Hyung Jun Ahn

Dark quenched matrix metalloproteinase fluorogenic probe for imaging osteoarthritis development in vivo.

The early detection of osteoarthritis (OA) is currently a key challenge in the field of rheumatology. Biochemical studies of OA have indicated that matrix metalloproteinase-13 (MMP-13) plays a central role in cartilage degradation. In this study, we describe the potential use of a dark-quenched fluorogenic MMP-13 probe to image MMP-13 in both in vitro and rat models. The imaging technique involved using a MMP-13 peptide substrate, near-infrared (NIR) dye, and a NIR dark quencher. The results from this study demonstrate that the use of a dark-quenched fluorogenic probe allows for the visual detection of MMP-13 in vitro and in OA-induced rat models. In particular, by targeting this OA biomarker, the symptoms of the early and late stages of OA can be readily monitored, imaged, and analyzed in a rapid and efficient fashion. We anticipate that this simple and highly efficient fluorogenic probe will assist in the clinical management of patients with OA, not only for early diagnosis but also to assess individual patient responses to new drug treatments.

Nanobubbles from Gas-Generating Polymeric Nanoparticles: Ultrasound Imaging of Living Subjects†

Ultrasound (US) is a noninvasive biomedical imaging modality that is widely available, inexpensive, safe, and provides real-time imaging and diagnosis. US contrast enhancement improves the image quality because of acoustic impedance mismatch, which can occur when a liquid or gas is encapsulated within shell materials or when liquid or solid particles with a high density difference are brought together. Microbubbles were primarily used as US contrast agents for blood flow imaging, however, development of metabolic or molecular US imaging that employs microbubbles has been limited because sub-microbubbles or nanoparticles that are appropriate for tissue penetration have poor echogenic sensitivity, while nanoparticles can not be visualized at the resolution of US imaging instruments (typically 50 to 100 mm). Recently, nanoparticles have been employed in numerous biomedical applications. Nanoparticles bypass the reticuloendothelial system, and allow prolonged circulation and passive localization within tumor vasculature by tissue extravasation and enhanced permeation and retention (EPR). Moreover, nanoparticles can be modified by addition of ligands that increase nanoparticle affinity toward specific target sites, such as tumors. In addition, the insertion of protease cleavage sites can provide activatable imaging probes, thus allowing simultaneous molecular imaging and therapeutics. The need to use nanosized carriers for in vivo imaging and therapy has led to the development of laser-induced photoacoustic US and to the exploration of materials-based approaches that employ perfluorocarbonencapsulated lipid-based nanoparticles and polymeric micelles. Herein, we describe the generation of nanobubbles, which can be imaged by US and are derived from gas-generating polymeric nanoparticles (GGPNPs). The proposed mechanism involves localization of echogenic GGPNPs in a tumor and coalescence of generated nanobubbles, followed by fusion of nanobubbles into microbubbles. Significantly, we produced GGPNP nanobubbles without encapsulation of a gas precursor (perfluorocarbon). Instead, in our system, the GGPNP carbonate side chain is degraded to form carbon dioxide. The mechanism appears to involve diffusion of water into the GGPNPs, cleavage of the carbonate side chains by hydrolysis, and formation of carbon dioxide nanobubbles on the GGPNP surface, followed by expansion or coalescence of nanobubbles into microbubbles. The resulting microbubbles exhibit resonance under a US field (Figure 1a). Polyesters with carbonate side chains (poly(BL-PO)) were synthesized by ring-opening copolymerization of gbutyrolactone and propylene oxide using samarium diiodide as initiator (Figure S1b in the Supporting Information). The carbonate side chains were created by conjugation with hydroxy groups of hydroxy g-butyrolactone and cholesteryl chloroformate or ethyl chloroformate (Figure S1–S3 in the Supporting Information). The copolymerization ratios of cholesteryl carbonate g-butyrolactone and ethyl carbonate g-butyrolactone were varied with respect to that of propylene oxide. The resulting chemical structures are shown in Figure 1b. The initial feed ratios of three monomers were 1:2:1 for poly(BL-PO). The actual ratio of three monomer units in the poly(BL-PO) was confirmed as 1:1.74:0.81 by H NMR spectroscopy (see Figure S4 in the Supporting Information). This result shows that the compositions of the copolymers were well-matched to the initial feed ratio. The presence of the cholesteryl group means that the relative proportions of cholesteryl carbonate and ethyl carbonate determine the physical stability of the polymer. The hydrolyzed particles produce biocompatible water, carbon dioxide, cholesterol, and ethanol. To prove the biocompatibility of our particles, we examined their effect on cell viability. At a concentration of up to 1 mgmL , our particles were not cytotoxic (see Figure S8 in the Supporting Information). Nanoparticles generated from poly(BL-PO) (1:2:1) were termed GGPNPs. Dynamic light scattering (DLS) indicated that the average GGPNP diameter was 581 nm (Figure S6 in the Supporting Information), and transmission electron microscopy (TEM) indicated that the GGPNP morphology was spherical and the diameter was less than 500 nm (Figure 1c). The size appeared larger by DLS probably [*] Dr. E. Kang, H. S. Min, Dr. H. J. Ahn, Dr. I. C. Yoon, Dr. K. Choi, Dr. K. Kim, Dr. I. C. Kwon Biomedical Research Center Korea Institute of Science and Technology 39-1 Hawolgok-dong, Seongbuk-gu, Seoul 136 791 (Korea) Fax: (+82)2-958-5909 E-mail: ikwon@kist.re.kr

Chimeric Capsid Protein as a Nanocarrier for siRNA Delivery: Stability and Cellular Uptake of Encapsulated siRNA

For the efficient cytoplasmic delivery of siRNA, we designed a chimeric capsid protein composed of a capsid shell, integrin targeting peptide, and p19 RNA binding protein. This recombinant protein assembled into a macromolecular container-like structure with capsid shell and provided a nanocarrier for siRNA delivery. Our capsid nanocarriers had dual affinity both for siRNA within the interior and integin receptors on the exterior, and the capsid shell structure allowed the encapsulated siRNAs to be protected from the external nucleases, leading to the enhanced stability of siRNA in serum conditions. The capsid nanocarriers could complex with siRNA in a size-dependent and sequence-independent manner and showed the pH-dependent complexing/dissocation behaviors with siRNA. Moreover, RGD peptides on the exterior surface of the capsid shell enabled the capsid nanocarriers to deliver siRNA into the cytosol of the target cells. Here, we demonstrated the superior efficiency of our siRNA/capsid nanocarrier complexes in RFP gene silencing, compared to untreated cells. These results provide an alternative approach to enhancing the stability of siRNA as well as to achieving targeted siRNA delivery.

Designed Nanocage Displaying Ligand-Specific Peptide Bunches for High Affinity and Biological Activity

Protein-cage nanoparticles are promising multifunctional platforms for targeted delivery of imaging and therapeutic agents owing to their biocompatibility, biodegradability, and low toxicity. The major advantage of protein-cage nanoparticles is the ability to decorate their surfaces with multiple functionalities through genetic and chemical modification to achieve desired properties for therapeutic and/or diagnostic purposes. Specific peptides identified by phage display can be genetically fused onto the surface of cage proteins to promote the association of nanoparticles with a particular cell type or tissue. Upon symmetrical assembly of the cage, peptides are clustered on the surface of the cage protein in bunches. The resulting PBNC (peptide bunches on nanocage) offers the potential of synergistically increasing the avidity of the peptide ligands, thereby enhancing their blocking ability for therapeutic purposes. Here, we demonstrated a proof-of-principle of PBNCs, fusing the interleukin-4 receptor (IL-4R)-targeting peptide, AP-1, identified previously by phage display, with ferritin-L-chain (FTL), which undergoes 24-subunit assembly to form highly stable AP-1-containing nanocage proteins (AP1-PBNCs). AP1-PBNCs bound specifically to the IL-4R-expressing cell line, A549, and their binding and internalization were specifically blocked by anti-IL-4R antibody. AP1-PBNCs exhibited dramatically enhanced binding avidity to IL-4R compared with AP-1 peptide, measured by surface plasmon resonance spectroscopy. Furthermore, treatment with AP1-PBNCs in a murine model of experimental asthma diminished airway hyper-responsiveness and eosinophilic airway inflammation along with decreased mucus hyperproduction. These findings hold great promise for the application of various PBNCs with ligand-specific peptides in therapeutics for different diseases, such as cancer.

Tumor-specific delivery of siRNA using supramolecular assembly of hyaluronic acid nanoparticles and 2b RNA-binding protein/siRNA complexes

Anticancer therapeutics delivering exogenous siRNA have been explored to suppress the tumor-associated genes, but several limitations of siRNA delivery such as tumor-targeted delivery, controlled siRNA release at the sites of interest, or instabilities of siRNA in physiological fluids should be preferentially addressed for its clinical applications. As an attempt to meet these criteria, we designed a supramolecular assembly, which was composed of cholesterol-bearing hyaluronic acid (HA-Chol) conjugates and 2b RNA-binding protein (2b)/siRNA complexes. In contrast to the traditional siRNA polyplexes using electrostatic interactions, HA-Chol nanoparticles, as a results of self-assembly of HA-Chol conjugates, provide the hydrophobic core that acts as the container for 2b protein/siRNA complexes, where a high affinity of 2b protein for siRNA could neutralize the negative-charged siRNA. Here, we investigated the potential of HA-Chol/2b/siRNA complexes as the siRNA carriers that provide encapsulation, protection, and targeted delivery of siRNA. The HA-Chol nanoparticles could selectively deliver 2b protein/siRNA complexes to the tumor cells with up-regulated CD44 receptors and suppress the expression of target gene. The pH-associated binding properties of siRNA for 2b proteins allowed the controlled release of siRNA in the endocytic compartments, and ultimately the released siRNA could obtain the RNAi acitivities in the cells, whereas the encapsulated 2b proteins still stayed within the HA-Chol nanoparticles. Our delivery systems demonstrate the promising potential of the efficient siRNA carriers in the anticancer therapeutic applications.

Systemic delivery of siRNA by chimeric capsid protein: Tumor targeting and RNAi activity in vivo

Recently, we reported that a chimeric capsid protein assembled into a macromolecular container-like structure with capsid shell and the resulting siRNA/capsid nanocarrier complexes efficiently suppressed RFP gene expression in the cell culture system. To extend RNAi to the in vivo applications, we here demonstrated that the siRNA/capsid nanocarrier complexes could have tumor-specific targeting ability in vivo as well as the increased stability of siRNA during body circulation. When systemically administered, our siRNA/capsid nanocarrier complexes delivered siRNA to tumor tissues and efficiently suppressed RFP gene expression in tumor-bearing mice. The enhanced longevity of siRNA in vivo could be explained by shielding effect derived from the capsid shell, where the encapsulated siRNAs are protected from nucleases in plasma. The multivalent RGD peptides on shell surface, as a result of self-assembling of capsid protein subunits, showed efficient delivery of siRNA to the tumor tissues in vivo, due to the RGD-mediated binding to integrin receptors overexpressed on tumor cells. Moreover, the prolonged in vivo circulation time of our siRNA/capsid nanocarrier complexes increased the potential to serve as siRNA carriers for optimal in vivo RNAi. These results provide an alternative approach to systemically deliver siRNA to the tumor sites as well as to enhance the stability of siRNA in vivo. Therefore, our results revealed the promising potential of our capsid nanocarrier system as a therapeutic siRNA carrier for cancer treatment.

Design of a platform technology for systemic delivery of siRNA to tumours using rolling circle transcription

For therapeutic applications of siRNA, there are technical challenges with respect to targeted and systemic delivery. We here report a new siRNA carrier, RNAtr NPs, in a way that multiple tandem copies of RNA hairpins as a result of rolling circle transcription (RCT) can be readily adapted in tumour-targeted and systemic siRNA delivery. RNAtr NPs provide a means of condensing large amounts of multimeric RNA transcripts into the compact nanoparticles, especially without the aid of polycationic agents, and thus reduce the risk of immunogenicity and cytotoxicity by avoiding the use of synthetic polycationic reagents. This strategy allows the design of a platform technology for systemic delivery of siRNA to tumour sites, because RCT reaction, which enzymatically generates RNA polymers in multiple copy numbers at low cost, can lead to directly accessible routes to targeted and systemic delivery. Therefore, RNAtr NPs suggest great potentials as the siRNA therapeutics for cancer treatment.

A monitoring method for Atg4 activation in living cells using peptide-conjugated polymeric nanoparticles

To date, several principal methods are presently used to monitor the autophagic process, but they have some potential experimental pitfalls or limitations that make them not applicable to living cells. In order to improve on the currently developed detection methods for autophagy, we report here fluorescent peptide-conjugated polymeric nanoparticles loaded with a lysosome staining dye in their core. The fluorescent peptide is designed to be specifically cleaved by the Atg4 cysteine protease, which plays a crucial role in autophagy activation. In this study, we demonstrate that peptide-conjugated polymeric nanoparticles can be used to visualize Atg4 activity in both cell-free and cell culture systems. The fluorescence imaging of cells incubated with nanoparticles demonstrates that Atg4 activity is activated in the autophagy-induced conditions, but suppressed in the autophagy-inhibited conditions. These results indicate that Atg4 activity is correlated with autophagic flux through its own regulatory pathway. Therefore, our strategy provides an alternative detection method that can clearly distinguish between an “autophagy active” and “autophagy inactive” state in cultured cells. As our nanoparticles are highly cell-permeable and biocompatible, this detection system has general applicability to living cells and can be extended to cell-based screening to evaluate newly developed compounds.

Optimization of matrix metalloproteinase fluorogenic probes for osteoarthritis imaging.

Among the classical collagenases, matrix metalloproteinase-13 (called MMP-13, collagenase-3) is one of the most important components for cartilage destruction of osteoarthritis (OA) developments. Despite many efforts, the detection methods of MMP-13 activity have been met with limited success in vivo, in part, due to the low sensitivity and low selectivity by homology of MMP family. Previously, we demonstrated the use of strongly dark-quenched fluorogenic probe allowed for the visual detection of MMP-13 in vitro and in OA-induced rat models. In this study, we described the optimization of MMP-13 fluorogenic probe for OA detection in vivo. Three candidate probes demonstrated recovered fluorescent intensity proportional with MMP-13 concentrations, respectively; however, Probe 2 exhibited both high signal amplification and selective recognition for MMP-13, not MMP-2 and MMP-9 in vitro. When Probe 2 was applied to OA-induced rat models, clear visualization of MMP-13 activity in OA-induced cartilage was obtained. Optimized MMP-13 fluorogenic probe can be applied to detect and image OA and have potential for evaluating the in vivo efficacy of MMP-13 inhibitors which are being tested for therapeutic treatment of OA.

The incorporation of GALA peptide into a protein cage for an acid-inducible molecular switch.

Caged proteins have been utilized as a biological container in a wide range of applications from material science to biomedicine, and GALA peptide has been known to undergo coil-to-helix transition upon the increased acidity. In this study, GALA synthetic peptide is incorporated to cage protein by genetic modification. Our engineered caged scaffold retains intact at the physiological pH but dissociate completely at pH 6.0, and the dissociated subunits are re-assembled simply by neutralization to biological pH. This acid-induced dissociation has the potential as molecular switch in vivo as well as in vitro so that the acid-sensitive caged proteins are applicable to drug delivery system for acidic target sites such as tumor. Since our design depends on the conformational transition of GALA peptide, not on removal of characteristic interface observed only in viral capsid-like protein, non-viral caged proteins can also be engineered to have molecular switching function. Therefore, this design for acid-sensitive scaffold would broaden the width of applications in nanotechnology including biomimetic material synthesis and biomedicine.