Preparation and characterization of mucus-penetrating papain/poly(acrylic acid) nanoparticles for oral drug delivery applications

  • Christiane Müller
  • Katharina Leithner
  • Sabine Hauptstein
  • Fabian Hintzen
  • Willi Salvenmoser
  • Andreas Bernkop-Schnürch
Research Paper

Abstract

Particle diffusion through the intestinal mucosal barrier is restricted by the viscoelastic and adhesive properties of the mucus gel layer, preventing their penetration to the underlying absorptive endothelial cells. To overcome this natural barrier, we developed nanoparticles which have a remarkable ability to cleave mucoglycoprotein substructures responsible for the structural and rheological properties of mucus. After rheological screening of various mucolytic proteases, nanoparticles composed of poly(acrylic acid) and papain were prepared and characterized regarding particle size and zeta potential. Analysis of nanoparticles showed mean diameters sub-200 nm (162.8–198.5 nm) and negative zeta potentials advancing the mobility in mucus gel. Using diffusion chamber studies and the rotating diffusion tubes method, we compared the transport rates of papain modified (PAPC) and unaltered poly(acrylic acid) (PAA) particles through freshly excised intestinal porcine mucus. Results of the diffusion assays demonstrated strongly enhanced permeation behavior of PAPC particles owing to local mucus disruption by papain. Improved transport rates, reduction in mucus viscosity and the retarded release of hydrophilic macromolecular compounds make proteolytic enzyme functionalized nanoparticles of substantial interest for improved targeted drug delivery at mucosal surfaces. Although cytotoxicity tests of the nanoparticles could not be performed, safety of papain and PAA was already verified making PAPC particles a promising candidate in the pharmaceutical field of research. The focus of the present study was the development of particles which penetrate the mucus barrier to approach the underlying epithelium. Improvements of particles that penetrate the mucus followed by cell uptake in this direction are ongoing.

Keywords

Oral drug delivery Mucus barrier Mucus-penetrating particles Poly(acrylic acid) Papain 

References

  1. Bernkop-Schnürch A (2000) Chitosan and its derivatives: potential excipients for peroral peptide delivery systems. Int J Pharm 194(1):1–13. doi:10.1016/s0378-5173(99)00365-8 CrossRefGoogle Scholar
  2. Bernkop-Schnürch A, Weithaler A, Albrecht K, Greimel A (2006) Thiomers: preparation and in vitro evaluation of a mucoadhesive nanoparticulate drug delivery system. Int J Pharm 317(1):76–81. doi:10.1016/j.ijpharm.2006.02.044 CrossRefGoogle Scholar
  3. Bradford MM (1976) Rapid and sensitive method for quantitation of microgram quantities of protein utilizing principle of protein–dye binding. Anal Biochem 72(1–2):248–254. doi:10.1006/abio.1976.9999 CrossRefGoogle Scholar
  4. Carlsson N, Borde A, Wolfel S, Akerman B, Larsson A (2011) Quantification of protein concentration by the Bradford method in the presence of pharmaceutical polymers. Anal Biochem 411(1):116–121. doi:10.1016/j.ab.2010.12.026 CrossRefGoogle Scholar
  5. Cone RA (2009) Barrier properties of mucus. Adv Drug Deliv Rev 61(2):75–85. doi:10.1016/j.addr.2008.09.008 CrossRefGoogle Scholar
  6. Crater JS, Carrier RL (2010) Barrier properties of gastrointestinal mucus to nanoparticle transport. Macromol Biosci 10(12):1473–1483. doi:10.1002/mabi.201000137 CrossRefGoogle Scholar
  7. Cu Y, Saltzman WM (2009) Controlled surface modification with poly(ethylene)glycol enhances diffusion of PLGA nanoparticles in human cervical mucus. Mol Pharm 6(1):173–181. doi:10.1021/mp8001254 CrossRefGoogle Scholar
  8. da Silva CR, Oliveira MBN, Motta ES, de Almeida GS, Varanda LL, de Padula M et al (2010) Genotoxic and cytotoxic safety evaluation of papain (Carica papaya L.) using in vitro assays. J Biomed Biotechnol. doi:10.1155/2010/197898
  9. Dautzenberg H, Hartmann J, Grunewald S, Brand F (1996) Stoichiometry and structure of polyelectrolyte complex particles in diluted solutions, Berichte Der Bunsen-Gesellschaft-Physical Chemistry. Chem Phys 100:1024–1032Google Scholar
  10. Dawson M, Krauland E, Wirtz D, Hanes J (2004) Transport of polymeric nanoparticle gene carriers in gastric mucus. Biotechnol Prog 20:851–857CrossRefGoogle Scholar
  11. Dünnhaupt S, Barthelmes J, Hombach J, Sakloetsakun D, Arkhipova V, Bernkop- Schnürch A (2011) Distribution of thiolated mucoadhesive nanoparticles on intestinal mucosa. Int J Pharm 408:191–199CrossRefGoogle Scholar
  12. Emerich DF, Thanos CG (2007) Targeted nanoparticle-based drug delivery and diagnosis. J Drug Target 15(3):163–183. doi:10.1080/10611860701231810 CrossRefGoogle Scholar
  13. Gauthier MA, Klok HA (2010) Polymer–protein conjugates: an enzymatic activity perspective. Polym Chem 1(9):1352–1373. doi:10.1039/c0py90001j CrossRefGoogle Scholar
  14. Grabovac V, Guggi D, Bernkop-Schnurch A (2005) Comparison of the mucoadhesive properties of various polymers. Adv Drug Deliv Rev 57(11):1713–1723. doi:10.1016/j.addr.2005.07.006 CrossRefGoogle Scholar
  15. Hoyer H, Schlocker W, Krum K, Bernkop-Schnürch A (2008) Preparation and evaluation of microparticles from thiolated, polymers via air jet milling. Eur J Pharm Biopharm 69:476–485CrossRefGoogle Scholar
  16. Itoyama K, Tanibe H, Hayashi T, Ikada Y (1994) Spacer effects on enzymatic-activity of papain immobilized onto porous chitosan beads. Biomaterials 15(2):107–112. doi:10.1016/0142-9612(94)90258-5 CrossRefGoogle Scholar
  17. Izumi T, Hirata M, Takahashi K, Kokufuta E (1994) Complexation of papain with strong polyanions and enzymatic-activities of the resulting complexes. J Macromol Sci A31(1):39–51. doi:10.1080/10601329409349716 Google Scholar
  18. Kilara A, Shahani KM, Wagner FW (1977) Preparation and properties of immobilized papain and lipase. Biotechnol Bioeng 19(11):1703–1714. doi:10.1002/bit.260191109 CrossRefGoogle Scholar
  19. Lai SK, Wang YY, Hanes J (2009) Mucus-penetrating nanoparticles for drug and gene delivery to mucosal tissues. Adv Drug Deliv Rev 61:158–171CrossRefGoogle Scholar
  20. Li G, Raman VK, Xie WC, Gross RA (2008) Protease-catalyzed co-oligomerizations of l-leucine ethyl ester with l-glutamic acid diethyl ester: sequence and chain length distributions. Macromolecules 41(19):7003–7012. doi:10.1021/ma800946d CrossRefGoogle Scholar
  21. Majima Y, Inagaki M, Hirata K, Takeuchi K, Morishita A, Sakakura Y (1988) The effect of an orally-administered proteolytic-enzyme on the elasticity and viscosity of nasal mucus. Arch OtoRhinoLaryngol 244(6):355–359. doi:10.1007/bf00497464 CrossRefGoogle Scholar
  22. Marschütz MK, Bernkop-Schnürch A (2002) Thiolated polymers: self-crosslinking properties of thiolated 450 kDa poly(acrylic acid) and their influence on mucoadhesion. Eur J Pharm Sci 15(4):387–394. doi:10.1016/s0928-0987(02)00025-8 CrossRefGoogle Scholar
  23. Mitchel REJ, Chaiken IM, Smith EL (1970) Complete amino acid sequence of papain—additions and corrections. J Biol Chem 245(14):3485Google Scholar
  24. Nordman H, Davies JR, Herrmann A, Karlsson NG, Hansson GC, Carlstedt I (1997) Mucus glycoproteins from pig gastric mucose: identification of different mucin populations from the surface epithelium. Biochem J 326:903–910Google Scholar
  25. Norris DA, Sinko PJ (1997) Effect of size, surface charge, and hydrophobicity on the translocation of polystyrene microspheres through gastrointestinal mucin. J Appl Polym Sci 63(11):1481–1492. doi:10.1002/(sici)1097-4628(19970314)63:11 CrossRefGoogle Scholar
  26. Olmsted SS, Padgett JL, Yudin AI, Whaley KJ, Moench TR, Cone RA (2001) Diffusion of macromolecules and virus-like particles in human cervical mucus. Biophys J 81(4):1930–1937CrossRefGoogle Scholar
  27. Peppas NA, Hansen PJ, Buri PA (1984) A theory of molecular-diffusion in the intestinal mucus. Int J Pharm 20(1–2):107–118. doi:10.1016/0378-5173(84)90222-9 CrossRefGoogle Scholar
  28. Rosenthal M, Traut HF (1951) The mucolytic action of papain for cell concentration in the diagnosis of gastric cancer. Cancer 4(1):147–149. doi:10.1002/1097-0142(195101)4:1 CrossRefGoogle Scholar
  29. Sangeetha K, Abraham TE (2006) Chemical modification of papain for use in alkaline medium. J Mol Catal B 38(3–6):171–177. doi:10.1016/j.molcatb.2006.01.003 CrossRefGoogle Scholar
  30. Schlamowitz M, Peterson LU (1959) Studies on the optimum pH for the action of pepsin on native and denaturated bovine serum albumin and bovine hemoglobin. J Biol Chem 234(12):3137–3145Google Scholar
  31. Shu SJ, Sun L, Zhang XG, Wu ZM, Wang Z, Li CX (2011) Polysaccharides-based polyelectrolyte nanoparticles as protein drugs delivery system. J Nanopart Res 13:3657–3670CrossRefGoogle Scholar
  32. Sipos T, Merkel JR (1970) An effect of calcium ions on activity, heat stability, and structure of trypsin. Biochemistry 9 (14):2766–2775. doi:10.1021/bi00816a003 Google Scholar
  33. Soppimath KS, Aminabhavi TM, Kulkarni AR, Rudzinski WE (2001) Biodegradable polymeric nanoparticles as drug delivery devices. J Control Release 70(1–2):1–20. doi:10.1016/s0168-3659(00)00339-4 CrossRefGoogle Scholar
  34. Spackman DH, Stein WH, Moore S (1960) Disulfide bonds of ribonuclease. J Biol Chem 235(3):648–659Google Scholar
  35. Tang BC, Dawson M, Lai SK, Wang YY, Suk JS, Yang M, Zeitlin P, Boyle MP, Fu J, Hanes J (2009) Biodegradable polymer nanoparticles that rapidly penetrate the human mucus barrier. Proc Natl Acad Sci USA 106(46):19268–19273. doi:10.1073/pnas.0905998106 CrossRefGoogle Scholar
  36. Thaurer MH, Deutel B, Schlocker W, Bernkop-Schnürch A (2009) Development of nanoparticulate drug delivery systems based on thiolated poly(acrylic acid). J Microencapsul 26:187–194CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2012

Authors and Affiliations

  • Christiane Müller
    • 1
  • Katharina Leithner
    • 1
  • Sabine Hauptstein
    • 1
  • Fabian Hintzen
    • 1
  • Willi Salvenmoser
    • 2
  • Andreas Bernkop-Schnürch
    • 1
  1. 1.Department of Pharmaceutical Technology, Institute of PharmacyCenter for Molecular Biosciences Innsbruck, University of Innsbruck, CCB—Centrum for Chemistry und BiomedicineInnsbruckAustria
  2. 2.Department for Evolutionary Developmental Biology, Institute of Zoology and Center for Molecular BiosciencesUniversity of InnsbruckInnsbruckAustria

Personalised recommendations