Kensuke Osada

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).

Polymeric micelles from poly(ethylene glycol)-poly(amino acid) block copolymer for drug and gene delivery

Dramatic advances in biological research have revealed the mechanisms underlying many diseases at the molecular level. However, conventional techniques may be inadequate for direct application of this new knowledge to medical treatments. Nanobiotechnology, which integrates biology with the rapidly growing field of nanotechnology, has great potential to overcome many technical problems and lead to the development of effective therapies. The use of nanobiotechnology in drug delivery systems (DDS) is attractive for advanced treatment of conditions such as cancer and genetic diseases. In this review paper for a special issue on biomaterial research in Japan, we discuss the development of DDS based on polymeric micelles mainly in our group for anti-cancer drug and gene delivery, and also address our challenges associated with developing polymeric micelles as super-functionalized nanodevices with intelligent performance.

Drug and gene delivery based on supramolecular assembly of PEG-polypeptide hybrid block copolymers

Recently, polypeptide hybrid polymers, particularly poly(ethylene glycol) (PEG)-polypeptide block copolymers, have been attracting significant interest for polymeric therapeutics, such as drug and gene delivery systems, utilizing their most relevant feature, that is the formation of micelles with a distinguished core-shell architecture. Of particular interest in the polypeptides is that a variety of functional groups, such as carboxyl groups and amino groups, are available as a side chain, and that they have propensities of low toxicity and biodegradability. The segregated polypeptide core of the micelle embedded in the hydrophilic palisade serves as a reservoir for a variety of drugs as well as of genes with diverse characteristics. The micelles have been developed with various functions, such as biocompatibility, stimuli- and environment-sensitivity, and targetability, aimed at their clinical use. Smart micelles have emerged as promising carriers that enhance the effect of drugs and genes with minimal side effects. In this review, recent advances in drug and gene delivery by polypeptide hybrid micelles, mostly accomplished in our group, are comprehensively described. Focus is placed on the design of PEG-polypeptide hybrid block copolymers, starting from the development of the drug-loading micelle systems to current efforts to establish a gene delivery system with a polyion complex (PIC) micelle, one of the most attractive topics in nanomedicine.

Targeted polymeric micelles for siRNA treatment of experimental cancer by intravenous injection

Small interfering ribonucleic acid (siRNA) cancer therapies administered by intravenous injection require a delivery system for transport from the bloodstream into the cytoplasm of diseased cells to perform the function of gene silencing. Here we describe nanosized polymeric micelles that deliver siRNA to solid tumors and elicit a therapeutic effect. Stable multifunctional micelle structures on the order of 45 nm in size formed by spontaneous self-assembly of block copolymers with siRNA. Block copolymers used for micelle formation were designed and synthesized to contain three main features: a siRNA binding segment containing thiols, a hydrophilic nonbinding segment, and a cell-surface binding peptide. Specifically, poly(ethylene glycol)-block-poly(L-lysine) (PEG-b-PLL) comprising lysine amines modified with 2-iminothiolane (2IT) and the cyclo-Arg-Gly-Asp (cRGD) peptide on the PEG terminus was used. Modification of PEG-b-PLL with 2IT led to improved control of micelle formation and also increased stability in the blood compartment, while installation of the cRGD peptide improved biological activity. Incorporation of siRNA into stable micelle structures containing the cRGD peptide resulted in increased gene silencing ability, improved cell uptake, and broader subcellular distribution in vitro and also improved accumulation in both the tumor mass and tumor-associated blood vessels following intravenous injection into mice. Furthermore, stable and targeted micelles inhibited the growth of subcutaneous HeLa tumor models and demonstrated gene silencing in the tumor mass following treatment with antiangiogenic siRNAs. This new micellar nanomedicine could potentially expand the utility of siRNA-based therapies for cancer treatments that require intravenous injection.

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.

Polyplex micelles prepared from ω-cholesteryl PEG-polycation block copolymers for systemic gene delivery.

Polyplex micelles formed with plasmid DNA (pDNA) and poly(ethylene glycol) (PEG)-block-poly{N-[N-(2-aminoethyl)-2-aminoethyl]aspartamide} [PAsp(DET)] exhibit effective endosomal escaping properties based on di-protonation of diamine side chains with decreasing pH, which improves their transfection efficiency and thus are promising candidates for local in vivo gene transfer. Here, PEG-PAsp(DET) polyplex micelles were further improved as in vivo systemic vectors by introduction of cholesterol (Chole) into the ω-terminus of PEG-PAsp(DET) to obtain PEG-PAsp(DET)-Chole. Introduction of the cholesterol resulted in enhanced association of block copolymers with pDNA, which led to increased stability in proteinous medium and also in the blood stream after systemic injection compared to PEG-PAsp(DET) micelles. The synergistic effect between enhanced polymer association with pDNA and increased micelle stability of PEG-PAsp(DET)-Chole polyplex micelles led to high in vitro gene transfer even at relatively low concentrations, due to efficient cellular uptake and effective endosomal escape of block copolymers and pDNA. Finally, PEG-PAsp(DET)-Chole micelles achieved significant suppression of tumor growth following intravenous injection into mice bearing a subcutaneous pancreatic tumor using therapeutic pDNA encoding an anti-angiogenic protein. These results suggest that PEG-PAsp(DET)-Chole micelles can be effective systemic gene vectors for treatment of solid tumors.

Bundled Assembly of Helical Nanostructures in Polymeric Micelles Loaded with Platinum Drugs Enhancing Therapeutic Efficiency against Pancreatic Tumor

Supramolecular assemblies of amphiphilic block copolymers having polypeptide segments offer significant advantages for tailoring spatial arrangement based on secondary structures in their optically active backbones. Here, we demonstrated the critical effect of α-helix bundles in cisplatin-conjugated poly(L- (or D-)glutamate) [P(L(or D)Glu)-CDDP] segment on the packaging of poly(ethylene glycol) (PEG)-P(L(or D)Glu)-CDDP block copolymers in the core of polymeric micelles (CDDP/m) and enhanced micelle tolerability to harsh in vivo conditions for accomplishing appreciable antitumor efficacy against intractable pancreatic tumor by systemic injection. CDDP/m prepared from optically inactive PEG-poly(D,L-glutamate) (P(D,LGlu)), gradually disintegrated in the bloodstream, resulting in increased accumulation in liver and spleen and reduced antitumor efficacy. Alternatively, CDDP/m from optically active PEG-P(L(or D)Glu) maintained micelle structure during circulation, and eventually attained selective tumor accumulation while reducing nonspecific distribution to liver and spleen. Circular dichroism and small-angle X-ray scattering measurements indicated regular bundled assembly of α-helices in the core of CDDP/m from PEG-P(L(or D)Glu), which is suggested to stabilize the micelle structure against dilution in physiological condition. CDDP/m suffered corrosion by chlorides in medium, yet the optically active micelles with α-helix bundles kept the micelle structure for prolonged time, with slowly releasing unimers and dimers from the surface of the bundled core in an erosion-like process, as verified by ultracentrifugation analysis. This is in sharp contrast with the abrupt disintegration of CDDP/m from PEG-P(D,LGlu) without secondary structures. The tailored assembly in the core of the polymeric micelles through regular arrangement of constituting segments is key to overcome their undesirable disintegration in bloodstream, thereby achieving efficient delivery of loaded drugs into target tissues.

Enhanced in vivo Magnetic Resonance Imaging of Tumors by PEGylated Iron-Oxide-Gold Core-Shell Nanoparticles with Prolonged Blood Circulation Properties

High-density poly(ethylene glycol) (PEG)-coated iron-oxide-gold core-shell nanoparticles (AuIONs) were developed as T(2) -weighted magnetic resonance imaging (MRI) contrast agents for cancer imaging. The PEG-coated iron-oxide-gold core-shell nanoparticles (PEG-AuIONs) were approximately 25 nm in diameter with a narrow distribution. Biodistribution experiments in mice bearing a subcutaneous colon cancer model prepared with C26 murine colon adenocarcinoma cells showed high accumulation of the PEG-AuIONs within the tumor mass and low nonspecific accumulation in the liver and spleen, resulting in high specificity to solid tumors. T(2) -weighted MR images following intravenous injection of PEG-AuIONs showed selective negative enhancement of tumor tissue in an orthotopic pancreatic cancer model prepared with MiaPaCa-2 human pancreatic adenocarcinoma cells. These results indicate that PEG-AuIONs are a promising MRI contrast agent for diagnosis of malignant tumors, including pancreatic cancer.

Targeted gene delivery by polyplex micelles with crowded PEG palisade and cRGD moiety for systemic treatment of pancreatic tumors

Adequate retention in systemic circulation is the preliminary requirement for systemic gene delivery to afford high bioavailability into the targeted site. Polyplex micelle formulated through self-assembly of oppositely-charged poly(ethylene glycol) (PEG)-polycation block copolymer and plasmid DNA has gained tempting perspective upon its advantageous core-shell architecture, where outer hydrophilic PEG shell offers superior stealth behaviors. Aiming to promote these potential characters toward systemic applications, we strategically introduced hydrophobic cholesteryl moiety at the ω-terminus of block copolymer, anticipating to promote not only the stability of polyplex structure but also the tethered PEG crowdedness. Moreover, Mw of PEG in the PEGylated polyplex micelle was elongated up to 20 kDa for expecting further enhancement in PEG crowdedness. Furthermore, cyclic RGD peptide as ligand molecule to integrin receptors was installed at the distal end of PEG in order for facilitating targeted delivery to the tumor site as well as promoting cellular uptake and intracellular trafficking behaviors. Thus constructed cRGD conjugated polyplex micelle with the elevated PEG shielding was challenged to a modeled intractable pancreatic cancer in mice, achieving potent tumor growth suppression by efficient gene expression of antiangiogenic protein (sFlt-1) at the tumor site.

Quantized Folding of Plasmid DNA Condensed with Block Catiomer into Characteristic Rod Structures Promoting Transgene Efficacy

Highly regulated folding of plasmid DNA (pDNA) through polyion complexation with the synthetic block catiomer, poly(ethylene glycol)-block-poly(L-lysine) (PEG-PLys), was found to occur in such a way that rod structures are formed with a quantized length of 1/2(n + 1) of the original pDNA length folding by n times. The folding process of pDNA was elucidated with regard to rigidity of the double-stranded DNA structure and topological restriction of the supercoiled closed-circular form, and a mechanism based on Eulers buckling theory was proposed. Folded pDNA exhibited higher gene expression efficiency compared to naked pDNA in a cell-free transcription/translation assay system, indicating that the packaging of pDNA into a polyion complex core surrounded by a PEG palisade is a promising strategy for constructing nonviral gene carrier systems. Extension of this finding may provide a reasonable model to further understand the packaging mechanism of supercoiled DNA structures in nature.