山崎 裕一

Muscle-targeted drug delivery is a major challenge in nanomedicine. The extravasation of nanomedicines (or nanoparticles) from the bloodstream into muscle tissues is hindered by the continuous endothelium, the so-called blood-muscle barrier. This study aimed to evaluate the optimal size of macromolecular drugs for extravasation (or passive targeting) into muscle tissues. We constructed a size-tunable polymeric delivery platform as a polymeric nanoruler by grafting poly(ethylene glycol)s (PEGs) onto the poly(aspartic acid) (PAsp) backbone. A series of PEG-grafted copolymers (gPEGs) with a narrow size distribution between 11 and 32 nm in hydrodynamic diameter (DH) were prepared by changing the molecular weight of the PEGs. Biodistribution analyses revealed that accumulation amounts of gPEGs in the muscle tissues of normal mice tended to decrease above their size of-15 nm (or-11 nm for the heart). The gPEGs accumulated in the skeletal muscles of Duchenne muscular dystrophy model mice (mdx mice) at a 2-3-fold higher level than in the skeletal muscles of normal mice. At the same time, there was a reduced accumulation of gPEGs in the spleen and liver. Intravital confocal laser scanning microscopy and immunohistochemical analysis showed extravasation and locally enhanced accumulation of gPEGs in the skeletal muscle of mdx mice. This study outlined the pivotal role of macromolecular drug size in muscle-targeted drug delivery and demonstrated the enhanced permeability of 11-32 nmsized macromolecular drugs in mdx mice.

We previously synthesized cysteine-installed C-terminally PEGylated oligolysines with 20 amino acid residues to form cross-linked polymeric micelles (PMs) with luciferase-coding plasmid DNA as a candidate for artificial gene vectors. Luciferase gene expression in HeLa cells mediated by PEG-CK18C, PEG-CK9CK9, and PEG-K9CK9C was reported to be 35-, 5.4-, and 1.3-fold higher than that mediated by cysteine-uninstalled PEGylated oligolysine PEG-K-20, respectively. However, after the publication, the survival rate of HeLa cells used in the previous study was found to be lower than usual when subcutaneously implanted into mice to create a xenograft model. In this study, to re-examine the peptide sequence-dependent gene expression, gene expression efficacy mediated by PEG-peptide PMs was compared with the PM cellular uptake results using newly obtained HeLa cell lines and the additional cell lines Huh-7, PANC-1, and BxPC3. As a result, PEG-K9CK9C PMs mediated the maximum gene expression in all cell lines, and the corresponding cellular uptake was also obtained. Therefore, we concluded that our previous results were erroneously obtained due to normality-depleted HeLa cells. A comparison of physicochemical characterizations, gene expression efficacy, and cellular uptake of PEG-peptide PMs is discussed in detail.

Development of efficient delivery vehicles for in vitro transcribed mRNA (IVT mRNA) is currently a major challenge in nanomedicines. For systemic mRNA delivery, we developed a series of cationic amphiphilic polyaspartamide derivatives (PAsp(DET/R)s) carrying various alicyclic (R) moieties with diethylenetriamine (DET) in the side chains to form mRNA-loaded polyplexes bearing stability under physiological conditions and possessing endosomal escape functionality. While the size and zeta-potential of polyplexes were comparable among various PAsp(DET/R)s, the transfection efficiencies of polyplexes were considerably varied due to difference in the R moieties of PAsp(DET/R)s and were described by an octanol-water (or buffer at pH 7.3) distribution coefficient (logD7.3). The critical logD7.3 for the efficient in vitro transfection of mRNA was indicated at -2.7 to -1.8. The polyplexes with logD7.3 > -1.8 elicited the much higher in vitro transfection efficiencies. After systemic administration, the polyplexes with logD7.3 from -1.8 to -1.3 elicited the significant mRNA expression specifically in the lungs. The highest mRNA expression in the lungs was achieved by a polyaspartamide derivative having a cyclohexylethyl group (PAsp(DET/CHE)), which induced more than 10-fold increase in mRNA transfection efficiency compared to commercially available lipid nanoparticles. The higher mRNA expression by polyplexes in the lungs was explained well by the preferential lung accumulation of intact mRNA, as determined by quantitative real-time PCR. Our results demonstrate that PAsp(DET/R)s are a promising synthetic material for the enhanced systemic IVT mRNA delivery.

To stabilize small interfering RNA (siRNA) in the bloodstream for systemic RNAi therapeutics, we previously fabricated ultrasmall siRNA nanocarriers that were sub-20 nm in hydrodynamic diameter, named as unit polyion complexes (uPICs), using two-branched poly(ethylene glycol)-b-poly(L-lysine) (bPEG-PLys). The blood retention time of uPICs is dramatically increased in the presence of free bPEG-PLys, suggesting dynamic stabilization of uPICs by free bPEG-PLys based on their equilibrium. Herein, we examined how the degree of polymerization of PLys (DPPLys) affected the dynamic stability of uPICs in the bloodstream during prolonged circulation. We prepared a series of bPEG-PLys with DPPLys values of 10, 13, 20, 40, and 80 for the uPIC formation and siRNA with 40 negative charges. These bPEG-PLys were then evaluated in physicochemical characterization and pharmacokinetic analyses. Structural analyses revealed that the uPIC size and association numbers were mainly determined by the molecular weights of PEG and DPPLys, respectively. Under bPEG-PLys-rich conditions, the hydrodynamic diameters of uPICs were 15-20 nm, which were comparable to that of the bPEG block (i.e., similar to 18 nm). Importantly, DPPLys significantly affected the association constant of bPEG-PLys to siRNA (K-a) and blood retention of free bPEG-PLys. A smaller DPPLys resulted in a lower K-a and a longer blood retention time of free bPEG-PLys. Thus, DPPLys can control the dynamic stability of uPICs, i.e., the balance between K-a and blood concentration of free bPEG-PLys. Ultimately, the bPEG-PLys with DPPLys values of 14 and 19 prolonged the blood circulation of siRNA-loaded uPICs with relatively small amounts of free bPEG-PLys. This study revealed that the uPIC formation between siRNA and bPEG-PLys can be controlled by their charges, which may be helpful for designing PIC-based delivery systems.

Oligolysine-based PEG-peptides with 15 or 20 amino acid residues including two cysteines were synthesized to formulate cross-linked polyplex micelles (PMs) incorporating luciferase-coding plasmid DNA (pDNA). Two cysteine residues were separately allocated at the C-terminal, center, or N-terminal of peptide moieties. Although TEM observation showed that all PEG-peptides condensed pDNA into rod-like or toroidal morphologies, the rod length distribution of PMs was affected by both the amino acid sequence and the peptide length of PEG-peptides. In comparison to the cysteine-uninstalled PEG-peptides, the cysteine-installed PEG-peptides exhibited a reductive environment-responsive pDNA release, which was observed in a gel retardation assay. From physicochemical characterizations, a relationship between the amino acid sequence and the in vitro gene expression efficacy of PMs in a cell-free protein synthesis system has been clearly demonstrated. Finally, the cell-based assay using HeLa cells has been tested, and the differences between both results of cell-free and cell-based systems are discussed.

Structural stability of polyplex micelles (PMs), prepared from plasmid DNA (pDNA) and poly(ethylene glycol)-b-poly(L-lysine) block catiomer (PEG-PLys), was evaluated in terms of their resistance against shear stress. When exposed to shear stress at magnitudes typically present in the blood stream, structural deterioration was observed in PMs owing to the partial removal of PEG-PLys strands. Eventually, impaired PEG coverage of the polyplex core led to accelerated degradation by nucleases, implying that structural deterioration by shear stress in blood stream may be a major cause of rapid clearance of PMs from blood circulation. To address this issue, introduction of disulfide crosslinking into the PM core was shown to be an efficient strategy, which successfully mitigated unfavorable effects of shear stress. Furthermore, improved in vivo blood retention profile and subsequently enhanced antitumor efficacy in systemic treatment of pancreatic adenocarcinoma were confirmed for the crosslinked PMs loaded with pDNA encoding an anti-angiogenic protein, suggesting that high stability under the shear stress during blood circulation may be a critical factor in systemically applicable gene delivery systems. (C) 2017 Elsevier Ltd. All rights reserved.

Much attention has been devoted to precise control of the size and morphology in nanosized molecular assemblies for a wide range of materials applications. Recently, we reported observing submicron/nanosized polyion complex vesicles (Nano-PICsomes) with a narrow size distribution, synthesized using specific types of homocatiomers and polyethylene glycol (PEG)-based block aniomers. However, only one example of Nano-PICsomes has been reported to date. Here, the role of the chemical composition of PEG-based block aniomers and the chemical structures of the side chains of homocatiomers were carefully examined to better understand the formation of Nano-PICsomes. Transmission electron microscopy and dynamic light scattering analyses of Nano-PICsomes revealed that a longer length of ionic segments in the block aniomers or a PEG weight fraction (f(PEG)) <10%, is required for the selective formation of Nano-PICsomes, whereas polymer combinations with f(PEG) >10% produced spherical micelles. In addition, the homocatiomers containing longer aliphatic side chains (e. g., five or six carbon atoms) favored the formation of Nano-PICsomes, whereas those containing shorter aliphatic side chains produced irregularly shaped PIC micelles. Accordingly, f(PEG) and the length of the side chain were found to be the key factors that control the morphologies of Nano-PICsomes. Insights gained from this study can broaden the spectrum of the design of Nano-PICsomes for use in a diverse range of material applications.

Understanding the dynamic behavior of molecular self-assemblies with higher-dimensional structures remains a key challenge to obtaining well-controlled and monodispersed structures. Nonetheless, there exist few systems capable of realizing the mechanism of supramolecular polymerization at higher dimensions. Herein, we report the unique self-assembling behavior of polyion complexes (PICs) consisting of poly(ethylene glycol)-polyelectrolyte block copolymer as an example of two-dimensional supramolecular living polymerization. Monodispersed and submicrometer unilamellar PIC vesicles (nano-PICsomes) displayed time-dependent growth while maintaining a narrow size distribution and a unilamellar structure. Detailed analysis of the system revealed that vesicle growth proceeded through the consumption of unit PICs (uPICs) composed of a single polycation/polyanion pair and was able to restart upon the further addition of isolated uPICs. Interestingly, the resulting vesicles underwent dissociation into uPICs in response to mechanical stress. These results clearly frame the growth as a two-dimensional supramolecular living polymerization of uPICs.

Small interfering RNA (siRNA) has great therapeutic potential for the suppression of proteins associated with disease, but delivery methods are needed for improved efficacy. Here, we investigated the properties of micellar siRNA delivery vehicles prepared with poly(ethylene glycol)-block-poly(L-lysine) (PEG-b-PLL) comprising lysine amines modified to contain amidine and thiol functionality. Lysine modification was achieved using 2-iminothiolane (2-IT) [yielding PEG-b-PLL(N2IM-IM)] or dimethyl 3,3'-clithiobispropionimidate (DTBP) [yielding PEG-b-PLL(MPA)], with modifications aimed to impart disulfide cross-linking ability without compromising cationic charge. These two lysine modification reagents resulted in vastly different chemistry contained in the reacted block copolymer, which affected micelle formation behavior and stability along with in vitro and in vivo performance. Amidines formed with 2-IT were unstable and rearranged into a noncharged ring structure lacking free thiol functionality, whereas amidines generated with DTBP were stable. Micelles formed with siRNA and PEG-b-PLL(N2IM-IM) at higher molar ratios of polymer/siRNA, while PEG-b-PLL(MPA) produced micelles only near stoichiometric molar ratios. In vitro gene silencing was highest for PEG-b-PLL(MPA)/siRNA micelles, which were also more sensitive to disruption under disulfide-reducing conditions. Blood circulation was most improved for PEG-b-PLL(N2IM-IM)/ siRNA micelles, with a circulation half-life 3 x longer than naked siRNA. Both micelle formulations are promising for siRNA delivery applications in vitro and in vivo.

We describe the development and application of intravital confocal micro-videography to visualize entrance, distribution, and clearance of drugs within various tissues and organs. We use a Nikon A1R confocal laser scanning microscope system attached to an upright ECLIPSE FN1. The Nikon A1R allows simultaneous four channel acquisition and speed of 30 frames per second while maintaining high resolution of 512 x 512 scanned points. The key techniques of our intravital imaging are (1) to present a flat and perpendicular surface to the objective lens, and (2) to expose the subject with little or no bleeding to facilitate optical access to multiple tissues and organs, and (3) to isolate the subject from the body movement without compressing the blood vessels, and (4) to insert a tail vein catheter for timed injection without moving the subject. Ear lobe dermis tissue was accessible without surgery. Liver, kidney, and subcutaneous tumor were accessed following exteriorization through skin incision. In order to image initial extravasations of compounds into tissue following intravenous injection, movie acquisition was initialized prior to drug administration. Our technique can serve as a powerful tool for investigating biological mechanisms and functions of intravenously injected drugs, with both spatial and temporal resolution. (C) 2010 Optical Society of America

An siRNA-grafted polymer through disulfide linkage was prepared to improve the physicochemical properties and transfection efficacies of the polyion complexes (PICs) as a nanocarrier of siRNA. The siRNA-grafted polymer formed stable PICs due to its larger numbers and higher density of anionic charges compared with monomeric siRNA, leading to effective internalization by cultured cells. Following the endosomal escape of the PIC, the disulfide linkage of the siRNA-grafted polymer allowed efficient siRNA release from the PIC under intracellular reductive conditions. Consequently, the PIC from the siRNA-grafted polymer showed a potent gene silencing effect without cytotoxicity or immunogenicity, demonstrating a promising feature of the siRNA-grafted polymer to construct the PIC-based nanocarrier for in vivo siRNA delivery. (c) 2010 Elsevier Ltd. All rights reserved.

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 Euler's 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.

Silica-coating of positively charged polyplexes was demonstrated through silicic acid condensation to improve the polyplexes for enhanced complex stability and transfection efficiency Silicic acid was efficiently condensed by polycations to form a silica network in the polyplex through electrostatic interaction and hydrogen bonding. The silica-coated (SC) polyplexes had an anionic surface charge of -20 mV and were 10-20 nm larger in size compared to the non-silica-coated control (+33 4 mV, 106 nm). Silica-coating significantly improved the polyplex stability against both dissociations by counter polyanion exchange and aggregation by salt The silica network was dissolved to form silicic acid by removing free silicic acid based on the equilibrium, SiO2 + 2H(2)O reversible arrow Si(OH)(4). Indeed, dialysis of the SC polyplex solution against excess silica-free buffer permitted plasmid DNA release from the silica-coated polyplex, indicating the reversible nature of the silica-layer. The SC polyplex achieved significantly higher transfection efficiency without serious cytotoxicity compared to the polyplex without silica-coating Detailed examinations of transfection using SC polyplexes revealed that the enhanced transfection efficiency was because of facilitated endosomal escape, possibly due to the protonation of the silica in acidic endosomal compartments. These findings demonstrate the utility of the silica-coating technique for polyplex-mediated gene delivery (C) 2010 Elsevier Ltd All rights reserved.

Isothermal titration calorimetry (ITC) was carried out to explore the condensation process of plasmid DNA (pDNA) molecules induced by poly(ethylene glycol)-poly(L-lysine) block copolymer (PEG-PLL) as a condensing agent. The ITC curves measured can be divided into two distinctive endothermic binding processes: the first was the binding of PEG-PLL to the elongated pDNA, and the second was the binding that accompanied the pDNA conformational transition. The thermodynamic parameters were obtained by fitting each ITC curve using our recently developed fitting method. The binding of PEG-PLL to the pDNA was accompanied by a small increase in enthalpy, a large increase in entropy, and a large decrease in free energy. The binding stabilized as the polymerization degree of PLL on PEG-PLL increased and the salt concentration decreased. Changes in the thermodynamic parameters are discussed in relation to both the polymerization degree of PLL on PEG-PLL and the salt concentration.

Fabrication of monodispersed, submicrometer-sized vesicles (nanosomes) that form through self-assembly possessing a thin and permeable membrane remains a significant challenge. Conventional fabrication of nanosomes through self-assembly of amphiphilic molecules often requires cumbersome processes using organic solvents combined with physical procedures (e.g., sonication, thermal treatment, and membrane filtration) to obtain unilamellar structures with a controlled size distribution. Herein, we report the first example of spontaneously formed submicrometer-sized unilamellar polyion complex vesicles (Nano-PICsomes) via self-assembly of a pair of oppositely charged PEG block aniomer and homocatiomer in an aqueous medium. Detailed dynamic light scattering and transmission electron microscopic analysis revealed that vesicle sizes can be controlled in the range of 100-400 nm with a narrow size distribution, simply by changing the total polymer concentration. Also, each Nano-PICsome was composed of a uniform single PIC membrane, the thickness of which is around 10-15 nm, regardless of its size. Fluorescence correlation spectroscopy measurement verified that Nano-PICsomes were able to encapsulate water-soluble fluorescent macromolecules in the inner water phase and release them slowly into the exterior. Moreover, cross-linking of the vesicle membrane allows tuning of permeability, enhancement in stability under physiological conditions, and preservation of size and structure even after freeze-drying and centrifugation treatment. Finally, Nano-PICsomes showed a long circulation time in the bloodstream of mice. Precise control of the particle size and structure of hollow capsules through simple aqueous self-assembly and easy modification of their properties by cross-linking is quite novel and fascinating in terms of ecological, low-cost, and low-energy fabrication processes as well as the potential utility in the biomedical arena.

A novel inorganic-organic hybrid nanocomposite is formed in situ using a simple and straightforward method. Conjugate of short interfering RNA (siRNA) duplex with poly(ethylene glycol) via a disulfide linkage (PEG-SS-siRNA) is demonstrated to regulate the crystal growth of calcium phosphate (CaP), yielding a monodispersed nanocomposite. The resultant nanocomposite can be utilized as nanocarriers for siRNA delivery.

Multipotent mesenchymal stem cells (MSCs) are one of the most powerful tools in regeneration medicine. Their low differentiation efficiency, however, limits further application of MSCs in clinical therapy. Here we report that a much higher multipotent differentiation efficiency of MSCs to adult cells can be achieved using a 3D spheroid culture method based on photolithography and micropatterning techniques. MSC spheroid of precise dimension and uniform quality cultured on the microdomain substrates was prepared first, and then was induced into adipocytes and osteoblasts. Both gene expression results from RT-PCR and morphology observation results revealed that the 3D spheroid culture method could greatly improve differentiation efficiency. Gene expression profiles obtained from gene microarray analysis confirmed the high differentiation efficiency and revealed that MSCs induced in 3D spheroid culture system regulated gene expression not only by increasing the expression levels of genes related to adipogenesis and osteogenesis, but also by down-regulating the gene maintaining MSCs' self-renewal phenotypes. We conclude that our 3D spheroid culture system contributes to an optimization for efficient differentiation of MSCs, offers insight into the mechanism of efficient differentiation of engineered 3D culture system, and has promise for wide applications in regeneration medicine and drug discovery fields. (C) 2009 Elsevier Ltd. All rights reserved.

The acidic pH-sensitivity of polyion complex vesicles (PICsomes) was investigated, using dynamic light scattering (DLS) and confocal laser scanning microscopy (CLSM). PICsomes showed pH-dependent and reversible structural transition, and also underwent a change in permeability by sensing acidic pH. Increased membrane permeability at the pH corresponding to cellular endosomes may be useful for future applications of PICsomes as a delivery vehicle of biologically active compounds to intracellular compartment.

A core-shell-type polyion complex (PIC) micelle with a disulfide cross-linked core was prepared through the assembly of iminothiolane-modified poly(ethylene glycol)-block-poly(L lysine) [PEG-b-(PLL-IM)] and siRNA at a characteristic optimum mixing ratio. The PIC micelles showed a spherical shape of similar to 60 nm in diameter with a narrow distribution. The micellar structure was maintained at physiological ionic strength but was disrupted under reductive conditions because of the cleavage of disulfide cross-links, which is desirable for siRNA release in the intracellular reductive environment. Importantly, environment-responsive PIC micelles achieved 100-fold higher siRNA transfection efficacy compared with non-cross-linked PICs prepared from PEG-b-poly(L-lysine), which were not stable at physiological ionic strength. PICs formed with PEG-b-(PLL-IM) at nonoptimum ratios did not assemble into micellar structure and did not achieve gene silencing following siRNA transfection. These findings show the feasibility of core cross-linked PIC micelles as carriers for therapeutic siRNA and show that stable micellar structure is critical for effective siRNA delivery into target cells.

Polyplexes assembled from poly(aspartamide) derivatives bearing 1,2-diaminoethane side chains, [PAsp(DET)] display amplified in vitro and in vivo transfection activity with minimal cytotoxicity. To elucidate the molecular mechanisms involved in this unique function of PAsp(DET) polyplexes, the physicochemical and biological properties of PAsp(DET) were thoroughly evaluated with a control bearing 1,3-diaminopropane side chains, PAsp(DPT). Between PAsp(DET) and PAsp(DPT) polyplexes, we observed negligible physicochemical differences in particle size and zeta-potential. However, the one methylene variation between 1,2-diaminoethane and 1,3-diaminopropane drastically altered the transfection profiles. In sharp contrast to the constantly high transfection efficacy of PAsp(DET) polyplexes, even in regions of excess polycation to plasmid DNA (pDNA) (high N/P ratio), PAsp(DPT) polyplexes showed a significant drop in the transfection efficacy at high N/P ratios due to the progressively increased cytotoxicity with N/P ratio. The high cytotoxicity of PAsp(DPT) was closely correlated to its strong destabilization effect on cellular membrane estimated by hemolysis, leakage assay of cytoplasmic enzyme (LDH assay), and confocal laser scanning microscopic observation. Interestingly, PAsp(DET) revealed minimal membrane destabilization at physiological pH, yet there was significant enhancement in the membrane destabilization at the acidic pH mimicking the late endosomal compartment (pH similar to 5). Apparently, the pH-selective membrane destabilization profile of PAsp(DET) corresponded to a protonation change in the flanking diamine unit, i.e., the monoprotonated gauche form at physiological pH and diprotonated anti form at acidic pH. These significant results suggest that the protonated charge state of 1,2-diaminoethane may play a substantial role in the endosomal disruption. Moreover, this novel approach for endosomal disruption neither perturbs the membranes of cytoplasmic vesicles nor organelles at physiological pH. Thus, PAsp(DET) polyplexes, residing in late endosomal or lysosomal states, smoothly exit into the cytoplasm for successful transfection without compromising cell viability.

Thiolated c(RGDfK)-poly(ethylene glycol)-block-poly(lysine) (PEG-PLys), a novel block polymer that has a cyclic RGD peptide in the PEG terminus and thiol groups in the PLys side chain, was prepared and applied to the preparation of targetable disulfide cross-linked polyplex micelles through ion complexation with plasmid DNA (pDNA). The obtained polyplex micelles achieved remarkably enhanced transfection efficiency against cultured HeLa cells possessing alpha(v)beta(3) integrin receptors, which are selectively recognized by cyclic RGD peptides, demonstrating the synergistic effect of cyclic RGD peptide ligands on the micelle surface and disulfide cross-links in the core to exert the smooth release of pDNA in the intracellular environment via reductive cleavage. This enhancement was not due to an increase in the uptake amount of polyplex micelles but to a change in their intracellular trafficking route. Detailed confocal laser scanning microscopic observation revealed that polyplex micelles with cyclic RGD peptide ligands were distributed in the perinuclear region in the early stages preferentially through caveolae-mediated endocytosis, which may be a desirable pathway for avoiding the lysosomal degradation of delivered genes. Hence, this approach to introducing ligands and cross-links into the polyplex micelles is promising for the construction of nonviral gene vectors that enhance transfection by controlling intracellular distribution.

PEG-based polyplex micelles, which can detach the surrounding PEG chains responsive to the intracellular reducing environment, were developed as nonviral gene vectors. A novel block catiomer, PEG-SS-P[Asp(DET)], was designed as follows: (i) insertion of biocleavable disulfide linkage between PEG and polycation segment to trigger PEG detachment and (ii) a cationic segment based on poly(aspartamide) with a flanking N-(2-aminoethyl)-2-aminoethyl group, P[Asp(DET)], in which the Asp(DET) unit acts as a buffering moiety inducing endosomal escape with minimal cytotoxicity. The polyplex micelles from PEG-SS-P[Asp(DET)] and plasmid DNA (pDNA) stably dispersed in an aqueous medium with a narrowly distributed size range of similar to 80 nm due to the formation of hydrophilic PEG palisades while undergoing aggregation by the addition of 10 mM dithiothreitol (DTT) at the stoichiometric charge ratio, indicating the PEG detachment from the micelles through the disulfide cleavage. The PEG-SS-P[Asp(DET)l micelles showed both a 1-3 orders of magnitude higher gene transfection efficiency and a more rapid onset of gene expression than PEG-P[Asp(DET)] micelles without disulfide linkages, due to much more effective enclosomal escape based on the PEG detachment in endosome. These findings suggest that the PEG-SS-P[Asp(DET)] micelle may have promising potential as a nonviral gene vector exerting high transfection with regulated timing and minimal cytotoxicity.

For the development of polyplex systems showing a high transfection efficacy without a large excess of polycations, a lysine (Lys) unit as a DNA anchoring moiety was introduced into the amino acid sequence in poly(ethylene glycol)-b-cationic poly(N-substituted asparagine) with a flanking N-(2-aminoethyl)-2-aminoethyl group (PEG-b-Asp(DET)) resulting in PEG-b-P[Lys/Asp(DET)], in which the Asp(DET) unit acts as a buffering moiety inducing endosomal escape with minimal cytotoxicity. PEG-b-P[Lys/Asp(DET)]/DNA polyplexes exhibited a narrow size distribution of approximately 90 nm without secondary aggregates at the stoichiometric N/P 1, suggesting the formation of PEG-shielded polyplex micelles. The introduction of Lys units into the catiomer sequence facilitated cellular uptake and a 100-fold higher level of gene expression with PEG-b-P[Lys/Asp(DET)]/DNA polyplex micelles prepared even at a lowered N/P 2, possibly due to the enhanced association power of the anchoring Lys units.

PEG-coated P-FeOOH nanoparticles were prepared through electrostatic complex formation of iron oxide nanoparticles with poly(ethylene glycol)-poly(aspartic acid) block copolymer [PEG-P(Asp)] in distilled water. By dynamic light scattering (DLS) measurement, the nanopaticle size was determined to be 70 nm with narrow distribution. The FT-IR and zeta potential experimental results proved that PEG-PAsp molecules bound to the surface of the iron oxide nanoparticles via the coordination between the carboxylic acid residues in the PAsp segment of the block copolymer and the surface Fe of the P-FeOOH nanoparticles. The PEG-coated nanoparticles revealed excellent solubility and stability in aqueous solution as well as in physiological saline. In vivo MRI experiments on tumor-bearing mice demonstrated that the PEG-coated nanoparticles prepared by the current approach achieved an appreciable accumulation into solid tumor, suggesting their potential utility as tumor-selective MRI contrast agents. (c) 2007 Elsevier B.V. All rights reserved.

A novel sensing method based on surface plasmon resonance (SPR) was developed for the highly sensitive quantification of low molecular weight (LMW) analytes (colloidal Au replacement assay). Gold nanoparticles (diameter = 20 nm) functionalized with lactosyl-poly(ethylene glycol) (PEG) were prepared and were specifically adsorbed onto a Ricinus communis agglutinin (RCA120)-immobilized SPR sensor chip surface. Subsequent injection of free d-galactose elicited the elution of the preadsorbed lactosyl-PEGylated gold nanoparticles in a manner proportional to the galactose concentration, achieving a substantial and quantitative analysis over a wide range of galactose concentrations (0.1-50 ppm). This method of d-galactose sensing through the substituted elution of preadsorbed nanoparticles from the sensor chip surface would be applicable for the highly sensitive SPR quantification of various LMW analytes, which are known to be difficult to detect by the conventional SPR sensing regime.

Novel thermosensitive polyion complex (PIC) micelles were prepared in an aqueous medium based on the complexation of a pair of oppositely charged block ionomers, poly(2-isopropyl-2-oxazoline)-b-poly(amino acid)s (PiPrOx-b-PAA), containing thermosensitive PiPrOx segments. The controlled synthesis of PiPrOx-b-PAA was achieved via the ring-opening anionic polymerization of N-carboxyanhydrides (NCA) of either epsilon-benzyloxycarbonyl-L-lysine (Lys(Z)-NCA) or beta-benzyl-L-aspartate (BLA-NCA) with omega-amino-functionalized PiPrOx macroinitiators and the subsequent deprotection reaction under acidic or basic conditions. Gel permeation chromatography (GPC) and H-1 NMR spectroscopy revealed that the syntheses of two block ionomers, poly(2-isopropyl-2-oxazoline)-b-poly(L-lysine) [PiPrOx-P(Lys)] and poly(2-isopropyl-2-oxazoline)-b-poly(aspartic acid) [PiPrOx-P(Asp)], proceeded almost quantitatively to give samples with a narrow molecular weight distribution (M-w/M-n <= 1.2). The mixing of these two oppositely charged block ionomers in an aqueous medium led to the spontaneous formation of PIC micelles, which was confirmed by dynamic light scattering (DLS) and transmission electron microscopy (TEM). The PIC micelles were spherical particles with a narrow distribution in the range of the measured concentration (0.125-1 mg/mL) and were stable without any secondary aggregates. Furthermore, the PIC micelles had a constant cloud-point temperature (T-cp) of similar to 32 degrees C under physiological conditions regardless of the total concentration, suggesting that the concentration factor is almost negligible with respect to the T-cp of the micelles presumably because of the increased local concentration of the PiPrOx segments in the shell layer. These PIC micelles have a promising application as a size-regulated smart nanocontainer loading charged compounds as well as bearing a thermosensitive outer shell that is useful for physical affinity control.

The therapeutic usefulness of macromolecular drugs such as plasmid DNA is often limited by the inefficient transfer of macromolecules to the cytosol. Photochemical internalization (PC) technology, in which the endosomial escape of DNA or its complex is assisted by co-incubated photosensitizers that photodamage endosome membrane, offers a solution for this problem. A series of poly(ethylene glycol) (PEG)-based block polycatiomers with increasing number of ethylenediamine repeating unit at side chain of polycatiomers were complexed with pDNA to form the PEGylated polyplexes as a biocompatible gene carrier. Dendrimeric plithalocyanine (DPc)-incorporated micelle was used to assist the gene transfer of these polyplexes in a light-inducible manner. As a result, the light-inducible transfection activity was significantly enhanced as the number of amino group at the side chain of PEG-b-polycatiomer increased. The polyplex from PEG-b-polycatiomer having the longest ethylenediamine structure achieved approximately 1000-fold enhancement of transfection upon photoirradiation. This result supports the underlying hypothesis that photochemical transfection and proton sponge effect of polycations can work synergistically to enhance the transfection efficiency. With careful balance between photochemical transfection enhancement and cytotoxicity, PEG-b-polycatiomers used in this study might be a potential candidate for in vivo PCI-mediated gene transfer. (c) 2006 Elsevier B.V. All rights reserved.

A novel curve fitting model was developed for the isothermal titration calorimetry (ITC) of a cationic ligand binding to DNA. The ligand binding often generates a DNA conformational change from an elongated random coil into a compact collapsed form that is referred to as "DNA condensation". The ligand binding can be classified into two regimes having different binding constants K-i, i.e., the binding to an elongated DNA chain with a binding constant K-1 and with K-2 that occurred during the conformational transition. The two-variable curve fitting models are usually bound by a strict regulation on the difference in the values of the binding constants K-1 > K-2. For the DNA condensation, however, the relationships for K-1 and K-2 are still unclear. The novel curve fitting model developed in this study takes into account this uncertainty on the relationship of the binding constants and is highly flexible for the two-variable binding constant system.

Nonviral gene vectors from synthetic catiomers (polyplexes) ore a promising alternative to viral vectors. In particular, many recent efforts have been devoted to the construction of biocompatible polyplexes for in vivo nonviral gene therapy. A promising approach in this regard is the use of poly(ethylene glycol) (PEG)based block cotiomers, which form a nanoscaled core-shell polyplex with biocompatible PEG palisades. In this study, a series of PEG-based block cotiomers with different amine functionalities were newly prepared by a simple and affordable synthetic procedure based on on aminolysis reaction, and their utility as gene carriers was investigated. This study revealed that the block catiomers carrying the ethylenediamine unit at the side chain are capable of efficient and less toxic transfection even toward primary cells, highlighting critical structural factors of the cationic units in the construction of polyplex-type gene vectors. Moreover, the availability of the polyplex micelle for transfection with primary osteoblasts will facilitate its use for bone regeneration in vivo mediated by nonviral gene transfection.

We report the novel preparation route of organic-inorganic hybrid nanocarriers entrapping Gd-DTPA (diethylenetriamino penta-acetic acid) based on the self-assembly of the block aniomer, poly(ethylene glycol)-block- poly(methacrylic acid), with calcium phosphate crystals as pH responsive MRI contrast agent.

In this study, we prepared a polymeric micelle containing plasmid DNA with both a high stability and a buffering capacity in endosomes. This micelle is composed of tribrock copolymers with two types of cationic units, lysine and a aspartamide derivative with diethylentriamine moiety (Asp(DET)). As a result, the transfection efficiency of the micelles from the triblock copolymers (PEG-b-PAsp(DET)-b-PLys) was one-two orders of magnitude higher than that of the micelles from the diblock coplymers. On the other hand, the transfection efficiency of the triblock copolymers (PEG-b-PLys-b-PAsp(DET)) was much lower than that of the diblock copolymer micelles. These results indicate that the micelle structures are different according to the arrangement of the cationic units.

There are many obstacles for successful in vivo gene delivery with non-viral gene vectors, e.g., the stability during the blood circulation and the specific internalization into the target site. Thus, in this study, we functionalized the polyion complex (PIC) micelles composed of plasmid DNA (pDNA) and poly(ethylene glycol)-poly(L-lysine) (PEG-PLL) block copolymers as follows to clear these obstacles. Firstly, disulfide-crosslinks were introduced into the core of the PIC micelles to acquire a high stability in the extracellular medium and an efficient release of pDNA in the intracellular reductive environment. Secondly, cyclic RGD peptide ligands were introduced into the surface of the PIC micelles for selective transfection into the cells with αvβ3 integrin receptors, such as vascular endothelial cells, and HeLa cells.

Polyion complex (PIC) micelles composed of plasmid DNA (pDNA) and PEG-poly(L-lysine) block copolymers have desirable properties for DNA delivery system, such as high colloidal stability and low interaction with blood components. Nevertheless, the PIC micelles need more stability during the blood circulation and the specific internalization into the target site. Thus, disulfide cross-links were introduced into the PIC micelle's core to acquire a high stability in the extracellular medium and an efficient release of pDNA in the intracellular compartment. And then, cyclic RGD peptide ligands were introduced into the PIC micelle's surface for selective transfection of HeLa cells.

Novel block copolymers with disulfide linkages between PEG and polycalion (PEG-SS-PAsp(DET)) were synthesized to develop intraccllular environment-sensitive polyion complex (PIC) micelles. The PIC micelles formed between the copolymers and plasmid DNA (pDNA) revealed 1-2 order of magnitude higher gene transfection efficiency against HeLa cells than the PIC micelles from the block copolymers without the disulfide linkage.

To develop a gene vector with a low cytotoxicity and high transfection efficiency, we prepared and characterized the polyplexes from three types of polycations which have poly-beta-benzyl-L-aspartate in the main chain and ethylenediamine units in the side chain.

To design polymeric micelles with a high stability and transfection efficiency via buffering effect, we synthesized novel PEG-b-random polycations with two types of cationic moieties, lysine and Asp(DET). As a result, the transfection efficiency of the micelles from the polycations with 47 and 71 % of Lys units in the random segment was two orders of magnitude higher than that of the micelles from PEG-PAsp(DET), and further, the enhanced transfeciton efficiency of the random systems was maintained even after the preincubation with serum proteins.

Novel ternary complex of triblock copolymer, pDNA and anionic dendrimer phthalocyanine was developed for systemic delivery application of photochemical transfection. There was 59 fold of transfection efficiency enhancement.

In this study, we synthesized poly (2-ethyl-2-oxazolines) (PEtOx). And using thermosensitive polymer (poly (2-isopropyl-2-oxazolines) (PiPrOx)), we prepared a series of block copolymers composed of PEtOx and PiPrOx. These copolymers form the aggregates by the hydrophobic interaction near the LCST We investigated the thermosensitive behavior of diblock copolymer (PEtOx (44)-PiPrOx (44)). The aggregates formed at the temperature near the LCST were observed by TEM. We confirmed the aggregates formation responding the temperature.

A stable, freeze-dried formulation consisting of a core-shell-type polyplex with a poly(ethylene glycol) (PEG) shell (polyplex micelles) was prepared from a polyion complex of plasmid DNA (pDNA) and thiolated PEG-poly(L-lysine) block copolymers. The use of lyoprotectants was avoided by crosslinking the core with disulfide bonds. The crosslinked polyplex micelles (CPMs) showed excellent stability during freeze-drying and reconstitution processes, which is in sharp contrast with the formation of visible agglomerates from the non-crosslinked polyplex micelles (NCPMs) after a similar process. A thiolation degree higher than 13% of the lysine residues was required to achieve sufficient tolerability of the CPMs during the freeze-drying/reconstitution cycle. Dynamic light scattering measurements and atomic force microscopy observations demonstrated that the original size and shape of the CPMs with a thiolation degree of higher than 13% were maintained even after the freeze-drying. Furthermore, the CPMs reconstituted from the freeze-dried state achieved a transfection efficiency as high as that of the original samples. The intravenous injection of the CPM with a thiolation degree of 37% into mice via the orbital vein led to an appreciably uniform gene expression of a yellow fluorescence protein variant (Venus) in the liver, while no gene expression was observed in the case of the free pDNA injection. The procedure of disulfide crosslinking of the polyplex micell core allows the preparation of non-viral gene vectors as a powder formulation without the use of any lyoprotectants. This achievement is certainly useful for pharmaceutical applications and exhibits many advantages, including easy concentration adjustments of dosing samples, long-term storage stability, and large-scale production reproducibility.

A novel cytoplasmic delivery system of antisense oligodeoxynucleotide (asODN) was developed by assembling a PEG-asODN conjugate with disulfide linkage (smart linkage) (PEG-SS-asODN) into polyion complex (PIC) micelles through the complexation with branched poly(ethylenimine) (B-PEI). The PIC micelle thus prepared showed a significant antisense effect against luciferase gene expression in HuH-7 cells, far more efficient than nonmicelle systems (asODN and PEG-SS-asODN in free form) and PIC micelle encapsulating the conjugate without the disulfide linkage. Use of poly(l-lysine) (PLL) instead of the B-PEI for PIC micellization led to a substantial decrease in the antisense effect. These results indicate that the PIC micelles formulated from PEG-SS-asODN conjugate and B-PEI is successfully transported from the endosomal compartment into the cytoplasm by the buffering effect of the B-PEI, releasing hundreds of active asODN molecules via cleavage of the disulfide linkage into the cellular interior, responding to a high glutathione concentration in the cytoplasmic compartment. Furthermore, the type of smart linkage (glutathione-sensitive SS linkage vs pH-sensitive linkage) in the conjugates substantially affected the antisense effect of the PIC micelles, depending on the nature of the counter polycation (B-PEI vs PLL. © 2005 American Chemical Society.

To improve the stability of lysozyme-incorporated polyion complex (PIC) micelles in physiological condition, three types of hydrophobic groups, including phenyl (Phe), naphthyl (Nap), and pyrenyl (Py) terminal groups, were separately introduced to the omega-end of poly(ethylene glycol)-poly(alpha,beta-aspartic acid) block copolymers (PEG-P(Asp)). The goal was to enhance association forces between the enzyme, lysozyme, and PEG-P(Asp) carriers. Introduction of these hydrophobic groups significantly decreases micellar critical association concentration and increases the micellar tolerability against increasing NaCl concentrations. Particularly, PIC micelles formed from PEG-P(Asp) with Py groups was most stable against increasing NaCl concentrations up to 0.1 M. Significant deviation from a spherical shape for the micelles was also observed for the PEG-P(Asp)-Py system, consistent with an increased association number.

An A-B-C type triblock copolymer, tandemly aligning two types of polycations with different pKa values in a single polymer strand, was developed for the construction of novel polyplex micelles, satisfying a high DNA condensing ability as well as a proton buffering activity directed to elevating gene transfection. The micelle might feature the distinctive three-layered structure, where an inner polyplex layer of condensed pDNA with poly(l-lysine) (pKa approximately 9.4) as the C segment is successively wrapped with an intermediate layer of poly[(3-morpholinopropyl)aspartamide] (B segment) with a comparatively low pKa of approximately 6.2, to provide a buffering effect, and an outer PEG layer (A segment) as a biocompatible palisade.

5 Conclusions: In this chapter, recent trends in the development of supramolecular nanocarriers of genes and related compounds, based on cationic copolymers, was highlighted. PEG is widely used as the non-ionic and hydrophilic parts of these non-viral gene carriers, as it has been approved by the FDA for use in pharmaceutical formulations. The PEGylation of proteins and peptide drugs has led to an improvement of their pharmacokinetics, proving the validity of using PEG to improve drug efficacy. Similarly, it is expected that PEGylation of gene carriers offers great promise for their optimization. Recent progress in the development of novel synthetic backbones for various kinds of PEG conjugates as well as preparation procedures for sophisticated polyplexes further extend the applicability of PEGylated gene carriers. PEG conjugates possessing moieties that are responsive to changes in their chemical and biological environment act as environmentally responsive polyplexes that can release therapeutic genes in response to external stimuli. Surface modification of polyplexes using ligands attached to the distal end of the PEG segment significantly enhances the internalization efficiency of polyplexes into the cytoplasm. In addition, various functional counterparts to the PEG segment are also available due to the recent development of regulated polymerization techniques. Currently, numerous studies are focusing on polyplex-based PEG-polycation copolymers with respect to their safety and gene transfection efficiency. Investigations of block- and graft-copolymer-based gene delivery systems have progressed rapidly, and is expected to lead to progress in engineered syntheses of PEG-based block and graft copolymers.

The binding of poly (ethylene glycoD - poly (L-lysine) (PEG - PLL) block copolymer to DNA was studied by isothermal titration calorimetry Two distinctive binding processes with endothermic character were observed for PEG-PLL binding to DNA: the one attributed to the binding of PEG-PLL to DNA without DNA condensation, and the other is the process accompanied with DNA condensation. The thermodynamic parameters are obtained by fitting each ITC curve using new fitting method. Based on the thermodynamic parameters, it becomes clear that both the binding of PEG-PLL to DNA and the conformational change of DNA are accompanied not only due to the large increase of entropy, which may be attributed to the release of low molecular counterions bound to in the vicinity of DNA and PEG-PLL, but also the large decrease of free energy which may be attributed to the electrostatic interaction of PEG-PLL with DNA.

Novel ternary complex of triblock copolymer, pDNA and anionic dendrimer phthalocyanine was developed for systemic delivery application of photochemical transfection. There was 25 times higher transfection efficiency compared to non-irradiated control.