Atsushi Harada

Block copolymer micelles for drug delivery: Design, characterization and biological significance

Recently, colloidal carrier systems have been receiving much attention in the field of drug targeting because of their high loading capacity for drugs as well as their unique disposition characteristics in the body. This paper highlights the utility of polymeric micelles formed through the multimolecular assembly of block copolymers as novel core-shell typed colloidal carriers for drug and gene targeting. The process of micellization in aqueous milieu is described in detail based on differences in the driving force of core segregation, including hydrophobic interaction, electrostatic interaction, metal complexation, and hydrogen bonding of constituent block copolymers. The segregated core embedded in the hydrophilic palisade is shown to function as a reservoir for genes, enzymes, and a variety of drugs with diverse characteristics. Functionalization of the outer surface of the polymeric micelle to modify its physicochemical and biological properties is reviewed from the standpoint of designing micellar carrier systems for receptor-mediated drug delivery. Further, the distribution of polymeric micelles is described to demonstrate their long-circulating characteristics and significant tumor accumulation, emphasizing their promising utility in tumor-targeting therapy. As an important perspective on carrier systems based on polymeric micelles, their feasibility as non-viral gene vectors is also summarized in this review article.

Polyion complex micelles from plasmid DNA and poly(ethylene glycol)-poly(L-lysine) block copolymer as serum-tolerable polyplex system: Physicochemical properties of micelles relevant to gene transfection efficiency

Polyion complex (PIC) micelles composed of the poly(ethylene glycol)-poly(L-lysine) (PEG-PLL) block copolymer and plasmid DNA (pDNA) were investigated in this study from a physicochemical viewpoint to get insight into the structural feature of the PIC micellar vector system to show practical gene transfection efficacy particularly under serum-containing medium. The residual ratio (r) of the lysine units in PEG-PLL to the phosphate units of pDNA in the system significantly affects the size of the PIC micelles evaluated from dynamic light scattering, being decreased from approximately 120 to 80 nm with an increase in the r value for the region with r > or = 1.0. The zeta potential of the complexes slightly increased with r in the same region, yet maintained a very small absolute value and leveled off to a few mV at r approximately 2.0. These results suggest that the micelles are most likely to take the core-shell structure with dense PEG palisades surrounding the PIC core to compartmentalize the condensed pDNA. Furthermore, an increasing r value in the region of r > or = 1 induces a rearrangement of the stoichiometric complex formed at r=1.0 to the non-stoichiometric complex composed of the excess block copolymer. The association number of pDNA and the block copolymer in the micelle was estimated from the apparent micellar molecular weight determined by static light scattering measurements, indicating that a single pDNA molecule was incorporated in each of the micelles prepared from the PEG (Mw=12,000 g/mol)-PLL (polymerization degree of PLL segment: 48) (12-48) block copolymer at r=2.0. These 12-48/pDNA micelles showed a gene expression comparable to the lipofection toward cultured 293 cells, though 100 microM chloroquine was required in the transfection medium. Notably, even in the presence of serum, the PIC micelles achieved appreciable cellular association to attain a high gene expression, which is in sharp contrast with the drastic decrease in the gene expression for lipoplex system in the presence of serum. A virus-comparable size (approximately 100 nm) with a serum-tolerable property of the PIC micelles indeed suggests their promising feasibility as non-viral gene-vector systems used for clinical gene therapy.

In situ single cell observation by fluorescence resonance energy transfer reveals fast intra-cytoplasmic delivery and easy release of plasmid DNA complexed with linear polyethylenimine

The investigation into the intracellular mechanisms for gene expression has acquired great impetus for the improvement of the transfection efficiency by a non‐viral gene delivery system.

Physicochemical properties and nuclease resistance of antisense-oligodeoxynucleotides entrapped in the core of polyion complex micelles composed of poly(ethylene glycol)–poly(l-Lysine) block copolymers

In this study, the physicochemical properties of polyion complex (PIC) micelles formed from antisense-oligodeoxynucleotides (antisense-ODN) and poly(ethylene glycol)-poly(L-lysine) block copolymers (PEG-PLL) were investigated to utilize them as a novel formulation for antisense-ODN delivery. Angular and concentration dependences of the diffusion coefficient of PIC micelles were evaluated by dynamic light scattering. Results suggested that the formed PIC micelles may have spherical shape with core-shell structure, in which the PIC core formed from antisense-ODN and PLL segment was surrounded by a PEG shell. The average radius of PIC micelles was dependent on the chain length of the PLL segment and was not influenced by the change in the length of ODN molecules at least in the range between 15 and 20 base pairs. Critical association concentration (cac) of PIC micelles was then determined from a profile of light scattering intensity versus concentration (Debye plots). Cac is ca. 0.20 mg/ml, which is low enough to ensure the micelle stability in very diluted condition as is the case with systemic injection into the blood compartment for antisense-ODN therapy. Furthermore, the stability of antisense-ODN against deoxyribonuclease I (DNase I) attack was evaluated using capillary gel electrophoresis, revealing that the complexation of antisense-ODN with PEG-PLL effectively prohibited DNase I attack. These characteristics of the PIC micelle system highlight its promising feature as ODN carrier used in the field of targeting therapy.

Temperature-related change in the properties relevant to drug delivery of poly(ethylene glycol)-poly(D,L-lactide) block copolymer micelles in aqueous milieu

The block copolymers of poly(ethylene glycol) and poly(D,L-lactide) (PEG-PDLLA), the latter having a glass transition temperature (T(g)) around the physiological condition, was self-assembled into polymer micelles with a narrow and unimodal distribution in aqueous milieu either by dialysis or by the ultrasonication-aided dispersion method. The 1H NMR measurement of the PEG-PDLLA micelles in D(2)O revealed a gradual increase in the chain mobility of PDLLA segment in the core of the micelles at a temperature range above the T(g) of PDLLA. The critical association concentration (c.a.c.) of the PEG-PDLLA micelles was determined at various temperatures (25-55 degrees C) using pyrene as a probe to monitor the change in the polarity of the microenvironment in the micelle. An Arrhenius plot of the c.a.c. (ln(c.a.c.) versus 1/T) exhibited a break near T(g) of PDLLA. In sharp contrast with the linear decrease in ln(c.a.c.) versus 1/T in the region above the T(g), there was observed an almost constant c.a.c. (7-8 mg/l) regardless of the temperature change below the T(g). Furthermore, the chain exchange reaction between micelles was investigated based on the migration of the end-tagged block copolymers (alpha-lactosyl-PEG-PDLLA and omega-pyrenyl-PEG-PDLLA). The change in the binding affinity of the fluorescent micelles toward the RCA-1 lectin immobilized column was monitored with time to estimate the chain exchange. Consequently, appreciable acceleration in the chain exchange rate was revealed by increasing the surrounding temperature indicating the core mobility to be a substantial factor for inter-micellar chain migration. These results indicate that the engineering of the thermal characteristics of the core-forming segment of the block copolymer should be one of the crucial factors for optimizing the properties of the polymer micelles used for drug delivery.

Pronounced activity of enzymes through the incorporation into the core of polyion complex micelles made from charged block copolymers.

Compartmentalization of enzymes in the nanometric-scaled container, to improve their stability and availability, has recently attracted a strong interest in the field of pharmaceutics. In this study, the enzymatic activity of lysozyme in the core of polyion complex (PIC) micelles, which were formed from egg white lysozyme and poly(ethylene glycol)-poly(alpha,beta-aspartic acid) block copolymer (PEG-P(Asp)), was evaluated using a colorimetric method. Apparent enzymatic activity of lysozyme entrapped in the core of PIC micelles remarkably increased compared to that of free lysozyme, which is mainly attributed to a decrease in the observed Michaelis constant (K(m,obs)). The reciprocal of the K(m,obs) values nicely correlated to the corona thickness of PIC micelles, suggesting that the corona layer of PIC micelle may act as the reservoir of the substrate, p-nitrophenyl penta-N-acetyl-beta-chitopentaoside. This result indicates that the enzymatic activity can be controlled by changing the corona thickness of PIC micelles through a variation in the mixing ratio of PEG-P(Asp) to lysozyme. This type of PIC micelle system entrapping enzyme in the core might be useful for the design of diagnostic as well as targetable therapeutic systems of enzyme including antibody-directed enzyme prodrug therapy (ADEPT).

Formation of Stable and Monodispersive Polyion Complex Micelles in Aqueous Medium from Poly(L-lysine) And Poly(Ethylene Glycol)-Poly(Aspartic Acid) Block Copolymer

Abstract In this study, the formation of polyion complex micelles from a pair of poly(L-lysine) homopolymers (P(Lys)) and poly(ethylene glycol)-poly(aspartic acid) block copolymers (PEG-P(Asp)) with varying chain length was demonstrated in aqueous medium. There exists the lower critical chain length in the charged segments of both P(Lys) and PEG-P(Asp) to form stable polyion complex micelles in nanometric scale. The scaled average characteristic line width (ΓTK2) was independent on the detection angles for all combinations, suggesting that the formed polyion complex micelles may have a spherical shape. Furthermore, the transitional diffusion coefficient (DT) had no concentration dependence, indicating the micelle system was free from secondary aggregates (the cluster of micelles). It is of interest that the micellar size was almost constant (ca. 50 nm) regardless of the change in the chain length of the charged segments. Size distribution was extremely narrow, and the values of variance μ2/Γ 2) were alway...

New macromolecular micelles based on degradable amphiphilic block copolymers of malic acid and malic acid ester

To study the behaviour of polymeric materials under in-vivo conditions, degradable macromolecular micelles based on amphiphilic block copolymers of poly(β-malic acid) as hydrophilic units and poly(β-malic acid alkyl esters) as hydrophobic blocks are studied. First three β-substituted β-lactones, benzyl malolactonate, butyl malolactonate, and butyl 3-methylmalolactonate were prepared, starting from aspartic acid. A prepolymer based on benzyl malate units was synthesized by anionic ring-opening polymerization of benzyl malolactonate. Then the carboxylic end groups of this prepolymer were used as initiator for the polymerization of the second lactone, e. g. butyl malolactonate or butyl 3-methylmalolactonate. The prepolymer and block copolymers have been characterized by 1H NMR and size exclusion chromatography (SEC). Degradable macromolecular micelles were prepared from the block copolymers by two different methods and characterized by dynamic light scattering and fluorescence measurements using pyrene as a fluorescence probe. It was shown that these amphiphilic degradable copolymers form stable micelles under physiological conditions (10–2M phosphate buffered solution, PBS, pH 7.4 with 0.15 M NaCl). Moreover, it was displayed that the characteristics of these macromolecular micelles, especially the critical micellar concentration (cmc), are depending on the chain length of both blocks and on the chemical structure of the hydrophobic block. A very important conclusion of this study is, that micelle formation is dependent on the pH of the medium. Therefore, besides the fact that such micelles are potentially degradable into non-toxic low molecular weight molecules, their properties and stability were proven to be pH-dependent. This property can lead development of an “intelligent” drug carrier able to release the entrapped biologically active molecule depending on the pH values.

Improvement of retroviral vectors by coating with poly(ethylene glycol)-poly (L-lysine) block copolymer (PEG-PLL)

Although some cationic reagents, such as polybrene, improve gene transduction in vitro, their use in vivo is prohibited due to their toxicity to the exposed cells. This paper demonstrates that a new cationic reagent, poly(ethylene glycol)‐poly(L‐lysine) block copolymer (PEG‐PLL), improves gene transduction with retroviral vectors without increasing cell toxicity.

Polyion complex micelles with core-shell structure : Their physicochemical properties and utilities as functional materials

Polyion complex (PIC) micelles were found to form from mixtures of a charged block copolymer with oppositely charged compounds including synthetic ionomers, surfactants, enzymes and DNA. This paper highlights unique physicochemical properties of PIC micelles, including their extremely narrow distribution and the strict chain length recognition. The features of PIC micelles made from a pair of oppositely charged block copolymers and enzyme were described focusing their utilities as functional materials including vehicles for enzyme delivery and sensoring devices for diagnosis.