Self-adjuvanted hyaluronate--antigenic peptide conjugate for transdermal treatment of muscular dystrophy.

Duchenne's muscular dystrophy (DMD) is a neuromuscular disorder accompanied with muscle weakness and wasting. Since myostatin was reported to be a key regulator of muscle wasting, myostatin inhibitors have been investigated as therapeutic candidates for the treatment of muscular diseases. Here, we report an antigenic peptide of myostatin fragment (MstnF) conjugated to hyaluronate (HA) with a low molecular weight (MW, 17 kDa) for transdermal immunotherapy of DMD. Facilitating the transdermal delivery, the low MW HA showed a boosting effect on the immunization of MstnF possibly by engaging both toll-like receptors and cluster of differentiation 44 (CD44). In vivo two-photon microscopy clearly visualized the effective transdermal penetration of HA-MstnF conjugates into deep intact skin layers. The transdermal immunization of mdx mice significantly increased antibody titers against myostatin. Furthermore, the mdx mice immunized with HA-MstnF conjugates resulted in statistically significant improvement in the biochemical and pathological status of skeletal musculature as well as functional behaviors.

Genetically engineered mesenchymal stem cell therapy using self-assembling supramolecular hydrogels.

Stem cell therapy has attracted a great deal of attention for treating intractable diseases such as cancer, stroke, liver cirrhosis, and ischemia. Especially, mesenchymal stem cells (MSCs) have been widely investigated for therapeutic applications due to the advantageous characteristics of long life-span, facile isolation, rapid proliferation, prolonged transgene expression, hypo-immunogenicity, and tumor tropism. MSCs can exert their therapeutic effects by releasing stress-induced therapeutic molecules after their rapid migration to damaged tissues. Recently, to improve the therapeutic efficacy, genetically engineered MSCs have been developed for therapeutic transgene expression by viral gene transduction and non-viral gene transfection. In general, the number of therapeutic cells for injection should be more than several millions for effective cell therapy. Adequate carriers for the controlled delivery of MSCs can reduce the required cell numbers and extend the duration of therapeutic effect, which provide great benefits for chronic disease patients. In this review, we describe genetic engineering of MSCs, recent progress of self-assembling supramolecular hydrogels, and their applications to cell therapy for intractable diseases and tissue regeneration.

Bioimaging of Hyaluronate-Interferon α Conjugates Using a Non-Interfering Zwitterionic Fluorophore.

We conducted real-time bioimaging of the hyaluronate-interferon α (HA-IFNα) conjugate using a biologically inert zwitterionic fluorophore of ZW800-1 for the treatment of hepatitis C virus (HCV) infection. ZW800-1 was labeled on the IFNα molecule of the HA-IFNα conjugate to investigate its biodistribution and clearance without altering its physicochemical and targeting characteristics. Confocal microscopy clearly visualized the effective in vitro cellular uptake of the HA-IFNα conjugate to HepG2 cells. After verifying the biological activity in Daudi cells, we conducted the pharmacokinetic analysis of the HA-IFNα conjugate, which confirmed its target-specific delivery to the liver with a prolonged residence time longer than that of PEGylated IFNα. In vivo and ex vivo bioimaging of the ZW800-1-labeled HA-IFNα conjugate directly showed real-time biodistribution and clearance of the conjugate that are consistent with the biological behaviors analyzed by an enzyme-linked immunosorbent assay. Furthermore, the elevated level of OAS1 mRNA in the liver confirmed in vivo antiviral activity of HA-IFNα conjugates. With the data taken together, we could confirm the feasibility of ZW800-1 as a biologically inert fluorophore and target-specific HA-IFNα conjugate for the treatment of HCV infection.

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.

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-ß3 (TGF-ß3) 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-ß3 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.

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-gold nanoparticle/interferon α complex for targeted treatment of hepatitis C virus infection.

Gold nanoparticles (AuNPs) have been extensively investigated as an emerging delivery carrier of various biopharmaceuticals. Instead of nonspecific polyethylene glycol (PEG) conjugated interferon α (IFNα) for the clinical treatment of hepatitis C virus (HCV) infection, in this work, a target-specific long-acting delivery system of IFNα was successfully developed using the hybrid materials of AuNP and hyaluronic acid (HA). The HA-AuNP/IFNα complex was prepared by chemical binding of thiolated HA and physical binding of IFNα to AuNP. According to antiproliferation tests in Daudi cells, the HA-AuNP/IFNα complex showed a comparable biological activity to PEG-Intron with a highly enhanced stability in human serum. Even 7 days postinjection, HA-AuNP/IFNα complex was target-specifically delivered and remained in the murine liver tissue, whereas IFNα and PEG-Intron were not detected in the liver. Accordingly, HA-AuNP/IFNα complex significantly enhanced the expression of 2',5'-oligoadenylate synthetase 1 (OAS1) for innate immune responses to viral infection in the liver tissue, which was much higher than those by IFNα, PEG-Intron, and AuNP/IFNα complex. Taken together, the target-specific HA-AuNP/IFNα complex was thought to be successfully applied to the systemic treatment of HCV infection.

Flt1 peptide-hyaluronate conjugate micelle-like nanoparticles encapsulating genistein for the treatment of ocular neovascularization.

Flt1 peptide of GNQWFI is an antagonistic peptide for vascular endothelial growth factor receptor 1 (VEGFR1 or Flt1). In this work, Flt1 peptide-hyaluronate (HA) conjugates were successfully synthesized and the resulting micelle-like nanoparticles were exploited to encapsulate genistein, an inhibitor of tyrosine-specific protein kinases, for the treatment of ocular neovascularization. The mean diameter of genistein-loaded Flt1 peptide-HA conjugate micelles was measured to be 172.0±18.7 nm, with a drug-loading efficiency of 40-50%. In vitro release tests of genistein from the genistein-loaded Flt1 peptide-HA conjugate micelles exhibited the controlled release for longer than 24h. In vitro biological activity of genistein/Flt1 peptide-HA micelles was corroborated from the synergistic anti-proliferation of human umbilical vein endothelial cells (HUVECs). Furthermore, we could confirm the anti-angiogenic effect of genistein/Flt1 peptide-HA micelles from the statistically significant suppression of corneal neovascularization in silver nitrate cauterized corneas of SD rats. The retinal vascular hyperpermeability was also drastically reduced by the treatment in diabetic retinopathy model rats.

In vivo real-time confocal microscopy for target-specific delivery of hyaluronic acid-quantum dot conjugates.

Hyaluronic acid (HA), which is a biocompatible, biodegradable, and linear polysaccharide in the body, has been widely used for various biomedical applications. In this work, real-time bioimaging for target-specific delivery of HA derivatives was carried out using quantum dots (QDs). In vitro confocal microscopy of HA-QD conjugates confirmed the intracellular delivery of HA derivatives to B16F1 cells with HA receptors by HA-receptor-mediated endocytosis. Furthermore in vivo real-time confocal microscopy of HA-QD conjugates successfully visualized the target specific delivery and accumulation of HA-QD conjugates from the fluorescence-labeled blood vessels to the liver tissues. The authors could confirm the feasibility of HA derivatives as a target-specific intracellular drug-delivery carrier for the treatment of liver diseases and the in vivo real-time confocal microscopy as a new bioimaging tool for various drug-delivery applications. FROM THE CLINICAL EDITOR: This study demonstrates the possibility of labeling hyaluronic acid with quantum dots for visualization and for targeted intracellular drug delivery in liver disease models.

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.

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.

Target specific hyaluronic acid-interferon alpha conjugate for the treatment of hepatitis C virus infection.

Interferon alpha (IFNα) conjugated with polyethylene glycol (PEG) has been widely used for the treatment of hepatitis C virus (HCV) infection as a once-a-week injection formulation. However, the PEGylated IFNα has a low efficacy of ca. 39% and a side effect after repeated injections possibly due to the non-specific delivery with PEGylation. In this work, target specific long-acting hyaluronic acid-interferon alpha (HA-IFNα) conjugate was successfully developed for the treatment of HCV infection. HA-IFNα conjugate was synthesized by coupling reaction between aldehyde modified HA and the N-terminal group of IFNα. The IFNα content could be controlled in the range of 2-9 molecules per single HA chain with a bioconjugation efficiency higher than 95%. According to in vitro anti-proliferation assay using Daudi cells, HA-IFNα conjugate showed a comparable biological activity to PEG-Intron. In vivo real-time bioimaging confirmed the target specific delivery of near-infrared fluorescence (NIRF) dye labeled HA-IFNα conjugate to the liver in mice. In addition, pharmacokinetic analysis revealed the enhanced residence time longer than 4 days. After tail-vein injection, HA-IFNα conjugate induced ca. 60% higher expression of 2',5'-oligoadenylate synthetase 1 (OAS 1) for innate immune responses to viral infection in the murine liver tissues than IFNα and PEG-Intron.

Theranostic systems assembled in situ on demand by host-guest chemistry.

Theranostic systems have been explored extensively for a diagnostic therapy in the forms of polymer conjugates, implantable devices, and inorganic nanoparticles. In this work, we report theranostic systems in situ assembled by host-guest chemistry responding to a request. As a model theranostic system on demand, cucurbit[6]uril-conjugated hyaluronate (CB[6]-HA) was synthesized and decorated with FITC-spermidine (spmd) and/or formyl peptide receptor like 1 (FPRL1) specific peptide-spmd by simple mixing in aqueous solution. The resulting (FITC-spmd and/or peptide-spmd)@CB[6]-HA was successfully applied to the bioimaging of its target-specific delivery to B16F1 cells with HA receptors and its therapeutic signal transduction with elevated Ca(2+) and phosphor-extracellular signal-regulated kinase (pERK) levels in FPRL1-expressing human breast adenocarcinoma (FPRL1/MCF-7) cells. Finally, we could confirm in vitro and in vivo stability of the highly specific host-guest interaction. The on-demand theranostic platform technology using host-guest chemistry can be exploited for various bioimaging, biosensing, drug delivery, and tissue engineering applications.

Target specific systemic delivery of TGF-ß 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-ß 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.

Identification of a novel recombinant mutation in Korean patients with Gaucher disease using a long-range PCR approach.

Gaucher disease (GD) is an autosomal recessive, lysosomal disorder caused by mutations in the gene for the ß-glucocerebrosidase (GBA) enzyme. Presence of the non-functional GBAP pseudogene, which shares high sequence similarity with the functional GBA gene, has made it difficult to carry out molecular analyses of GD, especially recombinant mutations. Using a long-range PCR approach that has been skillfully devised for the easy detection of GBA recombinant mutations, we identified four recombinant mutations including two gene conversion alleles, Rec 1a and Rec 8a, one reciprocal gene fusion allele, Rec 1b, and one reciprocal gene duplication allele, Rec 7b, in Korean patients with GD. Rec 8a, in which the GBAP pseudogene sequence from intron 5 to exon 11 is substituted for the GBA gene is a novel recombinant mutation. All mutations were confirmed by full sequencing of PCR amplicons and/or Southern blot analysis. These results indicate that the usage of long-range PCR may allow the rapid and accurate detection of GBA recombinant mutations and contribute to the improvement of genotyping efficiency in GD patients.

A novel homozygous MMP2 mutation in a patient with Torg-Winchester syndrome.

Torg-Winchester syndrome (OMIM 259600) is an autosomal recessive multicentric osteolysis disorder. Mutations in the gene for matrix metalloproteinase 2 (MMP2) are involved in its pathogenesis. This is the first report of Torg-Winchester syndrome in east Asians. A 31-year-old female Korean patient had the typical clinical phenotypes of the syndrome, including shortening of trunk and limbs and severe osteolysis resulting in extremely small hands and feet. In addition, she had cord compression at the cervico-medullary junction, as well as lumbar dural ectasia. Molecular analysis revealed a novel homozygous missense mutation of MMP2, c.1217G>A (p.G406D). Gelatin zymography demonstrated a complete loss of the MMP2 activity of the mutation. Our results provide insights into the clinical and radiological features and pathogenic mechanisms of the syndrome.

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 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.

The fabrication, characterization and application of aptamer-functionalized Si-nanowire FET biosensors.

An aptamer-functionalized silicon-nanowire (Si-NW) field effect transistor (FET) biosensor was successfully fabricated, characterized and applied to real-time electrical detection of binding with the target protein for biomedical applications. Surface modifications were carried out using 3-aminopropyl diethoxysilane and succinic anhydride to introduce amine and carboxyl groups onto Si substrates. Anti-thrombin aptamers with 5'-end amine groups were chemically grafted onto the surface-modified Si substrates through amide bond formation. Atomic force microscopic (AFM) analyses confirmed the successful immobilization of anti-thrombin aptamers on Si-NWs and their binding with thrombin samples. The anti-thrombin aptamers bound to Si-NWs through the linker appeared to have a mean height of approx. 4 nm and the thrombin/aptamer complex to have a mean height of approx. 8 nm. Fluorescence micrographs visualized the FITC-labeled thrombin after binding to anti-thrombin aptamers immobilized on Si-NWs. Furthermore, the anti-thrombin Si-NW FET biosensor was successfully applied to the real-time detection of electronic signals during and after binding with a thrombin sample at a concentration of approx. 330 pmol l(-1) and the thrombin in blood samples.