Drug Delivery and Translational Research

, Volume 7, Issue 4, pp 529–543 | Cite as

Enhancing thermal stability of a highly concentrated insulin formulation with Pluronic F-127 for long-term use in microfabricated implantable devices

  • Jason Li
  • Michael K. Chu
  • Brian Lu
  • Sako Mirzaie
  • Kuan Chen
  • Claudia R. Gordijo
  • Oliver Plettenburg
  • Adria Giacca
  • Xiao Yu WuEmail author
Original Article


Development of highly concentrated formulations of protein and peptide drugs is a major challenge due to increased susceptibility to aggregation and precipitation. Numerous drug delivery systems including implantable and wearable controlled-release devices require thermally stable formulations with high concentrations due to limited device sizes and long-term use. Herein we report a highly concentrated insulin gel formulation (up to 80 mg/mL, corresponding to 2200 IU/mL), stabilized with a non-ionic amphiphilic triblock copolymer (i.e., Pluronic F-127 (PF-127)). Chemical and physical stability of insulin was found to be improved with increasing polymer concentration, as evidenced by reduced insulin fibrillation, formation of degradation products, and preserved secondary structure as measured by HPLC and circular dichroism spectroscopy, respectively. This formulation exhibits excellent insulin stability for up to 30 days in vitro under conditions of continuous shear at 37 °C, attributable to the amphiphilic properties of the copolymer and increased formulation viscosity. The mechanism of stabilizing insulin structure by PF-127 was investigated by coarse-grained molecular dynamics (CG-MD), all-atom MD, and molecular docking simulations. The computation results revealed that PF-127 could reduce fibrillation of insulin by stabilizing the secondary structure of unfolded insulin and forming hydrophobic interaction with native insulin. The gel formulations contained in microfabricated membrane-reservoir devices released insulin at a constant rate dependent on both membrane porosity and copolymer concentration. Subcutaneous implantation of the gel formulation-containing devices into diabetic rats resulted in normal blood glucose levels for the duration of drug release. These findings suggest that the thermally stable gel formulations are suitable for long-term and implantable drug delivery applications.


Long-term thermal stability Highly concentrated insulin formulation Implantable drug delivery device Diabetes Effect of amphiphilic triblock copolymer Molecular docking Coarse-grained molecular dynamics 



This work was supported partially by the Ontario Research Fund-Research Excellence (ORF-RE) Nanomaterials grant (No. RE03-058) in partnership with Sanofi Aventis and the Equipment Grants from the Natural Sciences and Engineering Research Council (NSERC) of Canada to X.Y. Wu. The NSERC CGS scholarship to J. Li and OGS Scholarship and Ben Cohen top-up awards to both J. Li and M. Chu are also acknowledged. S. Mirzaie is supported by an Islamic Azad University, Sanandaj Branch, scholarship.

Compliance with ethical standards

Animal studies

All institutional and national guidelines for the care and use of laboratory animals were followed.

Conflict of interest

The authors declare that they have no conflict of interest.


  1. 1.
    Chu MK, Chen J, Gordijo CR, Chiang S, Ivovic A, Koulajian K, et al. In vitro and in vivo testing of glucose-responsive insulin-delivery microdevices in diabetic rats. Lab Chip. 2012;12:2533–9.CrossRefPubMedGoogle Scholar
  2. 2.
    Chu MK, Gordijo CR, Li J, Abbasi AZ, Giacca A, Plettenburg O, et al. In vivo performance and biocompatibility of a subcutaneous implant for real-time glucose-responsive insulin delivery. Diabetes Technol Ther. 2015;17:255–67.CrossRefPubMedGoogle Scholar
  3. 3.
    Gordijo CR, Koulajian K, Shuhendler AJ, Bonifacio LD, Huang HY, Chiang S, et al. Nanotechnology-enabled closed loop insulin delivery device: in vitro and in vivo evaluation of glucose-regulated insulin release for diabetes control. Adv Funct Mater. 2011;21:73–82.CrossRefGoogle Scholar
  4. 4.
    Gordijo CR, Shuhendler AJ, Wu XY. Glucose-responsive bioinorganic nanohybrid membrane for self-regulated insulin release. Adv Funct Mater. 2010;20:1404–12.CrossRefGoogle Scholar
  5. 5.
    Li J, Chu MK, Gordijo CR, Abbasi AZ, Chen K, Adissu HA, et al. Microfabricated microporous membranes reduce the host immune response and prolong the functional lifetime of a closed-loop insulin delivery implant in a type 1 diabetic rat model. Biomaterials. 2015;47:51–61.CrossRefPubMedGoogle Scholar
  6. 6.
    Yu W, Jiang G, Liu D, Li L, Chen H, Liu Y, et al. Fabrication of biodegradable composite microneedles based on calcium sulfate and gelatin for transdermal delivery of insulin. Mater Sci Eng C. 2017;71:725–34.CrossRefGoogle Scholar
  7. 7.
    Yu W, Jiang G, Liu D, Li L, Tong Z, Yao J, et al. Transdermal delivery of insulin with bioceramic composite microneedles fabricated by gelatin and hydroxyapatite. Mater Sci Eng C. 2017;73:425–8.CrossRefGoogle Scholar
  8. 8.
    Prausnitz MR. Microneedles for transdermal drug delivery. Adv Drug Deliv Rev. 2004;56:581–7.CrossRefPubMedGoogle Scholar
  9. 9.
    McBride SA, Tilger CF, Sanford SP, Tessier PM, Hirsa AH. Comparison of human and bovine insulin amyloidogenesis under uniform shear. J Phys Chem B. 2015;119:10426–33.CrossRefPubMedGoogle Scholar
  10. 10.
    Sluzky V, Tamada JA, Klibanov AM, Langer R. Kinetics of insulin aggregation in aqueous solutions upon agitation in the presence of hydrophobic surfaces. Proc Natl Acad Sci U S A. 1991;88:9377–81.CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Nielsen L, Khurana R, Coats A, Frokjaer S, Brange J, Vyas S, et al. Effect of environmental factors on the kinetics of insulin fibril formation: elucidation of the molecular mechanism. Biochemistry. 2001;40:6036–46.CrossRefPubMedGoogle Scholar
  12. 12.
    Brange J, Andersen L, Laursen ED, Meyn G, Rasmussen E. Toward understanding insulin fibrillation. J Pharm Sci. 1997;86:517–25.CrossRefPubMedGoogle Scholar
  13. 13.
    Huus K, Havelund S, Olsen HB, van de Weert M, Frokjaer S. Chemical and thermal stability of insulin: effects of zinc and ligand binding to the insulin zinc-hexamer. Pharm Res. 2006;23:2611–20.CrossRefPubMedGoogle Scholar
  14. 14.
    Huus K, Havelund S, Olsen HB, van de Weert M, Frokjaer S. Thermal dissociation and unfolding of insulin. Biochemistry. 2005;44:11171–7.CrossRefPubMedGoogle Scholar
  15. 15.
    Vinther TN, Norrman M, Ribel U, Huus K, Schlein M, Steensgaard DB, et al. Insulin analog with additional disulfide bond has increased stability and preserved activity. Protein Sci. 2013;22:296–305.CrossRefPubMedGoogle Scholar
  16. 16.
    Rajpar SF, Foulds IS, Abdullah A, Maheshwari M. Severe adverse cutaneous reaction to insulin due to cresol sensitivity. Contact Dermatitis. 2006;55:119–20.CrossRefPubMedGoogle Scholar
  17. 17.
    Zhang L, Zhu W, Song L, Wang Y, Jiang H, Xian S, et al. Effects of hydroxylpropyl-beta-cyclodextrin on in vitro insulin stability. Int J Mol Sci. 2009;10:2031–40.CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Dumortier G, Grossiord JL, Agnely F, Chaumeil JC. A review of poloxamer 407 pharmaceutical and pharmacological characteristics. Pharm Res. 2006;23:2709–28.CrossRefPubMedGoogle Scholar
  19. 19.
    Almeida H, Amaral MH, Lobao P, Sousa Lobo JM. Applications of poloxamers in ophthalmic pharmaceutical formulations: an overview. Expert Opin Drug Deliv. 2013;10:1223–37.CrossRefPubMedGoogle Scholar
  20. 20.
    Escobar-Chavez JJ, Lopez-Cervantes M, Naik A, Kalia YN, Quintanar-Guerrero D, Ganem-Quintanar A. Applications of thermo-reversible pluronic F-127 gels in pharmaceutical formulations. J Pharm Pharm Sci. 2006;9:339–58.PubMedGoogle Scholar
  21. 21.
    Taluja A, Bae YH. Role of a novel excipient poly(ethylene glycol)-b-poly(L-histidine) in retention of physical stability of insulin in aqueous solutions. Pharm Res. 2007;24:1517–26.CrossRefPubMedGoogle Scholar
  22. 22.
    Wang Y, Gao J-Q, Li F, S-H RI, Liang W-Q. Triblock copolymer Pluronic®F127 sustains insulin release and reduces initial burst of microspheres—in vitro and in vivo study. Colloid Polym Sci. 2006;285:233–8.CrossRefGoogle Scholar
  23. 23.
    Nasir F, Iqbal Z, Khan A, Khan JA, Khan A, Khuda F, et al. Development and evaluation of pluronic- and methylcellulose-based thermoreversible drug delivery system for insulin. Drug Dev Ind Pharm. 2014;40:1503–8.CrossRefPubMedGoogle Scholar
  24. 24.
    Barichello JM, Morishita M, Takayama K, Nagai T. Absorption of insulin from pluronic F-127 gels following subcutaneous administration in rats. Int J Pharm. 1999;184:189–98.CrossRefPubMedGoogle Scholar
  25. 25.
    Pillai O, Panchagnula R. Transdermal delivery of insulin from poloxamer gel: ex vivo and in vivo skin permeation studies in rat using iontophoresis and chemical enhancers. J Control Release. 2003;89:127–40.CrossRefPubMedGoogle Scholar
  26. 26.
    Barichello JM, Morishita M, Takayama K, Chiba Y, Tokiwa S, Nagai T. Enhanced rectal absorption of insulin-loaded Pluronic F-127 gels containing unsaturated fatty acids. Int J Pharm. 1999;183:125–32.CrossRefPubMedGoogle Scholar
  27. 27.
    Das N, Madan P, Lin S. Development and in vitro evaluation of insulin-loaded buccal Pluronic F-127 gels. Pharm Dev Technol. 2010;15:192–208.CrossRefPubMedGoogle Scholar
  28. 28.
    Das N, Madan P, Lin S. Statistical optimization of insulin-loaded Pluronic F-127 gels for buccal delivery of basal insulin. Pharm Dev Technol. 2012;17:363–74.CrossRefPubMedGoogle Scholar
  29. 29.
    Morishita M, Barichello JM, Takayama K, Chiba Y, Tokiwa S, Nagai T. Pluronic F-127 gels incorporating highly purified unsaturated fatty acids for buccal delivery of insulin. Int J Pharm. 2001;212:289–93.CrossRefPubMedGoogle Scholar
  30. 30.
    Bedrov D, Ayyagari C, Smith GD. Multiscale modeling of poly (ethylene oxide)-poly (propylene oxide)-poly (ethylene oxide) triblock copolymer micelles in aqueous solution. J Chem Theory Comput. 2006;2:598–606.CrossRefPubMedGoogle Scholar
  31. 31.
    Fischer J, Paschek D, Geiger A, Sadowski G. Modeling of aqueous poly (oxyethylene) solutions. 2. Mesoscale simulations. J Phys Chem B. 2008;112:13561–71.CrossRefPubMedGoogle Scholar
  32. 32.
    Hatakeyama M, Faller R. Coarse-grained simulations of ABA amphiphilic triblock copolymer solutions in thin films. Phys Chem Chem Phys. 2007;9:4662–72.CrossRefPubMedGoogle Scholar
  33. 33.
    Hezaveh S, Samanta S, De Nicola A, Milano G, Roccatano D. Understanding the interaction of block copolymers with DMPC lipid bilayer using coarse-grained molecular dynamics simulations. J Phys Chem B B. 2012;116:14333–45.CrossRefGoogle Scholar
  34. 34.
    Lee H, de Vries AH, Marrink S-J, Pastor RW. A coarse-grained model for polyethylene oxide and polyethylene glycol: conformation and hydrodynamics. J Phys Chem B. 2009;113:13186–94.CrossRefPubMedPubMedCentralGoogle Scholar
  35. 35.
    Nawaz S, Redhead M, Mantovani G, Alexander C, Bosquillon C, Carbone P. Interactions of PEO–PPO–PEO block copolymers with lipid membranes: a computational and experimental study linking membrane lysis with polymer structure. Soft Matter. 2012;8:6744–54.CrossRefGoogle Scholar
  36. 36.
    Otto DP, De Villiers MM. The experimental evaluation and molecular dynamics simulation of a heat-enhanced transdermal delivery system. AAPS PharmSciTech. 2013;14:111–20.CrossRefPubMedGoogle Scholar
  37. 37.
    Wood I, Martini M, Albano J, Cuestas M, Mathet V, Pickholz M. Coarse grained study of pluronic F127: comparison with shorter co-polymers in its interaction with lipid bilayers and self-aggregation in water. J Mol Struct. 2016;1109:106–13.CrossRefGoogle Scholar
  38. 38.
    Johnson WC. Analyzing protein circular dichroism spectra for accurate secondary structures. Proteins. 1999;35.Google Scholar
  39. 39.
    Greenfield NJ. Using circular dichroism spectra to estimate protein secondary structure. Nat Protoc. 2007;1:2876–90.CrossRefGoogle Scholar
  40. 40.
    Sreerama N, Woody RW. Estimation of protein secondary structure from circular dichroism spectra: comparison of CONTIN, SELCON, and CDSSTR methods with an expanded reference set. Anal Biochem. 2000;287:252–60.CrossRefPubMedGoogle Scholar
  41. 41.
    Marrink SJ, Risselada HJ, Yefimov S, Tieleman DP, De Vries AH. The MARTINI force field: coarse grained model for biomolecular simulations. J Phys Chem B. 2007;111:7812–24.CrossRefPubMedGoogle Scholar
  42. 42.
    Dehnavi E, Fathi-Roudsari M, Mirzaie S, Arab SS, Siadat SOR, Khajeh K. Engineering disulfide bonds in Selenomonas ruminantium β-xylosidase by experimental and computational methods. Int J Biol Macromolec. 2017;95:248–55.CrossRefGoogle Scholar
  43. 43.
    Ivanova MI, Sievers SA, Sawaya MR, Wall JS, Eisenberg D. Molecular basis for insulin fibril assembly. Proc Natl Acad Sci U S A. 2009;106:18990–5.CrossRefPubMedPubMedCentralGoogle Scholar
  44. 44.
    Berhanu WM, Masunov AE. Controlling the aggregation and rate of release in order to improve insulin formulation: molecular dynamics study of full-length insulin amyloid oligomer models. J Mol Model. 2012;18:1129–42.CrossRefPubMedGoogle Scholar
  45. 45.
    Choi JH, May BC, Wille H, Cohen FE. Molecular modeling of the misfolded insulin subunit and amyloid fibril. Biophys J. 2009;97:3187–95.CrossRefPubMedPubMedCentralGoogle Scholar
  46. 46.
    Kabsch W, Sander C. Dictionary of protein secondary structure: pattern recognition of hydrogen-bonded and geometrical features. Biopolymers. 1983;22:2577–637.CrossRefPubMedGoogle Scholar
  47. 47.
    Van Der Spoel D, Lindahl E, Hess B, Groenhof G, Mark AE, Berendsen HJ. GROMACS: fast, flexible, and free. J Comput Chem. 2005;26:1701–18.CrossRefPubMedGoogle Scholar
  48. 48.
    Parrinello M, Rahman A. Polymorphic transitions in single crystals: a new molecular dynamics method. J Appl Phys. 1981;52:7182–90.CrossRefGoogle Scholar
  49. 49.
    Froimowitz M. HyperChem: a software package for computational chemistry and molecular modeling. BioTechniques. 1993;14:1010–3.PubMedGoogle Scholar
  50. 50.
    Yang C, Lu D, Liu Z. How PEGylation enhances the stability and potency of insulin: a molecular dynamics simulation. Biochemistry. 2011;50:2585–93.CrossRefPubMedGoogle Scholar
  51. 51.
    Trott O, Olson AJ. AutoDock Vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading. J Comput Chem. 2010;31:455–61.PubMedPubMedCentralGoogle Scholar
  52. 52.
    Humphrey W, Dalke A, Schulten K. VMD: visual molecular dynamics. J Mol Graph. 1996;14:33–8.CrossRefPubMedGoogle Scholar
  53. 53.
    Scott G, Waite S, McGinley M. Separation of insulin degradation products with Jupiter Proteo. Phenomenex Inc Application Note TN-1022 2014.Google Scholar
  54. 54.
    Sabokdast M, Habibi-Rezaei M, Poursasan N, Sabouni F, Ferdousi M, Azimzadeh-Irani E, et al. Insulin glycation coupled with liposomal lipid peroxidation and microglial cell death. RSC Adv. 2015;5:33114–22.CrossRefGoogle Scholar
  55. 55.
    Gong H, He Z, Peng A, Zhang X, Cheng B, Sun Y, et al. Effects of several quinones on insulin aggregation. Sci Rep. 2014;4:5648.CrossRefPubMedPubMedCentralGoogle Scholar
  56. 56.
    Li D, Liu L, Yu H, Zhai Z, Zhang Y, Guo B, et al. A molecular simulation study of the protection of insulin bioactive structure by trehalose. J Mol Model. 2014;20:1–7.Google Scholar
  57. 57.
    Blundell T, Dodson G, Hodgkin D, Mercola D. Insulin: the structure in the crystal and its reflection in chemistry and biology by. Adv Protein Chem. 1972;26:279–402.CrossRefGoogle Scholar
  58. 58.
    Rafikova ER, Kurganov BI, Arutyunyan AM, Kust SV, Drachev VA, Dobrov EN. A mechanism of macroscopic (amorphous) aggregation of the tobacco mosaic virus coat protein. Int J Biochem Cell Biol. 2003;35:1452–60.CrossRefPubMedGoogle Scholar
  59. 59.
    Sawaya MR, Sambashivan S, Nelson R, Ivanova MI, Sievers SA, Apostol MI, et al. Atomic structures of amyloid cross-β spines reveal varied steric zippers. Nature. 2007;447:453–7.CrossRefPubMedGoogle Scholar

Copyright information

© Controlled Release Society 2017

Authors and Affiliations

  • Jason Li
    • 1
  • Michael K. Chu
    • 1
  • Brian Lu
    • 1
  • Sako Mirzaie
    • 2
  • Kuan Chen
    • 1
  • Claudia R. Gordijo
    • 1
  • Oliver Plettenburg
    • 3
    • 4
  • Adria Giacca
    • 5
  • Xiao Yu Wu
    • 1
    Email author
  1. 1.Advanced Pharmaceutics and Drug Delivery Laboratory, Leslie L. Dan Faculty of PharmacyUniversity of TorontoTorontoCanada
  2. 2.Department of BiochemistryIslamic Azad UniversitySanandajIran
  3. 3.Institute of Medicinal Chemistry, Helmholtz Zentrum MünchenDeutsches Forschungszentrum für Gesundheit und Umwelt (GmbH)NeuherbergGermany
  4. 4.Institute of Organic ChemistryLeibniz Universität HannoverHannoverGermany
  5. 5.Department of Physiology, Faculty of MedicineUniversity of TorontoTorontoCanada

Personalised recommendations