Peroxynitrite (ONOO–) Redox Signaling Molecule-Responsive

Letter pubs.acs.org/macroletters

Peroxynitrite (ONOO−) Redox Signaling Molecule-Responsive Polymersomes Jian Zhang,† Jun Hu,*,‡ Wei Sang,†,‡ Jianbo Wang,§ and Qiang Yan*,† †

Department of Macromolecular Science, State Key Laboratory of Molecular Engineering of Polymers, Fudan University, Shanghai 200433, China ‡ State Key Lab of Polymer Physics and Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, China § Computer Science School, China Women’s University, Beijing 100101, China S Supporting Information *

ABSTRACT: Designing specific-responsive polymer nanocapsules toward a definite cell signaling molecule for targeted therapy faces a great challenge. Here we demonstrate that new block copolymer appended trifluoromethyl ketone side groups can chemoselectively respond to an endogenous redox biosignal, peroxynitrite (ONOO−), but shield the interference of other biogenic reactive oxygen, nitrogen, and sulfur species (ROS/RNS/RSS). The ONOO− signaling molecule is capable of triggering cascade oxidation−elimination reactions to cleave the side functionalities from the polymer chain, which induces a large alteration of the polymer amphiphilicity and further leads to controllable disassembly of their self-assembled vesicular structure. Modulating the ONOO− stimulus concentrations could readily control the vesicle dissociation rates for desirable drug delivery. We envisage that this polymer model would provide a new scenario to construct bioresponsive macromolecular systems for future biomedical nanotechnologies. a diffusion-controlled producing rate at ∼1 × 1010 M−1 s−1, but its steady-state concentration in cells is only nanomolar level.20,21 After being aware of its crucial biological role, several probe molecules have been developed to detect ONOO−.22−26 However, constructing ONOO−-responsive polymer systems remains elusive thus far. This is understandable since there are a series of endogenous redox species such as peroxide (H2O2), NO, and glutathione (GSH) coexisting in cells. Conventional redox-sensitive polymers containing the sulfide/disulfide bond,27,28 selenide linkage,29 ferrocene,30 boronic ester,31 and proline oligomer32 are incapable of selectively differentiating ONOO− from its redox analogues. Hence, exploring specific novel polymer architectures responding to this biosignal and discovering their self-assembly mechanism will take a step in realizing the ultimate goal of precision-responsive macromolecules. Herein, we design a new phenyl methacrylate monomer bearing a special trifluoromethyl ketone moiety (TFK) and its block copolymer, poly(ethylene oxide)-b-poly(trifluoro-3-oxobutyl phenyl methacrylate (PEO45-b-PMATFKx, x = 27, 69, and 84: the details of synthesis and characterization are in the Supporting Information, Figures S1−S16 and Table S1). Since the monomer is composed of a strong electron-withdrawing

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iomolecule-responsive polymer vesicles, used as a kind of burgeoning nanocapsules, have sparked significant attention in recent years.1−3 Because most diseases are implicated in the aberrant secretion of biomolecules, imparting biomolecular responsivity to polymer systems is favorable to targeted drug delivery and nanodiagnosis. Up to now, some pioneered studies focused on using macromolecular-type biogenic substances (enzymes and DNA) as irritants to construct smart assemblies.4−10 Besides these biomacromolecules, we often overlook those more ubiquitous small-molecule biosignals, such as reactive species, metabolites, and neurotransmitters. Recently, several nascent works have achieved success: Yuan et al. prepared CO2-triggered polymersomes;11 Zhao and coworkers made artificial polymers responsive to adenosine triphosphate (ATP);12 and Davis and our group discovered nitric oxide (NO)13−15 and hydrogen sulfide (H 2S) 16 gasotransmitter responsive copolymers, respectively. Despite an imperative demand to enrich the family of biosignal-sensitive nanosized systems for specific cell treatment, creating synthetic polymer structures to selectively sense the signaling molecule is still a great challenge. Peroxynitrite (ONOO−), a redox signaling molecule generated from the reaction of NO and superoxide, is attracting increasing interest due to its central pathogenic factor.17 Cell misregulation of ONOO− is related to numerous diseases including neurodegeneration, angiocardiopathy, ischemia, and cancers.18,19 ONOO− is an intracellular short-lived species with © XXXX American Chemical Society

Received: June 20, 2016 Accepted: July 12, 2016

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DOI: 10.1021/acsmacrolett.6b00474 ACS Macro Lett. 2016, 5, 919−924

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ACS Macro Letters

Scheme 1. Schematic Illustration of (a) ONOO−-Induced Oxidation−Elimination Reaction, (b) ONOO− Signaling Molecule Responsive Block Copolymer (PEO-b-PMATFK), and Their Controllable Self-Assembly and Disassembly

Figure 1. (a) UV−vis spectra changes by mixing PEO-b-PMATFK (1.0 × 10−3 g/L, containing 3 μM TFK groups) and ONOO− (1 equiv) in PBS buffer solution (pH = 7.4) for 2 h (from pink to green curve). (b) Time-resolved 19F NMR spectra of PEO-b-PMATFK reacting with ONOO− in d8THF/D2O solvent (4/1, v/v). (c) 1H NMR spectra comparison among PEO-b-PMATFK at the beginning of ONOO− addition (top panel), the final products purified from solution after 2 h reaction with ONOO− (middle panel), and the commercial PEO-b-PMAA counterpart (down panel). The polymer samples were analyzed in CDCl3 solvent.

this reaction is only activated by ONOO−, as shown in Scheme 1a. With this in mind, we speculated that stimulated by ONOO− the polymer could be broken at the position of TFK side groups and prevent the disturbances from other biological interferents. This site-specific chemical cleavage may alter the

TFK unit and an adjacent phenyl ester spacer, it is prone that cascade oxidation−elimination reactions will occur in which the ketone is first oxidized to the dioxirane intermediate, and the latter undergoes an intramolecular elimination to unlock the ester bond and finally to afford spiroquinone.33 In particular, 920

DOI: 10.1021/acsmacrolett.6b00474 ACS Macro Lett. 2016, 5, 919−924

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ACS Macro Letters

Figure 2. TEM images of three PEO45-b-PMATFKx assemblies: (a) x = 27 for spherical micelles, (b) x = 69 for cylindrical fibers, and (c) x = 84 for polymersomes. (d) DLS data showing hydrodynamic diameters (Dh) of three different assemblies. The concentrations of three polymer samples were 6.0 × 10−3, 3.5 × 10−3, and 2.2 × 10−3 g/L, respectively (scale bar is 300 nm).

polymer displayed a single fluorine signal of trifluoromethyl ketone at δ = −79.3 ppm. After injecting ONOO−, the initial fluorine peak gradually decreased, while a new fluorine signal upshifted to δ = −84.3 ppm elevated (Figure 1b). This new signal corresponds to the chemical shift of 2-hydroxyl-2trifluoromethyl spiro-quinone (Figure S19). We also separated and purified the resulting polymers and used 1H NMR and gel permeation chromatography (GPC) to analyze. Without ONOO−, PEO-b-PMATFK showed two groups of strong proton peaks belonging to the aromatic region (δ = 6.80−7.22 ppm) and ethylidene (δ = 2.85−3.10 ppm), respectively. Upon exposure to ONOO− for 2 h, the above signals almost vanished, which indicates that the resulting polymer chain removes the majority of TFK side groups (Figure 1c, top and middle panel). In addition, we found that the obtained NMR curve is similar to commercial PEO-b-PMAA (Figure 1c, bottom panel). It means that triggered by ONOO− this amphiphilic PEO-b-PMATFK copolymer can unlock TFK functional groups to form totally water-soluble PEO-b-PMAA. GPC traces reveal that the molecular weight of the copolymer had a remarkable decrease from 26.3 kDa down to 11.7 kDa after stimulus (Figure S20). From this, the polymer cleavage efficiency was evaluated to be on average 82%. After understanding the cleavage process, we wanted to explore their self-assembly behavior. Self-assembled structures of block copolymers were prepared by the selective solvent method: briefly, PEO-b-PMATFK in THF solution (0.5 wt %, 4 mL) was slowly added to deionized water at a rate of 0.5 mL/h with stirring until the water content reached 45%. The suspension was dialyzed against water for 24 h to remove organic solvent, and then a transmission electron microscope (TEM) was used to visualize their morphologies. With an increase in the degree of polymerization of the PMATFK block,

balance of block copolymer amphiphilicity, further resulting in the disassembly of their self-assembled polymersomes (Scheme 1b). We first used a TFK-containing model compound, trifluoro(4-methoxyphenyl)butan-2-one, to survey whether it can react with ONOO−. It dissolved in THF/H2O solution (5 × 10−5 M, 1/9, v/v) and showed a shoulder absorption at 275 and 282 nm belonging to the band of the TFK group. In the presence of equal molar ONOO−, a weak band at 302 nm appeared, which corresponds to the ONOO−.34 After mixing for 30 min, these two groups of absorption bands dramatically decreased, whereas a new absorption at 246 nm was enhanced. It is consistent with a typical quinone band (Figure S17). Further NMR experiments confirm that the model compound treated upon ONOO− generated two products: one is 2-hydroxyl-2trifluoromethyl spiro-quinone, and the other is methacrylic acid (MAA).33 These indicate that ONOO− can induce a cleavage reaction so as to disconnect the phenyl ester bond (Figure S18). To further elucidate that the PEO-b-PMATFK polymer has similar reactivity, UV−vis and 19F NMR spectroscopy were employed to monitor the reactive kinetic process. Mixing PEOb-PMATFK (1.0 × 10 −3 g/L, containing TFK group concentration of 3 μM) with fresh ONOO− (1 equiv) in PBS buffer (pH = 7.4), it is clear that the mixture showed two strong absorptions at 278 and 302 nm ascribed to the polymer and ONOO−, respectively. With increasing reaction time, the two initial absorption peaks were slowly attenuated by nearly 85%, accompanied by a strengthened quinoid peak at 246 nm (Figure 1a). It implies that ONOO− was continually consumed to react with the copolymers, leading to the formation of spiroquinone. The changes of time-resolved 19F NMR titration corroborated this result. In the absence of ONOO−, the 921

DOI: 10.1021/acsmacrolett.6b00474 ACS Macro Lett. 2016, 5, 919−924

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ACS Macro Letters

Figure 3. (a) DLS profiles showing the disassembly process of PEO45-b-PMATFK84 polymersomes upon ONOO−. (b−d) TEM images illustrating the polymersomes reacted with ONOO− for different times: (b) 0.6 h, (c) 1.0 h, and (d) 2.0 h. The ONOO− stimulus level is kept at 5 equiv to the copolymer TFK groups (scale bar is 300 nm).

by 88% from 262 nm up to 494 nm; in the second part, Dh shows a linear decline from 494 nm down to 5.6 nm. These findings suggest that the vesicle disassembly kinetics obeys a swelling-to-crack mechanism. We used TEM to track this process. Their assembling structure began to transform as ONOO− was added to the polymersomes. As shown in Figure 3b, reacting with ONOO− for 0.6 h, swollen polymersomes were dominant in solution. Their size rose on average 90% from 245 to 460 nm, and their volume expanded nearly 690%. We inferred that a portion of hydrophobic PMATFK segments converts to hydrophilic PMAA chains, resulting in a strong hydration effect within the vesicle membrane; as a consequence, the vesicles swell to minimize the interfacial free energy. Similar phenomena could be found in other polymer vesicle systems.16,36 Prolonging the stimulus time to 1 h, most of the copolymers were sheared off. Because the polymer hydrophilic−hydrophobic balance was broken, the swollen polymersomes fractured into cracked membranes, and there was no intact vesicle found in TEM (Figure 3c). Finally, upon 2 h of ONOO− trigger, only nanofragments whose size was smaller than 5 nm came into view, revealing entirely vesicular disassembly (Figure 3d). To aim to distinguish cell microenvironment differences, we expected that this disassembly owns ONOO− specificity. Besides the ONOO− signaling molecule, there are a group of redox signalsso-called reactive oxygen, nitrogen, and sulfur species [ROS: H2O2, superoxide (O2•−) and hydroxyl radical (•OH); RNS: NO and nitrogen dioxide (NO2); RSS: GSH and thiol]coexisting in cytosol. Moreover, their normal physiological concentrations are much higher than ONOO−. Thus, we suspected that the strong oxidative stress of these species may interfere with our assemblies to identify ONOO−. From the above experiments, we have known that the cleavage of

their geometry showed a widely adjustable phase diagram from small spherical micelles through cylindrical nanofibers with high length/diameter ratio further to large polymer vesicles (Figure 2a−c), which resembles the self-assembly rule of common amphiphilic block copolymers.35 From TEM images, the average sizes of these spheres, fibers, and vesicles were 31, 47, and 245 nm, respectively. The three kinds of aggregates whose hydrodynamic diameters (Dh), as determined by dynamic light scattering (DLS), were strongly dependent on the number of TFK repeating units were 38, 61, and 262 nm, respectively, which is in line with the TEM results (Figure 2d). We chose the vesicle system (PEO45-b-PMATFK84) for subsequent investigation. The stability of the vesicles was tested against glycerol to mimic the viscosity of cytoplasm condition and PBS buffer to mimic the physiological salt environment. As summarized, the aggregates had nearly no volume change in 25 wt % glycerol and swelled less than 10% in 20 mM PBS for 24 h, as measured by DLS (Table S2). The ONOO−-responsive vesicular disassembly was examined by the turbidity of aggregate solution under physiological conditions (pH = 7.4, Figure S21). By applying ONOO− (35 μM, 5 equiv) to our polymersomes (2.2 × 10−3 g/L, containing 7 μM of TFK groups), the optical transmittance at 550 nm increased from 23% to 94% within 2 h, as a result of the disruption of these assemblies into water-soluble PEO-b-PMAA unimers. The disassembly rate is ONOO− dose-dependent. Lowering the ONOO− concentration to 2 and 1 equiv, the rupture time of these polymersomes can be decelerated to 4 and 9 h, respectively. The vesicular Dh evolution plotted against reaction time reflected their dissociation process (Figure 3a). Notably, the DLS curve is discontinuous at a time of 0.6 h: one segment extends from start to 0.6 h, while the other covers the range from 0.6 to 2.0 h. In the first part, the Dh increases rapidly 922

DOI: 10.1021/acsmacrolett.6b00474 ACS Macro Lett. 2016, 5, 919−924

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ACS Macro Letters

conditions was depicted. In the absence of any stimuli, the HTloaded polymersomes gave a free-release profile (less than 8% within 16 h), indicating the stability of the vesicles. Addition of ONOO− to the nanocapsules could cleave the polymers, resulting in the leak of HT. When the concentration of ONOO− rises from 7 to 35 μM, a higher content of HT was released from 61%, 84%, to nearly 100%. It demonstrates that varying ONOO− stimulus level could modulate the vesicular disruption rates to realize controlled payload release. Moreover, only ONOO− triggers an effective release, but other biological irritants yielded negligible release quantities (Figure S23). In conclusion, we present a successful demonstration of a signaling molecule-responsive polymer system based on a trifluoromethyl ketone (TFK)-containing block copolymer. The incorporation of TFK side groups into block chains endows the copolymer with selective response capability toward a definite endogenous reactive species, peroxynitrite (ONOO−), while shielding the negative disturbances from other intracellular ROS, RNS, and RSS. The polymers enable self-assembly into polymersomes that can specifically disassemble by ONOO−-induced side group cleavage. Considering that ONOO− metabolic dysfunction involves a lot of severe diseases, these smart polymersomes have the potential to act as efficient nanocapsules for drug delivery. It is anticipated that this polymer model would offer a new perspective on smallmolecule bioactivator-responsive self-assembly systems.

polymer side groups can cause an UV−vis absorption change. On the basis of this fact, we mixed the vesicles with the above analytes at a higher concentration (350 μM, 50 equiv), incubating for half a day. If we defined ONOO− as 100% activity, other analytes displayed negligible disassembling ability (Figure 4 and Figure S22). These findings demonstrate that the PEO-b-PMATFK vesicles can specifically sense endogenous ONOO− and meanwhile inhibit other redox-active biomolecules.

Figure 4. Specific-responsive comparison of PEO-b-PMATFK vesicles with other biological ROS and RNS interferents. (The polymer concentration kept at 2.2 × 10−3 g/L, containing 7 μM TFK groups. ROS/RNS/RSS concentrations kept at 350 μM incubated for 12 h; ONOO− concentration was 35 μM for 2 h.)

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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/acsmacrolett.6b00474. Materials and methods; synthesis, characterization, and procedures; and supporting characterization (PDF)

Finally, to assess the possibility of utilizing these ONOO−sensitive vesicles as nanocapsules, we carried out a drug release experiment. It is known that the cell overproduction of ONOO− can cause serious blood vessel constriction and hypertension. Hydrochlorothiazide (HT), as a vasodilator, is used for treating hypertension. Hence, we attempted to encapsulate HT in polymersomes for potential therapy. For encapsulation, an aqueous solution of HT (200 μg/mL, 0.5 mL) was slowly added to polymer THF solution to induce coassembly. The resulting assemblies were purified by dialysis against PBS buffer (pH = 7.4, MWCO = 3.0 kDa) for 3 days to remove unloaded HT. The release amount could be recorded by fluorescence change at a diagnostic emission λem = 355 nm. As shown in Figure 5, the release process under variable

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the Grant of Chinese Recruitment Program of Global Experts (KHH1717002 and JIH1717005). REFERENCES

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Figure 5. Stimulus-triggered controllable cargo release of HT drug from PEO-b-PMATFK polymersomes under a variety of ONOO− concentrations: no stimulus (black diamond), 7 μM (green triangle), 14 μM (red circle), and 35 μM (blue square). 923

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DOI: 10.1021/acsmacrolett.6b00474 ACS Macro Lett. 2016, 5, 919−924