Yoon Young Kang

Current preclinical small interfering RNA (siRNA)-based conjugate systems for RNA therapeutics.

Recent promising clinical results of RNA therapeutics have drawn big attention of academia and industries to RNA therapeutics and their carrier systems. To improve their feasibility in clinics, systemic evaluations of currently available carrier systems under clinical trials and preclinical studies are needed. In this review, we focus on recent noticeable preclinical studies and clinical results regarding siRNA-based conjugates for clinical translations. Advantages and drawbacks of siRNA-based conjugates are discussed, compared to particle-based delivery systems. Then, representative siRNA-based conjugates with aptamers, peptides, carbohydrates, lipids, polymers, and nanostructured materials are introduced. To improve feasibility of siRNA conjugates in preclinical studies, several considerations for the rational design of siRNA conjugates in terms of cleavability, immune responses, multivalent conjugations, and mechanism of action are also presented. Lastly, we discuss lessons from previous preclinical and clinical studies related to siRNA conjugates and perspectives of their clinical applications.

Submicron-sized hydrogels incorporating cyclic dinucleotides for selective delivery and elevated cytokine release in macrophages.

UNLABELLED Despite the emerging evidences supporting the potential of cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) as a vaccine adjuvant, few properly designed micro-/nanocarriers for the delivery of cyclic dinucleotides have been developed. In this study, we formulated cGAMP within linear polyethyleneimine (LPEI)/hyaluronic acid (HA) hydrogels via inverse water-in-oil (W/O) emulsion/crosslinking. Spherical and cationic LPEI/HA hydrogels (LH gels) with a size of 455.3±3.1nm and a surface charge of 48.7±3.7mV were selectively and efficiently delivered into phagocytic macrophage cells, which are one type of antigen-presenting cells (APCs), but not into non-phagocytic fibroblast cells. LH gels incorporating cGAMP (LH/cGAMP gels) elicited excellent induction of the cytokines interferon-β (IFN-β) and interleukin-6 (IL-6). In particular, the amount of IFN-β released by LH hydrogels was significantly increased by 2.5-fold compared to that released by conventional cationic liposomes, such as Lipofectamine. In addition, fabricated LH gels showed superior biocompatibility in phagocytic cell lines and primary bone marrow-derived macrophages (BMDMs). After intramuscular injection with ovalbumin into C57BL/6 mice, LH/cGAMP gels exhibited significantly elevated levels of anti-ovalbumin total IgG in serum and IFN-β mRNA in spleens. Thus, the newly designed cGAMP-incorporating hydrogels can serve as safe and potent adjuvants for vaccination and immunotherapy. STATEMENT OF SIGNIFICANCE Since cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) was first found as a second messenger of immune signaling in human systems in February 2013 (Science, 15, 826), several scientific studies have been reported related to the potential of cGAMP as a vaccine adjuvant or additive for immunotherapy. However, only naked cGAMP without carriers were studied via intramuscular or intranasal administration so far. In our study, we first investigated the feasibility of polymeric hydrogels incorporating cGAMP in terms of selective uptake into phagocytic antigen presenting cells (APCs), induction of cytokines, production of target antibodies, and biocompatibility for vaccination and immunotherapy in vitro and in vivo. Therefore, we believe this manuscript would be of great interest to the biomaterial communities especially who are studying immunotherapy.

Evaluation of the Enhanced Antioxidant Activity of Curcumin within Exosomes by Fluorescence Monitoring

In this study, we compared the antioxidant activities of curcumin (Cur) and a Cur formulation using a fluorescence analysis assay. The Cur formulation was prepared by a simple incorporation of Cur into exosomes (EXO) to produce Cur/EXOs. Free Cur had a low fluorescence intensity in aqueous solution because of its poor stability as a result of its autoxidation, whereas a significantly higher fluorescence intensity was observed for Cur/EXOs. Compared to free Cur, the increased level of intact Cur in EXOs allowed for enhanced antioxidant activity in H2O2 scavenging activity and DPPH assays. Compared to Cur at high concentration (200 μM), Cur/ EXOs were significantly less cytotoxic. The antioxidant activity of Cur or Cur/EXOs in cells could be easily demonstrated by monitoring decreases in their fluorescence intensity. Following subcutaneous injection, the fluorescence intensities of Cur/EXOs were much higher than that of Cur, suggesting that Cur/EXOs improve Cur stability in vivo. Taken together, we have demonstrated the superiority of Cur/EXOs over free Cur in terms of aqueous stability and antioxidant activity using fluorescence monitoring both in vitro and in vivo.

Complexation of curcumin with 2-aminoethyl diphenyl borate and implications for spatiotemporal fluorescence monitoring

In this study, we successfully determined spatiotemporal distribution of curcumin in mice via simple and fast fluorescence detection of native curcumin and stabilized curcumin. We used 2-aminoethyl diphenyl borate (DPBA) as a stabilizer of curcumin, which binds to curcumin and enhances its aqueous stability. After intravenous injection, curcumin and DPBA-curcumin complexes showed similar fluorescence intensities in the brain, pancreas, lungs, and kidneys at 15min. However, stabilized DPBA-curcumin complexes exhibited much stronger fluorescent signals at metabolically active sites such as liver tissues than native curcumin. After incubation for 1-3h, native curcumin showed significantly rapid reduction of fluorescent signals, compared to DPBA-curcumin complexes, probably due to degradation and reduction. In addition, complicate extraction procedures inhibited precise fluorescent monitoring of unstable curcumin, which result in different biodistribution of curcumin before and after extraction. Direct fluorescent monitoring could allow evaluation of in vivo distribution and fate of curcumin, which could be also applied to diverse natural polyphenols with fluorescent signals.

Complementary analysis of curcumin biodistribution using optical fluorescence imaging and mass spectrometry

In this study, a complementary analysis was performed to improve the precision of the determination of in vivo curcumin biodistribution after intravenous administration. Overall, similar curcumin biodistribution profiles were obtained using optical fluorescence imaging and mass spectrometry. Poor curcumin accumulation was observed in the heart, spleen, and kidney. However, noticeable accumulation of curcumin in the brain was only observed using fluorescence imaging, probably owing to the insufficient extraction of curcumin from the brain for mass spectrometry. In addition, an exact and reliable measurement of curcumin accumulation in tissues such as the liver, gallbladder, and pancreas was performed using mass spectrometry because of high autofluorescence. Taken together, complementary analysis using optical fluorescence imaging and mass spectrometry allowed the precise determination of curcumin in each tissue. Furthermore, this complementary analytical strategy could be used to elucidate the in vivo distribution of a wide range of fluorescent polyphenols.

Comparative evaluation of cell- and serum-derived exosomes to deliver immune stimulators to lymph nodes

To determine whether exosomes are efficient carriers for immune stimulating molecules into lymph nodes, comparative studies of exosomes (EXOs) derived from different origins (cells and serums) in terms of physicochemical properties and delivery efficiency were performed. Serum-derived EXOs were of a preferable size and generated higher yields than RAW264.7 cell-derived exosomes (RAW-EXO). In particular, fetal bovine serum-derived exosomes (bo-EXO), with a size below 50 nm, were delivered not only to surface zones (subcapsular sinus (SCS) macrophage zone) but also to inner paracortex zones (T cell zone) of lymph nodes, which allowed an efficient delivery of immune stimulating molecules to antigen presenting cells and T cells. The encapsulation of immune stimulating biomolecules (monophosphoryl lipid A (MPLA) and CpG oligodeoxynucleotides (CpG ODN)) within EXOs greatly increased intracellular delivery to macrophages via phagocytic pathways, which induced higher TNF-α and IL-6 secretion than free MPLA and free CpG ODN. MPLA-incorporated exosomes activated and differentiated T cells after subcutaneous injection, which elevated cytokine IFN-γ and TNF-α induction for CD3+ T cells. Taken together, bo-EXOs might serve as efficient carrier systems of immune stimulators to lymph nodes for desired immune responses.

Enzymatically Produced miR34a Nanoparticles for Enhanced Antiproliferation Activity

Among a range of functional forms of RNA, microRNAs (miRNAs) have held a great promise for their natural ability to influence cellular behavior by regulating gene expression. In particular, it is revealed that miR34a has a function as a tumor suppressor by regulating silent information regulator 1 expression, causing antiproliferation of tumor. To utilize functional miR34a as an effective cancer therapeutics, an enzymatic approach to synthesize miR34a nanoparticles is adapted. The enzymatically self‐assembled miR34a nanoparticles have a high cargo capacity and a suitable size for cellular uptake. Indeed, the miR34a nanoparticles are delivered successfully to PC3 cells, human prostate cancer cells, and suppress proliferation and migration of those cells. These results suggest that the enzymatically produced miR34a nanoparticles can function as an effective tumor suppressor. Furthermore, it is expected that the enzymatic synthesis of miRNA nanoparticles can be adapted universally for delivery of other functional miRNAs as well.

CpG incorporated DNA microparticles for elevated immune stimulation for antigen presenting cells

As emerging evidence supports the immune stimulating capability of the CpG oligodeoxynucleotides (ODN), CpG-based adjuvants have been widely used. For efficient induction of immune responses, current issues affecting the use of nucleic acid-based adjuvants, e.g. stability in physiological conditions, delivery to immune cells, and successful release within the phagolysosome, should be addressed. Here, we present CpG-based DNA microparticles (DNA-MPs) fabricated by complementary rolling circle amplification (cRCA) as adjuvants for enhancing immune response and production of selective antibody production. Using cRCA method, the sizes of CpG-based DNA-MPs were finely controlled (0.5 and 1 μm) with superior and provided mismatched single stranded form of CpG ODN region for specific cleavage site by DNase II within the phagolysosome. Fabricated CpG-based 1 μm DNA-MPs (DNA-MP-1.0) were successfully internalized into primary macrophages and macrophage cell line (RAW264.7 cells), and elicited superior cytokine production e.g. TNF-α and IL-6, compared to conventional CpG ODNs. After in vivo administration of DNA-MP-1.0 with model antigen ovalbumin (OVA), significantly elevated OVA-specific antibody production was observed. With this in mind, DNA-MP-1.0 could serve as a novel type of adjuvant for the activation of macrophages and the following production of selective antibodies without any noticeable toxicity in vitro and in vivo.

Effects of curcumin-/boron-basedcompound complexation on antioxidant and antiproliferation activity

Simple and reproducible formulation strategies are needed to improve the bio-availability of curcumin. In this study, curcumin was successfully complexed with two boron-based compounds: 2-aminoethyl diphenyl borate (DPBA) and bortezomib (BTZ; Velcade®). In reverse-phase high-performance liquid chromatography, DPBA/curcumin complexes (DPBA/cur) showed delayed elution times compared to those of free curcumin. The UV–visible absorbance peak of DPBA/cur and BTZ and curcumin complexes (BTZ/cur) appeared redshifted. DPBA complexation has a negligible effect on the antioxidant and antiproliferation properties of curcumin for two types of cancer cells: MCF-7 and A549. Thus, curcumin complexation with boron-based compounds could be a method to enhance in vivo stability without loss of bioactivity (i.e., antioxidant and antiproliferation effects).

Analysis of the biodistribution of natural products in mice by using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Natural products originating from plants have various beneficial pharmacological effects, such as anticancer, antimicrobial, and anti-inflammatory activities, while being nontoxic. Therefore, tremendous efforts have been invested in understanding their bioactivities in the body to facilitate therapeutic target validation. However, such research is still challenging for certain natural products, such as flavonoids, which are rapidly metabolized in and eliminated from the human body. To investigate the bioactivities of such products, particularly in certain tissues, it is necessary to understand their biodistribution in vivo. In this respect, reliable analytical methods with simple and efficient procedures for the in vivo evaluation of natural small molecules are urgently required. In particular, mass spectrometry (MS) can be effectively used to analyze small molecules after tissue extraction, as MS has various advantages including accuracy, simplicity, and high sensitivity. Herein, we report the biodistribution of a natural small molecule by using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). After intravenously injecting gomisin H into a mouse as a model natural product, it was extracted from each organ and then analyzed by MALDI-TOF MS. The analysis showed that gomisin H accumulated mainly in the liver and relatively large amounts of the product existed in the kidney and brain compared to those in other tissues.