Sei Kwang Hahn

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.

Target specific intracellular delivery of siRNA/PEI-HA complex by receptor mediated endocytosis.

Hyaluronic acid (HA) plays important biological roles in tissue integrity, angiogenesis, wound healing, and cell motility through the interaction with receptors on cell membranes. In this work, we investigated the effect of HA modification on the receptor-mediated endocytosis labeling HA derivatives with quantum dots (QDots). HA-QDot conjugates with a degree of modification less than ca. 25 mol % appeared to be more efficiently taken up to B16F1 cells by HA receptor mediated endocytosis than QDots alone. On the basis of bioimaging study, polyethyleneimine, PEI-HA conjugate with 24.2 mol % PEI content was developed as a target specific intracellular delivery carrier of siRNA. The siRNA/PEI-HA complex exhibited higher gene silencing efficiency in B16F1 cells with HA receptors than siRNA/PEI complex. Anti-PGL3-Luc siRNA/PEI-HA complex appeared to silence PGL3-Luc gene in the range of 50%-85% depending on the serum concentration up to 50 vol %. According to in vivo biodistribution test, siRNA/PEI-HA complex accumulated mainly in the tissues with HA receptors such as liver, kidney, and tumor. Furthermore, intratumoral injection of anti-VEGF siRNA/PEI-HA complex resulted in an effective inhibition of tumor growth by the HA receptor mediated endocytosis to tumor cells in C57BL/6 mice. Considering all these results, anti-VEGF siRNA/PEI-HA complex was thought to be applied successfully as target specific antiangiogenic therapeutics for the treatment of diseases in the tissues with HA receptors, such as liver cancer and kidney cancer.

Hyaluronic Acid−Quantum Dot Conjugates for In Vivo Lymphatic Vessel Imaging

A simple and novel electrostatic coupling method is reported, which provides a hyaluronic acid-quantum dot conjugate (HA-QD) that is colloidally stable and size-tunable from 50 to 120 nm. The HA-QDs show cancer targeting efficiency, which suggests diagnostic and imaging applications. The conjugates are also demonstrated for the fluorescence staining capability for lymphatic vessels in vitro and in vivo. Using the HA-QDs in a small animal model, lymphatic vessels are visualized real-time in vivo for days. Comprehensive cytotoxicity evaluations are made for the conjugates and the unconjugated counterpart. The HA-QDs showcase the potentials toward cancer imaging and real-time visualization of changes in lymphatic vessels such as lymphangiogenesis.

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.

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.

Target specific tumor treatment by VEGF siRNA complexed with reducible polyethyleneimine–hyaluronic acid conjugate

Target specific delivery of small interfering RNA (siRNA) has been regarded as one of the most important technologies for the development of siRNA therapeutics. In this work, non-toxic low molecular weight (MW) polyethyleneimine (PEI, 2000 Da) was cross-linked with cystamine bisacrylamide (CBA) to prepare reducible PEI-SS in the body. Then, PEI-SS was conjugated with hyaluronic acid (HA) in the form of block-copolymer to enhance serum stability and facilitate target specific cellular uptake of siRNA by HA receptor mediated endocytosis. The cytotoxicity of (PEI-SS)-b-HA conjugate appeared to be negligible likely due to the degradation of PEI-SS to low MW PEI in the cytosol. Flow cytometric and confocal microscopic analyses confirmed the HA receptor mediated endocytosis of siRNA/(PEI-SS)-b-HA complex. The siRNA/(PEI-SS)-b-HA complex demonstrated an excellent in vitro gene silencing efficiency in the range of 50-80% reducing the mRNA expression level in the absence and presence of 50 vol% serum. Moreover, intra-tumoral injection of vascular endothelial growth factor (VEGF) siRNA/(PEI-SS)-b-HA complex resulted in dramatically inhibited tumor growth with reduced VEGF mRNA and VEGF levels in the tumors.

ELECTRICAL DETECTION OF VEGFS FOR CANCER DIAGNOSES USING ANTI-VASCULAR ENDOTHERIAL GROWTH FACTOR APTAMER-MODIFIED SI NANOWIRE FETS

We report the real-time, label-free and electrical detection of vascular endotherial growth factor (VEGF) for cancer diagnosis using anti-VEGF aptamer-modified Si nanowire field-effect transistors (SiNW-FETs). Specifically, the high quality single-crystalline SiNWs of both n-type and p-type characters were surface modified with the covalent immobilization of anti-VEGF aptamers, and they were turned into SiNW-FET biosensors for the VEGF detection. We show that the VEGF molecules consistently act on the gate dielectrics of both n-type and p-type SiNW-FETs as electrically positive point-charges; their recognition to anti-VEGF aptamers depletes (accumulates) the charge carriers in the p-type (n-type) SiNW-FETs and thus decreases (increases) the detection currents. The detection limit for VEGFs in this study was determined as 1.04nM and 104pM for the cases of n-type and p-type SiNW-FETs, respectively.

Effect of cross-linking reagents for hyaluronic acid hydrogel dermal fillers on tissue augmentation and regeneration.

A novel, biocompatible, and nontoxic dermal filler using hyaluronic acid (HA) hydrogels was successfully developed for tissue augmentation applications. Instead of using highly reactive cross-linkers such as divinyl sulfone (DVS) for Hylaform, 1,4-butanediol diglycidyl ether (BDDE) for Restylane, and 1,2,7,8-diepoxyoctane (DEO) for Puragen, HA hydrogels were prepared by direct amide bond formation between the carboxyl groups of HA and hexamethylenediamine (HMDA) with an optimized carboxyl group modification for effective tissue augmentation. The HA-HMDA hydrogels could be prepared within 5 min by the addition of HMDA to HA solution activated with 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC) and 1-hydroxybenzotriazole monohydrate (HOBt). Five kinds of samples, a normal control, a negative control, a positive control of Restylane, adipic acid dihydrazide grafted HA (HA-ADH) hydrogels, and HA-HMDA hydrogels, were subcutaneously injected to wrinkled model mice. According to the image analysis on dorsal skin augmentation, the HA-HMDA hydrogels exhibited the best tissue augmentation effect being stable longer than 3 months. Furthermore, histological analyses after hematoxylin-eosin (H&E) and Massons trichrome staining revealed the excellent biocompatibility and safety of HA-HMDA hydrogels. The dermal thickness and the dermal collagen density in wrinkled mice after treatment with HA-HMDA hydrogels for 12 weeks were comparable to those of normal mice. Compared with HA-DVS hydrogels and Restylane, the excellent tissue augmentation by HA-HMDA hydrogels might be ascribed to the biocompatible residues of amine groups in the cross-linker of HMDA. The HA-HMDA hydrogels will be investigated further as a novel dermal filler for clinical applications.

Nanographene oxide-hyaluronic acid conjugate for photothermal ablation therapy of skin cancer.

Melanoma skin cancer is one of the most dangerous skin cancers and the main cause of skin-cancer-related mortality. Hyaluronic acid (HA) has been used as an effective transdermal delivery carrier of chemical drugs and biopharmaceuticals. In this work, a nanographene oxide-HA conjugate (NGO-HA) was synthesized for photothermal ablation therapy of melanoma skin cancer using a near-infrared (NIR) laser. Confocal microscopy and ex vivo bioimaging clearly visualized the remarkable transdermal delivery of NGO-HA to tumor tissues in the skin of mice, which might be ascribed to highly expressed HA receptors and relatively leaky structures around tumor tissues, enabling the enhanced permeation and retention of nanoparticles. The NIR irradiation resulted in complete ablation of tumor tissues with no recurrence of tumorigenesis. The antitumor effect was confirmed by ELISA for caspase-3 activity and histological and immunohistochemical analyses with TUNEL assay for tumor apoptosis. Taken together, we could confirm the feasibility of transdermal NGO-HA for photothermal ablation therapy of melanoma skin cancers.