Jeong-A Yang

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.

In situ supramolecular assembly and modular modification of hyaluronic acid hydrogels for 3D cellular engineering.

A facile in situ supramolecular assembly and modular modification of biocompatible hydrogels were demonstrated using cucurbit[6]uril-conjugated hyaluronic acid (CB[6]-HA), diaminohexane-conjugated HA (DAH-HA), and tags-CB[6] for cellular engineering applications. The strong and selective host-guest interaction between CB[6] and DAH made possible the supramolecular assembly of CB[6]/DAH-HA hydrogels in the presence of cells. Then, the 3D environment of CB[6]/DAH-HA hydrogels was modularly modified by the simple treatment with various multifunctional tags-CB[6]. Furthermore, we could confirm in situ formation of CB[6]/DAH-HA hydrogels under the skin of nude mice by sequential subcutaneous injections of CB[6]-HA and DAH-HA solutions. The fluorescence of modularly modified fluorescein isothiocyanate (FITC)-CB[6] in the hydrogels was maintained for up to 11 days, reflecting the feasibility to deliver the proper cues for cellular proliferation and differentiation in the body. Taken together, CB[6]/DAH-HA hydrogels might be successfully exploited as a 3D artificial extracellular matrix for various tissue engineering applications.

Transdermal delivery of hyaluronic acid -- human growth hormone conjugate.

Hyaluronic acid (HA) is one of the major components of extracellular matrix (ECM). Keratinocyte and fibroblast are known to have HA receptors in the skin. Fibroblast also has human growth hormone (hGH) receptors. In this work, HA-hGH conjugate was developed as a receptor mediated transdermal delivery system of protein drugs. HA-hGH conjugate was synthesized by specific coupling reaction between aldehyde modified HA and the N-terminal amine group of hGH. We could confirm the proliferative effect of HA on keratinocyte and fibroblast, and the biological activity of HA-hGH conjugate in fibroblast with an elevated expression level of phosphorylated Janus kinase 2 (p-JAK2). Interestingly, fluorescence microscopy clearly visualized the dramatically enhanced penetration of HA-hGH conjugate through the dorsal skin of mice after topical treatment with FITC labeled HA-hGH conjugate. According to pharmacokinetic analysis, HA-hGH conjugate appeared to be delivered through the skin into the blood stream possibly by the receptor mediated transdermal delivery. This work confirms the feasibility of using the HA-hGH conjugate as a model system for the receptor mediated transdermal delivery of protein drugs and their further exploitation for various cosmetic and tissue engineering applications.

Injectable hyaluronic acid–tyramine hydrogels for the treatment of rheumatoid arthritis

Rheumatoid arthritis (RA) is a chronic inflammatory disease caused by inflammation of the synovial membrane, leading in turn to articular cartilage destruction. In this work, injectable tyramine modified hyaluronic acid (HA-Tyr) hydrogels were developed for the treatment of RA. HA-Tyr conjugate was synthesized by amide bond formation between carboxyl groups of HA and amine groups of tyramine. Then, HA-Tyr hydrogels were prepared by radical crosslinking reaction using H(2)O(2) and horse-radish peroxidase. Intra-articular injection of HA-Tyr hydrogels encapsulating dexamethasone (DMT) as a model drug resulted in successful treatment of RA with reduced interlukine-6, prostaglandin E2 and four types of cytokine levels in collagen-induced arthritis animal models. Histological analysis with hematoxylin and eosin (H&E) staining also confirmed the therapeutic effect of injectable HA-Tyr hydrogels with DMT. Taken together, the injectable HA-Tyr hydrogels were thought suitable to be developed as a therapeutically effective drug carrier for the treatment of RA.

3D Tissue Engineered Supramolecular Hydrogels for Controlled Chondrogenesis of Human Mesenchymal Stem Cells

Despite a wide investigation of hydrogels as an artificial extracellular matrix, there are few scaffold systems for the facile spatiotemporal control of mesenchymal stem cells (MSCs). Here, we report 3D tissue engineered supramolecular hydrogels prepared with highly water-soluble monofunctionalized cucurbit[6]uril-hyaluronic acid (CB[6]-HA), diaminohexane conjugated HA (DAH-HA), and drug conjugated CB[6] (drug-CB[6]) for the controlled chondrogenesis of human mesenchymal stem cells (hMSCs). The mechanical property of supramolecular HA hydrogels was modulated by changing the cross-linking density for the spatial control of hMSCs. In addition, the differentiation of hMSCs was temporally controlled by changing the release profiles of transforming growth factor-β3 (TGF-β3) and/or dexamethasone (Dexa) from the hydrolyzable Dexa-CB[6]. The effective chondrogenic differentiation of hMSCs encapsulated in the monoCB[6]/DAH-HA hydrogel with TGF-β3 and Dexa-CB[6] was confirmed by biochemical glycosaminoglycan content analysis, real-time quantitative PCR, histological, and immunohistochemical analyses. Taken together, we could confirm the feasibility of cytocompatible monoCB[6]/DAH-HA hydrogels as a platform scaffold with controlled drug delivery for cartilage regeneration and other various tissue engineering applications.

Target specific systemic delivery of TGF-β siRNA/(PEI-SS)-g-HA complex for the treatment of liver cirrhosis

A target specific systemic delivery system of siRNA therapeutics was successfully developed using reducible polyethyleneimine grafted hyaluronic acid [(PEI-SS)-g-HA] conjugates. The PEI-SS was synthesized by Michael addition of low molecular weight PEI (MW = 2000) with cystaminebisacrylamide (CBA), and grafted to carboxyl groups of HA via amide bond formation after activation with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and 1-hydroxybenzotriazole monohydrate (HOBt). The confocal microscopic and fluorometric analyses confirmed the effective cellular uptake of siRNA/(PEI-SS)-g-HA complex by HA receptor mediated endocytosis. In vitro gene silencing efficiency was ca. 80% in the presence of 10 vol% serum and ca. 50% in the presence of 50 vol% serum in B16F1 melanoma cells and activated hepatic stellate cells (HSCs). Furthermore, target specific systemic delivery of apolipoprotein B (ApoB) siRNA/(PEI-SS)-g-HA complex resulted in a drastically reduced ApoB mRNA level down to ca. 20% in a dose-dependent manner. Finally, TGF-β siRNA/(PEI-SS)-g-HA complex showed a feasible therapeutic effect on liver cirrhosis with a significantly reduced nodule formation, collagen content, and HSC number. The siRNA/(PEI-SS)-g-HA complex can be exploited for the target specific systemic treatment of various liver diseases.

Reducible hyaluronic acid-siRNA conjugate for target specific gene silencing.

Despite wide applications of polymer-drug conjugates, there are only a few polymer-siRNA conjugates like poly(ethylene glycol) conjugated siRNA. In this work, reducible hyaluronic acid (HA)-siRNA conjugate was successfully developed for target specific systemic delivery of siRNA to the liver. The conjugation of siRNA to HA made it possible to form a compact nanocomplex of siRNA with relatively nontoxic linear polyethyleneimine (LPEI). After characterization of HA-siRNA conjugate by size exclusion chromatography (SEC) and gel electrophoresis, its complex formation with LPEI was investigated with a particle analyzer. The HA-siRNA/LPEI complex had a mean particle size of ca. 250 nm and a negative or neutral surface charge at physiological condition. The reducible HA-siRNA/LPEI complex showed a higher in vitro gene silencing efficiency than noncleavable HA-siRNA/LPEI complex. Furthermore, after systemic delivery, apolipoprotein B (ApoB) specific HA-siApoB/LPEI complex was target specifically delivered to the liver, which resulted in statistically significant reduction of ApoB mRNA expression in a dose dependent manner. The HA-siRNA conjugate can be effectively applied as a model system to the treatment of liver diseases using various siRNAs and relatively nontoxic polycations.

Hyaluronic acid-tumor necrosis factor-related apoptosis-inducing ligand conjugate for targeted treatment of liver fibrosis.

Liver fibrosis is a chronic liver disease caused by viral infection and/or metabolic, genetic and cholestatic disorders. The inhibition of hepatic stellate cell (HSC) activation and the selective apoptosis of activated HSCs can be a good strategy to treat liver fibrosis. The activated HSCs are known to be more susceptible to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induced apoptosis than normal HSCs because death receptor 5 is overexpressed on the cell surface. In this work, a target-specific and long-acting hyaluronic acid (HA)-TRAIL conjugate was successfully developed for the treatment of liver fibrosis. The HA-TRAIL conjugate was synthesized by a coupling reaction between aldehyde-modified HA and the N-terminal amine group of TRAIL. The biological activity of the HA-TRAIL conjugate was confirmed by an in vitro anti-proliferation assay and caspase-3 expression in human colon cancer HCT116 cells. In vivo real-time bioimaging exhibited the target-specific delivery of near-infrared fluorescence dye-labeled HA-TRAIL conjugate to the liver in mice. According to pharmacokinetic analysis, the HA-TRAIL conjugate was detected for more than 4days after single intravenous injection into Sprague-Dawley (SD) rats. Finally, we could confirm the antifibrotic effect of HA-TRAIL conjugate in an N-nitrosodimethylamine-induced liver fibrosis model SD rats.

Supramolecular hydrogels for long-term bioengineered stem cell therapy.

Synthetic hydrogels have been extensively investigated as artificial extracellular matrices (ECMs) for tissue engineering in vitro and in vivo. Crucial challenges for such hydrogels are sustaining long-term cytocompatible encapsulation and providing appropriate cues at the right place and time for spatio-temporal control of the cells. Here, in situ supramolecularly assembled and modularly modified hydrogels for long-term engineered mesenchymal stem cell (eMSC) therapy are reported using cucurbit[6]uril-conjugated hyaluronic acid (CB[6]-HA), diaminohexane conjugated HA (DAH-HA), and drug-conjugated CB[6] (drug-CB[6]). The eMSCs producing enhanced green fluorescence protein (EGFP) remain alive and emit the fluorescence within CB[6]/DAH-HA hydrogels in mice for more than 60 d. Furthermore, the long-term expression of mutant interleukin-12 (IL-12M) by eMSCs within the supramolecular hydrogels results in effective inhibition of tumor growth with a significantly enhanced survival rate. Taken together, these findings confirm the feasibility of supramolecular HA hydrogels as 3D artificial ECMs for cell therapies and tissue engineering applications.