Polymeric Framboidal Nanoparticles Loaded with a Carbon Monoxide

Article pubs.acs.org/bc

Polymeric Framboidal Nanoparticles Loaded with a Carbon Monoxide Donor via Phenylboronic Acid-Catechol Complexation André J. van der Vlies,†,‡ Ryosuke Inubushi,‡ Hiroshi Uyama,‡ and Urara Hasegawa*,‡,§ †

Frontier Research Center, Graduate School of Engineering, ‡Department of Applied Chemistry, Graduate School of Engineering, and §Frontier Research Base for Young Researchers, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan S Supporting Information *

ABSTRACT: Carbon monoxide (CO) is an essential gaseous signaling molecule in the human body. Toward the controlled delivery of CO to the target tissues or cells, nanomaterialbased CO donors have attracted growing attention. Here, we present CO-releasing polymeric nanoparticles (CONPs) prepared by simple mixing of phenylboronic acid-containing framboidal nanoparticles with the catechol-bearing CO-donor Ru(CO)3Cl(L-DOPA) via phenylboronic acid-catechol complexation. The CONPs release CO in response to cysteine and suppress the production of the pro-inflammatory mediators interleukin 6 (IL-6) and nitric oxide (NO) in lipopolysaccharide (LPS)-stimulated murine macrophages. This CONP platform may show promise in therapeutic applications of CO.

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INTRODUCTION Carbon monoxide (CO), one of the products formed by heme degradation, has been recognized as an essential gaseous signaling molecule in the human body.1−3 Although this gas is generally known as a lethal gas, recent reports have shown that CO at physiologically relevant levels exhibits remarkable biological activity including anti-inflammatory, anti-apoptotic, antiproliferative, and cytoprotective effects.4−7 With the understanding of its physiological roles, CO has attracted considerable interest as a potential therapeutic agent to treat various pathological conditions such as rheumatoid arthritis, sepsis, chronic graft rejection, and ischemia/reperfusion injury.2 However, the use of CO for therapeutic applications is limited since direct inhalation of this gas can potentially cause toxicity by impairing oxygen transport due to its high affinity toward hemoglobin. To achieve safe and efficient administration of CO, considerable efforts have been made to develop compounds that release CO under physiological conditions (CO donors).8 So far, various transition metal−carbonyl complexes and boranocarbonate derivatives have been reported and used to explore the therapeutic potential of CO in vitro and in vivo.9−12 One of the most widely studied CO donors is Ru(CO)3Cl(glycinate) (1, CORM-3, Scheme 1).13,14 CORM-3 has good aqueous solubility and releases 1 equiv of CO by ligand substitution. It has been reported that CORM-3 elicits diverse CO-related biological activity including vasodilatory, antiinflammatory, antibacterial, and anti-apoptotic effects.15−18 In spite of the success in preclinical experiments of these CO donors, there are still several issues to overcome for therapeutic © XXXX American Chemical Society

Scheme 1. Chemical Structure of CORM-3 (1) and Its Catechol-Bearing Analogue (2)

use such as rapid renal clearance from the body, poorly controlled tissue distribution, and potential toxicity.19 In general, drug molecules smaller than about 5 nm are rapidly eliminated from the bloodstream by the kidney after systemic administration.20 In addition, it has been reported that small drugs can spread throughout the body immediately after injection, which may lower therapeutic efficacy and lead to increased risk of side effects in healthy tissues.21 Furthermore, transition metal-based CO donors have potential toxicity by inducing oxidative stress and cellular damage.22 The polymeric nanomedicine approach has been widely used in the field of drug delivery.23 It is well-known that the use of polymeric nanoparticles in the size range of 10−200 nm as drug carriers can prolong blood circulation time, improve stability and solubility, as well as reduce toxic side effects of drugs. Therefore, it can be envisioned that this approach may also show promise in CO delivery to address the problems associated with low-molecular-weight CO donors. We previously reported polymeric micelles prepared from Received: March 9, 2016 Revised: April 6, 2016

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DOI: 10.1021/acs.bioconjchem.6b00135 Bioconjugate Chem. XXXX, XXX, XXX−XXX

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Bioconjugate Chemistry

reaction conditions for CO-DOPA loading were optimized and the CO releasing properties of the CONPs were evaluated. Furthermore, the anti-inflammatory activity and cytotoxicity of the CONPs were assessed in murine macrophages.

amphiphilic triblock copolymers composed of a hydrophilic poly(ethylene glycol) (PEG) segment, a CO-donating segment bearing Ru(CO)3Cl(ornithinate) moieties, and a hydrophobic poly(n-butylacrylamide) segment.24 We showed that the micelles successfully attenuated the inflammatory response in human monocytes and showed reduced toxicity compared to the low-molecular-weight ruthenium carbonyl complex. Following our report, other nanomaterial-based CO donors have been developed using copolymers, iron oxide and silica nanoparticles, peptide amphiphile nanofibers, and proteins as scaffolds.25−31 We recently reported phenylboronic acid-containing framboidal nanoparticles (PBANPs) synthesized via aqueous dispersion polymerization of N-acryloyl-3-aminophenylboronic acid (PBAAM) using methoxy poly(ethylene glycol) acrylamide (PEGAM) as the polymerizable dispersant and N,N′methylenebis(acrylamide) (MBAM) as the cross-linker.32 Interestingly, these nanoparticles possess a unique framboidal morphology. The PBANPs showed reversible swelling behavior in response to changes in pH and fructose concentration due to the Lewis acid nature of PBA and complex formation between PBA and fructose. In addition, PBANPs could be loaded with a high amount of Alizarin Red S, a dye that interacts with PBA, compared to PBA-bearing spherical nanoparticles of equivalent size due to the higher surface area-to-volume ratio of the framboidal morphology. Furthermore, incorporation of a fluorescent monomer, Nile Blue acrylamide, and PEGAM with a reactive group at the distal end yielded fluorescent PBANPs bearing reactive groups that were further modified with mannose groups. These mannosylated fluorescent PBANPs showed mannose receptor-mediated endocytosis in murine macrophages.33 With these unique features, PBANPs could be useful as a functional nanoscaffold for drug delivery applications. Here, we report a new strategy to prepare a nanomaterialbased CO donor by using the PBA-catechol complexation to load a catechol-bearing CO donor in the PBANPs. It is wellknown that phenylboronic acids (PBA) form cyclic boronate esters with compounds having cis diol groups and have especially high affinity toward catechol-containing compounds.34 In this study, Ru(CO)3Cl(L-DOPA), a catecholbearing analogue of CORM-3 (2, CO-DOPA, Scheme 1), was synthesized and loaded to the PBANPs at physiological pH to yield CO donor-loaded PBANPs (CONPs) (Figure 1). The

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RESULTS AND DISCUSSION Synthesis of Ru(CO)3Cl(L-DOPA) (CO-DOPA). The catechol-bearing CO donor Ru(CO)3Cl(L-DOPA) (2, CODOPA) was synthesized by reacting tricarbonyldichlororuthenium(II) dimer [Ru(CO)3Cl2]2 (3) with L-DOPA (4) in methanol according to the procedure reported for CORM-313 with slight modification (Scheme 2). The compound was analyzed by ATR-IR, ICP-OES, and 1H NMR. The IR spectrum in the region 2300−1800 cm−1 showed the characteristic ν(CO) stretching vibrations of coordinated CO ligands and was similar to that of CORM-3 (Figure 2a and Figure S1). The ruthenium content of the complex was 24.0 wt % as measured by ICP-OES, which is in good agreement with the theoretical value calculated for CO-DOPA (24.2 wt %). Furthermore, the 1H NMR spectrum showed the protons due to the phenolic OH (2 protons) and aromatic ring (3 protons) of the catechol group with an integral value ratio of 2:3 (Figure 2b) indicating that both phenolic OH groups are not coordinated to the ruthenium center. These results show successful synthesis of CO-DOPA. Synthesis of CO-Releasing PBANPs (CONPs). Phenylboronic acid-containing nanoparticles (PBANPs) were synthesized by aqueous dipersion polymerization of N-acryloyl-3aminophenylboronic acid (PBAAM) in the presence of methoxy poly(ethylene glycol) acrylamide (PEGAM) as the polymerizable dispersant and N,N′-methylenebis(acrylamide) (MBAM) as the cross-linker as reported previously.32,33 The Zaverage diameter of the PBANPs was 85 nm according to dynamic light scattering (DLS). The polydipersity index (PDI) value was 0.05 showing that these particles had a narrow size distribution. Furthermore, the negatively stained transmission electron microscopy (TEM) image shows that the PBANPs had a framboidal morphology as reported previously (Figure S2).32,33 PBANPs were loaded with CO-DOPA via phenylboronic acid-catechol complexation to yield CO-releasing nanoparticles (CONPs) as shown in Scheme 3. PBANPs were mixed with CO-DOPA (2 equiv to PBA) in phosphate buffer (pH 7.4) at RT. At the indicated time points, the reaction mixture was ultrafiltered (MWCO 3000 Da) and the ruthenium concentration in the filtrate was measured by ICP-OES to quantify the amount of unbound CO-DOPA. As shown in Figure 3a, the loading amount increased to 0.7 mol/mol of PBA within 45 min and reached a plateau. To further optimize CO-DOPA loading, we investigated the effect of the CO-DOPA/PBA molar feed ratio. PBANPs were reacted with CO-DOPA at different concentrations (1, 2, and 5 equiv to PBA) in phosphate buffer solution (pH 7.4) at RT for 45 min. As shown in Figure 3b, the CO-DOPA/PBA molar feed ratio did not significantly affect the loading amount. To confirm that CO-DOPA is loaded to PBANPs via phenylboronic acid-catechol complexation, we evaluated the effect of pH on the amount of loaded CO-DOPA. As shown in Figure 3c, the loading amount increased with the increase of pH and reached 1 mol CO-DOPA/mol of PBA at pH 9.4. In general, the binding constants of boronic acids and diolcontaining compounds increase at higher pH. It has been reported that the binding constants of PBA and catechol are

Figure 1. Synthesis of CO-releasing nanoparticles (CONPs) and their anti-inflammatory activity. B


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Bioconjugate Chemistry Scheme 2. Synthesis of Ru(CO)3Cl(L-DOPA) (CO-DOPA)

Figure 2. Characterization of CO-DOPA. (a) ATR-IR in the spectral region 2300−1800 cm−1 showing the ν(CO) stretching vibrations of coordinated CO ligands. (b) 1H NMR in the spectral region 5−10 ppm showing the catechol protons of DOPA.

Scheme 3. Complexation of CO-DOPA with the PBA Moiety of PBANPs

Figure 3. Amount of CORM-DOPA loaded to PBANPs as a function of (a) reaction time (CO-DOPA/PBA molar ratio = 2, pH 7.4), (b) CODOPA/PBA molar feed ratio (reaction time: 45 min, pH 7.4), and (c) pH (CO-DOPA/PBA molar ratio = 2, reaction time: 45 min). Samples were collected and the amount of unbound CO-DOPA was determined by ICP-OES after ultrafiltration (n = 3).

500 M−1 at pH 7.0 and 3300 M−1 at pH 8.5.34 The pHdependence of the loading amount therefore indicates that CODOPA is bound to the PBANPs by PBA-catechol complexation. We next tested the stability of the CO-DOPA/PBA complex in the CONPs in the absence and presence of glucose as well as at different pH. It is known that the covalent bond between PBA and catechol is reversible and can be dissociated in the presence of other diol-containing compounds such as glucose.35 In addition, this interaction is favored at basic pH and becomes hydrolytically unstable at lower pH.35 Using CONPs prepared at pH 7.4 with a CO-DOPA/PBA ratio of 2, the release of CODOPA from CONPs was studied in phosphate buffer (pH 7.4) with and without glucose as well as acetate buffer (pH 5.0) at 37 °C. As shown in Figure 4, CONPs only released 17% of the loaded CO-DOPA after 1 week at pH 7.4. In the presence of glucose, the release of CO-DOPA was slightly accelerated and 37% of that originally loaded was released after 1 week. This result indicates that glucose displaces CO-DOPA by competing with binding to PBA. Furthermore, lowering the pH of the

Figure 4. Release of CO-DOPA from CONPs. CONPs were incubated in PBS (pH 7.4, circle), 100 mg/mL glucose-containing PBS (pH 7.4, triangle), and acetate buffer (pH 5.0, square) at 37 °C. At different time points, the samples were ultrafiltered and the amount of released CO-DOPA determined by ICP-OES (n = 3).

CONP solution to 5.0 led to 52% release within 2 d. It has been reported that the binding constants of PBA and catechol decreases from 500 to 31 M−1 by lowering the pH from 7.0 to C


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Bioconjugate Chemistry 5.8.34 Therefore, this result may be due to the reduced binding constant between CO-DOPA and PBA at lower pH. Although more CO-DOPA could be loaded at high pH (pH 9.4), CO-DOPA would be released at physiological pH 7.4 due to the pH-dependent interaction between CO-DOPA and PBA. In addition, metal carbonyl complexes are generally unstable when kept at basic pH for too long and lose their CO-releasing ability. Therefore, for further study, we used CONPs prepared at pH 7.4 (reaction time: 45 min, CO-DOPA/PBA molar ratio = 2). Structural Characterization of the CONPs. The size distribution of the CONPs was analyzed by DLS. As shown in Figure 5a, CONPs were monodisperse (PDI: 0.1) having a Z-

Figure 6. Characterization of CONPs. (a) TEM image of CONPs without staining. (b) ATR-IR spectra of CONPs and PBANPs in the spectral region 2500−1700 cm−1.

(data not shown). This result shows that the CO-DOPA is distributed throughout the CONPs. The presence of CODOPA in the nanoparticles was further confirmed by ATR-IR. As shown in Figure 6b, the carbonyl vibration bands of the coordinated CO ligands of CO-DOPA were clearly observed for CONPs, but not for PBANPs. CO Release Properties of CO-DOPA and CONPs. We first examined CO release from CO-DOPA and CONPs using the myoglobin (Mb) assay as reported previously.24 In this assay, the CO donor solution is added to the deoxy Mb solution in phosphate buffered saline (PBS) containing sodium dithionite (Na2S2O4), which reduces Mb to deoxy Mb and induces CO-release from the CO donor by ligand substitution. Deoxy Mb traps CO released from the donor to form carboxy Mb (MbCO) which can be quantified by UV−vis spectroscopy. As shown in Figure 7, CO-DOPA released 0.97 equiv of CO. Figure 5. Hydroynamic size and morphology of the CONPs. (a) Size distribution of PBANPs and CONPs as measured by DLS. (b) AFM image of CONPs. (c) TEM image of CONPs negatively stained with 0.5 wt % Preyssler-type potassium phosphotungstate solution.

average diameter of 188 nm. The increase in size compared to PBANPs (diameter: 85 nm) may be due to swelling of the PBANPs. Upon complexation with CO-DOPA, the PBA moiety becomes more hydrophilic due to the formation of an anionic tetrahedral form. In addition, the hydrophilic nature of the ruthenium carbonyl moiety, which is reported to be in anionic forms at pH 7.4 (Scheme S1),14 would also be responsible for swelling of the polymer network within the nanoparticles. The zeta-potential values of the PBANPs and CONPs were −5.9 and −4.9 mV, respectively, showing that complexation of the PBANPs with CO-DOPA does not significantly affect the surface charge of the nanoparticles. Furthermore, the morphology of CONPs was observed by atomic force microscopy (AFM) and negative-stain TEM. The results show that the CONPs retained the characteristic framboidal morphology as observed for the PBANPs (Figure 5b and c). The presence of CO-DOPA within the CONPs was confirmed by TEM without staining. Since ruthenium has high electron density, the nanoparticles loaded with CO-DOPA will appear as dark spots in the TEM image. As shown in Figure 6a, dark circular structures with a diameter of about 150 nm were visible, whereas we did not observe any such structures for PBANPs without CO-DOPA due to the low electron density of boron, carbon, nitrogen, and oxygen present in the PBANPs

Figure 7. CO release as measured by the myoglobin (Mb) assay. CODOPA (triangle) or CONPs (circle) were mixed with deoxyMb in PBS (pH 7.4) containing sodium dithionite. At different time points, the formation of carboxyMb was quantified by UV−vis spectroscopy (n = 3).

This result shows that CO-DOPA has the same CO-releasing ability as CORM-3 which has been reported to release 1 equiv of CO under the same condition.13 In the case of CONPs, the amount of released CO was 0.77 equiv within 2 h. To evaluate CO release under physiologically relevant conditions, we examined CO release from CO-DOPA and CONPs in the presence of cysteine in PBS and fetal bovine serum (FBS). These experiments were carried out in a cylindrical separable flask equipped with a CO detector and a test tube which was closed with a rubber septum for loading solutions via a syringe as we reported previously (Figure S3).24 As can be seen in Figure 8a, CO-DOPA released CO at pH 7.4 in a cysteine concentration-dependent manner. In the presence of 10 mM cysteine, CO-DOPA released 0.1 equiv of CO after 4 D


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reduced cysteine residues are abundant (at the millimolar level) inside cells,37 while the concentration of these thiol-containing compounds is much lower in human serum (about 8 μM for cysteine and 2 μM for glutathione).38,39 Therefore, CO-DOPA and CONPs may be sufficiently stable in the bloodstream and release CO only when taken up by cells. Anti-Inflammatory Effect of CO-DOPA and CONPs in the Lipopolysaccharide (LPS)-Induced Pro-Inflammatory Response of Murine Macrophages. The antiinflammatory effect of CO-DOPA and CONPs was assessed in murine RAW264.7 macrophages. The macrophages were stimulated with 0.1 μg/mL of lipopolysaccharide (LPS), a tolllike receptor 4 ligand that induces innate immune responses, in the presence of CO-DOPA, CONPs, and CORM-3 at different concentrations. After 24 h, we measured the concentration of the pro-inflammatory mediators interleukin-6 (IL-6) and nitric oxide (NO) in the culture medium by ELISA and Griess assay, respectively. As shown in Figure 9, IL-6 production was

Figure 8. CO release in the presence of cysteine as measured by a CO gas sensor. (a,b) Effect of cysteine on the CO release profiles of CODOPA and CONPs. (a) CO-DOPA and (b) CONPs were reacted with cysteine (0.1, 1, and 10 mM) in PBS (pH 7.4). (c,d) Stability of CO-DOPA and CONPs in the presence of FBS. (c) CORM-DOPA and (d) CONPs were incubated with 80% FBS in PBS (pH 7.4) for 2 h before adding cysteine to a final concentration of 10 mM. The experiments were conducted in a closed system and the CO concentration in the gas phase was monitored by a CO gas sensor. Concentration of CONPs and CO-DOPA: 1 mM. Figure 9. Anti-inflammatory effect of CO-DOPA and CONPs on the LPS-induced inflammatory response of RAW264.7 macrophages. Cells were cultured in medium containing 0.1 μg/mL LPS and CO-DOPA (circle), CONPs (triangle), and CORM-3 (square). After 1 d, the concentrations of the pro-inflammatory mediators (a) IL-6 and (b) nitric oxide (NO) in the culture medium were measured by ELISA and Griess assay, respectively. *p < 0.05, **p < 0.01, ***p < 0.001 versus CO-DOPA, n = 3.

h, while CO release was below the detection limit in the presence of 0.1 mM cysteine. This CO release profile is very similar to that measured for CORM-3 as shown in Figure S4. This shows that CO-DOPA has the same CO releasing characteristics as CORM-3. In the case of CONPs, cysteine concentration-dependent CO release was also observed, but CO release was slower. The amount of CO released from the CONPs after 4 h was 40% of that from CO-DOPA (Figure 8b). CO release from CORM-3 has been proposed to occur by substitution of the labile chloride or glycinate ligands by cysteine. The trans-labilizing effect of coordinated cysteine is believed to induce CO release.36 Because of the structural similarity we assume a similar mechanism for CO release from CO-DOPA and CONPs. The slow CO release from the CONPs compared to free CO-DOPA may relate to the hydrophobic surrounding within the nanoparticles which limit access of cysteine to CO-DOPA. We also studied CO release in the presence of FBS. We first incubated CO-DOPA and CONPs in 80% FBS/PBS for 2 h before adding cysteine to a final concentration of 10 mM. As shown in Figure 8c and d, both CO-DOPA and CONPs did not release CO in the presence of FBS, but CO release was observed upon the addition of cysteine. The amounts of CO released for CO-DOPA and CONPs were similar to that measured in the absence of FBS (Figure 8a and b). These results clearly show that both CO-DOPA and CONPs are stable and kept their CO release ability in the presence of FBS for at least 2 h. This cysteine-dependent CO release may be important for efficient delivery of CO to target cells. It is known that thiol compounds such as cysteine, glutathione, and proteins with

suppressed in the presence of the different CO donors. CODOPA showed significant reduction in the IL-6 production while CONPs showed a modest inhibitory effect on the IL-6 production. This result may relate to a slower CO release from CONPs as observed in Figure 8. Cytotoxicity of CO-DOPA and the CONPs in Murine Macrophages. Since transition metals are known to induce cellular damage and stress response,22 it is possible that the observed anti-inflammatory effects were due to the toxicity of the ruthenium complex itself and not related to CO released from the complex. To confirm that the observed antiinflammatory effect is not due to the cytotoxicity of CODOPA and the CONPs, we assessed the cell viability of murine RAW264.7 macrophages treated with these CO donors using the MTT assay. CO-DOPA did not show obvious toxicity up to 500 μM, and a slight reduction in cell viability (70%) was observed at 1 mM (Figure 10a). Interestingly, CO-DOPA showed much lower toxicity compared to CORM-3. In the case of CONPs, cell viability was above 80% even at 1 mM, showing that incorporation of CO-DOPA within the nanoparticles reduced the toxic effect of CO-DOPA (Figure 10b). Based on these results, we believe that the reduction of IL-6 and NO production is not caused by ruthenium itself, but by E


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On the other hand, due to its low molecular weight and amphiphilic nature, CO-DOPA is expected to cross cellular membranes and distribute within the cytoplasm. This difference in intracellular distribution between CO-DOPA and CONPs may also contribute to the different inhibitory effect on IL-6 and NO production.

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CONCLUSIONS CO-releasing nanoparticles (CONPs) were prepared by simple mixing of phenylboronic acid-containing framboidal nanoparticles (PBANPs) with the catechol-bearing CO-donor Ru(CO)3Cl(L-DOPA) (CO-DOPA) in aqueous buffer solutions. The CONPs had a framboidal morphology with a diameter of 188 nm. Both CO-DOPA and CONPs released CO in the presence of cysteine, but were stable in FBS. CONPs showed slower CO release compared to CO-DOPA. Furthermore, CO-DOPA and CONPs exerted anti-inflammatory effects in LPS-stimulated RAW264.7 macrophages. Because of the serum stability and cysteine-induced CO release, the CONPs may have potential in CO-based therapy.

Figure 10. Cytotoxicity of CONPs, CO-DOPA, and CORM-3 in RAW264.7 macrophages. (a) RAW264.7 macrophages were cultured in the presence of CO-DOPA (triangle) and CORM-3 (circle) for 1 d. (b) RAW264.7 macrophages were cultured in the presence of CODOPA (triangle), CONPs (circle), and PBANPs (square) for 1 d. Cell viability was assessed by the MTT assay. *p < 0.05, ***p < 0.001 (n = 3).

the CO released from CORM-3, CO-DOPA, and CONPs. It has been shown that CORM-3 reduces pro-inflammatory markers in LPS-stimulated macrophages.40 It was also shown that chemically inactivated CORM-3, i.e., a ruthenium complex no longer able to release CO, did not show any of these effects, pointing to CO being responsible for the observed bioactivity. Importantly, the cell viability was not affected by CORM-3, as well as its inactive form, indicating that the biological activity was not due to cytotoxicity of ruthenium present in the compounds. Since in our case cell viability was not affected at concentrations at which the anti-inflammatory effects were observed, we believe that the observed bioactivity is caused by CO and not by ruthenium. Cellular Uptake of the CONPs. To confirm that the CONPs are taken up by RAW264.7 macrophages, we prepared fluorescent Nile Blue-labeled PBANPs as previously reported33 and loaded these particles with CO-DOPA to yield fluorescent CONPs with a hydrodynamic diameter of 195 nm according to DLS. These fluorescent CONPs were then incubated with RAW264.7 macrophages for 2.5 h and their cellular uptake was observed by confocal laser scanning fluorescence microscopy (CLSFM). As shown in Figure 11, fluorescent spots were observed intracellularly indicating that CONPs were taken up by RAW264.7 macrophages and stayed within endo/lysosomes.

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EXPERIMENTAL PROCEDURES

Materials. Tricarbonyldichlororuthenium(II) dimer [Ru(CO)3Cl2]2, myoglobin from equine skeletal muscle and N,N′methylenebis(acrylamide) (MBAM) were purchased from Sigma-Aldrich. 3-aminophenylboronic acid, N-(1-naphthyl)ethylenediamine dihydrochloride, sulfanilamide, and ammonium persulfate (APS) were purchased from the Tokyo Chemical Industry. Tetrahydrofuran (THF), hexane, methanol, molecular sieves 3A, phosphate buffer solution (pH 7.4), and acetate buffer (pH 5.0) were purchased from Nacalai Tesque. Glycine, L-3,4-dihydroxyphenyl alanine (L-DOPA), sodium nitrite, D-glucose, and 28% sodium methoxide methanol solution were purchased from Wako Pure Chemicals. Nitric acid 65% (Suprapure), ruthenium ICP standard, and Amicon filter unit (MWCO 3000 Da) were purchased from Merck Millipore. Dulbecco’s phosphate buffered saline (PBS), fetal bovine serum (FBS), Dulbecco’s Modified Eagle’s Medium GlutaMAX, and penicillin−streptomycin, and Nunc MaxiSorp flat-bottom 96-well microplates were purchased from Thermo Fisher Scientific. Deuterated dimethyl sulfoxide (DMSO-d6) was purchased from Cambridge Isotope Laboratories. Mouse IL-6 DuoSet Economy Pack was purchased from R&D Systems. Spectra/Por 6 Standard regenerated celulose tubing (MWCO 2000 Da) was purchased from Spectrum Laboratories. 96-well microplates were purchased from Iwaki. MBAM was recrystallized from methanol. APS was recrystallized from ethanol/water (1:1). Methanol was kept over molecular sieves in an amber bottle. Molecular sieves were dried under reduced pressure at 160 °C for 4 h. N-Acryloyl-3-aminophenylboronic acid (PBAAM), methoxy poly(ethylene glycol) acrylamide (PEGAM), and CORM-3 were synthesized as reported previously.24,32 NMR Spectroscopy. 1H NMR spectra were acquired on a Bruker DPX400 NMR spectrometer. A total of 32 scans were collected and the d1 was set to 10 s. The chemical shifts are reported relative to the residual undeuterated NMR solvent signal. Dynamic Light Scattering (DLS) And Zeta Potential Measurements. Hydrodynamic diameter of the NPs in deionized water was obtained on an Otsuka ELSZ1000 instrument using polystyrene cuvettes. The mean diameter

Figure 11. Cellular uptake of CONPs. RAW264.7 macrophages were cultured in the (a) absence or (b) presence of Nile blue-labeled CONPs for 2.5 h and observed by CLSFM. Scale bars: 50 μm. F


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Bioconjugate Chemistry (Z-average) and polydispersity index (PDI = μ2/Γ2) were calculated by the cumulant method. Zeta potential of the PBANPs and CONPs were measured using a zeta potential accessory. Atomic Force Microscopy (AFM). A solution of PBANPs in water was dropped onto a fresh mica surface and then dried by blowing off the solution by a hand blower. Images were acquired on a Seiko Instruments SPA400 in dynamic force mode (DFM) using a Si probe with Al coating (SI-DF20, Seiko). Transmission Electron Microscopy (TEM). A solution of PBANPs or CONPs in water was placed onto a carbon coated 250 mesh copper grids and then dried by blotting the side of the grid with filter paper. The grids were negatively stained with 0.5 wt % Preyssler-type potassium phosphotungstate solution. Images were acquired on a Hitachi H-7650 TEM operating at 100 kV. UV−vis Spectroscopy. Spectra were obtained on a Hitachi U-2810 or a Tecan infinite M200 well plate reader. Attenuated Total Reflection Infrared Spectroscopy (ATR-IR). ATR-IR spectra were obtained on a Thermo Scientific Nicolet iS5 equipped with an iD5 universal ATR sampling accessory. Inductively Coupled Plasma Optical Emission Spectrometry (ICP-OES). The samples were dissolved in 65% nitric acid and digested overnight at RT. This solution was diluted with Milli-Q water. Ruthenium concentration was measured on a Shimadzu Sequential Plasma Emission Spectrometer ICPS-7510 instrument using the ruthenium ICP standard solution. Confocal Laser Scanning Fluorescence Microscopy (CLSFM). Fluorescent images were acquired on an Olympus FluoView FV1000-D confocal laser scanning fluorescence microscope equipped with 405, 473, 559, and 635 nm lasers. A 635 nm laser was used for excitation and a 655−755 nm barrier filter was used to observe Nile Blue fluorescence. Synthesis of Ru(CO)3Cl(L-DOPA) (2, CO-DOPA). [Ru(CO)3Cl2]2 (3) (129 mg, 0.25 mmol) and L-DOPA (4) (103 mg, 0.52 mmol) were placed in a Schlenk tube and dissolved in 60 mL of anhydrous methanol. After three vacuum-nitrogen purge cycles, 200 mg of 28% sodium methoxide methanol solution (1.04 mmol) was added under nitrogen flow. The reaction mixture was shielded from light and stirred for 18 h at RT. The bluish green solution was evaporated under reduced pressure at 40 °C to remove methanol. The resulting solid was dissolved in 40 mL of THF and filtered through paper filter. The clear solution was concentrated under reduced pressure at 40 °C and precipitated in 250 mL of hexane to yield a bluish green solid. Yield: 163 mg (76%). Synthesis of Phenylboronic Acid-Containing Nanoparticles (PBANPs). PBANPs were synthesized as reported previously.32,33 PBAAM (9.5 mg, 50 μmol), PEGAM (67.9 mg, 12.5 μmol), and MBAM (0.8 mg, 5 μmol) were dissolved in 18.8 mL of 0.1 M phosphate buffer solution (pH 7.4) and placed in a Schlenk tube. The solution was degassed by nitrogen bubbling for 30 min followed by three vacuumnitrogen purge cycles. After 30 min at 70 °C, a solution of APS (1.14 mg, 5 μmol) in Milli-Q water was added to the reaction mixture under nitrogen flow followed by three vacuum-nitrogen purge cycles. The reaction mixture was stirred at 70 °C for 24 h. After exposure to air and cooling down to RT, the solution was dialyzed against Milli-Q water (MWCO 2000 Da) and lyophilized.

Synthesis of CO-Releasing Nanoparticles (CONPs). A typical procedure is as follows: PBANPs (11.2 mg/mL, 7.2 mM PBA groups) were mixed with CO-DOPA (final concentration: 6.0 mg/mL, 14.4 mM) in 1 mL of 0.1 M phosphate buffer solution (pH 7.4) and stirred for 45 min at RT. The solution was ultrafiltered using an Amicon filtration unit (100 × g, 30 min) to remove unbound CO-DOPA. The ruthenium concentration of the filtrate was measured by ICP-OES to quantify the loading amount of CO-DOPA onto PBANPs. The concentrated sample solution was washed with Milli-Q water by ultrafiltration (3 × 1 mL) and lyophilized to yield CONP. Synthesis of Fluorescent PBANPs. Fluorescent PBANPs were synthesized by aqueous dispersion copolymerization of PBAAM and Nile Blue acrylamide (NBAM) (PBAAM/NBAM molar ratio = 5/1) in the presence of PEGAM and MBAM as previously reported.33 Synthesis of Fluorescent CONPs. Fluorescent CONPs were prepared in the same way as described above for CONPs by substituting the PBANPs with the fluorescent PBANPs. CO Release Measured by the Myoglobin (Mb) Assay. CO release was quantified by the Mb assay as reported previously.24 Briefly, 15 mL of 2 mg/mL Mb in PBS was degassed by nitrogen bubbling for 30 min and mixed with 1.5 mL of 24 mg/mL Na2S2O4 in degassed PBS. In a quartz cuvette 3.6 mL of this solution was mixed with 0.15 mL of CO-DOPA or CONPs in degassed PBS. The absorbance of 470−620 nm was recorded as a function of time. The concentration of MbCO was calculated using the following eq 1.24 ⎛ε A ⎞ εi [MbCO] = ⎜ d − 542 ⎟ · [Mb] + [MbCO] Ai ⎠ εd − εc ⎝ εi

(1)

where A542 and Ai are the absorbance values at 542 and 552 nm, εd, εc, and εi are the extinction coefficient of Mb at 542 nm, MbCO at 542 nm, and Mb and MbCO at 552 nm (the isosbestic point), respectively. [Mb] and [MbCO] are the concentration of Mb and MbCO. εd/εi and εc/εi are 0.836 and 1.227, respectively. CO Release in the Presence of Cysteine. The experiment was carried out in a cylindrical separable flask equipped with a Drager Pac7000 CO detector and a test tube (see Figure S3). A solution of CO-DOPA or CONPs in 2.0 mL of PBS was placed in a test tube. For the measurement in the presence of FBS, 0.4 mL of the CO-DOPA and CONP solutions were mixed with 1.6 mL of FBS. After closing the lid, 3.0 mL of cysteine in PBS (final cysteine concentration: 0.1, 1, 10 mM) was added through the septum using a syringe. The gas-phase CO concentration was recorded every 1 min. The amount of released CO (NCO [mol]) was calculated using the following eq 2.24 NCO =

⎛ Vg V⎞ + cVl = p⎜ + l⎟ RT k⎠ ⎝ RT

pVg

(2)

where p is the partial pressure of CO, Vg and Vl are the volume of the gas phase (292.5 mL) and liquid phase (5 mL), R is the gas constant (0.08205 L·atm·mol−1·K−1), T is the temperature, c is the CO concentration in the liquid phase, and k is Henry’s law constant of CO in water (1052.63 L·atm·mol−1 at 25 °C). Cell Culture. RAW264.7 cells were cultured in DMEM GlutaMAX supplemented with 10% FBS and 50 U/mL−50 μg/ mL penicillin−streptomycin in a CO2 incubator at 37 °C. Cells G


Article

Bioconjugate Chemistry were dislodged from the culture flask using a cell scraper and passaged when reaching 70−80% confluency. MTT Assay. RAW 264.7 macrophages were seeded at 5 × 104 cell/well in 96 well polystyrene plates in 100 μL of culture medium and cultured for 1 d. The medium was replaced with 90 μL of culture medium and 10 μL of CO-DOPA and CONPs solutions (in water) at different concentrations. Each condition was run in triplicate. After 1 d, the medium was replaced with 100 μL/well fresh medium containing 0.5 mg/mL MTT and then incubated for 2 h at 37 °C. Then, 100 μL of 0.1 g/mL sodium dodecyl sulfate in 0.01 M HCl (aq) was added to each well to lyse cells and solubilize the formazan crystals. The OD at 570 nm was measured with a plate reader. Statistical analysis was performed using the Student’s t test (n = 3). Anti-Inflammatory Effect in LPS-Induced Pro-Inflammatory Response in Murine Macrophages. RAW 264.7 macrophages were seeded in a 96-well plate (5 × 104 cells/ well) and cultured for 1 d. The medium was replaced with 100 μL/well of fresh culture medium containing 0.1 μg/mL LPS and the CO donors. After 24 h of culture, the medium was collected. IL-6 concentration in the medium was measured by ELISA. Statistical analysis was performed using the Student’s t test (n = 3). Griess Assay. Nitrite (NO 2 −) concentrations were measured by the Griess assay in transparent 96 well polystyrene plates. The samples were reacted with 50 μL of 2% (w/v) sulfanilamide in 5% HCl (aq) for 5 min before adding 50 μL 0.1% (w/v) N-(1-naphthyl)ethylenediamine dihydrochloride (aq). After 5 min, the absorbance at 550 nm was measured. To calculate the nitrite concentrations, a standard curve was obtained using 0−100 μM sodium nitrite solutions. Cellular Uptake of Fluorescent CONPs. RAW264.7 cells were seeded in a triple-well glass-based dish (5 × 104 cells/ well) with 200 μL medium and cultured for 1 day. The medium was replaced with 200 μL medium containing 0.8 mg/mL fluorescent CONPs. After 2.5 h of culture, the medium was removed and cells were washed with fresh medium before acquiring CLSFM images.

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Sadakane (Hiroshima University, Japan) for supplying Preyssler-type potassium phosphotungstate.

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ABBREVIATIONS CO, carbon monoxide; PBA, phenylboronic acid; PBANPs, phenylboronic acid-containing nanoparticles; CORM-3, carbon monoxide releasing molecule 3; L-DOPA, dopamine; CODOPA, a catechol-bearing analogue of CORM-3; CONPs, carbon monoxide-releasing nanoparticles; PBAAM, N-acryloyl3-aminophenylboronic acid; MBAM, N,N′-methylenebis(acrylamide); PEGAM, methoxy poly(ethylene glycol) acrylamide; PDI, polydipersity index; Mb, myoglobin; IL-6, interleukin 6; LPS, lipopolysaccharide

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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.bioconjchem.6b00135. IR spectra of CORM-3 and CO-DOPA, TEM image of the PBANPs, experimental setup to measure CO, CO release from CORM-3 and the different forms of CORM-3 in aqueous solution (PDF)

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REFERENCES

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by Grant-in-Aid for Challenging Exploratory Research, no. 26560241, from the Japan Society for the Promotion of Science and Research Grant from the Ogasawara Foundation for the Promotion of Science & Engineering, Japan. We thank Dr. Eiko Mochizuki (Osaka University, Japan) for the TEM measurements, and Prof. M. H


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