Ferrocene-Containing Polymer via the Biginelli Reaction for In Vivo

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Letter Cite This: ACS Macro Lett. 2019, 8, 639−645

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Ferrocene-Containing Polymer via the Biginelli Reaction for In Vivo Treatment of Oxidative Stress Damage Tengfei Mao,†,‡,§ Lei Yang,§,∥ Guoqiang Liu,† Yen Wei,† Yanzi Gou,*,‡ Jun Wang,*,‡ and Lei Tao*,†

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The Key Laboratory of Bioorganic Phosphorus Chemistry & Chemical Biology (Ministry of Education), Department of Chemistry, Tsinghua University, Beijing 100084, People’s Republic of China ‡ Science and Technology on Advanced Ceramic Fibers and Composites Laboratory, National University of Defense Technology, Changsha, 410073, People’s Republic of China ∥ Cancer Institute and Hospital, Peking Union Medical College and Chinese Academy of Medical Science, Beijing, 100021, People’s Republic of China S Supporting Information *

ABSTRACT: Small molecule antioxidants have little impact on oxidative stress in vivo because of their poor bioavailability. To explore an antioxidant for in vivo applications, a polymeric antioxidant containing a ferrocene moiety was developed. The ferrocenecontaining monomer was synthesized through the robust tricomponent Biginelli reaction with a high yield. The corresponding watersoluble copolymer was conveniently prepared via radical polymerization. Both the ferrocene moiety and the Biginelli structure (dihydropyrimidin-2(H)-one) contributed to the remarkable radical scavenging ability of this highly biocompatible copolymer. It was more efficient than traditional small molecule antioxidants at protecting cells against fatal oxidative stress. This copolymer also showed clear therapeutic activity in counteracting oxidation-induced acute liver damage in a live mouse model. Our study into functional organometallic polymers resulted in a promising polymeric biomaterial that may find therapeutic applications and have important implications in the fields of organic chemistry and polymer chemistry.

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been improved using polymers.8−11 Currently, clinical applications of antioxidants are drawing increased attention as the general public grows increasingly concerned about aging and health. Polymeric antioxidants have the potential to overcome inherent limitations of traditional antioxidants. Thus, the development of new polymeric antioxidants that are safe and have clear effects in vivo is important in terms of fundamental research and practical applications. The Biginelli reaction, as introduced by Pietro Biginelli in 1891, efficiently generates dihydropyrimidin-2(H)-one (DHPM) from the reaction of an aldehyde, β-ketoester, and (thiol)urea.12 The Biginelli reaction has been widely studied in organic chemistry and pharmaceutical chemistry because DHPM derivatives have many biological properties such as antioxidant, anti-inflammatory, anticancer, and antibacterial activities.13,14 Recently, this old multicomponent reaction (MCR) has been used to prepare new functional polymers.15−19 When thiourea and thiourea derivatives were used, the resulting polymers exhibited attractive radical scavenging (antioxidant) abilities.18,19 In a recent study, a polymer with DHPM side chains performed better than even superoxide dismutase (SOD)

xidative stress is caused by excess reactive oxygen/ nitrogen species (ROS/RNS). These species lead to disrupted redox signaling and/or oxidative damage to biomolecules, such as DNA, proteins, and lipids. Oxidative stress is believed to be closely related to aging and several degenerative diseases (such as Alzheimer’s and Parkinson’s diseases), depression, cancer, atherosclerosis, infections, and tissue damage.1−3 To treat or proactively prevent such diseases, large doses of antioxidant supplements have been prescribed to human subjects, including vitamins (A, C, and E), polyphenols, and carotenoids. Nevertheless, clinical trials have found little or no evidence to support the benefits of these dietary antioxidants, despite their considerable antioxidant ability in vitro.4−6 The basic properties of these antioxidants (such as high instability, rapid clearance from the body, and poor water solubility) are the possible reasons for their low bioavailability (the amount of a bioactive compound in the bloodstream), which may in turn explain their poor performances in suppressing the oxidative stress levels in patients.5,7 Polymers have been used to improve the bioavailability of traditional antioxidants. By covalent/noncovalent inclusion in water-soluble polymers, antioxidants can be easily dispersed in aqueous solutions with clearly enhanced stability and activity in vitro (examples include quercetin, vitamin C, and curcumin). In some in vivo cases, the bioavailability of antioxidants has also © 2019 American Chemical Society

Received: March 24, 2019 Accepted: May 16, 2019 Published: May 23, 2019 639

DOI: 10.1021/acsmacrolett.9b00210 ACS Macro Lett. 2019, 8, 639−645

Letter

ACS Macro Letters at protecting cells from fatal UV irradiation.19 This finding has opened new opportunities for exploring synthetic polymer antioxidants, which might even be superior to traditional antioxidants. To promote the in-depth research in this field, we report an antioxidative ferrocene-containing polymer, constructed from a monomer unit that was obtained through a Biginelli reaction, and its in vivo treatment of oxidative stress damage (Scheme 1).

consistent with the theoretical value (1:1:1). This ferrocene− DHPM monomer was copolymerized with commercially available poly(ethylene glycol methyl ether) methacrylate (PEGMA, Mn ∼ 950 g mol−1) via a convenient radical polymerization to produce the water-soluble copolymer (Figure 1a, labeled P1). The 1H NMR spectrum of P1 (Figure 1b’) shows characteristic peaks of the DHPM group (NH: 10.38, 9.38 ppm; PhCH: 4.94 ppm) and the methyl group in the PEG segments (CH3O: 3.23 ppm). The integral ratio between the PhCH methine and the methyl group in the PEG moiety (I4.94/I3.23 = 0.93:3) matched well with the theoretical value (1:3). The copolymer was found to have a high molecular weight (Mn(GPC) ∼ 61900 g mol−1; Figure 1b’, Table S1). Thus, the preparation of the desired ferrocene−DHPM copolymer was facile. 2,2′-Azinobis(3-ethylbenzothiazoline-6-sulfonate) cationic radical (ABTS+•) was used as a model radical to test the radical scavenging ability of P1 as literatures.24 Three polymers were prepared and used as the controls as follows: (1) a homopolymer of PEGMA (P(PEG), Figure 2a, Figure S1);

Scheme 1. Ferrocene-Containing Polymer for In Vivo Treatment of Oxidative Stress Damage

Ferrocene has been widely studied since 1951.20,21 It is a neutral, nontoxic, organometallic, and redox-active compound and a potential radical scavenger because of its unique electronabundant structure.22−25 Introducing a ferrocene moiety is an efficient strategy for increasing the antioxidant ability of many functional groups including DHPM group.24−26 In this study, ferrocenecarboxaldehyde, thiourea, and the commercially available monomer 2-(acetoacetoxy) ethyl methacrylate (AEMA) were used to prepare the ferrocene-containing monomer via the Biginelli reaction (Figure 1a). The target ferrocene−DHPM monomer was easily synthesized in 87% yield without column chromatography. From the 1 H NMR spectrum (Figure 1b), characteristic peaks of the DHPM group (NH: 10.41, 9.45 ppm; PhCH: 4.91 ppm) could be clearly identified. The integral ratio between the PhCH methine and the vinyl group (I4.91/I5.73/I6.04 = 1:1:1) was

Figure 2. (a) Four polymers used in the radical scavenging test. (b, b’, b”) Color change of ABTS+• solutions in the presence of polymers. (c) ABTS+• radical concentration over time in the presence of various test polymers (absorbance monitored at 734 nm). ABTS+• solution (190 μL, ∼ 66 μM) mixed with polymer solutions (10 μL, [pendant group]: 0.2 mM). Water served as the blank. Data presented as mean ± SD, n = 5.

(2) a ferrocene-containing copolymer containing ester linkages in place of the DHPM group (P2, Figures 2a and S2 and Table S1); and (3) a copolymer using benzaldehyde instead of ferrocenecarboxaldehyde for the Biginelli reaction (P3, Figures 2a and S3 and Table S1). Water served as a blank control, and the color change of ABTS+• was photographically captured over time. The radical scavenging ability of polymers followed the order of P1 > P3 > P2 > P(PEG) by direct visual observation (Figure 2b, b’, b”), which is consistent with quantitative data analysis from monitoring the absorbance at 734 nm (Figure 2c). The

Figure 1. (a) Preparation of the ferrocene−DHPM monomer and the ferrocene−DHPM-containing copolymer. (b, b’) 1H NMR spectra (DMSO-d6, 400 MHz) of the ferrocene−DHPM monomer and the related polymer (b: monomer; b’: polymer). 640

DOI: 10.1021/acsmacrolett.9b00210 ACS Macro Lett. 2019, 8, 639−645

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ACS Macro Letters radical scavenging capability of P1, P2, and P3 was evaluated (Figure S4). At the same concentrations, the radical quenching effect of P1 is greater than the sum of P2 and P3. This indicates the possible synergy effect between ferrocene and DHPM groups. We continued by evaluating the ability of P1 and several wellknown small molecule antioxidants (vitamin C, glutathione, and anthocyanidins; Figure 3a) to protect cells from oxidative stress.

Figure 3. (a) Water-soluble small molecular antioxidants. (b) Cytotoxicity of P1, vitamin C, glutathione, and anthocyanidins to L929 cells, 24 h culture. Data are represented as mean ± SD, n = 5.

Prior to the experiment, cytotoxicity of P1 and small molecular antioxidants were tested using a cell counting kit-8 (CCK-8) assay. The murine fibroblast cell line L929 was chosen as the model cell (Figure 3b). The IC50 value of anthocyanidins to L929 cells was 0.28 mg/mL, as calculated using SPSS 24.0. The IC50 values of vitamin C and glutathione to L929 cells were 1.80 and 5.50 mg/mL, respectively. In contrast, cultured cells retained ∼93% viability, even in 20 mg/mL of P1, indicative of the obvious advantage of P1 in terms of nontoxicity. tert-Butyl hydroperoxide (t-BHP) induces oxidative stress in different cell lines.27 Here, we tested the cytotoxicity of t-BHP to L929 cells (Figure S5a) and chose 200 μM of t-BHP as the test concentration because this t-BHP concentration resulted in clearly increased ROS concentrations (∼ 350%) and adequate cell necrosis (cell viability: ∼ 45%) (Figure S5b). Then, the viability of L929 cells in the presence of t-BHP and different antioxidants was tested. Only nontoxic concentrations of antioxidants to the L929 cells were used (P1: 20 mg/mL, vitamin C: 0.5 mg/mL, glutathione: 2 mg/mL, and anthocyanidins: 0.13 mg/mL). Cells cultured with t-BHP (200 μM) and no antioxidant acted as the control. Cells in culture medium only were used as the blank. Fluorescein diacetate/propidium iodide (FDA/PI) double staining was performed to simultaneously observe the living and dead cells (Figure 4). Compared with the blank (Figure 4a), t-BHP-induced oxidative stress led to obvious cell necrosis after a 24 h culture (Figure 4b). Anthocyanidins and vitamin C provided virtually

Figure 4. FDA/PI double staining of L929 cells in (a) culture medium only; (b) t-BHP (200 μM); (c) t-BHP and anthocyanidins (0.13 mg/ mL); (d) t-BHP and vitamin C (0.5 mg/mL); (e) t-BHP and glutathione (2 mg/mL); and (f) t-BHP and P1 (20 mg/mL). Scale bar = 100 μm.

zero protection to cells to against oxidative stress damage (Figure 4c, 4d). Cells cultured with vitamin C had even less viability than the control (Figure 4b,d). This may have resulted from a pro-oxidant effect of vitamin C28,29 or the inhibition of catalase by vitamin C30 or both. 641

DOI: 10.1021/acsmacrolett.9b00210 ACS Macro Lett. 2019, 8, 639−645

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ACS Macro Letters Glutathione provided remarkable protection against oxidative stress; nevertheless, few dead cells were observed (Figure 4e). In contrast, nearly all cells cultured with P1 survived the t-BHPinduced oxidative stress (Figure 4f). These results were in good agreement with quantitative data obtained from the CCK-8 assay (Figure S6), suggesting that P1 is better than traditional small molecule antioxidants at protecting cells against t-BHPinduced oxidative damage. The antioxidant effects of different antioxidants in cells were studied. When L929 cells were cultured with P1 (20 mg/mL), ROS concentrations in cells increased only slightly and still within safe levels (∼150%) (Figure S7). When the cells were cultured in the presence of P1 (20 mg/mL) and t-BHP (200 μM), the ROS concentrations in the cells plateaued at the level that cells could tolerate (∼180%), retaining ∼ 98% viability for the cells (Figure S7). This suggests P1 can protect cells by effectively reducing ROS concentrations to tolerable levels. Conversely, ROS concentrations in cells cultured with small molecule antioxidants continued to increase in the presence of tBHP (ROS peaks: vitamin C (∼330%, Figure S8); glutathione (∼280%, Figure S9); and anthocyanidins (∼330%, Figure S10)). This confirmed that the cytotoxicity of small molecular antioxidants limits their application to protect cells from oxidative stress. P1 had excellent nontoxicity compared with other antioxidants, which allowed it to be used in large amounts. CCl4 is a well-known hepatotoxin that induces acute or chronic liver injury and simulates the damage caused by oxidative stress and free radicals.31 Here, CCl4 was used as a source of oxidative stress in vivo, and an experiment was designed to evaluate the in vivo antioxidant capability of P1 by simulating a treatment of CCl4-induced acute liver injury. Silymarin, an excellent antioxidant and the active ingredient of several clinical medicines (Legalon SIL (Madaus) or Silimarit (Bionorica)) for liver disease, was used as a control. Prior to the experiment, safe doses and administration methods of both silymarin and P1 were evaluated. Silymarin solution (20 mg/mL) was prepared according to the literature32 and given to Balb/c mice (5−7 weeks old, 18−21 g, male and female) through intravenous tail injection or intraperitoneal injection. Mice died immediately after intravenous tail injections of 200 or 600 μL of silymarin solution. Five of six mice died within 24 h after intraperitoneal injection of 600 μL of silymarin solution. All mice survived intraperitoneal injection of 200 μL of silymarin solution. Meanwhile, all mice survived intravenous tail injection of 600 μL of P1 in a saline solution (20 mg/mL). Thus, the in vivo experiment was designed as follows: 30 Balb/c mice (5−7 week old, 18−21 g, male and female) were intraperitoneally injected a CCl4 solution (0.5% v/v in corn oil, 50 μL) and randomly assigned to five groups (six mice per group) for the following treatments: (1) no additional treatments after CCl4 administration; (2) intraperitoneal injection of silymarin solution (20 mg/mL, 200 μL); (3) intraperitoneal injection of P1 (20 mg/mL, 200 μL); (4) intravenous tail injection of P1 (20 mg/mL, 600 μL); and (5) intravenous tail injection of saline solution (600 μL). Mice were fasted overnight but given tap water ad libitum, then sacrificed 24 h after treatments. Blood was collected to measure serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels. Livers were isolated for analysis. The left liver lobes were used for histological studies using hematoxylin and eosin (H&E) staining (Figure 5), and the remaining liver portions were used to determine levels of SOD (a biomarker for ROS production) and malondialdehyde

Figure 5. (a−f) Histological images of liver sections from different groups (600× magnification): (a) Health controls; (b) group 1 (no additional treatment); (c) group 2 (CCl4 + intraperitoneal injection of silymarin (200 μL, 20 mg/mL)); (d) group 3 (CCl4 + intraperitoneal injection of P1 (200 μL, 20 mg/mL)); (e) group 4 (CCl4 + intravenous tail injection of P1 (600 μL, 20 mg/mL)); (f) group 5 (CCl4 + intravenous tail injection of saline (600 μL)); Scale bar = 20 μm; and (g) MDA and SOD levels in different groups. Data are represented as mean ± SD, n = 6. *p < 0.05, **p < 0.01.

(MDA; a biomarker for lipid peroxidation). Healthy mice were used as controls. Compared with the healthy liver tissue (Figure 5a), the liver tissue in the mice after CCl4 administration showed obvious morphological changes around the central veins. The hepatic cells lost their natural structure: vacuoles (black arrows) and inflamed cells (yellow arrows) can be clearly observed in the liver tissue (Figure 5b). After the intraperitoneal injection of silymarin (20 mg/mL, 200 μL), the liver tissue was not as damaged as the no-treatment group (Figure 5c). Similar results were observed when P1 was given via intraperitoneal injection (20 mg/mL, 200 μL; Figure 5d). However, P1 could be given to animals in a larger dose because of its excellent safety. Thus, when a large dose of P1 (20 mg/mL, 600 μL) was given via intravenous tail injection, most liver cells remained intact. There were significantly fewer vacuoles and inflamed cells than in mice given a small dose of silymarin or P1 (Figure 5e). This suggests that a large dose of P1 can effectively attenuate pathological changes induced by CCl4 because the intravenous tail injection of saline alone yielded no therapeutic effect (Figure 5f). SOD and MDA levels in the healthy mice group were 175.5 U/mg protein and 3.8 nmol/mg protein, respectively (Figure 642

DOI: 10.1021/acsmacrolett.9b00210 ACS Macro Lett. 2019, 8, 639−645

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ACS Macro Letters

polymer chemistry to achieve new functional polymers.44−49 This first ferrocene polymer via the Biginelli reaction might prompt a broad study of organometal compounds and MCRs in polymer science and lead to new organometallic polymers via different MCRs for biological and medical applications. Moreover, polymers with a potential to become therapeutic agents should be biodegradable. In future research, the combination of ferrocene-DHPM group used here with other polymerization methods (ring-open polymerization, polycondensation, etc.) might offer new polymeric antioxidants that are more biofriendly to control the levels of oxidative stress in vivo.

5g). After CCl4 administration, the SOD level reduced to 142.6 U/mg protein and the MDA level increased to 18.1 nmol/mg protein, indicative of severe liver damage. Intravenous tail injection of saline hardly affected the SOD level (143.9, p = 0.74, compared with the CCl4 only group) and slightly reduced the MDA level (16.8, p < 0.05, compared with the CCl4 only group) in mice. Intraperitoneal injection of silymarin also had negligible effects on the SOD level (147.1, p = 0.069, compared with the CCl4 only group) and a weak effect on the MDA level (15.9, p < 0.05, compared with CCl4 only group). Similar, albeit worse, results were obtained from the 200 μL of P1 group (SOD: 146.7, p = 0.41, MDA: 16.1, p = 0.071; compared with the CCl4 only group). When a large dose of P1 was given via intravenous tail injection, the SOD level was significantly higher than the CCl4 only group (160.1 U/mg protein) and the MDA level was significantly lower at 11.4 nmol/mg protein (p < 0.01, compared with the CCl4 only group). Serum ALT and AST levels (biochemical markers for early acute hepatic damage) were also measured (Table S2). CCl4 induced dramatic increases in ALT and AST levels (2348 ± 198 U/L and 1415 ± 258 U/L, respectively) compared with the health control group (ALT: 30 ± 8 U/L, AST: 72 ± 18 U/L). The increase of ALT and AST levels was slightly suppressed by a low dose of silymarin or P1 (silymarin: 17.6% and 25.1%, respectively; P1:16.8% and 14.7%, respectively); however, a large dose of P1 resulted in significant suppression of ALT and AST levels (48.7% and 50.4%, respectively). For healthy mice, intravenous tail injection of P1 (600 μL) and intraperitoneal injection of silymarin (200 μL) did not lead to obvious pathological changes in the liver tissue (Figure S11); meanwhile, only small changes in various biomarkers were detected (MDA, SOD, ALT, and AST) (Table S3). This confirms that the dose and administration methods in the present study were safe for mice and have no significant effect on the experimental results. Therefore, our study suggests that silymarin is effective in counteracting oxidative stress-induced liver damage. However, the intrinsic defects of silymarin (poor water solubility, low bioavailability, and potential toxicity) limit the administration method of silymarin, thus leading to unsatisfactory therapy effects. In contrast, the excellent biosafety of P1 ensured that a large dose of P1 could be safely administered. Therefore, P1 achieved superior therapy results in the treatment of acute liver injury than silymarin, demonstrating the great potential of polymer antioxidants for in vivo applications to inhibit oxidative stress-induced damage. In summary, we have developed a ferrocene and DHPMcontaining monomer via the Biginelli reaction. The resulting polymer product demonstrated better radical scavenging ability than polymers containing only ferrocene or DHPM groups alone. This confirmed that the ferrocene moiety can efficiently improve the antioxidant capability of DHPM. This ferrocene− DHPM polymer was relatively nontoxic and offered much better protection to cells against t-BHP induced oxidative stress than traditional small molecule antioxidants. In an in vivo mouse model experiment, this polymer effectively counteracted CCl4induced serious acute liver injury, the therapeutic effect was superior to that of silymarin, which is an active pharmaceutical ingredient in clinically prescribed medicines. This highlights the value of combining organometal compounds and MCRs to achieve new functional polymers with useful properties. Nowadays, polymers are considered as promising biomaterials as drug/gene carriers, tissue engineering scaffolds, and antibacterial surfaces.33−43 MCRs are playing new roles in

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsmacrolett.9b00210. Detailed methods to synthesize/analyze monomers and polymers, cell experiments, animal experiments, as well as supporting figures, schemes, and tables (PDF)

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AUTHOR INFORMATION

Corresponding Authors

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

Lei Tao: 0000-0002-1735-6586 Author Contributions §

These authors contributed equally to this manuscript.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was supported by the National Science Foundation of China (21574073) and the Postdoctoral Science Foundation of China (2014M552685). All in vivo experiments were performed under the technical guidelines for nonclinical studies, as issued by the China Food and Drug Administration. This study was approved by the ethics committee of the Chinese Academy of Medical Science Cancer Hospital (Permit No. NCL2018A167).

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REFERENCES

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DOI: 10.1021/acsmacrolett.9b00210 ACS Macro Lett. 2019, 8, 639−645

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DOI: 10.1021/acsmacrolett.9b00210 ACS Macro Lett. 2019, 8, 639−645