, yielding 0.39 g (47 %) of PMeOx-PDFEA 2:1 (MF1), 0.81 g (71 %) of PMeOx-PDFEA 1:1 (MF2) and 1.15 g (64 %) PMeOx-PDFEA 1:2 (MF3). Removal of copolymer chain-end groups. The end groups of macroCTA1 were removed by treating the polymer with an excess of AIBN in methanol heated to 50 °C for 3 h. The polymer was precipitated in diethyl ether and purified by gel filtration using Sephadex LH20 in methanol (yield ≥98%). The end groups of macroCTA2 were removed by aminolysis, followed by Michael addition, according to the described procedure.31 Then, the polymer was precipitated in diethyl ether and purified by gel filtration using Sephadex LH-20 in methanol (yield ≥98%). Figure S1 shows the disappearance of the CTA1 and CTA2 characteristic signals from the NMR spectra. 1H and

13

C NMR. 1H and

13

C NMR spectra were acquired using a Bruker Avance III 600

(or Avance DPX 300) spectrometer operating at 600.2 MHz (300.1 MHz). The width of the 1H NMR 90° pulse was 10 µs (15.6 µs), with a relaxation delay of 10 s and acquisition time of 2.18 s (4.95 s). All spectra are provided in the supplementary information (Figures S2S7). Block ratios in the copolymers were determined by 1H NMR (Figures S2-S7), using the methylene protons of the CHF2 group of PDFEA (δ = 5.9), the proton coupled with the carbon atom of the CHOH group of PHPMA (δ = 3.9) and protons of the CH3 group of MeOx (δ = 2.1), calculated by following equations (1, 2). 4 = 9 (1)


10

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Biomacromolecules

2 + 4 − 6 = 3 ∙ 6 (2) Size exclusion chromatography (SEC). The number-average molecular weight (Mn), weightaverage molecular weight (Mw) and polydispersity (Đ = Mw/Mn) of polymers were analyzed by SEC, using an HPLC Ultimate 3000 system (Dionex, USA) equipped with an SEC column (TSKgel SuperAW3000 150 × 6 mm, 4 µm). Three detectors, UV/Vis, refractive index (RI) Optilab®-rEX and multi-angle light scattering (MALS) DAWN EOS (Wyatt Technology Co., USA), were employed with a methanol and sodium acetate buffer (0.3 M, pH 6.5) mixture (80:20 w %, flow rate of 0.5 mL min−1) as the mobile phase. RI chromatograms are shown in the supplementary information (Figure S8). UV/VIS Spectrophotometry. Concentrations of end-groups in the polymers with attached CTA groups were measured using a UV/VIS spectrophotometer (Evolution 220 Spectrometer (Thermo

Scientific,

USA).

Cyano-4-(phenylcarbonothioylthio)pentanoic

acid

end-groups were determined at 334 nm (ε = 3.71 × 103 mol−1 cm−1), whereas cyano4[(dodecylsulfanylthiocarbonyl)sulfanyl] pentanoic acid end-groups were found at 308 nm (ε = 10.9 × 103 mol−1 cm−1). Dynamic light scattering (DLS). The hydrodynamic radii (Rh) and scattering intensity as a function of polymer temperature were determined by the DLS method using a Zetasizer NanoZS instrument, Model ZEN3600 (Malvern Instruments, UK). The apparent volumeweighted hydrodynamic diameter of particles, Rh, was measured at a scattering angle of θ = 173 °, and the DTS (Nano) program was used to evaluate the data. The measurement was conducted with a polymer concentration of 1, 5 or 10 mg mL−1 in phosphate buffered saline (150 mM, pH 7.4) at 8 – 50 °C; all solutions were filtered prior to measurement through a


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0.22µm PVDF syringe filter. The measurement step was 1 °C, and at each temperature, the sample was equilibrated for 5 min before measurement. The acquisition time was set to 5 min, which helped to thoroughly smooth out any fluctuations affecting the correlation curve. Static light scattering (SLS). The radii of gyration (Rg) and the molecular weights of particles (M) at body temperature were determined by the SLS method using an ALV-6000 (ALV-GmbH, Germany). The z-average radii of gyration of particles, z, and their weight average molecular

weights,

and concentrations

Mw, through

were the

determined analysis

from of

measurements

Zimm-like

plots

at

several

created

angles by

the

ALV / Static & Dynamic FIT and PLOT 4.31 10/01 programs. The measurements were conducted in phosphate buffered saline (150 mM, pH 7.4) with polymer concentrations of 2.5, 5.0, 7.5 and 12 mg mL−1 for HF3 and 1.0, 1.25, 1.5 and 2.0 mg mL−1 for MF3 at 37 °C; all solutions were filtered prior to measurement through a 0.22-µm PVDF syringe filter. The measurement angle range was 40° - 50° with a step of 10°. The experiment used 3 averaged measurements for each angle with the acquisition time set to 1 minute. This setup reduces the influence of random dust particles and other fluctuations on measurement and provides us with statistical error data. Transmission electron microscopy (TEM) and cryo-transmission electron microscopy (Cryo-TEM). Transmission electron microscopy (TEM) of nanoparticles was performed using a Tecnai G2 Spirit Twin 12 (FEI Company; Czech Republic) microscope equipped with a Cryo-holder (Gatan; CA, USA). The samples were prepared above their LCST temperatures as follows: In the first step, all solutions (polymer concentration 5 mg mL−1) and tools for sample preparation (tweezers, microscopic grids, etc.) were placed in an oven heated to 50 °C and left there for >15 min. In the second step, the solution at 50 °C was


12

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Biomacromolecules

processed in one of three ways: (a) 2 µL of the solution was deposited onto a TEM carbon-coated copper grid and left to evaporate in the heated oven at 50 °C; (b) 2 µL of the solution was deposited onto a TEM carbon-coated copper grid, left in the oven at 50 °C for 1 min, removed from the oven, and then the excess solvent was removed by touching the bottom of the copper grid with a small piece of filter paper; or (c) 3 µL of the solution was deposited onto a TEM holey carbon-coated copper grid, flash frozen in liquid ethane and kept under liquid nitrogen. More technical details about all of the above-listed preparation methods can be found in our previous papers.32, 33, 34 The dried samples from the first two methods (a, b) were observed using TEM microscopy at room temperature. The frozen samples from the third method (c) were observed using Cryo-TEM microscopy. All micrographs are bright field images taken at 120 kV. Critical association concentration (CAC). Solutions of the copolymers were prepared by direct dissolution in cold phosphate buffered saline (PBS, 150 mM, pH 7.4) at a concentration of 1 mg mL−1 PBS and subjected to serial dilution with concentrations down to 10–5 mg mL−1. Each sample was then prepared by the addition of 10 µL of pyrene solution (1.2 × 10–4 mol L−1 in methanol) to 2 mL of the diluted solution. The final concentration of pyrene in water thus reached 6 × 10–7 mol L−1. Fluorescence spectra of the samples were recorded (90 ° angle geometry, 1 × 1 cm quartz cell) with an FP-6200 spectrofluorometer (Jasco Europe, Italy), equilibrated for 10 min at 37 °C, using excitation at 333 nm. The intensities of the emission bands I1 at 372 nm and I3 at 383 were then evaluated, and their ratio was plotted versus the polymer concentration. The I1/I3 ratio remained constant at a certain polymer concentration, below which the pyrene remained in the aqueous phase. When pyrene was sequestered into the nanoparticle core, I1/I3 decreased.


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Page 14 of 42

The CAC was determined as the intersection between the plateau at I1/I3 ≈ 1 and the tangent of the curve where the I1/I3 ratio decreased with an increase in the copolymer concentration. T1 19F NMR relaxation time. The T1 measurements were performed by a Bruker Avance III 400 MHz spectrometer using an inversion recovery pulse sequence. The temperature of the sample was calibrated using 99.8 % CD3OD by measuring the chemical shift differences between the two 1H NMR signals.35 Particular care was taken to ensure that the sample reached thermal equilibrium before the data acquisition started (Table S1). Cytotoxicity. Cell lines: HeLa - human cervical carcinoma cell line (kindly provided by Dr. Melkova, First Faculty of Medicine, Charles University in Prague); J774A.1 - murine monocyte/macrophage cell line (ECACC General Cell collection, Public Health England, UK); HF - primary fibroblasts, SU-DHL-5 - human B lymphoblast cell line (ATCC, LGC Standards Sp. z.o.o. Poland). The cells were cultivated in DMEM and IMDM for SU-DHL-5 cells (Thermo Scientific, CR). The media were supplemented with heat-inactivated 10% fetal bovine serum (FBS), 100 U mL-1 penicillin and 100 µg mL-1 streptomycin. Cytotoxicity of polymer conjugates: in total, 5 × 103 cells for HeLa and 10 × 103 cells for J77A4A.1, SU-DHL-5 and HF per well, respectively, were seeded in 100 µL of media in 96-well flat-bottom plates (TPP, Czech Republic) 24 h before the addition of polymer conjugates. The concentrations of conjugates varied in the range of 0.002 – 1 mg L−1. The cells were cultivated for 72 h in 5 % CO2 at 37 °C. After that, 10 µL of AlamarBlue® cell viability reagent (Life Technologies, Czech Republic) was added to each well and incubated for 4 h at 37 °C. The active component of the AlamarBlue reagent, resazurin, was reduced to the highly fluorescent compound resorufin only in viable cells. Its fluorescence was detected using a Synergy Neo plate reader (Bio-Tek, Czech Republic) with excitation at 570 nm and emission


14

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Biomacromolecules

at 600 nm. As a control, cells cultivated in the medium without polymer conjugates were examined. The assay was repeated two times independently. Hemolysis. Fresh human blood was collected from a healthy volunteer into heparin-coated vacutainers (Becton Dickinson Czechia Ltd., Prague, Czech Republic). Plasma was removed by centrifugation at 3000 rpm for 10 min. The red blood cells (RBCs) were washed three times with Dulbecco's Phosphate-Buffered Saline (DPBS). For the hemolysis experiment, the RBC suspension was diluted with DPBS to the final concentration corresponding to full blood (1:49). To 0.3 mL of diluted RBCs, 1.2 mL of the appropriately diluted formulation in PBS was added. By this procedure, several dilutions of polymers were prepared (100, 200, 400 and 800 µg mL−1). Triton X-100 (1%, DPBS) and DPBS served as positive and negative controls, respectively. After incubation at 37 °C for 3, 6 or 24 h, the samples were centrifuged (3000 rpm, 10 min). Subsequently, supernatants were collected, and their absorbance values at 541 nm were measured on a Synergy H1 Hybrid Reader instrument (Biotek, Winooski, USA) using Nunc Cell Culture Microplates (Thermo Scientific Nunc, USA). The percent hemolysis of RBCs in each sample was calculated by the following equation (3):

Hemolysis =

sample absorbance – negative control ) 100 positive control – negative control

(3) Hemolysis assays were expressed as a percentage of the positive control (1% Triton X-100 in DPBS), which was considered to be 100 %. Hemoglobin release up to 2 % F was classified as non-hemolytic, according to the ASTM F756-08 standard.36 The result of this assay was obtained as a mean value from three parallel samples.


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1H and 19F MR imaging and spectroscopy (in vitro study). All imaging and spectroscopy experiments were performed on a 4.7 T MR Bruker spectrometer Bruker Biospec (Bruker BioSpin, Germany). MR imaging of HF3 and MF3 phantoms was performed using a wide-tuned 1

H-19F half-saddle homemade coil. Tubes containing polymer solutions with different

concentrations (3.3, 5, 10 and 50 g L−1) were examined. A 19F turbo spin echo sequence Rapid Acquisition with Relaxation Enhancement (RARE) for phantom imaging was used with these sequence parameters: repetition time (TR) = 2000 ms, effective echo time (TE) = 43.5 ms, turbo factor =16, number of acquisitions (NA) = 16, 128 and 256, field of view 5 cm, matrix 64 x 64, spatial resolution 0.6 x 0.6 mm2, slice thickness 4 mm, acquisition time (TA) = 2.8, 17.4 and 34.8 min). The temperature dependence of HF3 and MF3 was measured using a wide-tuned 1H-19F fiveturn homemade solenoid coil. For the temperature dependence experiment, a single pulse 19F spectroscopy sequence was used with these parameters: TR = 1069 ms, AC = 32, flip angle (FA) = 30°, TA = 34 s. The wire coil was twisted with a heating pipe with flowing hot water for precise phantom temperature adjustment. The temperature range was 30 – 45 °C. Each measurement was followed with a 5-min pause to achieve temperature equilibrium within the whole phantom volume. For verification of the results, measurements at 37 °C and 45 °C were repeated after 30 minutes.

Results and discussion Block

copolymers

poly[N-(2-hydroxypropyl)methacrylamide]-block-poly[N-(2,2-

difluoroethyl)acrylamide] (HF1-3) were prepared by two successive RAFT polymerizations


16

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Biomacromolecules

of the corresponding monomers with dithiobenzoate as the chain transfer agent (CTA1). Poly(2-methyl-2-oxazoline) (PMeOx) was prepared by living cationic ring opening polymerization using allyl-4-nitrophenyl-sulfonate as the initiator. The initiator-based allyl group at the polymer chain-end allows its further modification, e.g., with targeting units, another imaging modality or chelators.37,38 After the polymerization was complete, the cationic living chain-ends were treated with triethylammonium salt of trithiocarbonate. In this reaction, the chain transfer agent is introduced to the polymer to form macroCTA2, which was used for the RAFT polymerization of the second poly[N-(2,2-difluoroethyl)acrylamide] block (MF1-3 copolymers). The concentration of CTA in macroCTA1 and macroCTA2 was detected by

UV/VIS

at

334

nm

and

308

nm,

respectively.

After

the

polymerization

of poly[N(2,2difluoroethyl)acrylamide] block, both CTAs were removed to avoid the obvious effect of CTA end-groups on polymer thermoresponsivity. The dithiobenzoate was removed by radical reaction with AIBN, and trithiocarbonate was removed by aminolysis, followed by Michael addition (Scheme 1). Figure S1 shows the disappearance of the CTA1 and CTA2 characteristic signals from the NMR spectra.


17


Page 18 of 42

Scheme 1. Synthesis of diblock copolymers with PHPMA or PMeOx as hydrophilic blocks and PDFEA as the thermoresponsive block. Block ratios in the copolymers were determined by 1H NMR (Figures S2-S7), using the methylene protons of the CHF2 group of PDFEA (δ = 5.9), the proton coupled with the carbon atom of the CHOH group of PHPMA (δ = 3.9) and protons of the CH3 group of MeOx (δ = 2.1). Molecular weights and polydispersity were obtained using SEC (Table 1, Figure S8). Table 1. Physicochemical characteristics of the prepared first blocks and corresponding diblock copolymers and physical properties of assembled particles. Sample name

Polymer

Block ratiosa

MacroCTA1

PHPMA

-

HF1 HF2 HF3

PHPMAPDFEA

w % of fluorine

Mw (kDa)b

Mn (kDa)b

Ɖ (Mw/Mn)

13.3

12.7

1.05

CPT (°C)c

Rh ± σ (nm)c

2:1

6.7

17.6

15.8

1.11

37

123 ± 19

1:1

16.9

33.7

31.9

1.06

31

35 ± 5

1:2

20.7

51.5

48.3

1.07

23

77 ± 52


18

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Biomacromolecules

MacroCTA2

PMeOx

MF1 PMeOxPDFEA

MF2 MF3

a

-

8.1

7.2

1.12

2:1

10.1

12.7

11.9

1.07

31

670 ± 203

1:1

14.0

16.2

15.3

1.06

30

31 ± 12

1:2

18.7

24.5

22.8

1.08

33

47 ± 14

Determined by NMR spectroscopy. bDetermined by SEC. cDetermined by DLS.

The thermal response of the prepared copolymers in aqueous solutions was investigated by dynamic light scattering. Hydrodynamic radii (Rh) of the particles were measured in the temperature range from 10 °C to 50 °C. All copolymers exhibited temperature-dependent solution behavior; above a certain temperature, their individual molecules dissolved in PBS buffer started to assemble and form larger particles. This cloud-point temperature (CPT) was highly dependent on the ratio between homopolymer blocks for copolymers with PHPMA, while the copolymers with PMeOx showed no such trend (Table 1). The higher content of the more hydrophobic block should result in the lower CPT, but this effect is obviously also dependent on the type of hydrophilic monomer, as it was previously described for other systems.28 The concentration of the polymer seemed to have only a marginal influence on the CPT, even for ten-fold diluted samples, which is essential for the intended use where the sample is diluted with body fluids. The values of CPT, as well as Rh of the most prevalent particles in the solution at 37 °C, are listed in Table 1. The representative examples of size distributions are presented in Figures 1-2, while the other CPTs are illustrated in Figure S10. For the size distributions and dependence of CPTs on concentration for all samples and concentrations, see the supplementary information (Figure S9).


19


Page 20 of 42

The reversibility of these thermally dependent changes was investigated as well. After obtaining the data from the heating process, the polymer solution was cooled from 50 °C back to 10 °C. When the temperature dropped below the CPT, the particles disassemble rapidly into single molecules. Therefore, the nanoprecipitation was found to be fully reversible. This is important for the unimer-nanoparticle equilibrium-based degradability because polymers that crystallize after the initial phase separation from water (e.g., poly(2-isopropyl-2-oxazoline)) may become kinetically frozen and, in fact, become non-dissolvable even after cooling below the phase separation temperature.

Figure 1. Self-assembly of HF3 (1% polymer solution in 150 mM phosphate buffered saline, pH 7.4): 3D graph of the dependence of the size distribution on temperature (left) and CryoTEM image (right).


20

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Biomacromolecules

Figure 2. Self-assembly of MF1 (1% polymer solution in 150 mM phosphate buffered saline, pH 7.4): 3D graph of the dependence of the size distribution on temperature (left) and TEM image (right). Samples

of

HF3

and

MF3

(block

copolymers

with

a

block

ratio

of

1:2

hydrophobic/fluorinated) were further analyzed by static light scattering to obtain the molecular weights, radii of gyration and densities of the nanoparticles formed at 37 °C. The obtained data are listed in Table 2. Table 2. SLS data for selected samples. Molecular weight (Mw), hydrodynamic radius (Rh) and calculated density (ρ) of particles assembled from each block copolymer. Sample name

Polymer

Block ratiosa

Mw ·103 (kDa)b

Rg (nm)b

ρ (g cm-3)b

HF3

PHPMAPDFEA

1:2

66.8

106

0.022

MF3

PMeOxPDFEA

1:2

10.4

67

0.013

a

Determined by NMR spectroscopy. bDetermined by SLS


21


Page 22 of 42

The DLS and SLS results together with the transmission electron microscope (TEM) images provide comprehensive insight into the structures for each copolymer, HF3 and MF3, and suggest that the internal structure of the nanoparticles is that of a nanogel. The first indication is the size of the particles (by DLS and SLS), which cannot be so high that the system would be a micelle. Hence, we determined the density of the core (Table 2), which was very low, indicating that the system could be a nanogel or vesicle. Nevertheless, the samples cannot be vesicular particles because of the uniform polydispersity and uniform distribution of matter (by TEM). Another proof is the molecular weight of the particle after self-assembly (Table 2), which indicates that approx. 500 – 1000 polymer chains comprise one nanoparticle, while a micelle contains approx. 10 – 100 polymer chains. The morphology of the selfassembled polymer particles is summarized in Figures 1, 2 and S11. Both polymers (PMeOx-PDFEA and PHPMA-PDFEA) formed larger particles at a ratio of 2:1 (copolymers MF1, HF1) and smaller particles at a ratio of 1:2 (copolymers MF3, HF3). The particles of both polymers with a ratio of 1:1 were unstable during the TEM sample preparation. The size ratio between hydrophobic and hydrophilic blocks of amphiphilic copolymers has obviously significant influence on the morphology of particles they form in aqueous solutions. In the case of highly organized micelles and vesicles the block copolymers with smaller hydrophobic blocks form smaller micellar particles, whereas block copolymers with larger hydrophobic blocks form larger vesicular particles, as it has been previously described.39 This is in direct contrast to the behavior of our nanogel particles, which exhibit only limited degree of organization and form larger particles for polymers with smaller hydrophobic blocks and smaller particles for polymers with larger hydrophobic blocks. The formation of stable particles from amphiphilic block copolymers is a matter of equilibrium between forces holding


22

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Biomacromolecules

the polymer chains together and forces acting to solvate the chains with solvent molecules (e.g. water) and cause molecular dissolution.40 Based on our experimental results we hypothesize that our polymers with smaller hydrophobic blocks form particles, which could be described in a similar way as the plum pudding model of an atom. Most of the volume of the particles is formed by hydrated hydrophilic blocks whose chains are swollen and extended in aqueous environment, and within this hydrophilic gel (the “pudding”) smaller pockets of hydrophobic blocks (the “plums”) non-covalently bound together are placed, while their chains are contracted to minimize their interaction with water. Similar behavior can be observed in other copolymers.41 This morphological arrangement is made possible only by the favorable size ratio of hydrophilic to hydrophobic blocks in the polymer molecule. In the case of our other polymers in which the hydrophobic blocks are larger than the hydrophilic blocks there is not enough hydrophilic “pudding” covering the hydrophobic “plums” formed by the fluorinated blocks aggregating together to form an interconnecting net with the hydrophilic domains of other particles. In result, the hydrophilic blocks only cover the surface of the hydrophobic domains, which is why we obtain smaller particles for these polymers and block size ratios. The smaller particles of MF3 exhibited lower average sizes in comparison to the particles of HF3, which was in perfect agreement with the light scattering experiments. To summarize, the TEM and DLS measurements reveal that the copolymers with higher contents of thermoresponsive blocks form nanoparticles (Figure 1) in a size range that is well suited for imaging, e.g., of tumors or inflamed tissue (20 – 200 nm, approx. 80 nm in this case), due to the EPR effect.


23


Page 24 of 42

In order to verify the correctness of the TEM results showing the studied polymer systems after fast drying, we also performed Cryo-TEM microscopy visualizing the particles after fast freezing the solution. Figure 1 shows a Cryo-TEM micrograph of one selected system, HF3. The DLS measurements reveal that at temperatures below the cloud point temperature (CPT), there are always (with the exception of MF2) two dominant species present in the sample. Smaller entities (Rh about 3-7 nm) correspond to free polymers in solution, while the larger ones correspond to particles that are always present even in lower temperatures (Figures 1, 2, S10), resulting from the partial agglomeration between copolymers. However, the larger particles scatter light much more than smaller ones, so the content of larger particles can be neglected. At the CPT, the peak representing polymers in solution immediately converts into peaks corresponding to the nanoparticles due to nanoprecipitation, while the intensities of scattered light show an abnormal increase. This increase corresponds to phenomenon described as anomalous micellization behavior in diblock copolymer solutions.42, 43 The critical association concentration (CAC) is indispensable information about the stability of the self-assembled particles. Individual CACs are deduced from the charts shown in Figure S12. The resulting CAC for HF3 is 51.6 mg L−1 and for MF3 is 26.2 mg L−1. As nanogels are more appropriate for the bioimaging purposes due to their size, the 19F NMR, MRI, cytotoxicity and blood compatibility (i.e., hemolytic properties) were further studied for the micellar samples (PHPMA-PDFEA and PMeOx-PDFEA at a ratio of 1:2, HF3 and MF3). The 19F NMR T1 relaxation times (Table S2) were measured before and after nanoprecipitation. The T1 relaxation time dependence on the temperature is relatively low, showing only minor effects of nanoprecipitation on the relaxation behavior of the 19F nuclei. This information is vital for

the

potential

use

of

nanogels

as

contrast

agents

for

19

F

MR

imaging.


24

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Biomacromolecules

After the nanoprecipitation, the mobility of fluorine atoms remains high, so the fluorine signals in 19F NMR are still clearly visible. To assess the biological properties of synthesized polymers, their cytotoxicity and hemolytic properties were studied. The polymer conjugates did not show any significant cytotoxicity in the range of polymer concentrations tested (0.002 – 1 mg mL−1). The viability of cells incubated with even the highest polymer concentration (1 g L−1, corresponding to 5 g of polymer per human blood pool of approx. 5 L) was not decreased below 80% for the most sensitive cell line Su-DHL-5 and not below 90% for the rest of the cell lines (Figure S13). These data point towards the possible application of these polymers for their intended biomedical purposes. The interaction of the formulations with red blood cells (RBCs) as major blood components and one of the first contact partners after systemic administration was studied with regard to red blood cell hemolysis. The HF3 copolymer induced no hemolysis after a 3-, 6- or 24-hour incubation at 37 °C within the tested concentration range (i.e., 100 – 800 µg mL−1). The formulation MF3 caused no hemolysis after a 3-hour incubation within the tested concentrations. Long-term hemolytic studies (6 and 24 hours), however, revealed the beginning of hemolysis (i.e., values ranging from 2 to 4 %) in the case of samples with higher polymer concentrations (200, 400 and 800 µg mL−1). This effect is probably due to the amphiphilic character of the tested formulation. However, for comparison, negative controls revealed a mean hemolysis of approx. 1 %. Moreover, the MF3 formulation at the biologically relevant concentration of 100 µg mL−1 caused no hemolysis, even during the 6- and 24-hour incubation, suggesting that it is safe for RBCs. Hemolysis assay-related results are shown in Figure S14. The MR spectroscopy experiment showed the dependence of the 19F MR signal on temperature for both copolymer (HF3 and MF3) probes. HF3 possesses a stronger temperature dependence


25


Page 26 of 42

(Figure 3) in that the MR signal rises with increasing temperature. Compared to HF3, the temperature dependency for MF3 was not so significant (Figure 3); for 30 °C and higher temperatures, the MR signal was practically constant, with a difference lower than 1 %. For lower temperatures, the signal was higher. Repeated measurements at 37 °C and 45 °C confirmed the stability of probes, as the signal intensity did not change significantly. The resonance frequency for each measurement was stable, with frequency differences lower than 0.5 ppm. Signal was received from 550 µl solutions with polymer concentrations of 50 g L−1. For copolymer MF3, the abnormal signal level at 25 °C was due to a phase change. For imaging use, an appropriate MR image was collected for each temperature.

Figure 3. Temperature dependence. MR spectra and corresponding images of copolymer HF3 and MF3 phantoms are shown for various temperatures.


26

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Biomacromolecules

The MRI sensitivity of the probes was checked by measurement with various scan times. The MRI experiments proved the feasibility of in vitro visualization of these probes. Therefore, we supposed that these fluorine probes might also be used in in vivo experiments. Only phantoms with the highest concentration of polymers were detected within 2 minutes of scanning (Figure 4). For 17-minute scans, we were able to detect phantoms of all used concentrations; however, the

19

F signal from HF3 was higher than that of MF3, which corresponds

with the higher concentration of fluorine in the HF3 copolymer. The temperature change could have a high impact on T2 relaxation times due to the possible change in mobility of fluorine atoms, which is reflected in the unfavorable broadening of the fluorine signal.44 However in Figure 3, it can be seen that the broadening of signal, that is, the T2 change, is insignificant (e.g., 284 Hz at 30 °C and 255 Hz at 40 °C), due to sufficient solvation of fluorine atoms in the nanogels.

Figure 4. MR imaging of phantoms of HF3 and MF3 copolymers at 20 °C (polymer solution in 150 mM phosphate buffered saline, pH 7.4). In the first column, there are 2 min 8 s 19

F acquisition time images only. At the top of the second column, there is a 1H navigator image

overlapped with the 17 min 4 s

19

F red image. At the bottom of the second column, there is


27


Page 28 of 42

a 19F image after 17 min 4 s only. At the bottom of the third column, there is a 1H navigator image overlapped with the 34 min 8 s 19F red image. The non-uniformity of signals shown in Figure 4 is mainly caused by a surface radiofrequency coil that was used in the MR measurements. This coil has a high sensitivity but low homogeneity. Non-uniformity of 1H and

19

F (HF3; t = 17 min 4 s; signal on the right side) is

possibly caused by the sedimentation of the 19F polymer at the bottom of the tube, while the time for settling between protons and fluorine MRI is quite long and, in the case of HF3, which has a CPT very close to room temperature, sedimentation could appear. In addition, Figure S15 shows graphs with the signal to noise ratio (SNR) dependence on concentration. They show a linear dependence (that is, with higher concentrations, we observe higher 19F MR signals). Since the thermoresponsive nanogel shows fair sensitivity for

19

F MRI, mainly due to low

T2 changes and high concentrations of fluorine, it could be considered a universal system where a variety of further modification could create new applications. There are many previously published contrast agents for

19

F MRI with various types of stimuli responsivity, for example

temperature-sensitive liposomal contrast agents,18 pH-responsive star polymer nanoparticles45 or fluorinated dendrimers,46 ion-responsive contrast agents,47 etc. These systems have similar functions as smart drug carrier for simultaneous diagnostic and therapy (theranostics). In our approach, thermoresponsive function is a very simple way of preparing well defined nanoparticles in aqueous solution, without need of any other nanoparticle preparation technique. Conclusions We describe for the first time self-assembled thermoresponsive polymer nanogels suitable for

19

F MRI with a high loading of magnetically equivalent fluorine atoms. The structure

of the nanogels is based on amphiphilic copolymers containing two blocks: one hydrophilic biocompatible block and one fluorinated thermoresponsive block. Depending on the block ratio,


28

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Biomacromolecules

the size of the nanogels formed after heating from room to body temperature may be fine-tuned in a wide range. The in vitro

19

F MRI experiments reveal the excellent sensitivity

of the copolymer contrast agents, while the nanogels were proven to be non-cytotoxic for several cell lines. Associated content Supporting Information. Charts with 1H NMR spectra of diblock copolymers and 1H NMR spectra of CTA groups; summary from size exclusion chromatography (SEC); 3D graphs of the dependence of the size distribution on temperature measured by dynamic light scattering (DLS); charts of the dependence of relative count rates on temperature; transmission electron microscopy (TEM) micrographs; summary tables from 19F NMR (T1 relaxation time for different temperatures); charts showing the critical association concentration (CAC), summary of the cytotoxicity and hemolysis assays and the signal to noise ratio dependence on polymer concentration. Acknowledgements The authors acknowledge financial support from the Czech grant foundation (grant # 16-03156S). Author Jiri Trousil acknowledges financial support from Charles University (project no. SVV260440). References (1)

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Figure 1. Self-assembly of HF3 (1% polymer solution in 150 mM phosphate buffered saline, pH 7.4): 3D graph of the dependence of the size distribution on temperature (left) and CryoTEM image (right). 158x59mm (150 x 150 DPI)



Figure 2. Self-assembly of MF1 (1% polymer solution in 150 mM phosphate buffered saline, pH 7.4): 3D graph of the dependence of the size distribution on temperature (left) and TEM image (right). 155x60mm (150 x 150 DPI)


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Figure 3. Temperature dependence. MR spectra and corresponding images of copolymer HF3 and MF3 phantoms are shown for various temperatures. 214x109mm (150 x 150 DPI)



Figure 4. MR imaging of phantoms of HF3 and MF3 copolymers at 20 °C (polymer solution in 150 mM phosphate buffered saline, pH 7.4). In the first column, there are 2 min 8 s 19F acquisition time images only. At the top of the second column, there is a 1H navigator image overlapped with the 17 min 4 s 19F red image. At the bottom of the second column, there is a 19F image after 17 min 4 s only. At the bottom of the third column, there is a 1H navigator image overlapped with the 34 min 8 s 19F red image. 165x68mm (150 x 150 DPI)


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Graphical abstract 165x94mm (150 x 150 DPI)



Scheme 1. Synthesis of diblock copolymers with PHPMA or PMeOx as hydrophilic blocks and PDFEA as the thermoresponsive block.

164x73mm (150 x 150 DPI)


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Self-assembled Thermoresponsive Polymeric ... - ACS Publications

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