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A mild route to entrap papain into cross-linked PEG microparticles via visible light-induced inverse emulsion polymerization

  • Biomaterials
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Abstract

Entrapment of enzymes into a cross-linked network is an effective way to enable their recycling. In order to obtain a satisfied recovery of enzyme activity, a mild encapsulating condition is highly required due to the delicate nature of enzymes. Herein, a facile and mild visible light-induced inverse emulsion polymerization technique was developed for in situ entrapment of enzyme. In this method, poly (ethylene glycol) diacrylate (PEGDA) was dissolved in phosphate buffer saline and used as disperse phase, while continuous phase was composed of liquid paraffin, photoinitiators (isopropylthioxanthone and ethyl 4-dimethylaminobenzoate) and emulsifier (Span 80 and Tween 80). Under the irradiation of visible light, PEGDA could be cross-linkedly polymerized and formed microparticles with diameter ranged from 0.75 to 6.5 μm. When the glutaraldehyde cross-linked papain was added into disperse phase, it could be in situ entrapped into the microparticles after the visible light-induced inverse emulsion polymerization. The immobilized papain exhibited higher activity in a wide range of temperature and pH than free papain. Moreover, the immobilized papain could maintain 60% of its initial activity even after ten cycles of usage. This simple and mild strategy to in situ entrapment of enzymes has potential application in fields such as biocatalyst, biosensor and drug delivery.

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References

  1. Li J, Ma J, Jiang Y, Jiang T, Wang Y, Chen Y, Liu S (2016) Immobilizing enzymes in regular-sized gelatin microspheres through a membrane emulsification method. J Mater Sci 51(13):6357–6369. doi:10.1007/s10853-016-9932-5

    Article  Google Scholar 

  2. Sheldon RA (2007) Enzyme immobilization: the quest for optimum performance. Adv Synth Catal 349(8–9):1289–1307

    Article  Google Scholar 

  3. Tielmann P, Kierkels H, Zonta A, Ilie A, Reetz MT (2014) Increasing the activity and enantioselectivity of lipases by sol–gel immobilization: further advancements of practical interest. Nanoscale 6(12):6220–6228

    Article  Google Scholar 

  4. Zhu X, Ma Y, Zhao C, Lin Z, Zhang L, Chen R, Yang W (2014) A mild strategy to encapsulate enzyme into hydrogel layer grafted on polymeric substrate. Langmuir 30(50):15229–15237

    Article  Google Scholar 

  5. Grotzky A, Altamura E, Adamcik J, Carrara P, Stano P, Mavelli F, Nauser T, Mezzenga R, Schlüter AD, Walde P (2013) Structure and enzymatic properties of molecular dendronized polymer-enzyme conjugates and their entrapment inside giant vesicles. Langmuir 29(34):10831–10840. doi:10.1021/la401867c

    Article  Google Scholar 

  6. Schachschal S, Adler H-J, Pich A, Wetzel S, Matura A, van Pee K-H (2011) Encapsulation of enzymes in microgels by polymerization/cross-linking in aqueous droplets. Colloid Polym Sci 289(5):693–698. doi:10.1007/s00396-011-2392-1

    Article  Google Scholar 

  7. Si Tamaru, Kiyonaka S, Hamachi I (2005) Three distinct read-out modes for enzyme activity can operate in a semi-wet supramolecular hydrogel. Chem-A Eur J 11(24):7294–7304

    Article  Google Scholar 

  8. Ren C, Zhang J, Chen M, Yang Z (2014) Self-assembling small molecules for the detection of important analytes. Chem Soc Rev 43(21):7257–7266

    Article  Google Scholar 

  9. Cantone S, Ferrario V, Corici L, Ebert C, Fattor D, Spizzo P, Gardossi L (2013) Efficient immobilisation of industrial biocatalysts: criteria and constraints for the selection of organic polymeric carriers and immobilisation methods. Chem Soc Rev 42(15):6262–6276. doi:10.1039/C3CS35464D

    Article  Google Scholar 

  10. Cosnier S (1999) Biomolecule immobilization on electrode surfaces by entrapment or attachment to electrochemically polymerized films. A review. Biosens Bioelectron 14(5):443–456

    Article  Google Scholar 

  11. Alnaief M, Alzaitoun MA, García-González CA, Smirnova I (2011) Preparation of biodegradable nanoporous microspherical aerogel based on alginate. Carbohyd Polym 84(3):1011–1018. doi:10.1016/j.carbpol.2010.12.060

    Article  Google Scholar 

  12. Kabanov AV, Vinogradov SV (2009) Nanogels as pharmaceutical carriers: finite networks of infinite capabilities. Angew Chem Int Ed 48(30):5418–5429

    Article  Google Scholar 

  13. Donini C, Robinson D, Colombo P, Giordano F, Peppas N (2002) Preparation of poly (methacrylic acid-g-poly (ethylene glycol)) nanospheres from methacrylic monomers for pharmaceutical applications. Int J Pharm 245(1):83–91

    Article  Google Scholar 

  14. Zhang X, Malhotra S, Molina M, Haag R (2015) Micro-and nanogels with labile crosslinks–from synthesis to biomedical applications. Chem Soc Rev 44(7):1948–1973

    Article  Google Scholar 

  15. Bao S, Wu D, Su T, Wu Q, Wang Q (2015) Microgels formed by enzyme-mediated polymerization in reverse micelles with tunable activity and high stability. RSC Adv 5(55):44342–44345. doi:10.1039/C5RA02162F

    Article  Google Scholar 

  16. Fouassier JP, Allonas X, Lalevee J, Visconti M (2000) Radical polymerization activity and mechanistic approach in a new three-component photoinitiating system. J Polym Sci Pol Chem 38(24):4531–4541. doi:10.1002/1099-0518(20001215)38:24<4531:AID-POLA220>3.0.CO;2-U

    Article  Google Scholar 

  17. Zhang L, Ma Y, Zhao C, Zhu X, Chen R, Yang W (2015) Synthesis of pH-responsive hydrogel thin films grafted on PCL substrates for protein delivery. J Mater Chem B 3(39):7673–7681

    Article  Google Scholar 

  18. Zhao C, Lin Z, Yin H, Ma Y, Xu F, Yang W (2014) PEG molecular net-cloth grafted on polymeric substrates and Its bio-merits. Sci Rep 4:4982. doi:10.1038/srep04982

    Article  Google Scholar 

  19. Zhang H, Wu C, Zhang Y, White CJB, Xue Y, Nie H, Zhu L (2010) Elaboration, characterization and study of a novel affinity membrane made from electrospun hybrid chitosan/nylon-6 nanofibers for papain purification. J Mater Sci 45(9):2296–2304. doi:10.1007/s10853-009-4191-3

    Article  Google Scholar 

  20. Yoo G, Bong J-H, Kim S, Jose J, Pyun J-C (2014) Microarray based on autodisplayed Ro proteins for medical diagnosis of systemic lupus erythematosus (SLE). Biosens Bioelectron 57:213–218. doi:10.1016/j.bios.2014.02.018

    Article  Google Scholar 

  21. Vasconcellos FC, Goulart GA, Beppu MM (2011) Production and characterization of chitosan microparticles containing papain for controlled release applications. Powder Technol 205(1):65–70

    Article  Google Scholar 

  22. Müller C, Perera G, König V, Bernkop-Schnürch A (2014) Development and in vivo evaluation of papain-functionalized nanoparticles. Eur J Pharm Biopharm 87(1):125–131. doi:10.1016/j.ejpb.2013.12.012

    Article  Google Scholar 

  23. Sahoo B, Sahu SK, Bhattacharya D, Dhara D, Pramanik P (2013) A novel approach for efficient immobilization and stabilization of papain on magnetic gold nanocomposites. Colloid Surface B 101:280–289

    Article  Google Scholar 

  24. Homaei A (2015) Enhanced activity and stability of papain immobilized on CNBr-activated sepharose. Int J Biol Macromol 75:373–377. doi:10.1016/j.ijbiomac.2015.01.055

    Article  Google Scholar 

  25. Mahmoud KA, Lam E, Hrapovic S, Luong JH (2013) Preparation of well-dispersed gold/magnetite nanoparticles embedded on cellulose nanocrystals for efficient immobilization of papain enzyme. ACS Appl Mater Interfaces 5(11):4978–4985

    Article  Google Scholar 

  26. Yang Y-C, Deka JR, Wu C-E, Tsai C-H, Saikia D, Kao H-M (2017) Cage like ordered carboxylic acid functionalized mesoporous silica with enlarged pores for enzyme adsorption. J Mater Sci 52(11):6322–6340. doi:10.1007/s10853-017-0864-5

    Article  Google Scholar 

  27. Miyamoto D, Watanabe J, Ishihara K (2004) Effect of water-soluble phospholipid polymers conjugated with papain on the enzymatic stability. Biomaterials 25(1):71–76. doi:10.1016/S0142-9612(03)00474-5

    Article  Google Scholar 

  28. Barbosa O, Torres R, Ortiz C, Berenguer-Murcia A, Rodrigues RC, Fernandez-Lafuente R (2013) Heterofunctional supports in enzyme immobilization: from traditional immobilization protocols to opportunities in tuning enzyme properties. Biomacromol 14(8):2433–2462

    Article  Google Scholar 

  29. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72(1):248–254. doi:10.1016/0003-2697(76)90527-3

    Article  Google Scholar 

  30. Guo Y, Wang Z, Qu W, Shao H, Jiang X (2011) Colorimetric detection of mercury, lead and copper ions simultaneously using protein-functionalized gold nanoparticles. Biosens Bioelectron 26(10):4064–4069

    Article  Google Scholar 

  31. Nitsawang S, Hatti-Kaul R, Kanasawud P (2006) Purification of papain from Carica papaya latex: aqueous two-phase extraction versus two-step salt precipitation. Enzyme Microb Tech 39(5):1103–1107

    Article  Google Scholar 

  32. Bai H, Huang Z, Yang W (2009) Visible light-induced living surface grafting polymerization for the potential biological applications. J Polym Sci Pol Chem 47(24):6852–6862

    Article  Google Scholar 

  33. Graillat C, Pichot C, Guyot A, El Aasser M (1986) Inverse emulsion polymerization of acrylamide. I. Contribution to the study of some mechanistic aspects. J Polym Sci Pol Chem 24(3):427–449

    Article  Google Scholar 

  34. Benda D, Šňupárek J, Čermák V (2001) Oxygen inhibition and the influence of pH on the inverse emulsion polymerization of the acrylic monomers. Eur Polym J 37(6):1247–1253

    Article  Google Scholar 

  35. Wang S, Chen K, Li L, Guo X (2013) Binding between proteins and cationic spherical polyelectrolyte brushes: effect of pH, ionic strength, and stoichiometry. Biomacromol 14(3):818–827. doi:10.1021/bm301865g

    Article  Google Scholar 

  36. Birner-Grünberger R, Scholze H, Faber K, Hermetter A (2004) Identification of various lipolytic enzymes in crude porcine pancreatic lipase preparations using covalent fluorescent inhibitors. Biotechnol Bioeng 85(2):147–154

    Article  Google Scholar 

  37. Zhou Y-J, Hu C-L, Wang N, Zhang W-W, Yu X-Q (2013) Purification of porcine pancreatic lipase by aqueous two-phase systems of polyethylene glycol and potassium phosphate. J Chromatogr B 926:77–82

    Article  Google Scholar 

  38. Wan X, Liu T, Hu J, Liu S (2013) Photo-degradable, protein-polyelectrolyte complex-coated, mesoporous silica nanoparticles for controlled co-release of protein and model drugs. Macromol Rapid Comm 34(4):341–347

    Article  Google Scholar 

  39. Shakya AK, Sami H, Srivastava A, Kumar A (2010) Stability of responsive polymer–protein bioconjugates. Prog Polym Sci 35(4):459–486

    Article  Google Scholar 

  40. Bhardwaj A, Lee J, Glauner K, Ganapathi S, Bhattacharyya D, Butterfield DA (1996) Biofunctional membranes: an EPR study of active site structure and stability of papain non-covalently immobilized on the surface of modified poly (ether) sulfone membranes through the avidin-biotin linkage. J Membrane Sci 119(2):241–252

    Article  Google Scholar 

  41. Fernandez-Lafuente R, Rosell C, Rodriguez V, Guisan J (1995) Strategies for enzyme stabilization by intramolecular crosslinking with bifunctional reagents. Enzyme Microb Technol 17(6):517–523

    Article  Google Scholar 

  42. Mateo C, Abian O, Fernandez-Lafuente R, Guisan JM (2000) Increase in conformational stability of enzymes immobilized on epoxy-activated supports by favoring additional multipoint covalent attachment ☆. Enzyme Microb Technol 26(7):509–515

    Article  Google Scholar 

Download references

Acknowledgements

This work was financially supported by the National Natural Science Foundation of China (Grant Nos. 51521062, 51103009, 51473015, 51273012) and the Fundamental Research Funds for the Central Universities and Beijing Natural Science Foundation (Grant No. 2162035).

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Correspondence to Changwen Zhao or Wantai Yang.

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Wang, G., Chen, D., Zhang, L. et al. A mild route to entrap papain into cross-linked PEG microparticles via visible light-induced inverse emulsion polymerization. J Mater Sci 53, 880–891 (2018). https://doi.org/10.1007/s10853-017-1484-9

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