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Synthesis, Characterization, and Application of Long Acting Peptide - Hyaluronate Conjugate

Synthesis, Characterization, and Application of Long Acting Peptide - Hyaluronate Conjugate
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Bioconjugation technology using synthetic and natural polymers like poly(ethylene glycol) (PEG) and hyaluronic acid (HA) has been widely exploited for the development of long-acting biopharmaceuticals. The chemical attachment of polymers with the biopharmaceuticals, such as protein and peptide drugs, has been reported to increase the drug efficacy by reducing renal clearance, decreasing the immuno-response and alleviating the enzymatic degradation in the body. Recently, HA has been widely investigated as a novel drug carrier of various chemical and biopharmaceutical therapeutics. As a natural linear polysaccharide in the body, HA has several advantageous properties for biomedical applications, such as biocompatibility, biodegradability, non-immunogenicity, and non-toxicity. HA has been regarded as one of the best biomaterials in terms of safety issues. However, the short half life of HA should be overcome for long-term clinical applications. One of the main objectives in this study was to develop how to control the molecular degradation of HA derivatives for drug delivery applications. In Chapter 1, the characteristics of HA was introduced focusing on the biological functions and unique physico-chemical properties. In addition, the chemical modification of HA and the application of HA derivatives to drug delivery were described. In Chapter 2, a novel protocol to control the molecular degradation of HA was successfully developed for drug delivery applications. HA has a different conformational structure in water and in organic solvent. The carboxyl group of HA is known to be the recognition site of hyaluronidase and HA receptors. Based on these findings, HA was chemically modified by grafting adipic acid dihydrazide (ADH) to the carboxyl group of HA in the water to prepare HA-ADH_WATER and in the mixed solvent of water and ethanol to prepare degradation controlled HA-ADH_WATER/ETHANOL. To compare the degradation rate of the samples, three kinds of HA hydrogels were prepared by the crosslinking of HA-ADH_WATER or HA-ADH_WATER/ETHANOL with bis(sulfosuccinimidyl)suberate (BS3), and by the crosslinking of HA-OH with divinyl sulfone (DVS). In vitro and in vivo degradation tests showed that HA-DVS hydrogels were degraded the most rapidly, followed by HA-ADH_WATER hydrogels and HA-ADH_WATER/ETHANOL hydrogels. There was no adverse effect during and after the in vivo degradation tests. All of the HA hydrogel samples appeared to be biocompatible according to the histological analysis with hematoxylin-eosin and alcian blue staining. Based on these results, long acting HA - biopharmaceutical conjugates were developed after chemical modification of carboxyl groups of HA in the following studies. In Chapter 3, a new protocol for the synthesis of HA - peptide (CWRYMVm) conjugate for FPRL1 receptor was successfully developed for the treatment of inflammatory diseases such as sepsis. Aminoethyl methacrylated HA (HA-AEMA) was synthesized by the coupling reaction of tetrabutyl ammonium salt of HA (HA-TBA) and AEMA using benzotriazol-1-yloxy-tris(dimethyl-amino)phosphonium hexafluoro-phosphate (BOP) in dimethyl sulfoxide (DMSO). Then, HA-AEMA was conjugated with CWRYMVm in water via Michael addition reaction between methacryloyl group of HA-AEMA and thiol group of cysteine in CWRYMVm. The formation of HA-peptide conjugate was confirmed by 1H-NMR and gel permeation chromatography (GPC). The average number of conjugated peptide molecules could be controlled from 5 to 23 per single HA chain. The HA-peptide conjugate showed serum stability longer than four days. In vitro signal transduction activity of HA-peptide conjugate for FPRL1 receptor was confirmed from the elevated levels of phospho-extracellular signal-regulated kinase (pERK) and calcium ion in FPRL1 over-expressing RBL-2H3 cells. The partially decreased biological activity of HA-peptide conjugates by the steric hindrance of HA was recovered after its degradation by hyaluronidase treatment. In vivo tests of HA-peptide conjugate in murine model of sepsis confirmed the increased therapeutic effect after conjugation to HA. In Chapter 4, a novel protocol to synthesize anti-Flt1 peptide - HA conjugate was successfully developed for the treatment of ocular neovascularization. Using HA-TBA, water-insoluble anti-Flt1 peptide could be conjugated with HA in DMSO by the amide bond formation between carboxyl groups of HA and N-terminal amine groups of GGNQWFI. The formation of anti-Flt1 peptide - HA conjugate was confirmed by 1H-NMR and fluorometric analyses. The average number of grafted peptide molecules in anti-Flt1 peptide - HA conjugates could be controlled from 3 to 30 per single HA chain by changing the feeding amount of peptide for the conjugation reaction. The resulting HA - GGNQWFI conjugate self-assembled to form micelles in aqueous solution, as confirmed and characterized by transmission electron microscopy (TEM). According to in vitro biological activity tests, HA - GGNQWFI conjugate exhibited a dose-dependent inhibition effect on the binding of Flt1-Fc to VEGF165 coated on the well. Furthermore, in vivo biological activity of anti-Flt1 peptide - HA conjugate was confirmed from the inhibitory effect on corneal neovascularization in SD rats. VEGF receptor 2 expression in silver nitrate cauterized cornea was also reduced after treatment with anti-Flt1 peptide - HA conjugate. In addition, anti-Flt1 peptide - HA conjugate effectively inhibited retinal choroidal neovascuarization (CNV) in laser induced CNV model rats. In the diabetic retinopathy model rats, the retinal vascular permeability and the deformation of retinal vascular structure were significantly reduced after treatment with anti-Flt1 peptide - HA conjugates. Pharmacokinetic analysis confirmed the increased mean residence time of anti-Flt1 peptide after conjugation to HA longer than 2 weeks. In addition, anti-Flt1 peptide - HA conjugate also showed therapeutic effect on asthma reducing airway hyperresponsiveness (AHR) and lung inflammation evaluated by bronchoalveolar lavage (BAL) cellularity. Moreover, anti-Flt1 peptide - HA conjugate having a micelle structure in aqueous solution was developed as a novel drug carrier of various hydrophobic drugs such as genistein, dexamethasone, and doxorubicin. The hydrophobic drug - loaded micelles had a spherical shape with a diameter in the range of 100 ~ 300 nm, as characterized by dynamic light scattering (DLS) and TEM analyses. Anti-Flt1 peptide - HA conjugate capable of encapsulating hydrophobic drugs would be successfully exploited as a novel drug carrier for an anti-angiogenic cocktail therapy.
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