Yong-beom Lim

Biodegradable Polyester, Poly[α-(4-Aminobutyl)-l-Glycolic Acid], as a Non-Toxic Gene Carrier

AbstractPurpose. The aim of this study was to develop a non-toxic polymericgene carrier. For this purpose, biodegradable cationic polymer,poly[α-(4-aminobutyl)-l-glycolic acid] (PAGA) was synthesized. PAGA wasdesigned to have ester linkage because polyesters usually showbiodegradability. Methods. Degradation of PAGA in an aqueous solution was followedby matrix-assisted laser desorption/ionization time-of-flight mass spectrometry(MALDI-TOF MS). PAGA/DNA complexes were characterizedby gel electrophoresis, atomic force microscopy (AFM), dynamiclight scattering (DLS). The transfection was measured by using the β-galactosidase reporter gene. Results. PAGA was degraded in aqueous solution very quickly andthe final degradation product was a monomer (l-oxylysine). Formationof self-assembling biodegradable complexes between PAGA and DNAat a charge ratio 1:1 (+/−) was confirmed by gel band shift assay andAFM. In these studies, controlled release of DNA from the complexescould be seen. The complexes showed about 2-fold higher transfectionefficiency than DNA complexes of poly-l-lysine (PLL), a structuralanalogue of PAGA, which is the most commonly used poly-cation forgene delivery. The polymer did not show cytotoxicity, possibly becauseof its degradability and the biocompatibility of the monomer. Conclusions. The use of the biodegradable poly-cation, PAGA, as aDNA condensing agent will be useful in safe gene delivery.

Rod–coil block molecules: their aqueous self-assembly and biomaterials applications

Past decades have witnessed rapidly growing interest in nanometer-sized structures, which have great potential to be used in a variety of applications, such as electronics, sensors, coatings, and biomaterials. Supramolecular chemistry in particular has been actively applied to the development of such materials. Nanostructures can readily be accessed using bottom-up supramolecular approaches as they are composed of small molecules (supramolecular building blocks) requiring fewer steps to synthesize. Among various types of supramolecular building blocks, rod–coil molecules, due to their anisotropic molecular shape, are well-suited for tailoring nanostructural properties such as size and shape. This Feature Article highlights the self-assembly of rod–coil molecules in aqueous solution and introduces an emerging approach to the application of rod–coil nanostructures in biomaterials applications.

Filamentous Artificial Virus from a Self‐Assembled Discrete Nanoribbon

The creation of virus-like nanomaterials (artificial viruses) has been the subject of intensive research in the field of gene/ drug delivery because of their huge therapeutic potential. Some progress has been made in the field; however, compared to the natural viruses, which are evolution-tailored experts in gene delivery, the synthetic system is still far behind the natural system. One of the critical reasons for this shortcoming is that the size and shape of the artificial viruses, among the most important determinants of their efficiency, are very difficult to control. As the polyanionic nature of nucleic acids (DNA and RNA) prevents them from crossing the same charged cytoplasmic membrane barrier, a vector system is necessary to neutralize their charge and to install other functions, such as cell binding, endosome escape, and nucleus localization. Although there are serious safety concerns, such as immunogenicity and carcinogenesis, in the use of viral vectors, they are still far more widely used for gene therapy than nonviral vectors (artificial viruses) because of their higher efficiency. The basic principle of artificial virus formation is a condensation reaction which is induced by the attraction between oppositely charged molecules (for example, between cationic polymer and DNA). However, this polyion coupling generally leads to the formation of huge nanoaggregates that are highly heterogeneous in size and shape, and is uncontrollable in most cases. In regard to the size issue, a certain optimal size exists for the nanoparticulate delivery systems to work best. The shape of the nanoparticle can also be a crucial factor. For example, sometimes the cylindrical (that is, filamentous) nanostructure has certain advantages over the spherical one in that it persists longer in vivo, which might explain why many filamentous viruses exist in nature. Therefore, it is imperative to find a general strategy to control the size and shape of artificial viruses. Recent advances in supramolecular chemistry have made it possible, by rational design of building blocks, to control supramolecular architectures from spherical micelles, cylindrical micelles, vesicles, and toroids to nanotubes. Inspired by this elaboration, we envisioned that the control of the size and shape of artificial viruses would be possible if the preorganized supramolecular architectures used were robust polycationic scaffolds that remain unchanged after the formation of an interpolyelectrolyte complex (IPC) with negatively charged nucleic acids. Herein, we report a potentially generalizable strategy to create an artificial virus that memorizes the size and shape of its precursor. By using a preorganized supramolecular nanostructure as a template, we show that well-defined, discrete artificial viruses can be elaborated after IPC formation between the nanostructure and nucleic acids. This controlled feature and appropriate surface functionalization with multivalent carbohydrate ligands make the artificial virus highly efficient in the intracellular delivery of genes and drugs. With the aim of constructing filament-shaped, discrete artificial viruses, a b-sheet peptide-based supramolecular building block (Glu-KW) was designed (Figure 1a). To our knowledge the filament-shaped artificial virus is unprecedented. It has been shown that the combination of hydrophobic and electrostatic interactions produced by the alternating placement of hydrophobic and charged amino acids in b-sheet peptides promotes b-sheet interaction and subsequent self-assembly into bilayered filamentous nanostructures (b ribbon). Coupling of hydrophilic segments, such as polyethylene glycol, hydrophilic peptides, or carbohydrates, to b-sheet peptides has been reported to stabilize b-ribbon nanostructures by suppressing lateral aggregate formation. The Glu-KW structure is characterized by a b-sheetforming self-assembly segment, two linker segments, a nucleic acid-binding cationic segment, and a carbohydrate ligand segment. The b-sheet peptide segment consists of tryptophan–lysine–tryptophan–aspartic acid repeats, the amino acid configuration of which promotes b-sheet formation. The linker segments are designed to be flexible and nonionic by using glycine and serine residues. The eight lysine residues are placed between two linker segments to shield the cationic segment, upon self-assembly, from the b-ribbon surface. dGlucose is positioned at the outermost part of Glu-KW to render the b-ribbon surface charge-neutral and to increase the chances of b-ribbon binding to the cell surface through multivalent interactions with cell-surface glucose transporters (GLUTs). GLUTs are present in nearly all mammalian cells and overexpressed in most cancer cells. To address the question of whether Glu-KW forms bsheet-mediated nanostructures, the self-assembly of Glu-KW was investigated by circular dichroism (CD) and transmission [*] Dr. Y.-b. Lim, E. Lee, Y.-R. Yoon, Prof. M. Lee Center for Supramolecular Nano-Assembly and Department of Chemistry Yonsei University, Seoul 120-749 (Korea) Fax: (+82)2-393-6096 E-mail: mslee@yonsei.ac.kr Homepage: http://csna.yonsei.ac.kr

Stabilization of an α Helix by β‐Sheet‐Mediated Self‐Assembly of a Macrocyclic Peptide

Protein roll call: Peptide-based building blocks, in which both an alpha-helix-forming segment and a beta-sheet segment are located within a single macrocyclic structure, self-assemble into alpha-helix-decorated artificial proteins. This approach provides a starting point for developing artificial proteins that can modulate alpha-helix-mediated interactions occurring in a multivalent fashion.

Comparative studies on the genotoxicity and cytotoxicity of polymeric gene carriers polyethylenimine (PEI) and polyamidoamine (PAMAM) dendrimer in Jurkat T-cells.

A safe alternative to the viral system used in gene therapy is a nonviral gene delivery system. Although polyethylenimine (PEI) and polyamidoamine (PAMAM) dendrimer are among the most promising gene-carrier candidates for efficient nonviral gene delivery, safety concerns regarding their toxicity remain. The aim of this study was to scrutinize the underlying mechanism of the cytotoxicity and genotoxicity of PEI (25 kDa) and PAMAM (G4). To our knowledge, this is the first study to explore the genotoxic effect of polymeric gene carriers. To evaluate cell death by PEI and PAMAM, we performed propidium-iodide staining and lactate-dehydrogenase release assays. The genotoxicity of the polymers was measured by comet assay and cytokinesis-block micronucleus assay. PEI- and PAMAM-treated groups induced both necrotic and apoptotic cell death. In the comet assay and micronuclei formation, significant increases in DNA damage were observed in both treatments. We conclude that PEI and PAMAM dendrimer can induce not only a relatively weak apoptotic and a strong necrotic effect, but also a moderate genotoxic effect.

Self-assembled multivalent carbohydrate ligands

Materials that display multiple carbohydrate residues have gained much attention due to their potential to inhibit or modulate biological multivalent interactions. These materials can be grouped accordingly to the way they are prepared, as unimolecular or as self-assembled systems. Both systems take advantage of the fact that multivalent interactions have significantly higher binding affinity than the corresponding monovalent interactions. The self-assembled system is a more recent field of research compared to the unimolecular system. In this review, we describe current efforts to realize multivalent carbohydrate interactions from the perspective of synthetic self-assembled systems. We limit the scope to self-assembled systems that are stable, soluble in aqueous solution and morphologically discrete. We grouped them into two separate categories. In the first category carbohydrate ligands self-assemble onto a pre-organized nanostructure, and in the second carbohydrate-conjugated block molecules spontaneously assemble to construct morphologically distinct nanostructures.

Nanostructures of β-sheet peptides: steps towards bioactive functional materials

The design and construction of synthetic self-assembled nanostructures is, in a large part, inspired from elaborate nanostructures in biological systems. If we look at it from another angle, the self-assembled nanostructures are excellent scaffolds for exploring and modulating biological phenomena when they are suitably functionalized with bioactive molecules. Of the many types of molecular building blocks for self-assembly, peptide-based building blocks have the advantage in that their constituent amino acids are biocompatible and the sequence space of peptide chains is vast. This contribution highlights an emerging approach to the biomaterial application of artificially designed and functional β-sheet peptide self-assemblies.

The inhibition of prions through blocking prion conversion by permanently charged branched polyamines of low cytotoxicity.

Branched polyamines are effective in inhibiting prions in a cationic surface charge density dependent manner. However, toxicity associated with branched polyamines, in general, often hampers the successful application of the compounds to treat prion diseases. Here, we report that constitutively maintained cationic properties in branched polyamines reduced the intrinsic toxicity of the compounds while retaining the anti-prion activities. In prion-infected neuroblastoma cells, quaternization of amines in polyethyleneimine (PEI) and polyamidoamine (PAMAM) dendrimers markedly increased the nontoxic concentration ranges of the compounds and still supported, albeit reduced, an appreciable level of anti-prion activity in clearing prions from the infected cells. Furthermore, quaternized PEI was able to degrade prions at acidic pH conditions and inhibit the in vitro prion propagation facilitated by conversion of the normal prion protein isoform to its misfolded counterpart, although such activities were decreased by quaternization. Quaternized PAMAM was least effective in degrading prions but efficiently inhibited prion conversion with the same efficacy as unmodified PAMAM. Our results suggest that quaternization represents an effective strategy for developing nontoxic branched polyamines with potent anti-prion activity. This study highlights the importance of polyamine structural control for developing polyamine-based anti-prion agents and understanding of an action mechanism of quaternized branched polyamines.

Designer Nanorings with Functional Cavities from Self-Assembling β-Sheet Peptides

β-Barrel proteins that take the shape of a ring are common in many types of water-soluble enzymes and water-insoluble transmembrane pore-forming proteins. Since β-barrel proteins perform diverse functions in the cell, it would be a great step towards developing artificial proteins if we can control the polarity of artificial β-barrel proteins at will. Here, we describe a rational approach to construct β-barrel protein mimics from the self-assembly of peptide-based building blocks. With this approach, the direction of the self-assembly process toward the formation of water-soluble β-barrel nanorings or water-insoluble transmembrane β-barrel pores could be controlled by the simple but versatile molecular manipulation of supramolecular building blocks. This study not only delineates the basic driving force that underlies the folding of β-barrel proteins, but also lays the foundation for the facile fabrication of β-barrel protein mimics, which can be developed as nanoreactors, ion- and small-molecule-selective pores, and novel antibiotics.

Cyclic Peptide Facial Amphiphile Preprogrammed to Self-Assemble into Bioactive Peptide Capsules

Self-assembled peptide nanostructures have shown great potential as promising biomaterials. Peptides have the advantage of being intrinsically biocompatible materials. Peptides, as self-assembling building blocks, have mostly been designed to have head/tail molecular configuration. Since there is a great deal of interest in the precise control of a self-assembly process, novel building blocks significantly different from those with conventional head/tail configuration might provide ample opportunities for constructing elaborate, versatile, and smart nanostructures. Cyclic peptides, due to their constrained nature, are one of those self-assembly building blocks that have unique topological features compared with conventional linear (head/tail) peptides. Another important self-assembling building block that has a special architecture is the facial amphiphiles. In facial amphiphiles, the hydrophilic and -phobic groups are located on two opposite faces, rather than at two ends as in head/tail amphiphiles. Herein, we present an approach to construct a novel type of self-assembling building blocks in which the characteristics of cyclic peptides and facial amphiphiles are combined. The self-assembly behavior of this novel cyclic peptide facial amphiphile (CPFA) could be regulated through the judicious design of molecular structure, showing the power of this rational approach for precise nanostructural control. Capsules or vesicles are one of the most important nanostructural morphologies that can be utilized in many types of bioapplications, including drug, gene, and protein delivery. Capsules have hollow interior within which cargo molecules can be entrapped. Upon suitable functionalization of outer shell, capsules can be made to bind or enter the cells. Traditionally, self-assembled capsules have been fabricated by using amphiphilic molecules of head/tail configuration, such as amphiphilic lipids and block copolymers. The supramolecular morphology of the head/tail amphiphiles depends highly on the relative volume fraction between hydrophilic and -phobic blocks. Therefore, the capsule formation, in most cases, can be possible only by adjusting the volume fraction by trial and error. To devise a building block preprogrammed to have a predictable self-assembly property for capsule formation, a symmetrical CPFA with a C3-symmetric triskelion shape was designed to allow the positioning of building blocks in a precise geometry for closed-capsule structure formation. CPFA consists of even-numbered l-amino acids with alternating hydrophilic and -phobic side chains (Figure 1). In this type of a molecular arrangement, the side chains (R groups at the a-carbon atoms) adopt alternating axial and equatorial positions as a low-energy conformation. In contrast, cyclic peptide structures made up of alternating land d-amino acids, as shown in several synthetic cyclic peptides, can adopt a flat-ring structure as a low-energy conformation in which all of the side chains lie horizontal to the plane of the ring (equatorial). The six-residue CPFA consists of three arginines and three hydrophobic amino acids as hydrophilic and -phobic residues, respectively. The hydrophilic and -phobic residues are placed at alternating positions in order for the adjacent side chains to be located at different faces (axial or equatorial). The guanidinium group in arginine has been found to be a crucial residue for cell-surface binding and penetration of cell-penetrating peptides. 27] The hydrophobic amino acids are tyrosine derivatives modified with long alkyl chains. In addition to CPFA with the perhydrogenated alkyl chain (hCPFA), CPFA with the perfluorinated alkyl chain (fCPFA) was synthesized to take advantage of the “fluorophobic effect”. The fluorophobic effect refers to the super[a] E. K. Chung, Dr. E. Lee, Prof. Dr. M. Lee Center for Supramolecular Nano-Assembly and Department of Chemistry, Seoul National University Seoul 151-747 (Korea) Fax: (+82)2-393-6096 E-mail : myongslee@snu.ac.kr [b] Prof. Dr. Y.-b. Lim Department of Materials Science and Engineering Yonsei University, Seoul 120-749 (Korea) Fax: (+82)2-365-5882 E-mail : yblim@yonsei.ac.kr Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/chem.200903145.