Sustained gastrointestinal activity of dendronized polymer–enzyme conjugates

  • A Corrigendum to this article was published on 19 February 2016


Methods to stabilize and retain enzyme activity in the gastrointestinal tract are investigated rarely because of the difficulty of protecting proteins from an environment that has evolved to promote their digestion. Preventing the degradation of enzymes under these conditions, however, is critical for the development of new protein-based oral therapies. Here we show that covalent conjugation to polymers can stabilize orally administered therapeutic enzymes at different locations in the gastrointestinal tract. Architecturally and functionally diverse polymers are used to protect enzymes sterically from inactivation and to promote interactions with mucin on the stomach wall. Using this approach the in vivo activity of enzymes can be sustained for several hours in the stomach and/or in the small intestine. These findings provide new insight and a firm basis for the development of new therapeutic and imaging strategies based on orally administered proteins using a simple and accessible technology.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Figure 1: Gastric stabilization and retention of exogenous enzymes by polymer modification.
Figure 2: In vitro and in vivo characterization of mucoadhesion of dendronized polymer (PG1) and PDL.
Figure 3: Stability and activity of a PEP from MX and MX–polymer conjugates in simulated GI tract conditions.
Figure 4: In vivo activity of a PEP from MX and MX–polymer conjugates.
Figure 5: Activity of a PEP from MX conjugated to a dendronized polymer (MX–PG1) in the stomach.

Change history

  • 21 January 2016

    In the version of this Article originally published, the concentration of trypsin and chymotrypsin in the Methods section 'In vivo imaging of enzyme activity' should have read "0.4 mg ml-1 in 50 mM phosphate buffer pH 7". This error does not affect the conclusions and has been corrected in the online versions of the Article.


  1. 1

    Bulow, L. & Mosbach, K. Multienzyme systems obtained by gene fusion. Trends Biotechnol. 9, 226–231 (1991).

    CAS  Article  Google Scholar 

  2. 2

    Zaks, A. & Klibanov, A. M. Enzymatic catalysis in organic media at 100 °C. Science 224, 1249–1251 (1984).

    CAS  Article  Google Scholar 

  3. 3

    Klibanov, A. M. Improving enzymes by using them in organic solvents. Nature 409, 241–246 (2001).

    CAS  Article  Google Scholar 

  4. 4

    Swartz, M. A., Hirosue, S. & Hubbell, J. A. Engineering approaches to immunotherapy. Sci. Transl. Med. 4, 148rv9 (2012).

    Article  Google Scholar 

  5. 5

    Lele, B. S., Murata, H., Matyjaszewski, K. & Russell, A. J. Synthesis of uniform protein–polymer conjugates. Biomacromolecules 6, 3380–3387 (2005).

    CAS  Article  Google Scholar 

  6. 6

    Mahmoud, E. A., Sankaranarayanan, J., Morachis, J. M., Kim, G. & Almutairi, A. Inflammation responsive logic gate nanoparticles for the delivery of proteins. Bioconj. Chem. 22, 1416–1421 (2011).

    CAS  Article  Google Scholar 

  7. 7

    Zhu, G. Z., Mallery, S. R. & Schwendeman, S. P. Stabilization of proteins encapsulated in injectable poly(lactide-co-glycolide). Nature Biotechnol. 18, 52–57 (2000).

    CAS  Article  Google Scholar 

  8. 8

    Keefe, A. J. & Jiang, S. Poly(zwitterionic)protein conjugates offer increased stability without sacrificing binding affinity or bioactivity. Nature Chem. 4, 59–63 (2012).

    CAS  Article  Google Scholar 

  9. 9

    Frokjaer, S. & Otzen, D. E. Protein drug stability: a formulation challenge. Nature Rev. Drug Discov. 4, 298–306 (2005).

    CAS  Article  Google Scholar 

  10. 10

    Harris, J. M. & Chess, R. B. Effect of PEGylation on pharmaceuticals. Nature Rev. Drug Discov. 2, 214–221 (2003).

    CAS  Article  Google Scholar 

  11. 11

    Pinier, M., Fuhrmann, G., Verdu, E. & Leroux, J-C. Prevention measures and exploratory pharmacological treatments of celiac disease. Am. J. Gastroenterol. 105, 2551–2561 (2010).

    CAS  Article  Google Scholar 

  12. 12

    Sarkissian, C. N. et al. A different approach to treatment of phenylketonuria: phenylalanine degradation with recombinant phenylalanine ammonia lyase. Proc. Natl Acad. Sci. USA 96, 2339–2344 (1999).

    CAS  Article  Google Scholar 

  13. 13

    Enattah, N. S. et al. Identification of a variant associated with adult-type hypolactasia. Nature Genet. 30, 233–237 (2002).

    CAS  Article  Google Scholar 

  14. 14

    Leeds, J. S., Oppong, K. & Sanders, D. S. The role of fecal elastase-1 in detecting exocrine pancreatic disease. Nature Rev. Gastroenterol. Hepatol. 8, 405–415 (2011).

    CAS  Article  Google Scholar 

  15. 15

    Fuhrmann, G. & Leroux, J-C. In vivo fluorescence imaging of exogenous enzyme activity in the gastrointestinal tract. Proc. Natl Acad. Sci. USA 108, 9032–9037 (2011).

    CAS  Article  Google Scholar 

  16. 16

    Shan, L. et al. Structural basis for gluten intolerance in celiac sprue. Science 297, 2275–2279 (2002).

    CAS  Article  Google Scholar 

  17. 17

    Mitea, C. et al. Efficient degradation of gluten by a prolyl endoprotease in a gastrointestinal model: implications for coeliac disease. Gut 57, 25–32 (2008).

    CAS  Article  Google Scholar 

  18. 18

    Di Sabatino, A. & Corazza, G. R. Coeliac disease. Lancet 373, 1480–1493 (2009).

    Article  Google Scholar 

  19. 19

    Jabri, B. & Sollid, L. M. Tissue-mediated control of immunopathology in coeliac disease. Nature Rev. Immunol. 9, 858–870 (2009).

    CAS  Article  Google Scholar 

  20. 20

    Tack, G. J., Verbeek, W. H. M., Schreurs, M. W. J. & Mulder, C. J. J. The spectrum of coeliac disease: epidemiology, clinical aspects and treatment. Nature Rev. Gastroenterol. Hepatol. 7, 204–213 (2010).

    CAS  Article  Google Scholar 

  21. 21

    Sollid, L. M. Coeliac disease: dissecting a complex inflammatory disorder. Nature Rev. Immunol. 2, 647–655 (2002).

    CAS  Article  Google Scholar 

  22. 22

    Husby, S. et al. European Society for Pediatric Gastroenterology, Hepatology, and Nutrition guidelines for the diagnosis of coeliac disease. J. Pediatr. Gastroenterol. Nutr. 54, 136–160 (2012).

    CAS  Article  Google Scholar 

  23. 23

    Cerf-Bensussan, N., Matysiak-Budnik, T., Cellier, C. & Heyman, M. Oral proteases: a new approach to managing coeliac disease. Gut 56, 157–160 (2007).

    CAS  Article  Google Scholar 

  24. 24

    Guo, Y. et al. Tuning polymer thickness: synthesis and scaling theory of homologous series of dendronized polymers. J. Am. Chem. Soc. 131, 11841–11854 (2009).

    CAS  Article  Google Scholar 

  25. 25

    Grotzky, A., Nauser, T., Erdogan, H., Schlüter, A. D. & Walde, P. A fluorescently-labeled dendronized polymer–enzyme conjugate carrying multiple copies of two different types of active enzymes. J. Am. Chem. Soc. 134, 11392–11395 (2012).

    CAS  Article  Google Scholar 

  26. 26

    Grotzky, A., Manaka, Y., Kojima, T. & Walde, P. Preparation of catalytically active, covalent alpha-polylysine–enzyme conjugates via UV/vis-quantifiable bis-aryl hydrazone bond formation. Biomacromolecules 12, 134–144 (2011).

    CAS  Article  Google Scholar 

  27. 27

    Shan, L., Mathews, I. I. & Khosla, C. Structural and mechanistic analysis of two prolyl endopeptidases: role of interdomain dynamics in catalysis and specificity. Proc. Natl Acad. Sci. USA 102, 3599–3604 (2005).

    CAS  Article  Google Scholar 

  28. 28

    Liu, P. & Krishnan, T. R. Alginate-pectin-poly-L-lysine particulate as a potential controlled release formulation. J. Pharm. Pharmacol. 51, 141–149 (1999).

    CAS  Article  Google Scholar 

  29. 29

    Patel, M. M. et al. Mucin/poly(acrylic acid) interactions: a spectroscopic investigation of mucoadhesion. Biomacromolecules 4, 1184–1190 (2003).

    CAS  Article  Google Scholar 

  30. 30

    Ward, F. W. & Coates, M. E. Gastrointestinal pH measurement in rats: influence of the microbial flora, diet and fasting. Lab. Anim. 21, 216–222 (1987).

    CAS  Article  Google Scholar 

  31. 31

    Cook, M. T., Tzortzis, G., Charalampopoulos, D. & Khutoryanskiy, V. V. Microencapsulation of probiotics for gastrointestinal delivery. J. Control. Release 162, 56–67 (2012).

    CAS  Article  Google Scholar 

  32. 32

    Lehr, C-M., Poelma, F. G. J., Junginger, H. E. & Tukker, J. J. An estimate of turnover time of intestinal mucus gel layer in the rat in situ loop. Int. J. Pharm. 70, 235–240 (1991).

    CAS  Article  Google Scholar 

  33. 33

    Lai, S. K., Wang, Y-Y. & Hanes, J. Mucus-penetrating nanoparticles for drug and gene delivery to mucosal tissues. Adv. Drug Deliv. Rev. 61, 158–171 (2009).

    CAS  Article  Google Scholar 

  34. 34

    Ensign, L. M., Cone, R. & Hanes, J. Oral drug delivery with polymeric nanoparticles: the gastrointestinal mucus barriers. Adv. Drug Deliv. Rev. 64, 557–570 (2012).

    CAS  Article  Google Scholar 

  35. 35

    Orts Gil, G., Łosik, M., Schlaad, H., Drechsler, M. & Hellweg, T. Properties of pH-responsive mixed aggregates of polystyrene-block-poly(L-lysine) and nonionic surfactant in solution and adsorbed at a solid surface. Langmuir 24, 12823–12828 (2008).

    Article  Google Scholar 

  36. 36

    Nilsson, F. & Johansson, H. A double isotope technique for the evaluation of drug action on gastric evacuation and small bowel propulsion studied in the rat. Gut 14, 475–477 (1973).

    CAS  Article  Google Scholar 

  37. 37

    Torjman, M. C., Joseph, J. I., Munsick, C., Morishita, M. & Grunwald, Z. Effects of isoflurane on gastrointestinal motility after brief exposure in rats. Int. J. Pharm. 294, 65–71 (2005).

    CAS  Article  Google Scholar 

  38. 38

    Green, P. H. R. & Cellier, C. Celiac disease. New Engl. J. Med. 357, 1731–1743 (2007).

    CAS  Article  Google Scholar 

  39. 39

    Turner, J. R. Intestinal mucosal barrier function in health and disease. Nature Rev. Immunol. 9, 799–809 (2009).

    CAS  Article  Google Scholar 

  40. 40

    Blau, N., van Spronsen, F. J. & Levy, H. L. Phenylketonuria. Lancet 376, 1417–1427 (2010).

    CAS  Article  Google Scholar 

  41. 41

    Sarkissian, C. N., Kang, T. S., Gamez, A., Scriver, C. R. & Stevens, R. C. Evaluation of orally administered PEGylated phenylalanine ammonia lyase in mice for the treatment of phenylketonuria. Mol. Genet. Metab. 104, 249–254 (2011).

    CAS  Article  Google Scholar 

  42. 42

    Griffiths, P. C. et al. PGSE-NMR and SANS studies of the interaction of model polymer therapeutics with mucin. Biomacromolecules 11, 120–125 (2009).

    Article  Google Scholar 

  43. 43

    Vandamme, T. F. & Brobeck, L. Poly(amidoamine) dendrimers as ophthalmic vehicles for ocular delivery of pilocarpine nitrate and tropicamide. J. Control. Release 102, 23–38 (2005).

    CAS  Article  Google Scholar 

  44. 44

    DiMagno, E. P., Go, V. L. W. & Summerskill, W. H. J. Relations between pancreatic enzyme outputs and malabsorption in severe pancreatic insufficiency. New Engl. J. Med. 288, 813–815 (1973).

    CAS  Article  Google Scholar 

  45. 45

    Domínguez–Muñoz, J. E. Chronic pancreatitis and persistent steatorrhea: what is the correct dose of enzymes? Clin. Gastroenterol. Hepatol. 9, 541–546 (2011).

    Article  Google Scholar 

  46. 46

    Fieker, A., Philpott, J. & Armand, M. Enzyme replacement therapy for pancreatic insufficiency: present and future. Clin. Exp. Gastroenterol. 4, 55–73 (2011).

    PubMed  PubMed Central  Google Scholar 

  47. 47

    Hawker, C. J. & Wooley, K. L. The convergence of synthetic organic and polymer chemistries. Science 309, 1200–1205 (2005).

    CAS  Article  Google Scholar 

  48. 48

    Matyjaszewski, K. & Tsarevsky, N. V. Nanostructured functional materials prepared by atom transfer radical polymerization. Nature Chem. 1, 276–288 (2009).

    CAS  Article  Google Scholar 

  49. 49

    Gauthier, M. A. & Klok, H-A. Polymer–protein conjugates: an enzymatic activity perspective. Polym. Chem. 1, 1352–1373 (2010).

    CAS  Article  Google Scholar 

  50. 50

    Bertrand, N. & Leroux, J-C. The journey of a drug-carrier in the body: an anatomo-physiological perspective. J. Control. Release 161, 152–163 (2012).

    CAS  Article  Google Scholar 

Download references


We thank B. Castagner, K. Fuhrmann, S. T. Proulx and J. Scholl for their technical help. Financial support from the Swiss National Science Foundation (310030_135732) and IG Zöliakie der Deutschen Schweiz is acknowledged.

Author information




G.F., M.A.G. and J-C.L. designed and conceived the study; PW and ADS participated in the design of the dendronized polymers and coupling chemistry; G.F. and A.G. prepared and characterized conjugates in vitro with the help of R.L. and S.M.; G.F. conducted and analysed all in vivo experiments; P.L., B.Z. and H.Y. synthesized and contributed compounds; G.F., M.A.G., A.G., P.W., A.D.S. and J-C.L. co-wrote the paper. All authors discussed the results and implications and commented on the manuscript at all stages.

Corresponding author

Correspondence to Jean-Christophe Leroux.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary information

Supplementary information (PDF 6053 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Fuhrmann, G., Grotzky, A., Lukić, R. et al. Sustained gastrointestinal activity of dendronized polymer–enzyme conjugates. Nature Chem 5, 582–589 (2013).

Download citation

Further reading