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Polyvalent dendrimer glucosamine conjugates prevent scar tissue formation

Abstract

Dendrimers are hyperbranched macromolecules that can be chemically synthesized to have precise structural characteristics. We used anionic, polyamidoamine, generation 3.5 dendrimers to make novel water-soluble conjugates of D(+)-glucosamine and D(+)-glucosamine 6-sulfate with immuno-modulatory and antiangiogenic properties respectively. Dendrimer glucosamine inhibited Toll-like receptor 4–mediated lipopolysaccharide induced synthesis of pro-inflammatory chemokines (MIP-1α, MIP-1β, IL-8) and cytokines (TNF-α, IL-1β, IL-6) from human dendritic cells and macrophages but allowed upregulation of the costimulatory molecules CD25, CD80, CD83 and CD86. Dendrimer glucosamine 6-sulfate blocked fibroblast growth factor-2 mediated endothelial cell proliferation and neoangiogenesis in human Matrigel and placental angiogenesis assays. When dendrimer glucosamine and dendrimer glucosamine 6-sulfate were used together in a validated and clinically relevant rabbit model of scar tissue formation after glaucoma filtration surgery, they increased the long-term success of the surgery from 30% to 80% (P = 0.029). We conclude that synthetically engineered macromolecules such as the dendrimers described here can be tailored to have defined immuno-modulatory and antiangiogenic properties, and they can be used synergistically to prevent scar tissue formation.

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Figure 1: Polyvalent dendrimer glucosamine conjugates.
Figure 2: Inhibitory effect of dendrimer glucosamine on LPS-mediated chemokine and cytokine release (af) Dendrimer glucosamine-inhibited pro-inflammatory chemokine (MIP-1α, MIP-1β, IL-8) (ac) and cytokine (TNF-α, IL-1β, IL-6) (df) release from human PBMCs when added 30 min before, or 2 h or 4 h after LPS.
Figure 3: Cell type specific effect of dendrimer glucosamine.
Figure 4: Effect of dendrimer glucosamine on dendritic cell–mediated functions.
Figure 5: The anti-angiogenic activity of dendrimer glucosamine 6-sulfate.
Figure 6: Histological cross-sections of rabbit eyes at day 30.

References

  1. 1

    Tomalia, D.A., Naylor, A.M. & Goddard, W.A. III. Starburst dendrimers: molecular level control of size, shape, surface chemistry, topology and flexibility from atoms to macroscopic matter. Angew. Chem. Int. Edit. 29, 138–175 (1990).

    Article  Google Scholar 

  2. 2

    Esfand, R. & Tomalia, D.A. Laboratory synthesis of poly(amidoamine) (PAMAM) dendrimers. in Dendrimer and Other Dendritic Polymers (eds. Frechet, J.M.J. & Tomalia, D.A.) 587–604 (Wiley, New York, 2001).

    Google Scholar 

  3. 3

    Malik, N., Evagorou, E.G. & Duncan, R. Dendrimer-platinate: a novel approach to cancer chemotherapy. Anticancer Drugs 10, 767–776 (1999).

    CAS  Article  Google Scholar 

  4. 4

    Zanini, D. & Roy, R. Practical synthesis of starburst PAMAM α-thiosialodendrimers for probing multivalent carbohydrate-lectin binding properties. J. Org. Chem. 63, 3486–3491 (1998).

    CAS  Article  Google Scholar 

  5. 5

    Cloninger, M.J. Biological applications of dendrimers. Curr. Opin. Chem. Biol. 6, 742–748 (2002).

    CAS  Article  Google Scholar 

  6. 6

    Thornton, M., Barkley, L. & Shaunak, S. The anti-Kaposi's sarcoma and anti-angiogenic activity of sulphated dextrins. Antimicrob. Agents Chemotherapy 43, 2528–2533 (1999).

    CAS  Article  Google Scholar 

  7. 7

    Duncan, R. The dawning of polymer therapeutics. Nat. Rev. Drug Discov. 2, 347–360 (2003).

    CAS  Article  Google Scholar 

  8. 8

    Roy, R. Recent developments in the rational design of multivalent glycoconjugates. Top. Curr. Chem. 187, 24–274 (1997).

    Google Scholar 

  9. 9

    Mammen, M., Choi, S.-K. & Whitesides, G.M. Polyvalent interactions in biological systems: implications for design and use of multivalent ligands and inhibitors. Angew. Chem. Int. Edit. 37, 2754–2794 (1998).

    Article  Google Scholar 

  10. 10

    Roberts, J.C., Bhalgat, M.K. & Zera, R.T. Preliminary biological evaluation of polyamidoamine (PAMAM) starburst dendrimers. J. Biomed. Mater. Res. 30, 53–65 (1996).

    CAS  Article  Google Scholar 

  11. 11

    Jevprasesphant, R., Penny, J., Attwood, D., McKeown, N.B. & D'Emmanuele, A. Engineering of dendrimer surfaces to enhance transepithelial transport and reduce cytotoxicity. Pharm. Res. 20, 1543–1550 (2003).

    CAS  Article  Google Scholar 

  12. 12

    Malik, N. et al. Dendrimers: relationship between structure and biocompatibility in vitro, and preliminary studies on the biodistribution of 125I-labelled polyamidoamine dendrimers in vivo. J. Controlled Release 65, 133–148 (2002).

    Article  Google Scholar 

  13. 13

    Koyanagi, S., Tanigawa, N., Nakagawa, H., Soeda, S. & Shimeno, H. Oversulfation of fucoidan enhances its anti-angiogenic and anti-tumor activities. Biochem. Pharmacol. 65, 173–179 (2003).

    CAS  Article  Google Scholar 

  14. 14

    Brandtzaeg, P. et al. Net inflammatory capacity of human septic shock plasma evaluated by a monocyte based target cell assay: identification of interleukin-10 as a major functional deactivator of human monocytes. J. Exp. Med. 184, 51–60 (1996).

    CAS  Article  Google Scholar 

  15. 15

    Wang, Z.M., Liu, C. & Dziarski, R. Chemokines are the main pro-inflammatory mediators in human monocytes activated by Staphylococcus aureus, peptidoglycan, and endotoxin. J. Biol. Chem. 275, 20260–20267 (2000).

    CAS  Article  Google Scholar 

  16. 16

    Le Naour, F. et al. Profiling changes in gene expression during differentiation and maturation of monocyte derived dendritic cells using both oligonucleotide microarrays and proteomics. J. Biol. Chem. 276, 17920–17931 (2001).

    CAS  Article  Google Scholar 

  17. 17

    Mead, A.L., Wong, T.T.L., Cordeiro, M.F., Anderson, I.K. & Khaw, P.T. Evaluation of anti-TGF–β2 antibody as a new postoperative anti-scaring agent in glaucoma surgery. Invest. Ophthalmol. Vis. Sci. 44, 3394–3401 (2003).

    Article  Google Scholar 

  18. 18

    Khaw, P.T., Doyle, J.W., Sherwood, M.B., Smith, M.F. & McGorray, S. Effects of intra-operative 5-fluorouracil or mitomycin C on glaucoma filtration surgery in the rabbit. Ophthalmology 100, 367–372 (1993).

    CAS  Article  Google Scholar 

  19. 19

    Siriwardena, D. et al. Human anti-TGF-β2 monoclonal antibody—a new modulator of wound healing in trabeculectomy. Ophthalmology 109, 427–431 (2002).

    Article  Google Scholar 

  20. 20

    Spinks, R.L., Baker, S.N., Jackson, A., Khaw, P.T. & Lemon, R.N. The problem of dural scaring: a solution using 5-fluorouracil. J. Neurophysiol. 90, 1324–1332 (2003).

    CAS  Article  Google Scholar 

  21. 21

    The AGIS investigators. The advanced glaucoma intervention study (AGIS): 7. The relationship between control of intraocular pressure and visual field deterioration. Am. J. Ophthalmol. 130, 429–440 (2000).

  22. 22

    Chang, L., Crowston, J.G., Cordeiro, M.F., Akbar, A.N. & Khaw, P.T. The role of the immune system in conjunctival wound healing after glaucoma surgery. Surv. Ophthalmol. 45, 49–68 (2000).

    CAS  Article  Google Scholar 

  23. 23

    Wells, A.P., Cordeiro, M.F., Bunce, C. & Khaw, P.T. Cystic bleb formation and related complications in limbus versus fornix based conjunctival flaps in paediatric and young adult trabeculectomy with mitomycin-C. Ophthalmology 110, 2192–2197 (2003).

    Article  Google Scholar 

  24. 24

    Flo, F.H. et al. Involvement of TLR2 and TLR4 in cell activation by mannuronic acid polymers. J. Biol. Chem. 277, 35489–35495 (2002).

    CAS  Article  Google Scholar 

  25. 25

    Garcia-Ramallo, E. et al. Resident cell chemokine expression serves as the major mechanism for leucocyte recruitment during local inflammation. J. Immunol. 169, 6467–6473 (2002).

    CAS  Article  Google Scholar 

  26. 26

    Sweeney, E.A., Lortat-Jacob, H., Priestley, G.V., Nakamoto, B. & Papayannopoulou, T. Sulfated polysaccharides increase plasma levels of SDF-1 in monkeys and mice: involvement in mobilization of stem/progenitor cells. Blood 99, 44–51 (2002).

    CAS  Article  Google Scholar 

  27. 27

    Termeer, C. et al. Oligosaccharides of hyaluronan activate dendritic cells via toll-like receptor 4. J. Exp. Med. 195, 99–111 (2002).

    CAS  Article  Google Scholar 

  28. 28

    Lehto, M. & Jarvinen, M. Collagen and glycosaminoglycan synthesis of injured gastrocnemius muscle in rat. Eur. Surg. Res. 17, 179–185 (1985).

    CAS  Article  Google Scholar 

  29. 29

    Fukasawa, M., Bryant, S.M., Orita, H., Campeau, J.D. & DiZerega, G.S. Modulation of proline and glucosamine incorporation into tissue repair cells by peritoneal macrophages. J. Surg. Res. 46, 166–171 (1989).

    CAS  Article  Google Scholar 

  30. 30

    Schmidt, R.J., Spyratou, O. & Turner, T.D. Biocompatibility of wound management products: the effect of various monosaccharides on L929 and 2002 fibroblast cells in culture. J. Pharm. Pharmacol. 41, 781–784 (1989).

    CAS  Article  Google Scholar 

  31. 31

    Setnikar, I., Giachetti, C. & Zanolo, G. Absorption, distribution and excretion of radioactivity after a single intravenous or oral administration of [14C]glucosamine to the rat. Pharmatherapeutica 3, 538–550 (1984).

    CAS  PubMed  Google Scholar 

  32. 32

    Aghazadeh-Habashi, A., Sattari, S., Pasutto, F.M. & Jamali, F. Single dose pharmacokinetics and bioavailability of glucosamine in the rat. J. Pharm. Pharm. Sci. 5, 181–184 (2002).

    CAS  PubMed  Google Scholar 

  33. 33

    Setnikar, I. & Rovati, L.C. Absorption, distribution, metabolism and excretion of glucosamine sulfate. Arzneimitteforschung 51, 699–725 (2001).

    CAS  Google Scholar 

  34. 34

    Hoshino, K. et al. TLR4 deficient mice are hyporesponsive to LPS: evidence for TLR4 as the LPS gene product. J. Immunol. 162, 3749–3752 (1999).

    CAS  PubMed  PubMed Central  Google Scholar 

  35. 35

    Takeuchi, O. et al. Differential roles of TLR2 and TLR4 in recognition of gram-negative and gram-positive bacterial cell wall components. Immunity 11, 443–451 (1999).

    CAS  Article  Google Scholar 

  36. 36

    Gordon, S. Pattern recognition receptors: doubling up for the innate immune response. Cell 111, 927–930 (2002).

    CAS  Article  Google Scholar 

  37. 37

    Paterson, H.M. et al. Injury primes the innate immune system for enhanced toll-like receptor reactivity. J. Immunol. 171, 1473–1483 (2003).

    CAS  Article  Google Scholar 

  38. 38

    Gillitzer, R. & Goebeler, M. Chemokines in cutaneous wound healing. J. Leucocyte Biol. 69, 513–521 (2001).

    CAS  Google Scholar 

  39. 39

    Smiley, S.T., King, J.A. & Hancock, W.W. Fibrinogen stimulates macrophage chemokine secretion through TLR4. J. Immunol. 167, 2887–2894 (2001).

    CAS  Article  Google Scholar 

  40. 40

    Tumpey, T.M. et al. Absence of MIP-1α prevents the development of blinding herpes stromal keratitis. J. Virol. 72, 3705–3710 (1998).

    CAS  PubMed  PubMed Central  Google Scholar 

  41. 41

    Pye, D.A., Vives, R.R., Turnbull, J.E., Hyde, P. & Gallagher, J.T. Heparan sulfate oligosaccharides require 6-O-sulfation for promotion of basic fibroblast growth factor mitogenic activity. J. Biol. Chem. 273, 22936–22942 (1998).

    CAS  Article  Google Scholar 

  42. 42

    Lundin, L. et al. Selectively desulfated heparin inhibits fibroblast growth factor-induced mitogenicity and angiogenesis. J. Biol. Chem. 275, 24653–24660 (2000).

    CAS  Article  Google Scholar 

  43. 43

    Schlessinger, J. et al. Crystal structure of a ternary FGF-FGFR-heparin complex reveals a dual role for heparin in FGFR binding and dimerization. Mol. Cell 6, 743–750 (2000).

    CAS  Article  Google Scholar 

  44. 44

    Li, A., Dubey, S., Varney, M.L., Dave, B.J. & Singh, R.K. IL-8 directly enhanced endothelial cell survival, proliferation, and matrix metalloproteinases production and regulated angiogenesis. J. Immunol. 170, 3369–3376 (2003).

    CAS  Article  Google Scholar 

  45. 45

    Girard, J.P. & Springer, T.A. High endothelial venules: specialised endothelium for lymphocyte migration. Immunol. Today 16, 449–457 (1995).

    CAS  Article  Google Scholar 

  46. 46

    Choy, E.H. & Panayi, G.S. Cytokine pathways and joint inflammation in rheumatoid arthritis. N. Engl. J. Med. 344, 907–916 (2001).

    CAS  Article  Google Scholar 

  47. 47

    Johnson, G.B., Brunn, G.J. & Platt, J.L. An endogenous pathway to systemic inflammatory response syndrome (SIRS)-like reactions through Toll-like receptor 4. J. Immunol. 172, 20–24 (2004).

    CAS  Article  Google Scholar 

  48. 48

    Sgouras, D. & Duncan, R. Methods for the evaluation of biocompatibility of soluble synthetic polymers which have potential for biomedical use. 1. Use of tetrazolium-based colorimetric assay as a preliminary screen for evaluation of in vitro cytotoxicity. J. Mater. Sci. Mater. Med. 1, 61–68 (1990).

    CAS  Article  Google Scholar 

  49. 49

    Kubota, Y., Kleinman, H.K., Martin, G.R. & Lawley, T.J. Role of laminin and basement membrane in the morphological differentiation of human endothelial cells into capillary-like structures. J. Cell Biol. 107, 1589–1598 (1988).

    CAS  Article  Google Scholar 

  50. 50

    Cordeiro, M.F., Gay, J.A., & Khaw, P.T. Human anti-TGF-β antibody: a new glaucoma anti-scarring agent. Invest. Ophthalmol. Vis. Sci. 40, 2225–2234 (1999).

    CAS  PubMed  Google Scholar 

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Acknowledgements

This study was supported by grants to S.S. from the National Institutes of Health (1-R21-A144694-01), The Wellcome Trust (068309) and The Wolfson Foundation (PR/013217). P.K. is supported, in part, by Moorfields Trustees, The Michael & Ilse Katz Foundation and an Alcon Research Institute Award.

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Correspondence to Sunil Shaunak.

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S.S., E.G. and R.D. are inventors on a patent in which the US National Institutes of Health have an interest. This patent has been assigned to a new university spinout company called Polytherics in which The Wellcome Trust, Imperial College London, University College London, School of Pharmacy London, S.B. and S.S. hold equity.

Supplementary information

Supplementary Notes

Detailed conjugation and characterisation procedures (PDF 105 kb)

Supplementary Table 1

Matrigel assay (PDF 84 kb)

Supplementary Fig. 1

Glaucoma filtration surgery (PDF 140 kb)

Supplementary Fig. 2

Multigene chemokine plasmid for RT-PCR (PDF 297 kb)

Supplementary Methods

Real-time mRNA PCR for human chemokines and cytokines (PDF 100 kb)

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Shaunak, S., Thomas, S., Gianasi, E. et al. Polyvalent dendrimer glucosamine conjugates prevent scar tissue formation. Nat Biotechnol 22, 977–984 (2004). https://doi.org/10.1038/nbt995

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