Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics
Interaction of insulin with a triblock copolymer of PEG-(fumaric-sebacic acids)-PEG: Thermodynamic and spectroscopic studies
Introduction
Amphipathic polymeric carriers have been successfully used in delivery of proteins and peptides and to prolong their therapeutic effect [1]. Most of the protein delivery systems based on the synthetic polymers involve the use of organic solvents or heat that can cause protein denaturation and loss of bioactivity. Although most of the problems in loading proteins into their carriers can be overcome by the use of polymeric hydrogels, they are yet far from being commercially viable candidates. Besides, non-biodegradable polymers suffer from serious drawbacks like toxicity, accumulation in the body, and the need for surgical removal after their implantation [2], [3].
In this study, insulin was used as a model protein because it is one of the most used peptide drugs and it has become the standard treatment for diabetes [4]. Diabetes mellitus is a serious pathological ailment responsible for major health care problems all around the world costing billions of Dollars annually [2]. Currently, insulin is mostly administered by frequent daily subcutaneous injections. Therefore, there has been a great deal of effort, with some success, in developing controlled insulin delivery systems based on oral or injectable formulations [2], [4], [5].
Numerous strategies have been devised to prepare insulin carriers based on different polymer or copolymer compositions, including pH-responsive hydrogels made of poly(methacrylic acid-co-2-methacryloxyethyl glucoside) P(MAA-co-MEG) copolymers [4], as well as those made of graft copolymers of PMAA and polyethylene glycol (PEG) P(MAA-g-EG) [4], [6] for oral delivery; injectable biodegradable thermal gels made of poly(lactide-co-glycolide) and PEG (PLGA-PEG-PLGA) triblock copolymers (ReGel) [2], [5], [7]; triblock copolymers of polyethylene oxide and polypropylene oxide (PEO-PPO-PEO) [8]; thermo-sensitive biodegradable hydrogels made of di-acryloyled Pluronic F127-oligo (ε-caprolactone) (F127-oligoCL-DA) [9], in which F127 is a triblock copolymer of PEO-PPO-PEO; and biodegradable microspheres of a blend of poly(fumaric anhydride) and PLGA, 50:50 (poly FA: PLGA), as potential oral drug delivery systems [10]. Other examples have been reported in detail in [1], [3], [11], [12], [13], [14]. The main focus of research on these polymeric carriers has been to address either the release profile of insulin from these carriers (in vitro or in vivo) [1], [2], [3], [4], [5], [7], [9], [10], [12], [13] or their possible cytotoxic effects [11]. However, there have been few studies on possible consequent alterations in insulin structure upon its incorporation in such polymeric vehicles [5]. Since insulin and amphiphilic copolymers both possess hydrophobic and hydrophilic segments, it is expected that they are mutually compatible and may be advantageous to the efficacy of the resultant drug formulations.
To assess the possible consequences of such interactions, bovine and human insulins, which differ only in three of their amino acids [15], were each incorporated in the same biodegradable polymeric formulation and their structural and thermodynamic properties compared. The polymer chosen was a biodegradable triblock copolymer made of PEG and poly (fumaric acid-co-sebacoyl chloride) (PEG-co-P(FA/SC)-co-PEG). The biophysical techniques employed were: isothermal titration calorimetry (ITC), fluorescence spectroscopy, and circular dichroism (CD). ITC was used to study the binding thermodynamics. Fluorescence spectroscopy was applied to detect the tertiary structural changes of insulin. CD was also used to study the changes in the secondary structure and those of thermal stability of the protein.
Section snippets
Materials
Bovine insulin (from bovine pancreas, 28.7 U/mg) was purchased from Sigma (St. Louis, MO). Human medicinal insulin (regular) was obtained from the local representative of Novo Ind. (Copenhagen, Denmark) in Tehran. Polyethylene glycol-co-poly (fumaric acid-co-sebacoyl chloride)-co-polyethylene glycol (PEG-co-P(FA/SC)-co-PEG) triblock copolymer (MW of ca. 9500) was synthesized in our laboratory according to procedures previously reported [16]. All other chemicals were analytical grade obtained
Thermodynamic analysis using ITC data
Fig. 1 shows the ITC curves of the binding of the copolymer to bovine (A) and human (B) insulins at 25 °C. The raw ITC data are shown on the top, while the bottom of each figure (A) and (B) shows a plot of the heat flow per mole of the titrant versus the molar ratio of the titrant to protein for each injection, after subtraction of the background titration. It is observed that the binding of the copolymer to protein in both cases is exothermic. The best fit for the integrated heat is obtained
Discussion
This study shows that the combination of fluorescence and CD spectroscopies and ITC are valuable tools in unraveling the minute differences between the interaction of bovine and human insulins with the triblock copolymer under identical conditions.
Conclusions
The far-UV CD as well as fluorescence transitions in both bovine and human insulins, upon addition of the triblock copolymer, resemble each other. Results also indicate the reduced accessibility of tyrosines upon interaction with the copolymer. What is more, the ITC data reveal that the copolymer has a rather weak interaction with these two proteins, and that the association process is exothermic. Results of thermal denaturation studies suggest that the copolymer slightly reduces the thermal
Acknowledgments
The authors express their appreciation to the Research Council of University of Tehran for their financial support. We gratefully acknowledge Mrs. Adeleh Divsalar for help with running the ITC experiments and analyzing the data.
References (28)
- et al.
A novel microgel and associated post- fabrication encapsulation technique of proteins
J. Control. Release
(2005) - et al.
In vitro release behavior and stability of insulin in complexation hydrogels as oral drug delivery carriers
Int. J. Pharm.
(2003) - et al.
Biodegradable copolymers for delivery of proteins and water-insoluble drugs
J. Control. Release
(2001) - et al.
Phase equilibrium and insulin partitioning in aqueous two-phase systems containing block copolymers and potassium phosphate
Fluid Phase Equilib.
(2004) - et al.
Synthesis of a chemically-crosslinked thermo-sensitive hydrogel film and in situ encapsulation of model protein drugs
React. Funct. Polym.
(2006) - et al.
Nanosized insulin-complexes based on biodegradable amine-modified graft polyesters poly[vinyl-3-(diethylamino)-propylcarbamate-co-(vinyl acetate)-co-(vinyl alcohol)]-graft-poly (l-lactic acid): protection against enzymatic degradation, interaction with Caco-2 cell monolayers, peptide transport and cytotoxicity
Eur. J. Pharm. Biopharm.
(2007) Thermo- and pH-responsive polymers in drug delivery
Adv. Drug Deliv. Rev.
(2006)- et al.
pH/temperature-sensitive polymers for macromolecular drug loading and release
J. Control. Release
(1994) - et al.
Biodegradable micelles/polymersomes from fumaric/sebacic acids and poly (ethylene glycol)
Biomaterials
(2003) - et al.
Early events in the fibrillation of monomeric insulin
J. Biol. Chem.
(2005)
Stimulation of insulin fibrillation by urea-induced intermediates
J. Biol. Chem.
Prediction of the association state of insulin using spectral parameters
J. Pharm. Sci.
Glucose sensing based on the intrinsic fluorescence of sol–gel immobilized yeast hexokinase
Anal. Biochem.
Thermochemistry of the specific binding of C12 surfactants to bovine serum albumin
Biochem. Biophys. Acta
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