Polymer–protein hybrid scaffolds as carriers for CORM-3: platforms for the delivery of carbon monoxide (CO)

Abstract

Recognised as a signalling molecule in mammals, carbon monoxide (CO) has a number of beneficial effects, including anti-inflammatory, anti-apoptotic, anti-proliferative and cytoprotective properties, and has been evaluated in clinical trials as a novel therapy. However, the clinical use of CO gas has been hampered due to safety issues and difficulties with delivery to specific sites of action. These challenges have led chemists to explore CO-releasing molecules (CORMs) as an alternative to administration of CO gas. [fac-RuCl(κ²-H₂NCH₂CO₂)(CO)₃] (CORM-3), (tricarbonylchloro(glycinato)ruthenium(II)), in particular, has gained increasing attention in biological studies due to its commercial water-soluble CORM and its remarkable biological effects. However, this compound possesses some unfavourable characteristics that have limited its clinical applications, including short CO-releasing half-life, poor stability and low cellular uptake efficiency. These issues can be prevented by incorporating CORM-3 into macromolecular scaffolds. In this study, we present for the first time the use of a polymer–protein hybrid system as a CORM-3 carrier. Firstly, the pyridyl disulfide-functional polymer (2-hydroxy ethyl acrylate) was prepared via Reversible Addition-Fragmentation chain-Transfer (RAFT) polymerisation. The obtained polymer was subsequently immobilised to protein, bovine serum albumin (BSA), via a “grafting to” approach. CORM-3 was then incorporated onto the protein–polymer conjugate BSA–P(HEA) via coordination interaction with histidine residues on BSA. A myoglobin assay demonstrated the CORM-3-functionalised polymer–protein conjugate released CO in a controlled manner in a buffer (pH 7.4) solution, providing slower release compared with CORM-3.

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Introduction

Carbon monoxide has potential as a therapeutic agent owing to its beneficial effects in mammals, including anti-inflammatory, anti-apoptotic, anti-proliferative, anti-microbial and cytoprotective effects.^1–8 However, the challenge of storing and delivering CO to the target tissue in a controlled and measurable fashion has hindered its clinical use because CO is a gaseous molecule and toxic at high concentrations. Thus, CORMs (CO releasing molecules)^4,6 have been explored as a safer and more convenient system for delivering CO. Among the early-developed and extensively studied CORMs in biological systems is CORM-3 (tricarbonylchloro(glycinato)ruthenium(II)). This compound exerts similar effects to those observed for CO inhalation in a multitude of animal models of inflammatory disease,^9–11 cardiovascular disease^12,13 as well as organ transplantation and preservation.¹⁴ Clinical applications are limited, however, owing to major problems associated with this compound, including short CO-releasing half-life, poor stability in aqueous environments and low cellular uptake efficiency. These issues can be addressed by conjugating CORM-3 to macromolecular scaffolds.

Proteins have gained increasing attention as CORM carriers owing to their unique characteristics, namely low toxicity, stability, biodegradability, non-immunogenicity and their ease of preparation.^16–19 CORM-3 can be attached to protein via a coordination interaction with histidine (His) residues on the protein.^15,20 His-15 in hen egg white lysozyme (HEWL) was demonstrated to react selectively with Ru^II(CO)₂ fragments derived from CORM-3 in aqueous solutions (Scheme 1).¹⁵ Bernardes group employed protein bovine serum albumin (BSA) as a scaffold for CO delivery both in vitro and in vivo.²¹ The CO-releasing protein-based system was prepared by the reaction of the hydrolytic composition product of CORM-3 with histidine residues exposed on the surface of BSA in aqueous solution. Nondenaturating nanoelectrospray ionisation MS (native MS) demonstrated 7 out of 16 His residues per BSA molecule reacted with CORM-3.²¹ The prepared CO-releasing material supressed the production of pro-inflammatory cytokines, including interleukin (IL)-6, IL-10, and tumour necrosis factor (TNF)-a, by cancer cells.²¹

Scheme 1 Reaction of CORM-3 with the single His–protein HEWL in aqueous aerobic solution. Adapted from ref. 15.

The CXC chemokine IL-8, which plays an important role in inflammatory response,²² was also used as a CO carrier.²³ The CO-releasing moiety cis-[Ru(CO)₂]²⁺ was reacted with selective His33 residues of IL-8 for 30 min in phosphate buffer solution (PBS) to yield IL-8-Ru(CO)₂. The metalloprotein was found to release CO spontaneously in live HeLa cells and was non-toxic. Notably, neither the introduction of ruthenium carbonyl fragment nor the CO release from IL-8-Ru(CO)₂ altered IL-8's neutrophil chemotactic activity. Therefore, protein shows promise for the safe and controlled delivery of CO into living cells or tissue.

Most protein-based drugs, however, exhibit rapid clearance and non-specific distribution of the drug, resulting in an inability to achieve sustained drug release.^18,24–26 Incorporation of protein onto polymers to form protein–polymer conjugates can provide enhanced therapeutic effectiveness over the native protein, prolonging half-life of the protein in the blood stream, enhancing protein stability, decreasing immunogenicity and increasing the hydrodynamic diameter of the protein.^27,28 Building upon the unique characteristics of protein–polymer conjugates, we aimed to develop a new CO delivery system using protein-based polymeric nanoparticles. To the best of our knowledge, no previous attempt has been made to explore polymer–protein hybrid scaffolds as platforms for CO delivery in biological systems.

In this article, we report for the first time the synthesis of protein-derived polymeric material for the delivery of CO. Firstly, pyridyl disulfide-functional polymer (2-hydroxy ethyl acrylate) (P(HEA)) was prepared via reversible addition–fragmentation chain-transfer (RAFT) polymerisation and subsequently reacted with BSA through a reversible disulfide bond. The successful conjugation of polymer to BSA was analysed via gel permeation chromatography (GPC) analysis and sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). CORM-3 was incorporated into the protein–polymer conjugate via coordination interaction to histidine residues on BSA. In comparison with free CORM-3, BSA–polymer-based CORM displayed slower CO release as observed by myoglobin assay.

Results and discussion

Preparation of pyridyl disulfide functional polymers P(HEA) and P(HEA) conjugated protein

To build a stable well-defined polymer–protein conjugate, we employed a “grafting to” approach involving a reactive group on the polymer coupling to a specific functional amino acid on the protein. Free thiols on cysteine residues^29,30 are common targets for site specific conjugations. Several thiol-reactive groups have been used for modification of free thiols on proteins, including maleimides,^31–34 vinyl sulfones,^35,36 and thiol–disulfides.^29,37–41 The pyridyl disulfide group is a particularly attractive functional group in bioconjugation procedures due to the fast formation of reversible disulfide linkages in thiol–disulfide exchange reactions. We have designed a polymer with a pyridyl dilsulfide group to conjugate with the cysteine residue on protein BSA (Cys-34) through a reversible disulfide bond. Utilising an Ellman's assay, we determined that 59 mol% of thiol residue was oxidised leaving 41 mol% of free thiol residue on BSA available for conjugation (Fig. S8, ESI†).

Extensively used in drug delivery and bioconjugation,^42,43 RAFT polymerisation is a robust method providing excellent control over molecular weight and the ability to introduce a variety of different moieties onto the polymer chain end. We chose a pyridyl disulfide functionalised RAFT agent for the polymerisation of 2-hydroxyethyl acrylate (HEA) affording a biocompatible and water soluble polymer^44–49 with a pyridyl disulfide end group (Scheme 2). The polymerisation was conducted in DMF at 70 °C using the [HEA] : [PDS-RAFT] : [AIBN] ratio of 200 : 1 : 0.1. The monomer conversion reached 90% after 2 h, which was calculated from ¹H-NMR spectroscopy based on the integral ratio of the vinyl proton peak of monomer at 5.9 ppm to the ester signal at 4.2 ppm of crude reaction mixture. The resultant homopolymer P(HEA) had a number average molecular weight (M_n) of 23 300 g mol⁻¹ and a dispersity (Đ) of 1.20 as measured by gel permeation chromatography (GPC) analysis. The P(HEA) polymer structure was confirmed by ¹H-NMR spectroscopy (Fig. 1) with characteristic signals at 4.2 and 3.8 ppm attributed to CH₂O ester and CH₂OH, respectively (as listed in Fig. 1) and pyridyl disulfide functional RAFT signals at 7.2–8.5 ppm attributed to phenyl protons on the pyridine group and the signal at 0.87–0.92 ppm corresponding to a methyl proton CH₃ in the trithiocarbonate unit (–SCH₂CH₂CH₂C̲H̲₃) (Fig. 1).

Scheme 2 Schematic diagram for preparation of CORM-3 functionalised polymer–protein hybrid BSA–P(HEA)-CORM-3.

Fig. 1 ¹H-NMR spectra of P(HEA) using PDS-RAFT as RAFT agent.

The functionalised polymer P(HEA) was subsequently reacted with BSA – the conjugation reaction was carried out in PBS (pH 8.2) for 24 h employing a large excess of polymer ([BSA] : [P(HEA)] = 1 : 30) to guarantee complete conjugation. After dialysis against deionised (DI) water (MWCO = 50 K), the sample was analysed by GPC and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The retention time of the polymer–protein conjugate P(HEA)–BSA, measured by GPC, shifted to the left (Fig. S5, ESI†) compared with the native protein BSA, confirming the successful attachment of polymer to protein. Additionally, the polymer–protein conjugate was observed at higher molecular weight (Fig. 2, lane 3 and 4) than the native BSA (Fig. 2, lane 2) as shown by SDS-PAGE. The final product comprised a mixture of BSA–P(HEA) and unreacted BSA (as observed in Fig. 2, lane 3) owing to the fact that the commercial BSA contained a mixture of oxidised and non-oxidised cysteine residues.^40,50 This was also confirmed by Ellman's assay, which showed that 41% of the Cys-34 on BSA was available for conjugation (Fig. S8, ESI†). After reaction with polymer, we did not observe free thiol, which suggests that one P(HEA) polymer was conjugated to BSA. In the presence of 2-mercaptoethanol (reducing conditions), the disulfide bond between the protein and polymer was cleaved, hence, the reduced BSA–P(HEA) (Fig. 2, lane 6) stayed at the same position compared to reduced BSA (Fig. 2, lane 5). Both aqueous GPC and SDS-PAGE indicated the successful conjugation of polymer with a pyridyl disulfide chain end to protein BSA through a reversible disulfide bond.

Fig. 2 SDS-PAGE visualised by Coomassie blue staining. Lane (1): protein marker, (2): non-reduced BSA; (3): non-reduced BSA–P(HEA); (4): non-reduced BSA–P(HEA)-CORM-3; (5): reduced BSA; (6): reduced BSA–P(HEA); (7): reduced BSA–P(HEA)-CORM-3. Note: the amount of protein in each sample is approximately 0.5 mg mL⁻¹.

Attachment of CORM-3 to polymer–protein conjugate BSA–P(HEA)

CORM-3 has been demonstrated to react with BSA via a coordination interaction with histidine (His) residues on the protein.^15,20,21,23 Therefore, the polymer–protein hybrid BSA–P(HEA) can interact with CORM-3. The CO-releasing macromolecule was simply prepared by mixing BSA–P(HEA) with 50 equivalents of CORM-3 in phosphate buffer solution (PBS, pH 7.4) for 1 h. The mixture was then dialysed against DI water to remove unreacted CORM-3. After purification, the IR spectrum of the CORM-3 conjugated polymer–protein hybrid vehicle (Fig. 3) showed an additional characteristic C=O band in the region from 1900–2100 cm⁻¹ compared with the blank polymer–protein conjugate, confirming attachment of CORM-3 to the polymer–protein conjugate BSA–P(HEA). To determine the number of His residues on BSA–P(HEA) reacted with CORM-3, the ruthenium content was measured using Inductively Coupled Plasma Optical Emission Spectrometry (ICP-OES). ICP-OES analysis showed that Ru content in BSA–P(HEA)-CORM-3 was 0.69%, which corresponds to an average of 6 out of 16 His residue on BSA being modified and is consistent with the data reported by Bernardes and co-workers.²¹ In addition, successful conjugation of CORM-3 to the polymer–protein hybrid (Fig. 2) was demonstrated by SDS-PAGE. As expected, in both non-reducing conditions and reducing conditions, the CORM-3 functionalised polymer–protein hybrid BSA–P(HEA)-CORM-3 (Fig. 2, lane 4 and lane 7) appeared at a higher molecular weight peak compared with blank BSA–P(HEA) (Fig. 2, lane 3 and lane 6). It should be noted that a reaction between CORM-3 and a protein–polymer hybrid BSA–P(HEA) that contained a mixture of modified BSA–P(HEA) and unmodified BSA would result in a mixture of modified BSA–P(HEA)-CORM-3 and BSA-CORM-3.

Fig. 3 ATR-FTIR spectra of polymer P(HEA), protein–polymer hybrid BSA–P(HEA) and CORM-3 conjugated protein–polymer hybrid BSA–P(HEA)-CORM-3.

Myoglobin assay

In order to investigate whether polymer–protein conjugate BSA–P(HEA)-CORM-3 could be used as a novel macromolecular carrier of CORM-3 in vitro, the CO-release properties of polymer–protein based-CORM-3 were monitored spectrophotometrically by measuring the conversion of deoxymyoglobin (Deoxy-Mb) to carboxymyoglobin (MbCO). As Deoxy-Mb has strong affinity for CO, it can bind CO to generate MbCO.⁵¹ The spectral changes of reduced myoglobin solution with the addition of small aliquots of CO-releasing macromolecule BSA–P(HEA)-CORM-3 were recorded for an extended period of time at room temperature. Fig. 4 showed the changes in the UV-Vis spectra during the evolution from Deoxy-Mb to MbCO for 80 min. CORM-3 rapidly decomposes in aqueous solution and spontaneously releases CO with a half-life of only a few minutes in aqueous solution.^51,52 The half-life of CORM-3 conjugated BSA was around 7.5 min (Fig. S7, ESI†), while CORM-3 conjugated polymer–protein hybrid liberated CO slowly with a half-life of approximately 30 min. In comparison with BSA-CORM-3 (Fig. S7, ESI†) the protein–polymer-based CORM displayed slower CO release, clearly demonstrating the advantage of our approach.

Fig. 4 (A) UV-Vis spectra change of a solution of reduced horse skeletal muscle myoglobin in the presence of BSA–P(HEA)-CORM-3 (5 µM) in 0.1 M phosphate buffer solution (B) CO release from BSA–P(HEA)-CORM-3 (5 µM) versus time in reduced myoglobin solution.

Conclusion

Our data describe a novel and simple approach for the delivery of CO utilising a polymer–protein material as the carrier. The CO-releasing polymer–protein material showed significant benefits over CORM-3 alone, including increased half-life of CORM-3. The half-life of CO from CORM-3 is very short (less than 1 min) while CORM-3 conjugated polymer–protein hybrid liberated CO slowly with a half-life of approximately 30 min. These features highlight the value of continued development of polymer–protein hybrid particles as macromolecular scaffolds for controlled delivery of CO.

Acknowledgements

CB thanks the Australian Research Council (ARC) for his Future Fellowship (FT120100096).

References

1. X. Ji, K. Damera, Y. Zheng, B. Yu, L. E. Otterbein and B. Wang, J. Pharm. Sci., 2016, 105, 406–416.
2. F. Zobi, Future Med. Chem., 2013, 5, 175–188.
3. D. Nguyen and C. Boyer, ACS Biomater. Sci. Eng., 2015, 1, 895–913.
4. R. Motterlini, B. E. Mann and R. Foresti, Expert Opin. Invest. Drugs, 2005, 14, 1305–1318.
5. R. Motterlini and L. E. Otterbein, Nat. Rev. Drug Discovery, 2010, 9, 728–743.
6. S. Garcia-Gallego and G. J. Bernardes, Angew. Chem., Int. Ed. Engl., 2014, 53, 9712–9721.
7. D. Nguyen, N. N. M. Adnan, S. Oliver and C. Boyer, Macromol. Rapid Commun., 2016, 37, 739–744.
8. D. Nguyen, T.-K. Nguyen, S. A. Rice and C. Boyer, Biomacromolecules, 2015, 16, 2776–2786.
9. S. Mizuguchi, J. Stephen, R. Bihari, N. Markovic, S. Suehiro, A. Capretta, R. F. Potter and G. Cepinskas, Am. J. Physiol.: Heart Circ. Physiol., 2009, 297, H920–H929.
10. K. Tsoyi, T. Y. Lee, Y. S. Lee, H. J. Kim, H. G. Seo, J. H. Lee and K. C. Chang, Mol. Pharmacol., 2009, 76, 173–182.
11. S. Lancel, S. M. Hassoun, R. Favory, B. Decoster, R. Motterlini and R. Neviere, J. Pharmacol. Exp. Ther., 2009, 329, 641–648.
12. J. E. Clark, P. Naughton, S. Shurey, C. J. Green, T. R. Johnson, B. E. Mann, R. Foresti and R. Motterlini, Circ. Res., 2003, 93, e2–e8.
13. Y. Guo, A. B. Stein, W. J. Wu, W. Tan, X. Zhu, Q. H. Li, B. Dawn, R. Motterlini and R. Bolli, Am. J. Physiol.: Heart Circ. Physiol., 2004, 286, H1649–H1653.
14. A. Bagul, S. A. Hosgood, M. Kaushik and M. L. Nicholson, Transplantation, 2008, 85, 576–581.
15. T. Santos-Silva, A. Mukhopadhyay, J. D. Seixas, G. J. L. Bernardes, C. C. Romão and M. J. Romão, J. Am. Chem. Soc., 2011, 133, 1192–1195.
16. A. O. Elzoghby, W. M. Samy and N. A. Elgindy, J. Controlled Release, 2012, 157, 168–182.
17. L. Chen, G. E. Remondetto and M. Subirade, Trends Food Sci. Technol., 2006, 17, 272–283.
18. A. O. Elzoghby, W. M. Samy and N. A. Elgindy, J. Controlled Release, 2012, 161, 38–49.
19. S. Gunasekaran, S. Ko and L. Xiao, J. Food Eng., 2007, 83, 31–40.
20. T. Santos-Silva, A. Mukhopadhyay, J. D. Seixas, G. J. L. Bernardes, C. C. Romao and M. J. Romao, Curr. Med. Chem., 2011, 18, 3361–3366.
21. M. Chaves-Ferreira, I. S. Albuquerque, D. Matak-Vinkovic, A. C. Coelho, S. M. Carvalho, L. M. Saraiva, C. C. Romão and G. J. L. Bernardes, Angew. Chem., Int. Ed., 2015, 54, 1172–1175.
22. D. J. Brat, A. C. Bellail and E. G. Van Meir, Neuro-Oncology, 2005, 7, 122–133.
23. I. S. Albuquerque, H. F. Jeremias, M. Chaves-Ferreira, D. Matak-Vinkovic, O. Boutureira, C. C. Romao and G. J. L. Bernardes, Chem. Commun., 2015, 51, 3993–3996.
24. L. R. Brown, Expert Opin. Drug Delivery, 2005, 2, 29–42.
25. V. P. Torchilin and A. N. Lukyanov, Drug Discovery Today, 2003, 8, 259–266.
26. B. J. Bruno, G. D. Miller and C. S. Lim, Ther. Delivery, 2013, 4, 1443–1467.
27. N. V. Katre, Adv. Drug Delivery Rev., 1993, 10, 91–114.
28. P. Caliceti and F. M. Veronese, Adv. Drug Delivery Rev., 2003, 55, 1261–1277.
29. I. Cobo, M. Li, B. S. Sumerlin and S. Perrier, Nat. Mater., 2015, 14, 143–159.
30. E. M. Pelegri-O'Day, E.-W. Lin and H. D. Maynard, J. Am. Chem. Soc., 2014, 136, 14323–14332.
31. Y. Jiang, M. Liang, D. Svejkar, G. Hart-Smith, H. Lu, W. Scarano and M. H. Stenzel, Chem. Commun., 2014, 50, 6394–6397.
32. G. Mantovani, F. Lecolley, L. Tao, D. M. Haddleton, J. Clerx, J. J. Cornelissen and K. Velonia, J. Am. Chem. Soc., 2005, 127, 2966–2973.
33. E. Bays, L. Tao, C. W. Chang and H. D. Maynard, Biomacromolecules, 2009, 10, 1777–1781.
34. M. Li, P. De, H. Li and B. S. Sumerlin, Polym. Chem., 2010, 1, 854–859.
35. G. N. Grover, S. N. S. Alconcel, N. M. Matsumoto and H. D. Maynard, Macromolecules, 2009, 42, 7657–7663.
36. T. Shimoboji, E. Larenas, T. Fowler, A. S. Hoffman and P. S. Stayton, Bioconjugate Chem., 2003, 14, 517–525.
37. K. L. Heredia, D. Bontempo, T. Ly, J. T. Byers, S. Halstenberg and H. D. Maynard, J. Am. Chem. Soc., 2005, 127, 16955–16960.
38. R. M. Broyer, G. N. Grover and H. D. Maynard, Chem. Commun., 2011, 47, 2212–2226.
39. L. Tao, J. Liu and T. P. Davis, Biomacromolecules, 2009, 10, 2847–2851.
40. C. Boyer, V. Bulmus, J. Liu, T. P. Davis, M. H. Stenzel and C. Barner-Kowollik, J. Am. Chem. Soc., 2007, 129, 7145–7154.
41. B. Yang, Y. Zhao, S. Wang, Y. Zhang, C. Fu, Y. Wei and L. Tao, Macromolecules, 2014, 47, 5607–5612.
42. C. Boyer, V. Bulmus, T. P. Davis, V. Ladmiral, J. Liu and S. Perrier, Chem. Rev., 2009, 109, 5402–5436.
43. A. Gregory and M. H. Stenzel, Expert Opin. Drug Delivery, 2011, 8, 237–269.
44. Y. Chan, T. Wong, F. Byrne, M. Kavallaris and V. Bulmus, Biomacromolecules, 2008, 9, 1826–1836.
45. D. Popescu, R. Hoogenboom, H. Keul and M. Moeller, Polym. Chem., 2010, 1, 878–890.
46. K. Bian and M. F. Cunningham, Macromolecules, 2005, 38, 695–701.
47. W. Steinhauer, R. Hoogenboom, H. Keul and M. Moeller, Macromolecules, 2010, 43, 7041–7047.
48. O. V. Khutoryanskaya, Z. A. Mayeva, G. A. Mun and V. V. Khutoryanskiy, Biomacromolecules, 2008, 9, 3353–3361.
49. A. Vallés Lluch, A. Campillo Fernández, G. Gallego Ferrer and M. Monleón Pradas, J. Biomed. Mater. Res., Part B, 2009, 90, 182–194.
50. J. Janatova, J. K. Fuller and M. J. Hunter, J. Biol. Chem., 1968, 243, 3612–3622.
51. R. Motterlini, J. E. Clark, R. Foresti, P. Sarathchandra, B. E. Mann and C. J. Green, Circ. Res., 2002, 90, E17–E24.
52. M. Desmard, R. Foresti, D. Morin, M. Dagouassat, A. Berdeaux, E. Denamur, S. H. Crook, B. E. Mann, D. Scapens, P. Montravers, J. Boczkowski and R. Motterlini, Antioxid. Redox Signaling, 2011, 16, 153–163.

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Footnote

† Electronic supplementary information (ESI) available: Additional characterisation, Ellman's assay and UV spectroscopy spectra. See DOI: 10.1039/c6ra21703f

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