Self-Assembled, Biodegradable Magnetic Resonance Imaging Agents

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Self-Assembled, Biodegradable Magnetic Resonance Imaging Agents: Organic Radical-Functionalized Diblock Copolymers Julian M. W. Chan,† Rudy J. Wojtecki,† Haritz Sardon,‡ Ashlynn L. Z. Lee,§ Cartney E. Smith,∥ Artem Shkumatov,∥ Shujun Gao,§ Hyunjoon Kong,∥ Yi Yan Yang,§ and James L. Hedrick*,† †

IBM Almaden Research Center, 650 Harry Road, San Jose, California 95120, United States POLYMAT, University of the Basque Country UPV/EHU Joxe Mari Korta Center, Avda. Tolosa 72, 20018 Donostia-San Sebastián, Spain § Institute of Bioengineering and Nanotechnology, 31 Biopolis Way, Singapore 138669, Singapore ∥ Department of Chemical and Biomolecular Engineering, Carl R. Woese Institute for Genomic Biology, University of Illinois at Urbana−Champaign, 600 South Mathews Avenue, Urbana, Illinois 61801, United States ‡

S Supporting Information *

ABSTRACT: We report the design, synthesis, and evaluation of biodegradable amphiphilic poly(ethylene glycol)-b-polycarbonate-based diblock copolymers containing pendant persistent organic radicals (e.g., PROXYL). These paramagnetic radicalfunctionalized polymers self-assemble into micellar nanoparticles in aqueous media, which preferentially accumulate in tumor tissue via the enhanced permeability and retention (EPR) effect. Through T1 relaxation NMR studies, as well as magnetic resonance imaging (MRI) studies on mice, we show that these nanomaterials are effective as metal-free, biodegradable MRI contrast agents. We also demonstrate anticancer drugs can be readily loaded into the nanoparticles, conferring therapeutic delivery properties in addition to their imaging properties making these materials potential theranostic agents in the treatment of cancer.

M

(ROS), which can also degrade radicals and lead to concurrent loss of imaging activity.12 Further challenges associated with imaging using nitroxyl radicals include limited in vivo lifetime, efficacy, and solubility. One of the more significant advances in this area has been the recent discovery of a water-soluble macromolecular organic radical contrast agent (ORCA) that could be used in vivo by Rajca et al.13 The authors demonstrated a reduction-resistant spirocyclohexyl nitroxide that were shown to be significantly more stable than nitroxyl radicals such as TEMPO and PROXYL that often succumb to physiological reducing agents. In this study, a PEG-functionalized fourth-generation poly(propylenimine) dendrimer-based material containing paramagnetic spirocyclohexyl nitroxide radicals was synthesized and studied. Importantly, the shielded organic radical provides enhanced radical half-life and extends the materials performance as a contrast agent for up to an hour in animal models. Subsequent reports employed a bottle-brush polymer topology strategy as a scaffold for the reduction-resistant nitroxides with

agnetic resonance imaging (MRI) is used as a standard level of care for noninvasive medical imaging of critical importance in the diagnosis of various human diseases.1 Today, the most commonly used MRI contrast agents are gadoliniumbased complexes containing the highly paramagnetic Gd3+ ion.2−4 While these are generally well-tolerated in the majority of patients, there have nevertheless been reports of side effects, including a serious condition known as nephrogenic systemic fibrosis.5 There has thus been significant interest in developing metal-free contrast agents based on paramagnetic organic radicals.6,7 For example, nitroxyl radicals such as TEMPO and PROXYL are stable spin-active species possessing unpaired electrons and the ability to provide imaging contrast by shortening the T1 relaxation of water in a manner analogous to paramagnetic Gd3+.8 Hence, these nitroxyl radicals have been used for imaging modalities such as MRI. These species, however, tend to be redox active and can be readily oxidized or reduced, complicating their utility in vivo.7,9−11 For example, cellular energy stems from electron transport via oxidation reactions, generating a reducing environment that will quench the nitroxyl radical to give an alkoxyamine. On the other hand, the regions around tumors and inflamed tissues present oxidizing environments due to reactive oxygen species © XXXX American Chemical Society

Received: December 5, 2016 Accepted: February 1, 2017

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DOI: 10.1021/acsmacrolett.6b00924 ACS Macro Lett. 2017, 6, 176−180

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ACS Macro Letters in vivo efficacy.14 While these seminal reports address the key barriers to an all-organic contrast agent, several challenges remain, including nondegradability, inherent toxicity, and synthetic complexity of the scaffold.15 Herein we report an alternative ORCA molecular design based on biodegradable and nontoxic components. Specifically, our system features poly(ethylene glycol)-b-polycarbonatebased (PEG-b-PC) diblock copolymers functionalized with the commercially available organic radicals PROXYL and TEMPO (Figure 1) as pendant substituents in a synthetically

simultaneously image the tumor as well as deliver drugs to a tumor site makes for a promising theranostic agent. The incorporation of PROXYL and TEMPO radicals onto the polymer backbone in the final step is the key part of our synthetic strategy, which sidesteps potential chemical incompatibilities with the radical centers that would lead to a loss of paramagnetic properties (Scheme 1). To do this, a pentaScheme 1. Synthesis of Radical-Functionalized PEGPolycarbonate Diblock Copolymers

Figure 1. Organic radical-functionalized diblock copolymer utilized as a biodegradable theranostic agent. (a) Schematic representation of diblock copolymer composed of a degradable aliphatic carbonate (PC) block with pendant nitroxide radicals and a poly(ethylene glycol) (PEG) block. (b) Interaction of water with the nitroxide radical causes significant relaxation, as measured by T1, enabling the diblock to function as a contrast agent. (c) The diblock copolymer self-assembles into a micelle where the hydrophobic PC block is shielded by the PEG block and allows for the loading of hydrophobic cargo into the micelle.

fluorophenyl carbonate monomer (MTC-OC6F5)19−21 was subjected to an organo-catalyzed ring-opening polymerization (ROP) promoted by a mono hydroxy-terminated 5 kDa-PEG macroinitiator to generate an amphiphilic block copolymer. Organic acids were surveyed, and the best catalyst for the controlled living polymerization of MTC-OC6F5 was found to be triflic acid.22 Narrow-disperse products of controlled molecular weights were obtained, which were readily functionalized in a postpolymerization step with either 3-(aminomethyl)-PROXYL or 4-amino-TEMPO to generate polymers P1 and P2, respectively.20,21 The postpolymerization strategy, together with the mild reaction conditions, avoids side reactions and preserves the integrity of the pendant radicals and polymer backbone (Scheme 1).20 The postpolymerization process was monitored by FTIR. When the postpolymerization was performed, there was a complete disappearance of the pentafluorophenyl ester (CO) stretch at 1760 cm−1. Two new bands appeared (amide I at 1650 cm−1 and amide II at 1550 cm−1), confirming successful formation of amide linkages (Figure S2). One key question with this strategy is whether the nitroxyl radicals, which are confined in the micellar core, can effectively relax the surrounding water molecules and provide sufficient imaging contrast. In principle, the micelle would require an efficient exchange of water (or the polymeric components) in order to act as an effective relaxation agent. Therefore, the relaxivity of polymer P1 was measured in a phosphate-buffered saline solution containing 5% by volume D2O. Under these conditions the polymer solution is turbid consistent with the self-assembly of micelles in aqueous environments. As a control, the T1 values of varying concentrations of PEG were also measured, however, as the concentration of PEG is increased, no significant change in T1 was observed (Figure 2a). However, P1 reduces the T1 of the solution by an order of magnitude at polymer concentrations as low as 2.7 mM, from a T1 value of 3.26 to 0.29 s. Given the narrow polydispersity of P1 (Đ = 1.2), the radical concentration can also be calculated at these low concentrations. P1 is capable of supplying a high localized concentration of an active relaxation agent while maintaining a low polymer concentration (Figure S3).

assessable/click strategy. While the ORCA examples described herein are not the most reduction-resistant radicals, this work highlights a supramolecular strategy for the self-assembly of diblock copolymers into micellar nanoparticles where the ORCAs are protected in a hydrophobic core.16 Our initial results on these assemblies as effective as MRI contrast agents in tissue in a hindlimb muscle animal model. In a separate animal model, anticancer drugs could be loaded into the nanoparticles, and the nanoparticles accumulated in tumor tissues after intravenous injection. Given the diagnostic and therapeutic capabilities demonstrated in these two animal models, these materials have a potential application as theranostic agents in the treatment of cancer. Results and discussion: This alternative ORCA molecular design features poly(ethylene glycol)-b-polycarbonate-based (PEG-b-PC) diblock copolymers17,18 functionalized with commercially available organic radicals PROXYL and TEMPO (Figure 1a,b) as pendant substituents. Through the judicious balance of hydrophobic (PC) and hydrophilic (PEG) block lengths, the amphiphilicity of these diblock copolymers were tuned to induce self-assembly into micelles in aqueous media.16 In this way, the active nitroxyl radicals on the hydrophobic block are shielded from fast reduction in vivo (e.g., by vitamin C) in order to enhance the lifetime of the contrast agent. Furthermore, the nanosize of these polymeric micelles allows for passive targeting of tumors via the enhanced permeation and retention effect (EPR). The hydrophobic core of the micelle also facilitates the encapsulation of a drug cargo (Figure 1c), adding a therapeutic function to its primary purpose as a contrast agent. This combined ability to 177

DOI: 10.1021/acsmacrolett.6b00924 ACS Macro Lett. 2017, 6, 176−180

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Furthermore, the biocompatibility of the polymer design is anticipated to enable higher loadings in vivo to achieve high contrast and shorter T1 values. MR phantom images were obtained for polymer P1. Images acquired on a 3 T clinical scanner demonstrated the ability of the probe to provide concentration-dependent, positive contrast against background. The T1 relaxivity of the material was found to be 0.22 mM−1 s−1, similar to other T1 relaxivity values reported for nitroxide-based compounds.12 Having demonstrated the ability of the polymer to enhance the signal in vitro, we then locally administered the contrast agent in the hindlimb muscle of a female C57BL/6J mouse. At a dose of 1 mmol/kg with respect to nitroxide radical, the high signal provided by the polymer was readily apparent in the region of injection (Figure 2b). This indicates the potential viability of the organic contrast agent as a probe to highlight areas of interest in vivo. Through dialysis, P1 was able to self-assemble into nanoparticles of size 103 ± 2 nm, PDI 0.45 ± 0.01, although P2 was found to be insoluble in water. Polymer P1 was used to

Figure 2. (a) Plot of 1/T1 values vs concentration of polymer P1 (blue) and a 5k PEG control (red). Dotted lines are the respective linear fits to the data. (b) T1 weighted MR image of the mouse hindlimb. The reagent was locally injected into a left hind limb. Yellow arrows (pointing to white regions) indicate the region where P1 was in highest concentration.

Figure 3. (a) Release profile of PTX from PTX-loaded P1 nanoparticles. (b) Viability of HepG2 cells after 48 h treatment with PTX-loaded P1 nanoparticles and free PTX at equivalent drug concentrations. (c) NIRF images of 4T1 tumor-bearing mice following intravenous administration of DiR-loaded P1 nanoparticles. 178

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in particular, negligible fluorescence signal was detected from the heart (Figure 3c). This targeted positioning of the nanoparticles into tumors can potentially translate into practical applications as an imaging probe for monitoring tumor location and progression. In conclusion, novel biodegradable and noncytotoxic paramagnetic diblock copolymers functionalized with persistent organic radicals have been successfully synthesized and tested as imaging agents. We showed that these radical-containing PEG-b-polycarbonates self-assemble into micellar nanoparticles that can accumulate in tumor tissue via the EPR effect. Besides offering promise as metal-free and biodegradable contrast agents for magnetic resonance imaging (MRI), as demonstrated in our mice models, we also showed that these nanomaterials are effective in encapsulating and delivering anticancer drugs to tumor sites. Based on their imaging and passive tumor-targeting abilities, as well as their ease of synthesis, biodegradability, and low cytotoxicity, the materials reported herein are potentially very useful as theranostic agents in the identification and treatment of cancers.

load paclitaxel (PTX), an extremely hydrophobic anticancer drug with low aqueous solubility (0.3 mg/L in water) and strong propensity for self-association to form large fibers.16 These PTX-loaded micelles were fabricated by simple selfassembly without the use of sonication or homogenization techniques. The encapsulation of PTX using polymer P1 greatly improved the aqueous solubility of the drug to 220 mg/ L and also resulted in significant reduction in both the size (30.0 ± 0.1 nm) and size distribution (0.198 ± 0.002) compared to the blank nanoparticles. These PTX-loaded nanoparticles are of optimum size (10−100 nm) to avoid clearance by the reticuloendothelial system (RES),23 and have a more favorable biodistribution into tumor tissues through the EPR effect.24 Moreover, in vitro drug release showed that PTX was released from P1 nanoparticles in a sustained manner over 100 h, with 72% of the total encapsulated PTX being released (Figure 3a). P1 also did not exhibit any hemolytic activity (∼0%) when tested against rat red blood cells, even at high polymer concentrations of up to 1000 mg/L (Figure S4). This is beneficial in ensuring that the nanoparticles will be able to function as drug carriers without inducing hemolytic damage during circulation or causing nonspecific cytotoxicity. Furthermore, polymer P1 showed negligible toxicity toward HepG2 cells even at a high polymer concentration of 500 mg/L with cell viability of ≥92% after 48 h treatment (Figure S4). However, when polymer P1 was used for encapsulating PTX, the drug-loaded nanoparticles induced high cytotoxicity due to anticancer effect of PTX with IC20 at a low concentration of 0.05 mg/L (Figure 3b). At equivalent PTX concentrations of 0.001 to 0.1 mg/L, the reduction in cell viability was lower for PTX-loaded nanoparticles as compared to free PTX. This is most likely due to a lower amount of drug being present in the culture medium as a result of the gradual release of the PTX from the nanoparticles. Noninvasive near-infrared fluorescence (NIRF) imaging was used to monitor the real-time in vivo biodistribution of the nanoparticles qualitatively. NIRF dyes have less interference from the background fluorescence of tissues due to the minimal absorption of NIR photons by water or hemoglobin.25 In this study, a NIRF molecule, DiR, was used as a model compound for encapsulation into the P1 nanoparticles (2.9 wt % DiR). The DiR-loaded nanoparticles were administrated to the 4T1 tumor-bearing mice via intravenous injections. After 1 h postinjection, the DiR-loaded nanoparticles were distributed throughout the animals, and appreciable contrast between subcutaneous tumor and normal tissues was observed from 24 h post-injection (Figure 3c). This passive tumor-targeting ability of P1 nanoparticles could be attributed to the extended circulation time of the nanoparticles and the EPR effect occurring within the tumor tissue, which increased the accumulation of the nanoparticles in the tumor. On day 7 post-injection, the mice were sacrificed and major organs such as heart, liver, spleen, lungs, and kidneys, as well as the tumor, were resected to evaluate the tissue distribution of DiR-loaded nanoparticles (Figure 3c). From qualitative analysis of the images, higher NIRF intensity was observed from the tumors of mice injected with the nanoparticles as compared to those injected with free DiR. This indicates that the fluorescence emission from the tumor occurs as a result of the accumulation of nanoparticles via the EPR effect. In mice treated with DiR-loaded nanoparticles, NIRF intensities were generally lower for normal tissues compared to the tumor, and

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MATERIALS AND METHODS

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ASSOCIATED CONTENT

Synthesis of Polymer P1 (PROXYL-functionalized) (Scheme 1). A 4 mL glass vial containing a magnetic stir-bar was charged with mPEG5k-b-poly(MTC-OC6F5), Mn = 11.3 KDa, PDI = 1.10 (GPC; 0.103 g, 0.133 mmol of MTC-OC6F5 moieties), anhydrous THF (1.0 mL), and triethylamine (0.0150 g, 0.148 mmol). Next, a solution of 3(aminomethyl)-PROXYL (0.0250 g, 0.146 mmol) in THF (0.5 mL) was added with stirring. The mixture was allowed to stir for 45 min at room temperature, after which it was pipetted into excess diethyl ether (16 mL) to precipitate the polymer as an off-white solid. The mixture was briefly sonicated and then centrifuged. The mother liquor was decanted off and more diethyl ether (20 mL) was added. A second round of sonication, centrifuging, and decanting afforded an off-white solid that was then dried under high vacuum for 24 h, Mn = 9.2 kDa, PDI = 1.23 (GPC). T1 Measurements. Methods. T1 measurements were conducted on the diblock copolymer P1 bearing pendant PROXYL radicals (Scheme 1). All experiments were carried out on a Bruker 300 MHz NMR using a saturation recovery pulse program. A total of nine T1 measurements were carried out in a phosphate-buffered saline solution with 5% (by volume) D2O at varying concentrations of spin-active polymer component (Table S1). To ensure that the relaxation of water was being reduced by the spin-active PROXYL moieties of the polymer, a control was performed using the hydrophilic poly(ethylene glycol) (PEG) component (i.e., 5k PEG). In this control T1 was measured as a function of increasing polymer concentration, a total of five T1 measurements were conducted with this control. Preparation of Blank Nanoparticles. The polymer P1 (10 mg) was dissolved in 2 mL of N,N-dimethylformamide (DMF) and transferred to a dialysis membrane tube with molecular weight cutoff (MWCO) of 1000 Da (Spectrum Laboratories, U.S.A.). The dialysis bag was immersed in 1 L of deionized (DI) water at 4 °C for 2 days. The dialysis medium was replaced at third, sixth and 24th hour with fresh DI water. At the end of the dialysis process, the resulting micelle solution was centrifuged at 4000 rpm for 5 min to remove large aggregates if present.

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsmacrolett.6b00924. Additional details including materials and methods (PDF). 179

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(21) Chan, J. M. W.; Ke, X.; Sardon, H.; Engler, A. C.; Yang, Y. Y.; Hedrick, J. L. Chem. Sci. 2014, 5 (8), 3294. (22) Sardon, H.; Engler, A. C.; Chan, J. M. W.; García, J. M.; Coady, D. J.; Pascual, A.; Mecerreyes, D.; Jones, G. O.; Rice, J. E.; Horn, H. W.; Hedrick, J. L. J. Am. Chem. Soc. 2013, 135 (43), 16235. (23) Rao, J. ACS Nano 2008, 2 (10), 1984. (24) Duncan, R. Nat. Rev. Drug Discovery 2003, 2 (5), 347. (25) Chen, X.; Conti, P. S.; Moats, R. A. Cancer Res. 2004, 64 (21), 8009.

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Julian M. W. Chan: 0000-0002-2734-6496 Haritz Sardon: 0000-0002-6268-0916 Hyunjoon Kong: 0000-0003-4680-2968 Yi Yan Yang: 0000-0002-1871-5448 James L. Hedrick: 0000-0002-3621-9747 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This study was supported by IBM Almaden Research Center, the Institute of Bioengineering and Nanotechnology (Biomedical Research Council, Agency for Science, Technology and Research, Singapore), National Institutes of Health (1R01 HL109192 to H.J.K.) and Chemistry−Biology Interface Training Grant 5T32-GM070421 to C.E.S.).

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REFERENCES

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DOI: 10.1021/acsmacrolett.6b00924 ACS Macro Lett. 2017, 6, 176−180