Effect of surface hydrophobicity of therapeutic protein loaded in polyelectrolyte nanoparticles on transepithelial permeability

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Oral delivery of protein drugs is greatly limited by low hydrophobicity, an important determinant for intestinal epithelial permeation and bioavailability. Herein, surface properties of recombinant erythropoietin were investigated using the fluorescent dye bis-ANS to monitor relative hydrophobicity for correlation with permeabilities with Caco-2 cells. At various pHs, bis-ANS fluorescence intensity indicated different surface hydrophobicities of erythropoietin molecules. Erythropoietin incorporated in chitosan or chitosan-trimethylchitosan (CS-TMC) nanoparticles prepared by polyelectrolyte complexation and ionotropic gelation with tripolyphosphate also showed different surface hydrophobicities. Chitosan nanoparticles with erythropoietin provided the most hydrophobic surface, followed by free erythropoietin (in water) and that loaded into CS-TMC nanoparticles. Chitosan nanoparticles were more effective than CS-TMC nanoparticles for permeation of erythropoietin across Caco-2 cell monolayers; the lowest permeability was shown by erythropoietin itself. Thus, hydrophilic protein molecules complexed with polyelectrolytes can provide more hydrophobic surfaces that enhance transepithelial permeability. This bis-ANS method also provides valuable information for the design of polyelectrolyte nanoparticules for oral delivery of protein drugs.

1. J. K. Ryu, H. S. Kim and D. H. Nam, Biotechnol. Bioprocess Engin., 17 (2012) 900-911; http://doi.org/10.1007/s12257-012-0095-1

2. J. Wang, V. Yadav, A. L. Smart, S. Tajiri and A. W. Basit, Toward oral delivery of biopharmaceuticals: an assessment of the gastrointestinal stability of 17 peptide drugs, Mol. Pharm. 12 (2015) 966–973; http://doi.org/10.1021/mp500809f

3. O. Zupančič and A. Bernkop-Schnürch, Lipophilic peptide character – What oral barriers fear the most?, J. Control. Release 255 (2017) 242–257; http://doi.org/10.1016/j.jconrel.2017.04.038

4. K. Park, I. C. Kwan and K. Park, Oral protein delivery: current status and future prospect, React. Funct. Polym. 71 (2011) 280–287; http://doi.org/10.1016/j.reactfunctpolym.2010.10.002

5. D. Vllasaliu, R. Exposito-Harris, A. Heras, L. Casettari, M. Garnett, L. Illum and S. Stolnik, Tight junction modulation by chitosan nanoparticles: Comparison with chitosan solution, Int. J. Pharm. 400 (2010) 183–193; http://doi.org/10.1016/j.ijpharm.2010.08.020

6. G. Camenisch, J. Alsenz, H. V. Waterbeemd and G. Folkers, Estimation of permeability by passive diffusion through Caco-cell monolayers using the drugs’ lipophilicity and molecular weight, Eur. J. Pharm. Sci. 6 (1998) 317–324; http://doi.org/10.1016/S0928-0987(97)10019-7

7. B. F. Choonara, Y. E. Choonara, P. Kumar, D. Bijukumar, L. C. du Toit and V. Pillay, A review of advanced oral drug delivery technologies facilitating the protection and absorption of protein and peptide molecules, Biotechnol. Adv. 32 (2014) 1269–1282; http://doi.org/10.1016/j.biotechadv.2014.07.006

8. T. Jung, W. Kamm, A. Breitenbach, E. Kaiserling, J. X. Xiao and T. Kissel, Biodegradable nanoparticles for oral delivery of peptides: is there a role for polymers to affect mucosal uptake? Eur. J. Pharm. Biopharm. 50 (2000) 147–160; http://doi.org/10.1016/S0939-6411(00)00084-9

9. P. Ahlin Grabnar and J. Kristl, The manufacturing techniques of drug-loaded polymeric nanoparticles from preformed polymers, J. Microencaps. 28 (2011) 323–335; http://doi.org/10.3109/02652048.2011.569763

10. J. Mirtič, J. Ilaš and J. Kristl, Influence of different classes of crosslinkers on alginate polyelectrolyte nanoparticle formation, thermodynamics and characteristics, Carbohydrate polymers 181 (2018) 93–102; http://doi.org/10.1016/j.carbpol.2017.10.040

11. A. T. Florence, Nanoparticle uptake by the oral route: Fulfilling its potential? Drug Discovery Today: Technologies 2 (2005) 75–81; http://doi.org/10.1016/j.ddtec.2005.05.019

12. L. Yin, J. Ding, C. He, L. Cui, C. Tang and C. Yin, Drug permeability and mucoadhesion properties of thiolated trimethyl chitosan nanoparticles in oral insulin delivery, Biomaterials 30 (2009) 5691–5700; http://doi.org/10.1016/j.biomaterials.2009.06.055

13. Y. Li, X. Chen and N. Gu, Computational investigation of interaction between nanoparticles and membranes: Hydrophobic/hydrophilic effect, J. Phys. Chem. B. 112 (2008) 16647–16653; http://doi.org/10.1021/jp8051906

14. J. Renukuntla, A. D. Vadlapudi, A. Patel, S. H. S. Boddu and A. K. Mitra, Approaches for enhancing oral bioavailability of peptides and proteins, Int. J. Pharm. 447 (2013) 75–93; http://doi.org/10.1016/j.ijpharm.2013.02.030

15. C. Contini, M. Schneemilch, S. Gaisford and N. Quirkedoi, Nanoparticle-membrane interactions, J. Exper. Nanosci. 13 (2018) 62–81; http://doi.org/10.1080/17458080.2017.1413253

16. Y. Xiao, M. R. Wiesner, Characterization of surface hydrophobicity of engineered nanoparticles, J. Hazard. Mater, 215–216 (2012) 146–151; http://doi.org/10.1016/j.jhazmat.2012.02.043

17. J. A. Patel and S. Garde, Efficient method to characterize the context-dependent hydrophobicity of proteins, Phys. Chem. B. 118 (2014) 1564–1573; http://doi.org/10.1021/jp4081977

18. A. Hawe, M. Sutter and W. Jiskoot, Extrinsic fluorescent dyes as tools for protein characterization, Pharm. Res. 25 (2008) 1487–1499; http://doi.org/10.1007/s11095-007-9516-9

19. D. Matulis, R. Lovrien, 1-Anilino-8-naphthalene sulfonate anion-protein binding depends primarily on ion pair formation, Biophys. J. 74 (1998) 422–429; http://doi.org/10.1016/S0006-3495(98)77799-9

20. B. Press and D. Di Grandi, Permeability for intestinal absorption: Caco-2 assay and related issues, Curr. Drug Metab. 9 (2008) 893–900; http://doi.org/10.2174/138920008786485119

21. I. Hubatsch, E. G. Ragnarsson and P. Artursson, Determination of drug permeability and prediction of drug absorption in Caco-2 monolayers, Nat. Protoc. 2 (2009) 2111–2119; http://doi.org/10.1038/nprot.2007.303

22. K. Berginc, S. Žakelj, L. Levstik, D. Uršič and A. Kristl, Fluorescin transport properties across artificial lipid membranes, Caco-2 cell monolayers and rat jejunum, Eur. J. Pharm. Biopharm. 66 (2007) 281–285; http://doi.org/10.2016/j.ejpb.2006.10.023

23. M. Cegnar, B. Podobnik, S. Caserman, M. Homar and J. Kerc, EPO Compositions for Oral Administration, WO2015/032973 (A1), 12 Mar 2015.

24. M. Marušič, T. Zupančič, G. Hribar, R. Komel, G. Anderluh and S. Caserman, The Caco-2 cell culture model enables sensitive detection of enhanced protein permeability in the presence of N-decyl-ß-D-maltopyranoside, New Biotechnologies 30 (2013) 507–515; http://doi.org/10.1016/j.nbt.2013.05.008

25. C. L. Cooper, P. L. Dubin, A. B. Kayitmazer and S. Turksen, Polyelectrolyte-protein complexes, Curr. Opin. Colloid. Interface Sci. 10 (2005) 52–78, http://doi.org/10.1016/j.cocis.2005.05.007

26. P. Maurel, Relevance of dielectric constant and solvent hydrophobicity to the organic solvent effect in enzymology, J. Biol. Chem. 253 (1978) 1671–1683.

27. E. Y. Chi, S. Krishnan, T. W. Randolph and J. F. Carpenter, Physical stability of proteins in aqueous solution: mechanism and driving forces in non-native protein aggregation, Pharm. Res. 20 (2003) 1325–1336; http://doi.org/10.1023/A:1025771421906

28. A. M. M. Sadeghi, F. A. Dorkoosh, M. R. Avadi, M. Weinhold, A. Bayat, F. Delie, R. Gurny, B. Larijani, M. Rafiee-Tehrani and H. E. Junginger, Permeation enhancer effect of chitosan and chitosan derivatives: Comparison of formulations as soluble polymers and nanoparticulate systems on insulin absorption in Caco-2 cells, Eur. J. Pharm. Biopharm. 70 (2008) 270–278; http://doi.org/10.1016/j.ejpb.2008.03.004.29

29. M. A. Mohammed, J. T. M. Syeda, K. M. Wasan and E. K. Wasan, An overview of chitosan nanoparticles and its application in non-parenteral drug delivery, Pharmaceutics 9 (2017) E53; http://doi.org/10.3390/pharmaceutics9040053

30. I. Pereira de Sousa, C. Steiner, M. Schmutzler, M. D. Wilcox, G. J. Veldhuis, J. P. Pearson, C. W. Huck, W. Salvenmoser and A. Bernkop-Schnürch, Mucus permeating carriers: formulation and characterization of highly densely charged nanoparticles, Eur. J. Pharm. Biopharm. 97 (2015) 273–279; http://doi.org/10.1016/j.ejpb.2014.12.024

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