Young Jik Kwon

Nanoantibiotics: a new paradigm for treating infectious diseases using nanomaterials in the antibiotics resistant era.

Despite the fact that we live in an era of advanced and innovative technologies for elucidating underlying mechanisms of diseases and molecularly designing new drugs, infectious diseases continue to be one of the greatest health challenges worldwide. The main drawbacks for conventional antimicrobial agents are the development of multiple drug resistance and adverse side effects. Drug resistance enforces high dose administration of antibiotics, often generating intolerable toxicity, development of new antibiotics, and requests for significant economic, labor, and time investments. Recently, nontraditional antibiotic agents have been of tremendous interest in overcoming resistance that is developed by several pathogenic microorganisms against most of the commonly used antibiotics. Especially, several classes of antimicrobial nanoparticles (NPs) and nanosized carriers for antibiotics delivery have proven their effectiveness for treating infectious diseases, including antibiotics resistant ones, in vitro as well as in animal models. This review summarizes emerging efforts in combating against infectious diseases, particularly using antimicrobial NPs and antibiotics delivery systems as new tools to tackle the current challenges in treating infectious diseases.

Efficient and targeted delivery of siRNA in vivo

RNA interference (RNAi) has been regarded as a revolutionary tool for manipulating target biological processes as well as an emerging and promising therapeutic strategy. In contrast to the tangible and obvious effectiveness of RNAi in vitro, silencing target gene expression in vivo using small interfering RNA (siRNA) has been a very challenging task due to multiscale barriers, including rapid excretion, low stability in blood serum, nonspecific accumulation in tissues, poor cellular uptake and inefficient intracellular release. This minireview introduces major challenges in achieving efficient siRNA delivery in vivo and discusses recent advances in overcoming them using chemically modified siRNA, viral siRNA vectors and nonviral siRNA carriers. Enhanced specificity and efficiency of RNAi in vivo via selective accumulations in desired tissues, specific binding to target cells and facilitated intracellular trafficking are also commonly attempted utilizing targeting moieties, cell‐penetrating peptides, fusogenic peptides and stimuli‐responsive polymers. Overall, the crucial roles of the interdisciplinary approaches to optimizing RNAi in vivo, by efficiently and specifically delivering siRNA to target tissues and cells, are highlighted.

Stimuli-responsive polymers and nanomaterials for gene delivery and imaging applications ☆

Multiple extra- and intracellular obstacles, including low stability in blood, poor cellular uptake, and inefficient endosomal escape and disassembly in the cytoplasm, have to be overcome in order to deliver nucleic acids for gene therapy. This review introduces the recent advances in tackling the key challenges in achieving efficient, targeted, and safe nonviral gene delivery using various nucleic acid-containing nanomaterials that are designed to respond to various extra- and intracellular biological stimuli (e.g., pH, redox potential, and enzyme) as well as external artificial triggers (e.g., light and ultrasound). Gene delivery in combination with molecular imaging and targeting enables diagnostic assessment, treatment monitoring and quantification of efficiency, and confirmation of cure, thus fulfilling the great promise of efficient and personalized medicine. Nanomaterials platform for combined imaging and gene therapy, nanotheragnostics, using stimuli-responsive materials is also highlighted in this review. It is clear that developing novel multifunctional nonviral vectors, which transform their physico-chemical properties in response to various stimuli in a timely and spatially controlled manner, is highly desired to translate the promise of gene therapy for the clinical success.

Enhanced antigen presentation and immunostimulation of dendritic cells using acid-degradable cationic nanoparticles

Abstract Acid-degradable cationic nanoparticles encapsulating a model antigen (i.e., ovalbumin) were prepared by inverse microemulsion polymerization with acid-cleavable acetal cross-linkers. Incubation of these degradable nanoparticles with dendritic cells derived from bone marrow (BMDCs) resulted in the enhanced presentation of ovalbumin-derived peptides, as quantified by B3Z cells, a CD8+ T cell hybridoma. The cationic nature of the particles contributed to the increased surface endocytosis (or phagocytosis) observed with BMDCs, which is the first barrier to overcome for successful antigen delivery. The acid sensitivity of the particles served to direct more ovalbumin antigens to be processed into the appropriately trimmed peptide fragments and presented via the major histocompatibility complex (MHC) class I pathway following hydrolysis within the acidic lysosomes. It was also shown that adjuvant molecules such as unmethylated CpG oligonucleotides (CpG ODN) and anti-interleukin-10 oligonucleotides (AS10 ODN) could be co-delivered with the protein antigen for maximized cellular immune response.

Enhanced detection of early-stage oral cancer in vivo by optical coherence tomography using multimodal delivery of gold nanoparticles

Contrast in optical coherence tomography (OCT) images can be enhanced by utilizing surface plasmon resonant gold nanoparticles. To improve the poor in vivo transport of gold nanoparticles through biological barriers, an efficient delivery strategy is needed. In this study, the improved penetration and distribution of gold nanoparticles were achieved by microneedle and ultrasound, respectively, and it was demonstrated that this multimodal delivery of antibody-conjugated PEGylated gold nanoparticles enhanced the contrast in in vivo OCT images of oral dysplasia in a hamster model.

Acid-responsive linear polyethylenimine for efficient, specific, and biocompatible siRNA delivery.

Efficient intracellular processes including cytosolic release and unpackaging of siRNA from the carrier in the cytoplasm are efficiency-determining steps in achieving successful gene silencing. In this study, acid-degradable ketalized linear polyethylenimine (KL-PEI) was synthesized for efficient, intracellular target-specific, and biocompatible siRNA delivery. The siRNA/KL-PEI polyplexes resulted in much higher RNA interference efficiency than unmodified L-PEI via selective cytoplasmic localization of the polyplexes and efficient disassembly of siRNA from the polyplexes, which were promoted upon acid-hydrolysis of amino ketal linkages. Confocal laser scanning microscopy demonstrated that siRNA was efficiently disassembled from the siRNA/KL-PEI polyplexes that were selectively localized in the cytoplasm. On the contrary, siRNA and unmodified linear PEI were colocalized in both the cytoplasm and the nucleus, and limited unpackaging of siRNA from the polyplexes was observed. In addition, ketalization further reduced the cytotoxicity of linear PEI but did not alter its serum-independent gene delivery efficiency. Therefore, KL-PEI is a promising nonviral vector for efficient and biocompatible siRNA delivery.

Controlled delivery of plasmid DNA and siRNA to intracellular targets using ketalized polyethylenimine.

A new polyethylenimine (PEI)-derived biodegradable polymer was synthesized as a nonviral gene carrier. Branches of PEI were ketalized, and capabilities of nucleic acid condensation and delivery efficiency of the modified polymers were compared with ones of unketalized PEI. Ketalized PEI was able to efficiently compact both plasmid DNA and siRNA into nucleic acids/ketalized PEI polyplexes with a range of 80-200 nm in diameter. Nucleic acids were efficiently dissociated from the polyplexes made of ketalized PEI upon hydrolysis. In vitro study also demonstrated that ketalization enhanced transfection efficiency of the polyplexes while reducing cytotoxicity, even at high N/ P ratios. Interestingly, transfection efficiency was found to be inversely proportional to molecular weights of ketalized PEI, while RNA interference was observed in the opposite way. This study implies that selective delivery of plasmid DNA and siRNA to the nucleus and the cytoplasm can be achieved by tailoring the structures of polymeric gene carriers.

Before and after Endosomal Escape: Roles of Stimuli-Converting siRNA/Polymer Interactions in Determining Gene Silencing Efficiency

Silencing the expression of a target gene by RNA interference (RNAi) shows promise as a potentially revolutionizing strategy for manipulating biological (pathological) pathways at the translational level. However, the lack of reliable, efficient, versatile, and safe means for the delivery of small interfering RNA (siRNA) molecules, which are large in molecular weight, negatively charged, and subject to degradation, has impeded their use in basic research and therapy. Polyplexes of siRNA and polymers are the predominant mode of siRNA delivery, but innovative synthetic strategies are needed to further evolve them to generate the desired biological and therapeutic effects. This Account focuses on the design of polymeric vehicles for siRNA delivery based on an understanding of the molecular interactions between siRNA and cationic polymers. Ideal siRNA/polymer polyplexes should address an inherent design dilemma for successful gene silencing: (1) Cationic polymers must form tight complexes with siRNA via attractive electrostatic interactions during circulation and cellular internalization and (2) siRNA must dissociate from its cationic carrier in the cytoplasm before they are loaded into RNA-induced silencing complex (RISC) and initiate gene silencing. The physicochemical properties of polymers, which dictate their molecular affinity to siRNA, can be programmed to be altered by intracellular stimuli, such as acidic pH in the endosome and cytosolic reducers, subsequently inducing the siRNA/polymer polyplex to disassemble. Specific design goals include the reduction of the cationic density and the molecular weight, the loss of branched structure, and changes in the hydrophilicity/hydrophobicity of the polymeric siRNA carriers, via acid-responsive degradation and protonation processes within the endosome and glutathione (GSH)-mediated reduction in the cytoplasm, possibly in combination with gradual stimuli-independent hydrolysis. Acetals/ketals are acid-cleavable linkages that have been incorporated into polymeric materials for stimuli-responsive gene and drug delivery. Tailoring the ketalization ratio and the molecular weight of ketalized branched PEI (K-BPEI) offers molecular control of the intracellular trafficking of siRNA/polymer polyplexes and, therefore, the gene silencing efficiency. The ketalization of linear PEI (K-LPEI) enhances gene silencing in vitro and in vivo by improving siRNA complexation with the polymer during circulation and cellular internalization, supplementing proton buffering efficiency of the polymer in the endosome, and facilitating siRNA dissociation from the polymer in the cytoplasm, in a serum-resistant manner. Spermine polymerization via ketalization and esterification for multistep intracellular degradations provides an additional polymeric platform for improved siRNA delivery and highly biocompatible gene silencing. The chemistry presented in this Account will help lay the foundation for the development of innovative and strategic approaches that advance RNAi technology.

Determination of Infectious Retrovirus Concentration from Colony-Forming Assay with Quantitative Analysis

ABSTRACT The colony formation assay is the most commonly used titration method for defining the concentration of replication-incompetent murine leukemia virus-derived retroviral vectors. However, titer varies with target cell type and number, transduction time, and concentration of polycation (e.g., Polybrene). Moreover, because most of the viruses cannot encounter target cells due to Brownian motion, their short half-lives, and the requirement for target cell division for activity, the actual infectious retrovirus concentration in the collected supernatant is higher than the viral titer. Here we correlate the physical viral particle concentration with the infectious virus concentration and colony formation titer with the help of a mathematical model. Ecotropic murine leukemia retrovirus supernatant, collected from the GP+E86/LNCX retroviral vector producer cell line, was concentrated by centrifugation and further purified by a sucrose density gradient. The physical concentration of purified viral vectors was determined by direct particle counting with an electron microscope. The concentrations of fresh and concentrated supernatant were determined by a quantitative reverse transcriptase activity assay. Titration of all supernatants by neomycin-resistant colony formation assay was also performed. There were 767 ± 517 physical viral particles per infectious CFU in the crude viral supernatant. However, the infectious viral concentration determined by mathematical simulation was 143 viral particles per infectious unit, which is more consistent with the concentration determined by particle counting in purified viral solution. Our results suggest that the mathematical model can be used to extract a more accurate and reliable concentration of infectious retrovirus.

Acid-transforming polypeptide micelles for targeted nonviral gene delivery

Efficient delivery of therapeutic genes requires overcoming key extracellular and intracellular barriers. These include stability during circulation, internalization by target cells, facilitated endosomal escape, and localization of genes in destined intracellular compartments (e.g., nucleus). Micelles that transform their structure in the mildly acidic endosome and release their cargo genes into the cytoplasm were synthesized by self-assembling DNA with PEG-conjugated poly(ketalized serine) [PEG-poly(kSer)]. It was confirmed that, upon acid-hydrolysis of ketal linkages, poly(kSer) converts to neutral and naturally occurring poly(serine), destabilizing PEG-poly(kSer)/DNA micelles. In vitro studies demonstrated that PEG-poly(kSer) micelles were able to transfect NIH 3T3 cells more efficiently than both PEG-poly(Lys)/DNA micelles and poly-L-lysine/DNA polyplexes through efficient DNA dissociation in the cytoplasm. In addition, the core of PEG-poly(kSer)/DNA micelles were cross-linked via acid-cleavable amine-bearing branches, and the resulting cross-linked PEG-poly(kSer)/DNA micelles showed improved transfection capability in the presence of serum. Conjugation of folic acids (FAs) at the PEG termini of the acid-transforming micelles resulted in selectively increased cellular internalization and transfection of FA receptor-expressing HeLa cells over NIH 3T3 cells, implicating the possibility of cancer-targeted gene delivery using FA-PEG-poly(kSer)/DNA micelles. This study demonstrates that the acid-transforming PEG-poly(kSer)/DNA micelles are promising nonviral vectors for stimuli-responsive, efficient, biocompatible, and targeted gene delivery.