Yan Lee

Charge‐Conversional Polyionic Complex Micelles—Efficient Nanocarriers for Protein Delivery into Cytoplasm

Special delivery! Polyionic complex (PIC) micelles that contain the charge-conversional moieties citaconic amide or cis-aconitic amide were developed for cytoplasmic protein delivery. The increase of the charge density on the protein cargo helped the stability of the PIC micelles without cross-linking, and the charge-conversion in endosomes induced the dissociation of the PIC micelles to result in efficient endosomal release (see picture).

Enhanced endosomal escape of siRNA-incorporating hybrid nanoparticles from calcium phosphate and PEG-block charge-conversional polymer for efficient gene knockdown with negligible cytotoxicity.

Development of safe and efficient short interfering RNA (siRNA) delivery system for RNA interference (RNAi)-based therapeutics is a current critical challenge in drug delivery field. The major barriers in siRNA delivery into the target cytoplasm are the fragility of siRNA in the body, the inefficient cellular uptake, and the acidic endosomal entrapment. To overcome these barriers, this study is presenting a hybrid nanocarrier system composed of calcium phosphate comprising the block copolymer of poly(ethylene glycol) (PEG) and charge-conversional polymer (CCP) as a siRNA vehicle. In these nanoparticles, the calcium phosphate forms a stable core to incorporate polyanions, siRNA and PEG-CCP. The synthesized PEG-CCP is a non-toxic endosomal escaping unit, which induces endosomal membrane destabilization by the produced polycation through degradation of the flanking cis-aconitylamide of CCP in acidic endosomes. The nanoparticles prepared by mixing of each component was confirmed to possess excellent siRNA-loading efficiency (∼80% of dose), and to present relatively homogenous spherical shape with small size. With negligible cytotoxicity, the nanoparticles efficiently induced vascular endothelial growth factor (VEGF) mRNA knockdown (∼80%) in pancreatic cancer cells (PanC-1). Confocal laser scanning microscopic observation revealed rapid endosomal escape of siRNA with the nanoparticles for the excellent mRNA knockdown. The results obtained demonstrate our hybrid nanoparticle as a promising candidate to develop siRNA therapy.

Efficient delivery of bioactive antibodies into the cytoplasm of living cells by charge-conversional polyion complex micelles

Antibodies are the most important component of humoral immunity, and can recognize and deactivate their corresponding extracellular antigens with outstanding selectivity. Moreover, the development of monoclonal and humanized antibodies has contributed greatly to the recent success of antibodies as biopharmaceuticals. However, the target of such antibodies is limited to the cell exterior because of the lack of a delivery system of antibodies into the interior of the cell. Although the detection or inactivation of an intracellular protein was partially accomplished by the intracellular expression of antibodies, the development of an efficient and safe delivery method of an antibody into living cells is required for further advances in therapeutics and bioanalysis. Various methods, such as microinjection, liposomes, cellpenetrating peptides, and even recombinant viruses, have been introduced; however, their general use is often limited because of the need for highly specialized devices, as well as the complexity and inefficiency of these methods. We recently developed a novel protein-delivery system into cytoplasm based on charge-conversional polyion complex (PIC) micelles. The charge density of a model protein, cytochrome c, can be temporarily increased by the modification of the e-amines of lysine residues into charge-conversional moieties, citraconic acid amide (Cit) or cis-aconitic acid amide (Aco) (Figure 1). As the positively charged lysines convert to the negatively charged carboxylic groups by this modification, the modified proteins become strongly anionic and the resulting charge density can be increased significantly to form stable PIC micelles with cationic block copolymers even at physiological salt concentrations. The charge-converted proteins and the cationic block in the copolymer form the core of the PIC micelle, and the polyethylene glycol (PEG) block forms the surface shell. After the PIC micelles were internalized to cells, the Cit and Aco rapidly degraded to reproduce the original lysines at the endosomal pH of 5.5. The dissociation of the PIC micelles follows the regeneration of the original protein to release the free cationic block copolymer, which induces the pH-dependent destabilization

Biosignal-sensitive polyion complex micelles for the delivery of biopharmaceuticals

The application of polyion complex (PIC) micelles into therapeutic fields is rapidly increasing due to simple and efficient encapsulation of biopharmaceuticals and outstanding biocompatibility among various polymer-based drug delivery carriers. Ionic biopharmaceuticals, such as DNA, RNA, and proteins can interact with ionic block copolymers to form PIC micelles with a core-shell structure. In this review, the development of smart PIC micelles that can respond to biosignals and the application of the biosignal-sensitive PIC micelles to the drug delivery are discussed. The change of ionic strength or pH-dependent protonation–deprotonation can be useful for the selective dissociation of PIC micelles because the ionic interaction between the block copolymer and counter-charged compounds is a main driving force for the formation of PIC micelles. The release of encapsulated biopharmaceuticals of PIC micelles can be effectively controlled by degradation of the chemical bonds in the block copolymer responding to the change of pH or reduction potential. Temperature-dependent hydrophilic–hydrophobic phase transition of block copolymers can also induce the destabilization of PIC micelles. Progress in smart PIC micelle as efficient, specific, and safe drug delivery system is indeed supported by the development of biosignal-sensitive block copolymers.

Introduction of stearoyl moieties into a biocompatible cationic polyaspartamide derivative, PAsp(DET), with endosomal escaping function for enhanced siRNA-mediated gene knockdown

Applications of siRNA for cancer therapy have been spotlighted in recent years, but the rational design of efficient siRNA delivery carriers is still controversial, especially because of possible toxicity of the carrier components. Previously, a cationic polyaspartamide derivative, poly{N-[N-(2-aminoethyl)-2-aminoethyl]aspartamide} (PAsp(DET)), was reported to exert high transfection efficacy for plasmid DNA with negligible cytotoxicity. However, its direct application for siRNA delivery was fairly limited due to the unstable polymer/siRNA complex formation. In this study, to overcome such instability, stearic acid as a hydrophobic moiety was conjugated to the side chain of PAsp(DET) with various substitution degrees. The stearoyl introduction contributed not only to siRNA complex formation with higher association numbers but also to complex stabilization. The obtained stearoyl PAsp(DET)/siRNA complex significantly accomplished more efficient endogenous gene (BCL-2 and VEGF) knockdown in vitro against the human pancreatic adenocarcinoma (Panc-1) cells than did the unmodified PAsp(DET) complex and commercially available reagents, probably due to the facilitated cellular internalization. This finding suggests that the hydrophobic PAsp(DET)-mediated siRNA delivery is a promising platform for in vivo siRNA delivery.

Biodegradable branched poly(ethylenimine sulfide) for gene delivery

We synthesized biodegradable b-PEIS (branched poly(ethylenimine sulfide)) by crosslinking linear PEIS. We controlled the degree of crosslinking and molecular weight by adjusting the amount of the crosslinker, bisepoxide. The b-PEIS was readily degradable under reductive conditions (5mm glutathione solution) and the degradation time was dependent on the degree of crosslinking. We controlled the molecular weights of the b-PEIS by regulating the amount of crosslinker and thus, the degree of crosslinking. Our titration data showed that there was almost no loss in buffering ability before or after bisepoxide crosslinking. We verified the degradation of this polymer by MALLS and gel electrophoresis, and confirmed that there was a high transfection efficiency and low cytotoxicity based on cellular data. Intracellular trafficking was observed by image restoration microscopy, demonstrating that b-PEIS does not accumulate in the cell interior.

A new biodegradable crosslinked polyethylene oxide sulfide (PEOS) hydrogel for controlled drug release

We developed a polyethylene glycol (PEG)-based biodegradable hydrogel through disulfide crosslinking of polyethylene oxide sulfide (PEOS). The crosslinking rate was highly dependent on temperature, and incubation at about 40-50 degrees C was required for efficient crosslinking. The crosslinked PEOS hydrogel showed glutathione-dependent dissolution and corresponding controlled release of a model drug-fluorescein isothiocyanate (FITC)-labeled dextran-because the disulfide bond, the main linker, is selectively degraded in response to the high concentration of glutathione. The temperature-sensitive crosslinking and the hydrogel formation have the potential for use as an injectable biogel precursor, which was confirmed by in situ gel formation in mice.

Effective healing of diabetic skin wounds by using nonviral gene therapy based on minicircle vascular endothelial growth factor DNA and a cationic dendrimer.

The development of an efficient method to improve the wound healing process is urgently required for diabetic patients suffering a threat of limb amputations. Various growth factors have been proposed for treatment; however, more research still has to be carried out to maintain their curative effect. In the present study, we describe a simple nonviral gene therapy method for improving wound healing.

pDNA/poly(L-lysine) Polyplexes Functionalized with a pH-Sensitive Charge-Conversional Poly(aspartamide) Derivative for Controlled Gene Delivery to Human Umbilical Vein Endothelial Cells.

An efficient endosome-escaping function was integrated into the polyplex of plasmid DNA (pDNA) with poly(L-lysine) (PLys) to improve its gene transfection efficiency through electrostatic coating with charge-conversional polymer (CCP). CCP showed charge-conversional function responding to endosomal pH, leading to the release of pDNA/PLys polyplex into the cytoplasm. The cells took up the intact CCP-integrated ternary polyplex, which exerted appreciably higher transfection efficiency with lower cytotoxicity than pDNA/PLys polyplex against human umbilical vein endothelial cells (HUVECs). This is consistent with the facilitated endosomal escape of the CCP-integrated ternary polyplex compared to the pDNA/PLys polyplex as directly observed with confocal laser-scanning microscopy.

Preparation of non-aggregated fluorescent nanodiamonds (FNDs) by non-covalent coating with a block copolymer and proteins for enhancement of intracellular uptake

Fluorescent nanodiamonds (FNDs) are very promising fluorophores for use in biosystems due to their high biocompatibility and photostability. To overcome their tendency to aggregate in physiological solutions, which severely limits the biological applications of FNDs, we developed a new non-covalent coating method using a block copolymer, PEG-b-P(DMAEMA-co-BMA), or proteins such as BSA and HSA. By simple mixing of the block copolymer with FNDs, the cationic DMAEMA and hydrophobic BMA moieties can strongly interact with the anionic and hydrophobic moieties on the FND surface, while the PEG block can form a shell to prevent the direct contact between FNDs. The polymer-coated FNDs, along with BSA- and HSA-coated FNDs, showed non-aggregation characteristics and maintained their size at the physiological salt concentration. The well-dispersed, polymer- or protein-coated FNDs in physiological solutions showed enhanced intracellular uptake, which was confirmed by CLSM. In addition, the biocompatibility of the coated FNDs was expressly supported by a cytotoxicity assay. Our simple non-covalent coating with the block copolymer, which can be easily modified by various chemical methods, projects a very promising outlook for future biomedical applications, especially in comparison with covalent coating or protein-based coating.