Eun Ju Oh

Target specific and long-acting delivery of protein, peptide, and nucleotide therapeutics using hyaluronic acid derivatives

Hyaluronic acid (HA) is a biodegradable, biocompatible, non-toxic, non-immunogenic and non-inflammatory linear polysaccharide, which has been used for various medical applications such as arthritis treatment, ocular surgery, tissue augmentation, and so on. In this review, the effect of chemical modification of HA on its distribution throughout the body was reported for target specific and long-acting delivery applications of protein, peptide, and nucleotide therapeutics. According to the real-time bio-imaging of HA derivatives using quantum dots (QDot), HA-QDot conjugates with 35mol% HA modification maintaining enough binding sites for HA receptors were mainly accumulated in the liver, while those with 68mol% HA modification losing much of HA characteristics were evenly distributed to the tissues in the body. The results are well matched with the fact that HA receptors are abundantly present in the liver with a high specificity to HA molecules. Accordingly, slightly modified HA derivatives were used for target specific intracellular delivery of nucleotide therapeutics and highly modified HA derivatives were used for long-acting conjugation of peptide and protein therapeutics. HA has been also used as a novel depot system in the forms of physically and chemically crosslinked hydrogels for various protein drug delivery. This review will give you a peer overview on novel HA derivatives and the latest advances in HA-based drug delivery systems of various biopharmaceuticals for further clinical development.

Single-File Diffusion of Protein Drugs through Cylindrical Nanochannels

A new drug delivery device using cylindrical block copolymer nanochannels was successfully developed for controlled protein drug delivery applications. Depending on the hydrodynamic diameter of the protein drugs, the pore size in cylindrical nanochannels could be controlled precisely down to 6 nm by Au deposition. Zero-order release of bovine serum albumin (BSA) and human growth hormone (hGH) by single-file diffusion, which has been observed for gas diffusion through zeolite pores, was realized up to 2 months without protein denaturation. Furthermore, a nearly constant in vivo release of hGH from the drug delivery nanodevice implanted to Sprague-Dawley (SD) rats was continued up to 3 weeks, demonstrating the feasibility for long-term controlled delivery of therapeutic protein drugs.

Hyaluronic acid–polyethyleneimine conjugate for target specific intracellular delivery of siRNA

A novel target specific small interfering RNA (siRNA) delivery system was successfully developed using polyethyleneimine (PEI)-hyaluronic acid (HA) conjugate. Anti-PGL3-Luc siRNA was used as a model system suppressing the PGL3-Luc gene expression. The siRNA/PEI-HA complex with an average size of ca. 21 nm appeared to be formed by electrostatic interaction between the negatively charged siRNA and the positively charged PEI of PEI-HA conjugate. The cytotoxicity of siRNA/PEI-HA complex to B16F1 cells was lower than that of siRNA/PEI complex according to the MTT assay. When B16F1 and HEK-293 cells were treated with fluorescein isothiocyanate (FITC) labeled siRNA/PEI-HA complex, B16F1 cells, with a lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1), showed higher green fluorescent intensity than HEK-293 cells because of the HA receptor mediated endocytosis of the complex. Accordingly, the PGL3-Luc gene silencing of anti-PGL3-Luc siRNA/PEI-HA complex was more efficient in B16F1 cells than in HEK-293 cells. In addition, the inhibited PGL3-Luc gene silencing effect in the presence of free HA in the transfection medium revealed that siRNA/HA-PEI complex was selectively taken up to B16F1 cells via HA receptor mediated endocytosis. All these results demonstrated that the intracellular delivery of anti-PGL3-Luc siRNA/PEI-HA complex could be facilitated by the HA receptor mediated endocytosis.

Long acting hyaluronate--exendin 4 conjugate for the treatment of type 2 diabetes.

Despite clinical exploitation of exendin 4 for the treatment of type 2 diabetes, the significantly short half-life requiring twice a day injection has limited the wide applications. In this work, a protocol for the synthesis of long acting hyaluronate (HA) - exendin 4 conjugate was successfully developed using Michael addition chemistry between vinyl sulfone modified HA (HA-VS) and thiolated exendin 4. The exendin 4 content could be controlled in the range of 5-30 molecules per single HA chain with a bioconjugation efficiency higher than 90%. The conjugation of exendin 4 with HA resulted in about 20 times improved in vitro serum stability maintaining the hypoglycemic and gluco-regulatory bioactivities of exendin 4. HA - exendin 4 conjugates showed excellent glucose-lowering capabilities in type 2 db/db mice demonstrating protracted hypoglycemic effect up to 3 days after a single subcutaneous injection. Furthermore, insulin immunohistochemical analysis of islets in db/db mice confirmed the improved insulinotropic activity of HA - exendin 4 conjugates. The HA - exendin 4 conjugates will be investigated further as a twice a week injection dosage form for clinical applications.

DNA/PEI/Alginate polyplex as an efficientin vivo gene delivery system

A novel non-viral gene delivery system comprised of a DNA/PEI/Alginate (DPA) polyplex was prepared and assessedin vitro andin vivo. Coating the positively charged DNA/PEI (DP) complex with a polyanion resulted in a high level ofin vitro reporter gene transfection in the presence of 50 vol% serum due to the minimized cytotoxicity of PEI and the reduced nonspecific interactions with serum components. Among the tested anionic polymers, which included sodium alginate, poly(methacrylic acid) and poly(acrylic acid), the sodium alginate showed the highest gene transfection efficiency. The DPA polyplex also showed a reduced level of erythrocyte aggregation in target cells when compared with the DP complex. According toin vivo studies in which reporter genes encoding green fluorescent protein (GFP) and luciferase were used, injection of the DPA polyplex into tumor cells in six week old female C57/BL6 mice resulted in a much higher level of GFP expression and approximately 7 fold higher luciferase expression than treatment with the DP complex. Taken together, these results demonstrated that the anionic alginate coating of the DP complex contributed to efficient gene deliveryin vitro andin vivo.

Synthesis, characterization, and preliminary assessment of anti-Flt1 peptide-hyaluronate conjugate for the treatment of corneal neovascularization

Anti-Flt1 peptide of GNQWFI has been reported to inhibit vascular endothelial growth factor receptor 1 (VEGFR1) - mediated endothelial cell migration and tube formation. In this work, a protocol to synthesize anti-Flt1 peptide-hyaluronate (HA) conjugate was successfully developed for the treatment of corneal neovascularization. Using tetrabutyl ammonium salt of HA (HA-TBA), water-insoluble anti-Flt1 peptide could be conjugated with HA in dimethyl sulfoxide (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 (1)H 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. According to in vitro biological activity tests, anti-Flt1 peptide-HA conjugate exhibited a significant inhibition effect on the binding of Flt1-Fc to VEGF(165) 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 silver nitrate cauterized corneas of SD rats. The VEGF receptor 2 expression was also reduced after treatment with anti-Flt1 peptide-HA conjugate. The water-soluble anti-Flt1 peptide-HA conjugate was thought to have a potential to be developed as anti-angiogenic therapeutics for the treatment of corneal neovascularization.

Anti-Flt1 peptide – Hyaluronate conjugate for the treatment of retinal neovascularization and diabetic retinopathy

Anti-angiogenic therapeutics has been investigated extensively for the treatment of retinal and choroidal vascular diseases, and diabetic retinopathy. Anti-Flt1 peptide of GNQWFI is an antagonistic peptide for vascular endothelial growth factor receptor 1 (VEGFR1 or Flt1) inhibiting VEGFR1-mediated endothelial cell migration and tube formation. In this work, anti-Flt1 peptide (GGNQWFI) was chemically conjugated to tetra-n-butyl ammonium modified hyaluronate (HA-TBA) via amide bond formation in dimethyl sulfoxide (DMSO) using benzotriazol-1-yloxy-tris(dimethylamino)phosphonium hexafluorophosphate (BOP). The resulting HA - GGNQWFI conjugate self-assembled to form micelle-like nanoparticles 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 VEGF(165) coated on the well. Furthermore, anti-Flt1 peptide - HA conjugate effectively inhibited retinal choroidal neovascularization (CNV) in laser induced CNV model rats. The retinal vascular permeability and the deformation of retinal vascular structure were also significantly reduced in diabetic retinopathy model rats after treatment with anti-Flt1 peptide - HA conjugate. Pharmacokinetic analysis confirmed the increased mean residence time of anti-Flt1 peptide after conjugation to HA longer than 2 weeks.

Signal Transduction of Hyaluronic Acid−Peptide Conjugate for Formyl Peptide Receptor Like 1 Receptor

Agonistic and antagonistic peptides for formyl peptide receptor like 1 (FPRL1) receptor have been investigated as novel drug candidates for inflammatory diseases such as sepsis, asthma, and rheumatoid arthritis. In this work, a novel protocol for the synthesis of hyaluronic acid (HA)-peptide (CWRYMVm) conjugate for FPRL1 receptor was successfully developed for further clinical applications of peptide drugs. Aminoethyl methacrylated HA (HAAEMA) was synthesized by the coupling reaction of tetrabutyl ammonium salt of HA (HA-TBA) and AEMA using benzotriazol-1-yloxy-tris(dimethylamino) phosphonium hexafluorophosphate (BOP) in dimethyl sulfoxide (DMSO). Then, HA-AEMA was conjugated with CWRYMVm in water via Michael addition reaction between methacrylate group of HA-AEMA and thiol group in cysteine. 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 the HA-peptide conjugate for FPRL1 receptor was confirmed from the elevated levels of phospho-extracellular signal-regulated kinase (pERK) and calcium ion in FPRL1 overexpressing 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.

Characterization of PEGylated Anti-VEGF Aptamers Using Surface Plasmon Resonance

ConclusionsSPR analysis for the assessment of anti-VEGF RNA aptamer binding affinity was successfully carried out before and after PEGylation. Anti-VEGF 2′-OMe RNA aptamer was synthesized and PEGylated through the amide bond formation between the succinimidyl group of mPEG-SPA and the terminal amine group of anti-VEGF 2′-OMe RNA aptamer. According to the SPR analysis using a BIAcore 2000 instrument, the binding affinity of anti-VEGF 2′-OMe-RNA aptamer to the VEGF (Kd=1.87×10−9 M) decreased significantly after PEGylation (Kd=8.×10−8 M). The PEGylated anti-VEGF RNA aptamer showed a slow adsorption and a rapid desorption. Interestingly, the anti-VEGF DNA aptamer having the same sequence with the anti-VEGF 2′-OMe RNA aptamer showed no binding affinity at all. The SPR method was thought to be usefully applied for the assessment of bio-conjugated aptamer therapeutics.

Real-time bioimaging of hyaluronic acid derivatives using quantum dots for biopharmaceutical delivery applications

Hyaluronic acid (HA), which has been used for various medical applications, is a biocompatible, biodegradable, and linear polysaccharide in the body. In this work, real-time bioimaging of HA derivatives was carried out using quantum dots (QDot) for target specific and long acting delivery applications of protein, peptide, and nucleotide therapeutics. In vitro bioimaging of HA-QDot conjugates successfully visualized the target specific delivery of HA derivatives to B16F1 cells with HA receptors by HA receptor mediated endocytosis, which reflects the feasibility of HA derivatives for intracellular delivery of biopharmaceuticals, especially nucleotide therapeutics. Furthermore, according to in vivo real-time bioimaging, HA-QDot conjugates with 35 mol% HA modification were mainly accumulated in the liver, while those with 68 mol% HA modification were evenly distributed to the tissues in the mice. Accordingly, slightly modified HA derivatives were used for target-specific delivery of biopharmaceuticals for the treatment of liver diseases and highly modified HA derivatives were used for long acting conjugation of peptide and protein therapeutics. These HA-based biopharmaceutical delivery systems can be exploited for further clinical development.