Skip to main content
SpringerLink
Log in

In Vitro and ex Vivo Intestinal Tissue Models to Measure Mucoadhesion of Poly (Methacrylate) and N-Trimethylated Chitosan Polymers

  • Published:
Pharmaceutical Research Aims and scope Submit manuscript

No Heading

Purpose.

The adhesion of a range of polymers based on poly(2-(dimethylamino-ethyl) methacrylate (pDMAEMA) was assessed using human mucus-secreting and non mucus-secreting intestinal cell monolayers, HT29-MTX-E12 (E12) and HT29 monolayers, as well as excised non-everted intestinal sacs from rats. Differentiation of mucoadhesion from bioadhesion was achieved by pre-treatment with the mucolytic agent, N-acetyl cysteine (NAC). Adherence of pDMAEMA polymers was compared to that obtained with the mucoadhesive, N-trimethylated chitosan (TMC).

Methods.

The quantity of adherent coumarin 343-conjugated polymers to HT29, E12, and intestinal sacs was measured by fluorescence. Confocal laser scanning microscopy (CLSM), light microscopy, and fluorescent microscopy were used to provide direct evidence. Measurements of transepithelial electrical resistance (TEER), permeability to FITC-dextran 4000 (FD-4), and the release of lactate dehydrogenase (LDH) were used to assess potential cytotoxicity of polymers.

Results.

Adherence of unquaternized and of 10%, 24%, and 32% methyl iodide-quaternized pDMAEMA polymers was measured in E12, HT29, and sacs. All pDMAEMA polymers showed significantly higher levels of adhesion to mucus (mucoadhesion) than to epithelium (bioadhesion). Colocalization of pDMAEMA with mucus was confirmed in E12 by microscopy. TMC showed equally high levels of mucoadhesion as unquaternized and 24% quaternized pDMAEMA, but displayed higher levels of bioadhesion. pDMAEMA-based polymers demonstrated lower levels of adherence to E12 and rat sacs in the presence of NAC, whereas adherence of TMC was unchanged. pDMAEMA significantly decreased the permeability of FD-4 across E12 monolayers and sacs and was less cytotoxic in E12 than in HT29. In contrast, TMC increased the permeability of FD-4 across E12 and sacs and was less cytotoxic in E12 than in HT29.

Conclusions.

Human mucus–producing E12 monolayers can be used to assess polymer mucoadhesion and give similar data to isolated rat intestinal sacs. pDMAEMA displayed similar levels of mucoadhesion and lower levels of bioadhesion than a chitosan derivative and it was not cytotoxic. pDMAEMA decreased FD-4 flux in the presence of mucus, whereas TMC increased it. The combination of mucus and methacrylate polymers appears to increase barrier function of the apical membrane.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

Abbreviations

CLSM:

confocal laser scanning microscopy

FD-4:

fluoresceinisothiocyanate-dextran (MW 4 kDa)

Mn:

average molecular weight number

Papp:

apparent permeability coefficient

PDi:

polydispersity index (molecular weight distribution)

pDMAEMA:

poly(2-(dimethylamino-ethyl) methacrylate

TEER:

transepithelial electrical resistance

TMC:

N-trimethylated chitosan

References

  1. 1. J. M. Gu, J. R. Robinson, and S. H. Leung. Binding of acrylic polymers to mucin/epithelial surfaces: structure-property relationships. Crit. Rev. Ther. Drug Carrier Syst. 5:21–67 (1988).

    Google Scholar 

  2. 2. A. Bernkop-Schnurch and G. Walker. Multifunctional matrices for oral peptide delivery. Crit. Rev. Drug Carrier Syst. 18:459–501 (2001).

    Google Scholar 

  3. 3. M. K. Chourasia and S. K. Jain. Pharmaceutical approaches to colon targeted delivery systems. J. Pharm. Pharm. Sci. 6:33–66 (2003).

    Google Scholar 

  4. 4. D. S. Jones, M. S. Lawlor, A. D. Woolfson, Rheological and mucoadhesive characterisation of polymeric systems composed of poly(methylvinylether-co-maleic anhydride) and poly(vinyl-pyrrolidone), designed as platforms for topical drug delivery. J. Pharm. Sci. 92:995–1007 (2003).

    Google Scholar 

  5. 5. J. Smith, E. Wood, and M. Dornish. Effect of chitosan on epithelial cell tight junctions. Pharm. Res. 21:43–49 (2004).

    Google Scholar 

  6. 6. L. Wu, O. Zaborina, A. Zaborin, E. B. Chang, M. Musch, C. Holbrook, J. Shapiro, J. R. Turner, G. Wu, K. Y. Lee, and J. C. Alverdy. High-molecular-weight polyethylene glycol prevents lethal sepsis due to intestinal Pseudomonas aeruginosa. Gastroenterology 126:488–498 (2004).

    Google Scholar 

  7. 7. S. Higo, K. Ori, H. Takeuchi, H. Yamamoto, T. Hino, Y. Kawashima. A novel evaluation method of gastric mucoadhesive property in vitro and the mucoadhesive mechanism of tetracy-cline-sucralfate acidic complex for the eradication of Helicobacter pylori. Pharm. Res. 21:413–419 (2004).

    Google Scholar 

  8. 8. M. Bogataj, T. Vovk, M. Kerec, A. Dimnik, I. Grabnar, and A. Mrhar. The correlation between zeta potential and mucoadhesion strength on pig vesical mucosa. Biol. Pharm. Bull. 26:743–746 (2003).

    Google Scholar 

  9. 9. C.-M. Lehr, J. A. Bouwstra, J. J. Tukker, and H. E. Junginger. Intestinal transit of bioadhesive microspheres in an in situ loop in the rat -A comparative study with copolymers and blends based on poly(acrylic acid). J. Control. Rel. 13:51–62 (1990).

    Google Scholar 

  10. 10. D. Quintanar-Guerrero, R. Villalobos-Garcia, E. Alvarez-Colin, and J. M. Cornejo-Bravo. In vitro evaluation of the bioadhesive properties of hydrophobic polybasic gels containing N,N-dimethylaminoethyl methacrylate-co-methyl methacrylate. Biomaterials 22:957–961 (2001).

    Google Scholar 

  11. 11. D. Snyman, J. H. Hamman, and A. F. Kotze. Evaluation of the mucoadhesive properties of N-trimethyl chitosan chloride. Drug Dev. Ind. Pharm. 29:61–69 (2003).

    Google Scholar 

  12. 12. S. Kockisch, G. D. Rees, S. A. Young, J. Tsibouklis, and J. D. Smart. In situ evaluation of drug-loaded microspheres on a mucosal surface under dynamic test conditions. Int. J. Pharm. 276:51–58 (2004).

    Google Scholar 

  13. 13. I. J. Hidalgo, T. J. Raub, and R. T. Borchardt. Characterisation of the human colon carcinoma cell line (Caco-2) as a model system for intestinal epithelial permeability. Gastroenterology 96:736–749 (1989).

    CAS  PubMed  Google Scholar 

  14. 14. T. Lesuffleur, A. Barbat, E. Dussaulx, and A. Zweibaum. Growth adaptation to methotrexate of HT-29 human colon carcinoma cells is associated with their ability to differentiate into columnar absorptive and mucus-secreting cells. Cancer Res. 50:6334–6343 (1990).

    Google Scholar 

  15. 15. A. Wikman. J, Karlsson, I. Carlstedt, and P. Artursson. A drug absorption model based on the mucus layer producing human intestinal goblet cell line HT29-H. Pharm. Res. 10:843–852 (1993).

    Google Scholar 

  16. 16. C. Meaney and C. O’Driscoll. Mucus as a barrier to the permeability of hydrophilic and lipophilic compounds in the absence and presence of sodium taurocholate micellar systems using cell culture models. Eur. J. Pharm. Sci. 8:167–175 (1999).

    Google Scholar 

  17. 17. E. Walter, S. Janich, B. J. Roessler, J. M. Hilfinger, and G. L. Amidon. HT29-MTX/Caco-2 cocultures as an in vitro model for the intestinal epithelium: in vitro-in vivo correlation with permeability data from rats and humans. J. Pharm. Sci. 85:1070–1076 (1996).

    Google Scholar 

  18. 18. I. Behrens, P. Stenberg, P. Artursson, and T. Kissel. Transport of lipophilic drug molecules in a new mucus-secreting cell culture model based on HT29-MTX cells. Pharm. Res. 18:1138–1145 (2001).

    Google Scholar 

  19. 19. I. Behrens, A. I. Pena, M. J. Alonso, and T. Kissel. Comparative uptake studies of bioadhesive and non-bioadhesive nanoparticles in human intestinal cell lines and rats: the effect of mucus on particle adsorption and transport. Pharm. Res. 19:1185–1193 (2002).

    Google Scholar 

  20. 20. R. R. Levine, W. F. McNary, P. J. Kornguth, and R. LeBlanc. Histological reevaluation of everted gut technique for studying intestinal absorption. Eur. J. Pharmacol. 9:211–219 (1970).

    Google Scholar 

  21. 21. L. Barthe, J. F. Woodley, S. Kenworthy, and G. Houin. An improved everted gut sac as a simple and accurate technique to measure paracellular transport across the small intestine. Eur. J. Drug Metab. Pharmacokinet. 23:313–323 (1998).

    Google Scholar 

  22. 22. A. L. Scheffner. The reduction in vitro in viscosity of mucoprotein solutions by a new mucolytic agent, N-acetyl-L-cysteine. Ann. N. Y. Acad. Sci. 106:298–310 (1963).

    Google Scholar 

  23. 23. P. van de Wetering, J. Y. Cherng, H. Talsma, D. J. Crommelin, and W. E. Hennink. 2-(Dimethylamino)ethyl methacrylate based (co)polymers as gene transfer agents. J. Control. Rel. 53:145–153 (1998).

    Google Scholar 

  24. 24. F. J. Verbaan, C. Oussoren, C. J. Snel, D. J. Crommelin, W. E. Hennink, and G. Storm. Steric stabilization of poly(2-(dimethylamino)ethyl methacrylate-based polyplexes mediates prolonged circulation and tumor targeting in mice. J. Gene Med. 6:64–75 (2004).

    Google Scholar 

  25. 25. S. B. Lee, R. R. Koepsel, S. W. Morley, K. Matyjaszewski, Y. Sun, and A. J. Russell. Permanent, nonleaching antibacterial surfaces. 1. Synthesis by atomic transfer radical polymerization. Biomacromolecules 5:877–882 (2004).

    Google Scholar 

  26. 26. A. P. Corfield, N. Myerscough, R. Longman, P. Sylvester, S. Arul, and M. Pignatelli. Mucins and mucosal protection in the gastrointestinal tract: new prospects for mucins in the pathology of gastrointestinal disease. Gut 47:589–594 (2000).

    Google Scholar 

  27. 27. D. M. Haddleton, M. C. Crossman, B. H. Dana, D. J. Duncalf, A. M. Heming, D. Kukulj, and A. J. Shooter. Atom transfer polymerization of methyl methacrylate mediated by alkylpyridyl-methanimine type ligands, copper(I) bromide, and alkyl halides in hydrocarbon solution. Macromolecules 32:2110–2119 (1999).

    Google Scholar 

  28. 28. C. Korzeniewski and D. M. Callewaert. An enzyme-release assay for natural cytotoxicity. J. Immunol. Methods 64:313–320 (1983).

    Google Scholar 

  29. 29. S. L. Gilat, A. Adronov, and J. M. J. Fréchet. Modular approach to the accelerated convergent growth of laser dye-labeled poly(aryl ether) dendrimers using a novel hypermonomer. J. Org. Chem. 64:7474–7484 (1999).

    Google Scholar 

  30. 30. A. B. Sieval, M. Thanou, A. F. Kotze, J. C. Verhoef, J. Brussee, and H. E. Junginger. Preparation and NMR characterization of highly substituted N-trimethyl chitosan chloride. Carbohydrate Polymers 36:157–165 (1998).

    Google Scholar 

  31. 31. M. Leonard, E. Creed, D. Brayden, and A. W. Baird. Evaluation of the Caco-2 monolayer as a model epithelium for iontophoretic transport. Pharm. Res. 17:1181–1188 (2000).

    Google Scholar 

  32. 32. D. Acilli, G. Menghi, G. Bonacucina, P. Di Martino, and G. F. Palmieri. Mucoadhesion dependence of pharmaceutical polymers on mucosa characteristics. Eur. J. Pharm. Sci. 22:225–234 (2004).

    Google Scholar 

  33. 33. S. E. Harding. Mucoadhesive interactions. Biochem. Soc. Trans. 31:1036–1041 (2003).

    Google Scholar 

  34. 34. H. K. Batchelor, D. Banning, P. W. Dettmar, F. C. Hampson, I. G. Jolliffe, and D. Q. Craig. An in vitro mucosal model for prediction of the bioadhesion of alginate solutions to the oesophagus. Int. J. Pharm. 238:123–132 (2002).

    Google Scholar 

  35. 35. J. Shimoda, H. Onishi, and Y. Machida. Bioadhesive characteristics of chitosan microspheres to the mucosa of rat small intestine. Drug Dev. Ind. Pharm. 27:567–576 (2001).

    Google Scholar 

  36. 36. M. P. Deacon, S. McGurk, C. J. Roberts, P. M. Williams, S. J. Tendler, M. C. Davies, S. S. Davis, and S. E. Harding. Atomic force microscopy of gastric mucin and chitosan mucoadhesive systems. Biochem. J. 348:557–563 (2000).

    Google Scholar 

  37. 37. A. Krauland and A. Bernkop-Schnurch. Thiomers: development and in vitro evaluation of peroral microparticulate peptide delivery system. Eur. J. Pharm. Biopharm. 57:181–187 (2004).

    Google Scholar 

  38. 38. A. F. Kotze, H. L. Luessen, B. J. de Leeuw, B. G. de Boer, J. C. Verhoef, and H. E. Junginger. N-trimethyl chitosan chloride as a potential absorption enhancer across mucosal surfaces: in vitro evaluation in intestinal epithelial cells (Caco-2). Pharm. Res. 14:1197–1202 (1997).

    Google Scholar 

  39. 39. A. F. Kotze, H. L. Luessen, A. G. de Boer, J. C. Verhoef, and H. E. Junginger. Chitosan for enhanced intestinal permeability: Prospects for derivatives soluble in neutral and basic environments. Eur. J. Pharm. Sci. 7:145–151 (1999).

    Google Scholar 

  40. 40. P. Calceti, S. Salmaso, G. Walker, and A. Bernkop-Schnurch. Development and in vivo evaluation of an oral insulin-PEG delivery system. Eur. J. Pharm. Sci. 22:315–323 (2004).

    Google Scholar 

  41. 41. S. E. Harding, S. S. Davis, M. P. Deacon, and I. Fiebrig. Biopolymer mucoadhesives. Biotechnol. Genet. Eng. Rev. 16:41–86 (1999).

    Google Scholar 

  42. 42. A. P. Corfield, D. Carroll, N. Myerscough, and C. S. Probert. Mucins in the gastrointestinal tract in health and disease. Front. Biosci. 6:D1321–D1357 (2001).

    Google Scholar 

  43. 43. J. H. Hamman, C. M. Schultz, and A. F. Kotze. N-trimethyl chitosan chloride: optimum degree of quaternization for drug absorption enhancement across epithelial cells. Drug Dev. Ind. Pharm. 29:161–172 (2003).

    Google Scholar 

  44. 44. C. Jonker, J. H. Hamman, and A. F. Kotze. Intestinal paracellular permeation enhancement with quaternised chitosan: in situ and in vitro evaluation. Int. J. Pharm. 238:205–213 (2002).

    Google Scholar 

  45. 45. R. K. Willits and W. M. Saltzman. Synthetic polymers alter the structure of cervical mucus. Biomaterials 22:445–452 (2001).

    Google Scholar 

  46. 46. V. M. Leitner, M. K. Marschutz, and A. Bernkop-Schnurch. Mucoadhesive and cohesive properties of poly (acrylic) acid-cysteine conjugates with regard to their molecular mass. Eur. J. Pharm. Sci. 18:89–96 (2003).

    Google Scholar 

  47. 47. S. N. E. Foster, J. P. Pearson, D. A. Hutton, A. Allen, and P. W. Dettmar. Interaction of polyacrylates with porcine pepsin and the gastric mucus barrier: a mechanism for mucosal protection. Clin. Sci. 87:719–726 (1994).

    Google Scholar 

  48. 48. M. S. Balda, J. A. Whitney, C. Flores, S. Gonzalez, M. Cereijido, and K. Matter. Functional dissociation of paracellular permeability and transepithelial electrical resistance and disruption of the apical-basolateral intramembrane diffusion barrier by expression of a mutant tight junction membrane protein. J. Cell Biol. 134:1031–1049 (1996).

    Google Scholar 

  49. 49. H. Lennernas, S. Nylander and A. L. Ungell. Jejunal permeability: a comparison between the Ussing chamber technique and single pass perfusion in humans. Pharm. Res. 14:667–671 (1997).

    Google Scholar 

  50. 50. S. Tavelin, V. Milovic, G. Ocklind, S. Olsson, and P. Artursson. A conditionally immortalized cell line for studies of intestinal drug transport. J. Pharm. Exp. Ther. 290:1212–1221 (1999).

    Google Scholar 

  51. 51. I. P. Kaur and R. Smitha. Penetration enhancers and ocular bioadhesives: two new avenues for ophthalmic drug delivery. Drug Dev. Ind. Pharm. 28:353–369 (2002).

    Google Scholar 

  52. 52. G. Di Colo, Y. Zambito, S. Burgalassi, A. Serafini, and M. F. Saettone. Effect of chitosan on in vitro release and ocular delivery of ofloxacin from erodible inserts based on poly(ethylene oxide). Int. J. Pharm. 248:115–122 (2002).

    Google Scholar 

  53. 53. J. P. Kraehenbuhl, E. Pringault, and M. R. Neutra. Review article: Intestinal epithelia and barrier functions. Aliment. Pharmacol. Ther. 11:3–9 (1997).

    Google Scholar 

  54. 54. S. Rosengren and K. E. Arfors. Polycations induce microvascular leakage of macromolecules in hamster cheek pouch. Inflammation 15:159–172 (1991).

    Google Scholar 

  55. 55. R. A. Jones, M. H. Poniris, and M. R. Wilson. pDMAEMA is internalised by endocytosis but does not physically disrupt endosomes. J. Control. Rel. 96:379–391 (2004).

    Google Scholar 

  56. 56. S. A. Cryan and C. M. O’Driscoll. Mechanistic studies on nonviral gene delivery to the intestine using in vitro differentiated cell culture models and an in vivo rat intestinal loop. Pharm. Res. 20:569–575 (2003).

    Google Scholar 

  57. 57. Y. Okawa, M. Kobayashi, S. Suzuki, and M. Suzuki. Comparative study of protective effects of chitin, chitosan, and N-acetyl chitohexaose against Pseudomonas aeruginosa and Listeria monocytogenes infections in mice. Biol. Pharm. Bull. 26:902–904 (2003).

    Google Scholar 

  58. 58. X. Wang, R. Andersson, V. Soltesz, W. Guo, and S. Bengmark. Water-soluble ethylhydroxyethyl cellulose prevents bacterial translocation induced by major liver resection in the rat. Ann. Surg. 217:155–167 (1993).

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Faculty of Veterinary Medicine, University College, Dublin, Ireland

    Simon Keely, Carolyn Wilson, Steve Carrington & David J. Brayden

  2. Conway Institute of Biomedical and Biomolecular Research, University College, Dublin, Ireland

    Simon Keely & David J. Brayden

  3. Department of Chemistry, University of Warwick, Coventry, UK

    Atvinder Rullay, Adrian Carmichael & David M. Haddleton

  4. Mucin Research Group, Clinical Science at South Bristol, Royal Infirmary, Bristol, UK

    Anthony Corfield

  5. Warwick Effect Polymers, Vanguard Centre, Science Park, Coventry, UK

    Adrian Carmichael

Authors
  1. Simon Keely
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Atvinder Rullay
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Carolyn Wilson
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Adrian Carmichael
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Steve Carrington
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Anthony Corfield
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. David M. Haddleton
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. David J. Brayden
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to David J. Brayden.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Keely, S., Rullay, A., Wilson, C. et al. In Vitro and ex Vivo Intestinal Tissue Models to Measure Mucoadhesion of Poly (Methacrylate) and N-Trimethylated Chitosan Polymers. Pharm Res 22, 38–49 (2005). https://doi.org/10.1007/s11095-004-9007-1

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11095-004-9007-1

Key words:

Search

Navigation