Kitae Park

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.

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–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.

Target-Specific Gene Silencing of Layer-by-Layer Assembled Gold–Cysteamine/siRNA/PEI/HA Nanocomplex

Target-specific intracellular delivery of small interfering RNA (siRNA) is regarded as one of the most important technologies for the development of siRNA therapeutics. In this work, a cysteamine modified gold nanoparticles (AuCM)/siRNA/polyethyleneimine (PEI)/hyaluronic acid (HA) complex was successfully developed using a layer-by-layer method for target-specific intracellular delivery of siRNA by HA receptor mediated endocytosis. Atomic force microscopic and zeta potential analyses confirmed the formation of a AuCM/siRNA/PEI/HA complex having a particle size of ca. 37.3 nm and a negative surface charge of ca. -12 mV. With a negligible cytotoxicity, AuCM/siRNA/PEI/HA complex showed an excellent target-specific gene silencing efficiency of ca. 70% in the presence of 50 vol % serum, which was statistically much higher than that of siRNA/Lipofectamine 2000 complex. In the competitive binding tests with free HA, dark-field bioimaging and inductively coupled plasma-atomic emission spectroscopy confirmed the target-specific intracellular delivery of AuCM/siRNA/PEI/HA complex to B16F1 cells with HA receptors. Moreover, the systemic delivery of AuCM/siRNA/PEI/HA complex using apolipoprotein B (ApoB) siRNA as a model drug resulted in a significantly reduced ApoB mRNA level in the liver tissue. Taken together, AuCM/siRNA/PEI/HA complex was thought to be developed as target-specific siRNA therapeutics for the systemic treatment of various liver diseases.

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.

Enhanced Production of Oceanic Dimethylsulfide Resulting from CO2-Induced Grazing Activity in a High CO2 World

Oceanic dimethylsulfide (DMS) released to the atmosphere affects the Earths radiation budget through the production and growth of cloud condensation nuclei over the oceans. However, it is not yet known whether this negative climate feedback mechanism will intensify or weaken in oceans characterized by high CO(2) levels and warm temperatures. To investigate the effects of two emerging environmental threats (ocean acidification and warming) on marine DMS production, we performed a perturbation experiment in a coastal environment. Two sets of CO(2) and temperature conditions (a pCO(2) of ∼900 ppmv at ambient temperature conditions, and a pCO(2) of ∼900 ppmv at a temperature ∼3 °C warmer than ambient) significantly stimulated the grazing rate and the growth rate of heterotrophic dinoflagellates (ubiquitous marine microzooplankton). The increased grazing rate resulted in considerable DMS production. Our results indicate that increased grazing-induced DMS production may occur in high CO(2) oceans in the future.

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.

Cationic solid lipid nanoparticles derived from apolipoprotein-free LDLs for target specific systemic treatment of liver fibrosis

Low density lipoprotein (LDL) plays an important role in transporting fat molecules including cholesterols in the body. In this work, cationic solid lipid nanoparticles (CSLNs), bioinspired and reconstituted from natural LDLs, were designed and applied to target specific systemic delivery of connective tissue growth factor siRNA (siCTGF) for the treatment of liver fibrosis. They could form a nuclease-resistant stable nano-complex with siRNA, which was efficiently internalized into cells achieving targeted gene silencing in the presence of serum with a remarkably low cytotoxicity. After intravenous injection, CSLN/siCTGF complex was target specifically delivered to the liver and resulted in a significant reduction in collagen content and pro-fibrogenic factors like tumor necrosis factor alpha (TNF-α), transforming growth factor beta (TGF-β), interleukin-6 (IL-6), and CTGF with the dramatic improvement of patho-physiological symptoms in liver fibrosis model rats. The bio-distribution study by fluorescence bioimaging and single-photon emission computed tomography (SPECT) confirmed the target specific delivery and accumulation of CSLN/siCTGF complexes to the liver tissues.

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.