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.
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References
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
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
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
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
Cone RA (2009) Barrier properties of mucus. Adv Drug Deliv Rev 61(2):75–85. doi:10.1016/j.addr.2008.09.008
Crater JS, Carrier RL (2010) Barrier properties of gastrointestinal mucus to nanoparticle transport. Macromol Biosci 10(12):1473–1483. doi:10.1002/mabi.201000137
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
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
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 100:1024–1032
Dawson M, Krauland E, Wirtz D, Hanes J (2004) Transport of polymeric nanoparticle gene carriers in gastric mucus. Biotechnol Prog 20:851–857
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 408:191–199
Emerich DF, Thanos CG (2007) Targeted nanoparticle-based drug delivery and diagnosis. J Drug Target 15(3):163–183. doi:10.1080/10611860701231810
Gauthier MA, Klok HA (2010) Polymer–protein conjugates: an enzymatic activity perspective. Polym Chem 1(9):1352–1373. doi:10.1039/c0py90001j
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
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 69:476–485
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
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
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
Lai SK, Wang YY, Hanes J (2009) Mucus-penetrating nanoparticles for drug and gene delivery to mucosal tissues. Adv Drug Deliv Rev 61:158–171
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
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
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
Mitchel REJ, Chaiken IM, Smith EL (1970) Complete amino acid sequence of papain—additions and corrections. J Biol Chem 245(14):3485
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 326:903–910
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
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):1930–1937
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
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
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
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):3137–3145
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 13:3657–3670
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
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
Spackman DH, Stein WH, Moore S (1960) Disulfide bonds of ribonuclease. J Biol Chem 235(3):648–659
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
Thaurer MH, Deutel B, Schlocker W, Bernkop-Schnürch A (2009) Development of nanoparticulate drug delivery systems based on thiolated poly(acrylic acid). J Microencapsul 26:187–194
Acknowledgments
This study was supported by the European Commission (EC). ALEXANDER (Mucus Permeating Nanoparticulate Drug Delivery Systems) is an Integrated Project founded within the Seventh Framework Programme of the EC (Grant Agreement Number 280761).
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Müller, C., Leithner, K., Hauptstein, S. et al. Preparation and characterization of mucus-penetrating papain/poly(acrylic acid) nanoparticles for oral drug delivery applications. J Nanopart Res 15, 1353 (2013). https://doi.org/10.1007/s11051-012-1353-z
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DOI: https://doi.org/10.1007/s11051-012-1353-z