Communication Cite This: Chem. Mater. 2018, 30, 4892−4896
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19
F Magnetic Resonance Imaging of Injectable Polymeric Implants with Multiresponsive Behavior
Ondrej Sedlacek,*,† Daniel Jirak,*,‡,¶ Andrea Galisova,‡ Eliezer Jager,† Jennifer E. Laaser,§ Timothy P. Lodge,§ Petr Stepanek,† and Martin Hruby*,†
Chem. Mater. 2018.30:4892-4896. Downloaded from pubs.acs.org by UNIV OF WATERLOO on 10/07/18. For personal use only.
†
Institute of Macromolecular Chemistry, v.v.i., Academy of Sciences of the Czech Republic, Heyrovsky Sq. 2, 162 06 Prague 6, Czech Republic ‡ Department of Diagnostic and Interventional Radiology, Institute for Clinical and Experimental Medicine, 140 21 Prague, Czech Republic ¶ Institute of Biophysics and Informatics, 1st Medicine Faculty Charles University, Salmovská 1, 12000 Prague 2, Czech Republic § Department of Chemistry and Department of Chemical Engineering & Materials Science, University of Minnesota, 207 Pleasant Street SE, Minneapolis, Minnesota 55455, United States S Supporting Information *
M
internal radiotherapy (brachytherapy) of solid tumors; here the radiolabeled thermoresponsive polymer is injected directly into the tumor, where it forms solid radioactive depot that locally burns the cancerous tissue.12 One of the main drawbacks of using simple thermosensitive polymers as injectable implants is the rapid obstruction of the injecting needle by phase-separated polymer. To overcome this, the LCST polymer can be injected in water-miscible biocompatible organic solvents, e.g., dimethyl sulfoxide (DMSO), and the phase transition occurs due to the solvent shift.12 Clearly, it would be desirable to avoid introduction of extraneous nonaqueous solvents. Our approach introduces pHresponsivity in addition to thermosensitivity, where the phase transition temperature of the polymer is strongly pHdependent. After injection of a slightly acidic polymer solution (pH ≈ 5, which is still biologically tolerated), the polymer is buffered in the physiological milieu (pH = 7.4), which triggers the drop in the transition temperature leading to polymer precipitation (Figure 1). The imaging of injectable thermoresponsive polymer implants by 19F MRI represents an attractive tool for straightforward and noninvasive monitoring of the implant localization, size, morphology and subsequent biodegradation. Such implants would not require introduction of any other label (e.g., fluorescent or radioactive), as the fluorinated polymer itself could be effectively imaged with a high contrast to noise ratio (CNR) due to the high local fluorine concentration. These materials would be particularly well suited as the scaffolds for the therapy of brain tumors (e.g., glioblastoma) due to the very local release of attached drug over a long time. However, the use of fluorinated thermoresponsive polymers in biomedical imaging as injectable MRI contrast agents has not been reported yet. Herein, we describe the in vivo 19F magnetic resonance imaging of injectable thermoresponsive polymer implants. These were subcutaneously and intramuscularly administered
agnetic resonance imaging (MRI) is an extremely useful noninvasive diagnostic imaging tool. In many cases, the use of contrast agents/probes is mandatory to fully exploit the diagnostic potential of MRI.1 Recently, MRI probes based on fluorinated compounds have emerged as highly promising contrast agents, because 19F, a natural monoisotope of fluorine, has resonance frequency close to that of hydrogen.2−4 Therefore, fluorinated probes can be detected by standard commercial MRI scanners with appropriate double-tuned 1 H/19F radiofrequency coils. Furthermore, there is essentially no background in fluorine-based images or spectra due to the nearly zero concentration of fluorine in living organisms. Though the 19F nucleus shows slightly lower sensitivity compared to hydrogen, it still has a high gyromagnetic ratio compared to other nuclei. This favors the use of fluorinated compounds as very potent MRI contrast agents, or probes to follow metabolic pathways.5 Despite its potential, 19F MRI is still used relatively rarely, mostly due to the limited availability, low content of fluorine in synthesized compounds, and/or unfavorable physical properties of the currently available fluorinated contrast agents. Therefore, the development of new fluorinated contrast agents for 19F is currently a great challenge and clinical need. Notably, 19F MRI could be beneficial for in vivo imaging of biological implants. An appealing strategy for the construction of such implants is the use of thermosensitive polymers exhibiting a lower critical solution temperature (LCST).6 In a typical use, these polymers are subcutaneously injected into the body at room temperature as aqueous solutions. Upon heating to body temperature, the polymer rapidly loses its hydration layer and precipitates, forming a solid implant without need of surgical intervention.7,8 The in situ aggregated polymer is in equilibrium with a low amount of dissolved polymer, which is subsequently excreted by the lymphatic system into urine (renal route) or feces (hepatobiliary route), causing gradual dissolution of the implant.9 Recently, these implants have been used as depot formulations for sustained release of hydrophobic drugs, which are entrapped in the depot by noncovalent interactions and are gradually released upon the depot degradation.10,11 Another useful application consists in the © 2018 American Chemical Society
Received: May 18, 2018 Revised: July 19, 2018 Published: July 20, 2018 4892
DOI: 10.1021/acs.chemmater.8b02115 Chem. Mater. 2018, 30, 4892−4896
Communication
Chemistry of Materials
Scheme 1. Synthesis of P(DFEAM-ImPAM) Copolymers F4, F8 and PDFEAM Homopolymer F0
Table 1. Characteristics of Synthesized (Co)polymers Polymer
Mwb (kDa)
Đb
ImPAM contenta (mol %)
F0 F4 F8
37.1 40.6 41.9
1.07 1.08 1.10
0 3.9 7.8
a
Determined by acid−base titration. bDetermined by SEC with light scattering detection.
were well-defined (dispersity Đ < 1.1) with average molar masses near 40 kDa. The acid−base titration of polymers below their LCST reveals the apparent pKa of the incorporated imidazole units in F4 (pKa = 6.95) and F8 (pKa = 6.75, Figure S3). These values are slightly lower than the reported pKa of 1ethylimidazole (pKa ∼ 7.3)15 due to the electrostatic repulsions of the charged neighboring imidazole groups. For the successful MRI visualization of phase-separated polymer, high mobility of the incorporated 19F atoms is needed. Unlike commonly used fluorinated polymers (e.g., polytetrafluoroethylene and polyvinylidene fluoride) that exhibit high crystallinity, which compromises the relaxivity of the fluorine atoms even in water,16 the amphiphilic character of our polymers ensures their high chain mobility even in the phaseseparated state. The intensity of the MRI signal then remains sufficient to achieve effective imaging even under in vivo conditions. The dual temperature/pH-responsive behavior of the synthesized polymers was studied by turbidimetry. As expected, the cloud-point temperature (TCP) of PDEAM homopolymer (F0) was nearly pH-independent (Figure 2). In contrast, incorporation of the imidazole groups into the F4 and F8 chains resulted in pH-dependent TCP values. At the physiological pH = 7.4, the TCP of both polymers remained around 22 °C. In acidic environment (pH = 5.1), the charged character of the polymer chains leads to an increase in TCP to 32 °C (F4) or 43 °C (F8), respectively. To be suitable as an 19 F MRI traceable injectable implant, the TCP of the polymer in acidic solution (during the in vivo administration) needs to be higher than the body temperature (∼37 °C). In such case, the polymer remains fully soluble without the risk of polymer precipitation in the needle leading to its obstruction. Therefore, polymer F8 was selected for further assessment. To gain more detailed insight into its phase separation, F8 was further investigated by dynamic light scattering (DLS). As DLS reveals assembly of the polymer aggregates at the molecular level, the values of TCP obtained as onset points of the increase in the scattering light intensity (21 °C at pH 7.4 and 42 °C at pH 5.1) are generally slightly lower than the corresponding values obtained by the turbidity measurements (Figure 2D). At temperatures well below TCP, free polymer chains of F8 are in coexistence with nanoparticles of apparent hydrodynamic diameter close to 40 nm (Figure 2C, S7,8). This
Figure 1. (A) Schematic illustration of the 19F MRI traceable implant formation. Fluorinated polymer is injected as an aqueous solution in acidic buffer and forms a solid implant upon change of pH in vivo. (B) Structure and dual pH-/thermoresponsive behavior of fluorinated polymer F8. (C) Macroscopic appearance of polymer F8 in buffers of different pH at 37 °C (cpol = 10 mg mL−1).
as an aqueous solution to form a traceable solid implant upon the change of pH. After implant formation, the polymer chain mobility remained sufficiently high to achieve a good 19F MRI signal. So far, the only described fluorinated thermosensitive homopolymer is poly(2,2-difluoroethylacrylamide) (PDFEAM), recently reported by Bak et al, which exhibits an LCST in water at ca. 23 °C, and negligible cytotoxicity.13 As PDFEAM itself cannot be injected to the body in its aqueous solution due to the rapid obstruction of the needle by phaseseparated polymer, we incorporated a small amount of pHresponsive imidazole units into the main backbone.14 This polymer can then be administered in slightly acidic conditions (pH = 5.1), where the imidazole units are protonated, which leads to an increase in the polymer LCST above body temperature. Upon injection, the body milieu buffers the polymer back to the physiological pH, leading to its precipitation by lowering the phase-transition temperature (Figure 1). Multi-stimuli-responsive fluorinated polymers were synthesized by reversible addition−fragmentation chain-transfer (RAFT) copolymerization of DFEAM with N-(3-imidazol-1ylpropyl)acrylamide (ImPAM) and 4-cyano-4[(dodecylsulfanylthiocarbonyl)sulfanyl] pentanoic acid as chain transfer agent (Scheme 1, Table 1; for the synthesis and characterization details, see Supporting Information). The resulting statistical copolymers differed in the content of the pH-responsive imidazole units (3.9 mol % for F4 and 7.8 mol % for F8). The same procedure was employed to prepare a non-pH-sensitive PDFEAM homopolymer F0. All polymers 4893
DOI: 10.1021/acs.chemmater.8b02115 Chem. Mater. 2018, 30, 4892−4896
Communication
Chemistry of Materials
Figure 3. In vitro magnetic resonance properties of F8 in buffered media (pH = 7.4). (A) 19F MR image (red) overlaid on 1H MR image (grayscale) of Eppendorf tube phantoms filled with F8 solution of different concentration; the numbers represent fluorine concentration in mM. The highest concentration corresponds to cpol = 100 mg mL−1. (B, C) Dependence of 19F MRS/MRI signal on temperature ([F] = 80 mM); error bars represent s.d. of the signal intensity within different slices; SNR = MRI signal-to-noise ratio.
Figure 2. Dual temperature/pH-responsive behavior of the (co)polymers in buffered solutions (cpol = 10 mg mL−1). The pHdependence of (A) the cloud-point temperature (TCP) of the (co)polymers determined by turbidimetry; (B) temperature-induced transmittance change (λ = 600 nm) of F8 at the indicated pH values; (C) hydrodynamic diameter (intensity-weighted) of F8 at 37 °C determined by dynamic light scattering; (D) temperature dependence of the scattered light intensity for F8.
with aqueous solution of polymer F8 (pH = 5.1) into both muscle and subcutaneous tissue. After a short time, the polymer precipitated due to the change of pH and formed a solid implant that was visualizable by 19F MRI (Figure 4A).
self-association process is expected due to the presence of the hydrophobic dodecyl-group at the end of the polymer chains, in analogy to a recently reported system based on poly(Nisopropylacrylamide).17−19 The presence of micelles significantly reduces the diffusion of the injected polymer in the tissue, fortuitously resulting in the increased homogeneity and reduced size of the formed implant. As the polymer temperature approaches TCP, the micelles become the only specimens detected in the sample. Finally, at the TCP, the polymer starts aggregating, leading to the increase in both scattered light intensity and the nanoparticle hydrodynamic size. The 19F magnetic resonance properties of F8 were examined at physiological pH 7.4 by both spectroscopy (MRS) and imaging (MRI) experiments in phantoms (Figure 3). The minimal concentration of F8 reliably detected by MRI within reasonable measurement conditions for in vivo experiments (e.g., t = 17 min) was cpol = 3 mg mL−1 (corresponding to a fluorine concentration [F] = 40 mM). As the polymer concentration in the typical injectable implant is expected to be higher, these results validate the use of F8 as a potential 19Ftraceable probe for in vivo experiments. Variable temperature MR experiments revealed a strong dependence of the 19F signal on temperature; we observed a lower MR signal intensity and higher peak line width with increasing temperature, presumably due to the phase-separation of the polymer which could affect 19F relaxation times (Figure S9). Nevertheless, the MRI/ MRS intensity of polymer F8 above its TCP remains high enough over the range of physiological temperatures for in vivo 19 F MRI measurements. This was further confirmed by 19F MR saturation recovery measurement of F8, revealing relatively low longitudinal relaxation time (T1) of 532 ms. The injectable polymer implants were also visualized by in vivo 19F MRI. Four healthy female Lewis rats were injected
Figure 4. In vivo 19F MRI of fluorinated implants formed after injection of F8 solution (200 μL; cpol = 100 mg mL−1; pH = 5.1) into healthy Lewis rats (n = 4). (A) 19F MR coronal images (red color) overlaid on 1H MR images (grayscale) of a rat injected with F8 into both the muscle (left) and subcutaneously (right). Slice thickness of 19 F was 13 mm. (B) 19F MR axial image of F8 implant (slice thickness of 19F image was 3 mm). (C) Volume change of the F8 polymer implants after in vivo administration as determined by 19F MRI. Error bars represent s.d. of volumes measured in different animals. 4894
DOI: 10.1021/acs.chemmater.8b02115 Chem. Mater. 2018, 30, 4892−4896
Communication
Chemistry of Materials *M. Hruby. E-mail:
[email protected].
Simultaneously, the anatomic images of the rats were acquired by standard 1H MRI. Strong 19F MR signals were detected in both injection sites in all animals for the whole experiment duration (2 months). This allows us to use higher resolution (slice thickness of 3 mm) that is usually desired for 19F in vivo experiments (Figure 4B), as well as detailed 3D imaging of the implants (Movie S1). The slow degradation of implants was confirmed using volumetry assessed by 19F MR signal; volume of implants slowly increases within the first days after the polymer administration (Figure 4C). This can be explained by the equilibrium of the phase-separated implant with the small part of dissolved polymer. This slowly diffuses into the surrounding tissues where it can, in the case of higher local concentration, precipitate again reshaping the implant with a larger size. At a further distance from the implant, the polymer concentration drops gradually, the polymer remains soluble and is continuously excreted from the organism. The implant volumes reached their maximum about 4 weeks following the probe administration, after which the volume decreased as the implant further physically degraded and was excreted. The kinetics of the implant degradation was generally independent of the administration site and was suitable for application as injectable depots for the sustained release of hydrophobic drugs or, in a radiolabeled form, as an injectable brachytherapy for solid tumors. To demonstrate the safety of the prepared implants, the body weights of the injected rats were measured. All animals gradually gained weight, and no signs of nonspecific toxicity or discomfort (often reflected in the loss of animal weight) were observed along the treatment, indicating excellent tolerance toward the prepared polymer implants in these applied doses. During the whole experiment, no foreign body response such as inflammation or any other physiological abnormality was observed by proton MRI (Figure S10). Moreover, levels of important biochemical blood parameters were in the physiological range. The results of this study indicate that these thermoresponsive fluorinated polymers can be effectively utilized for injectable solid implants, and that they can be efficiently visualized by 19F MRI technique. We provide here for the first time a proof-of-concept for a system that combines direct MR imaging of an injectable implant with a wide range of potential biological applications. The sufficient MRI sensitivity and slow degradation in animals suggest its theranostic potential, which may offer unprecedented progress in the field of next generation MRI-contrast agents.
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ORCID
Ondrej Sedlacek: 0000-0001-5731-2687 Jennifer E. Laaser: 0000-0002-0551-9659 Timothy P. Lodge: 0000-0001-5916-8834 Petr Stepanek: 0000-0003-1433-678X Martin Hruby: 0000-0002-5075-261X Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The work was supported by Czech Science Foundation (grant # 16-03156S), and by Ministry of Health of the Czech Republic (Institute for Clinical and Experimental Medicine− IKEM, Grant DRO IN # 00023001). Work at the University of Minnesota was supported by the National Science Foundation, through the University of Minnesota MRSEC (Award DMR1420013).
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.chemmater.8b02115. Detailed experimental methods, characterization of polymers, turbidimetry measurements, light scattering measurements, additional in vitro and in vivo MRI results (PDF) Movie S1: 3D structure of the F8 polymeric implant 31 days after injection (AVI)
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REFERENCES
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AUTHOR INFORMATION
Corresponding Authors
*O. Sedlacek. E-mail:
[email protected]. *D. Jirak. E-mail:
[email protected]. 4895
DOI: 10.1021/acs.chemmater.8b02115 Chem. Mater. 2018, 30, 4892−4896
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Chemistry of Materials (14) Bogomolova, A.; Kaberov, L.; Sedlacek, O.; Filippov, S.; Stepanek, P.; Král, V.; Wang, X.; Liu, S.; Ye, X.; Hruby, M. Double stimuli-responsive polymer systems: How to use crosstalk between pH-and thermosensitivity for drug depots. Eur. Polym. J. 2016, 84, 54−64. (15) Landquist, J.; Katritzky, A.; Rees, C. Comprehensive heterocyclic chemistry; Pergamon Press, 1984; Vol. 1, p 384. (16) Lammers, T.; Mertens, M. E.; Schuster, P.; Rahimi, K.; Shi, Y.; Schulz, V.; Kuehne, A. J.; Jockenhoevel, S.; Kiessling, F. Fluorinated polyurethane scaffolds for 19F magnetic resonance imaging. Chem. Mater. 2017, 29 (7), 2669−2671. (17) Du, J.; Willcock, H.; Patterson, J. P.; Portman, I.; O’Reilly, R. K. Self-Assembly of Hydrophilic Homopolymers: A Matter of RAFT End Groups. Small 2011, 7 (14), 2070−2080. (18) Li, Z.; Johnson, L. M.; Ricarte, R. G.; Yao, L. J.; Hillmyer, M. A.; Bates, F. S.; Lodge, T. P. Enhanced Performance of Blended Polymer Excipients in Delivering a Hydrophobic Drug through the Synergistic Action of Micelles and HPMCAS. Langmuir 2017, 33 (11), 2837−2848. (19) Zhou, C.; Hillmyer, M. A.; Lodge, T. P. Micellization and micellar aggregation of poly (ethylene-alt-propylene)-b-poly (ethylene oxide)-b-poly (N-isopropylacrylamide) triblock terpolymers in water. Macromolecules 2011, 44 (6), 1635−1641.
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DOI: 10.1021/acs.chemmater.8b02115 Chem. Mater. 2018, 30, 4892−4896
