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We show the result of pre-clinical study of NK911, a polymeric micelle carrier system for doxorubicin (DOX). The NK911 micelle carrier consists of polyethyleneglycol and conjugated doxorubicin-polyaspartic acid. It has high hydrophobic inner core, and therefore can entrap the sufficient amount of DOX. NK911 has a small particle size of about 40 nm in diameter that accumulates in tumor tissue by EPR effect showing much stronger activity than the free DOX. We plan to perform a clinical trial at National Cancer Center Hospital, Japan from 2001.

To achieve a combination of spatial specificity in a passive manner with a stimuli-responsive targeting mechanism, a temperature-responsive polymeric micelle is prepared using block copolymers of (poly(N-isopropylacrylamide-b-butylmethacrylate) (PIPAAm-PBMA)). The micelle inner core formed by self-aggregates of PBMA segments successfully loaded with a drug (adriamycin), and the outer shell of PIPAAm chains played a role of stabilization and initiation of micellar thermo-response. Optimum conditions were investigated for the micelle formation and drug loading into the inner cores in a view of micellar stability and function as drug carriers. Outer shell hydrophilicity that prevents inner core interaction with biocomponents and other micelles can be suddenly switched to hydrophobic at a specific site by local temperature increase beyond the LCST (lower critical solution temperature) (32.5 degrees C). These micelles showed reversible structural changes allowing drug release upon heating/cooling thermal fluctuations through the LCST. Polymeric micelles incorporated with adriamycin showed a dramatic thermo-responsive on/off switching behavior for both drug release and in vitro cytotoxicity according to the temperature responsive structural changes of a micellar shell structure. The reversible and sensitive thermo-response of the micelle opens up opportunities to construct a novel drug delivery system in conjunction with localized hyperthermia.

An anticancer drug adriamycin (ADR) was incorporated into polymeric micelles forming from poly(ethylene glycol)-poly(aspartic acid) block copolymer by chemical conjugation and physical entrapment. Structural stability of the polymeric micelles was found to be dependent on both the contents of chemically conjugated and physically entrapped ADR. The polymeric micelle with high contents of the chemically conjugated ADR and the physically entrapped ADR expressed very high in vivo antitumor activity against murine C 26 tumor, while the polymeric micelle with only the chemically conjugated ADR showed negligible in vivo activity. This indicates that the physically entrapped ADR played a major role in antitumor activity in vivo. For the polymeric micelle with the high ADR contents, it was found that a dimer of adriamycin molecules formed and that this dimer was physically entrapped in the inner core of the micelle as well as intact ADR.

Micelles of poly(ethylene oxide)-block-poly(β-benzyl- l -aspartate) (PEO-PBLA) were loaded with doxorubicin (DOX) and were characterized in relation to their use as drug vehicles. First, an oil-in-water emulsion method was developed to load DOX in PEO-PBLA micelles. The level of DOX in PEO-PBLA micelles was 5–12% w/w. Whereas the mean diameter of unloaded, PEO-PBLA micelles was ca., 19 nm, the mean diameter of PEO-PBLA micelles loaded with DOX was ≈37 nm. Minimal chemical degradation of DOX occurred as a result of loading in PEO-PBLA micelles. In addition, DOX in PEO-PBLA micelles was less susceptible to chemical degradation than free DOX in aqueous solution. There was evidence for retention of DOX in PEO-PBLA micelles even after freeze-drying and reconstitution in water. Lastly, PEO-PBLA micelles served as drug depots, slowly releasing DOX (days), even in the presence of 10% w/v serum albumin. The results suggest a number of pharmaceutical advantages of PEO-PBLA micelles for the delivery of DOX.

The success of most advanced drug delivery strategies requires development of sophisticated new site-specific carriers. Several new targeting methods use physical and chemical signals such as magnetic fields or changes in pH or temperature as targeting and triggering tools. In addition to site-specificity, the carrier should achieve passive targeting to evade the body's reticulo-endothelial system (RES) and exhibit long blood circulation times in order to efficiently distribute active drug to the site of action (active targeting). To fulfil these requirements, thermo-responsive polymeric micelles have been prepared from amphiphilic block copolymers composed of N -isopropylacrylamide (IPAAm) (a thermo-responsive outer shell) and styrene (St) (hydrophobic inner core). The polymeric micelle which is very stable in aqueous media was formed by the dialyzed method from DMF solution against water. The micelles have a unimodal size distribution (24±4 nm) and CMC was around 10 mg/l (ml→l). These micelles have a small diameter with a low critical micelle concentration, providing a carrier that may have long blood circulation times and a low RES uptake. When the temperature is increased above the transition temperature of the thermo-responsive block chains (32°C), the outer shell chains dehydrate and collapse, allowing aggregation between micelles and favoring binding interactions with cell membrane surfaces. Moreover, these changes are reversible. Hydrophobic molecules are shown to be incorporated into the inner hydrophobic core of the thermo-responsive micelles. Consequently, these micelles are valuable for site-specific delivery of drugs using changes in temperature as a trigger.

Sodium alginate forms a hydrogel upon contact with calcium ions in aqueous solution due to the physical crosslinking (chelation) between the carboxylate anions of guluronate units in alginate and the calcium ions. Alginate disintegration (dissolution) in phosphate buffered saline solution (pH 7.4, Ca 2+ -, Mg 2+ -free) occurs completely in a short time period after a certain lag time. Calcium ion release from the alginate gels is believed to be an influential factor in alginate dissolution. Using this alginate dissolution mechanism, we tried to release macromolecular dextran (nominal molecular weight of 145 000) in a pulsatile manner. As was expected, dextran with molecular weight of 145 000 was released completely in a short time range after a certain lag time. The lag time could be controlled by either alginate molecular weight, alginate concentration in the preparation, or gel beads size. The larger the diameter of the alginate gel beads, the later the observed release time onset. Thereafter dextran was released without changing release rate. Furthermore, we have succeeded to release dextran in a pulsatile fashion using calcium-alginate gel beads by mixing different bead sizes. These results indicate that pulsatile release of macromolecular drugs such as peptides and proteins could be achieved using calcium-alginate gel beads.

Cisplatin, an anticancer drug, was bound to aspartic acid residues of poly(ethylene glycol)-poly(aspartic acid) block copolymer (PEG-P(Asp)) by ligand substitution reaction at platinum atoms of cisplatin. At a molar ratio of cisplatin and the aspartic acid residue of 1:1, polymeric micelles were formed with an average diameter of 16 nm. A polymeric micelle fraction was easily purified by ultrafiltration, and a micellar structure of this fraction was stable in distilled water and NaCl solution at 37°C for 24 h. The polymeric micelle showed 1 8 to 1 5 cytotoxicity of intact cisplatin against murine B 16 melanoma cells during 24–72 h incubation. This suggests release of platinum complexes from the micelle.

An advanced insulin delivery system using a glucose-responsive polymer comprising both phenylboronic acid (PBA) and amino groups was developed and investigated for its efficacy at physiological pH. Stability of complexation of amine-containing PBA gel beads with gluconated insulin (G-Ins) was evaluated using a liquid chromatography system. The amino group presented in the PBA gel beads strongly affected G-Ins release from PBA groups at physiological pH due to an enhanced stability of the boronic acid complex with polyol in the presence of amine. Amine-containing PBA gel showed less rapid decrease of G-Ins in the column under buffer rinse over PBA gel. The released amount of G-Ins from amine-containing PBA gel is much greater than for the PBA gel, responding to the addition of glucose at physiological pH. The results of long-term release studies demonstrate that the amine-containing PBA gel has the capability to respond to glucose challenge over 120 h. Furthermore, a linearity of released G-Ins was observed with glucose concentration. These results demonstrate the feasibility of a glucose-responsive system to achieve insulin release in response to glucose under physiological conditions.

Our research is directed toward development and fundamental studies of biomedically relevant modulation systems using the temperature-responsive polymer, poly(N-isopropylacrylamide) (PIPAAm) functionalized with a carboxyl end group, as the switching element. This material has been attached to both biomolecules and solid surfaces to create new, modified bioconjugates and grafted surfaces, respectively. PIPAAm was used to introduce a reversible switching function correlated to hydration-dehydration changes of polymer chains in response to changes in temperature. The introduction of PIPAAm chains onto polymer surfaces is intended to impart temperature-responsive surface property changes. These surfaces were prepared using either PIPAAm with a terminal carboxyl end group or random copolymers of PIPAAm with acrylic acid. The effects of graft conformation on the dynamics of grafted PIPAAm species were investigated by means of aqueous dynamic contact angle measurement. Each PIPAAm-grafted surface showed completely hydrophilic nature below 20°C, however, these surfaces become hydrophobic above their critical temperature. The extent of hydrophobic property for multipoint-grafted surfaces was small compared to that of the terminally grafted surface. These hydrophilic/hydrophobic surface property changes affect the platelet contact activation. The increase in cytoplasmic [Ca2+]i in platelets was observed only when PIPAAm grafted surface become hydrophobic. The feasibility of cell activation/inactivation control by temperature-modulated surface changes is attractive as modulator for biomedical systems and this strategy may lead to development of new ‘intelligent materials' with specific temperature-modulated surface properties.

Electrochemical control of drug release was demonstrated by redox-active micelles of non-ionic surfactants having a ferrocenyl moiety (FPEG). The surfactants formed micelles at the concentrations above the critical micelle concentration (CMC = 0.1 mM) in normal saline solutions when they existed as the reduced form (FPEG), whereas the micelles were disassembled into monomers when they existed as the oxidized form (FPEG + ). This change was reversible and electrochemically controlled. The electroactivity of FPEG determined by cyclic voltammetry was quite sensitive to the aggregation states. Perylene, which was used as a model of hydrophobic drugs, could be solubilized in a 2 mM FPEG + normal saline solution, however, it was hardly solubilized in a 2 mM FPEG + solution that was made by oxidation of the former solution. Quick and discrete response for releasing perylene from the redox active micelles was achieved by using controlled potential bulk electrolysis method.

The conjugation reaction of adriamycin (ADR) to poly (ethylene oxide)-poly (aspartic acid) block copolymer (PEO-P(Asp)) was optimized in order to inhibit side reactions, which damage the ADR residues. By diminishing alkaline components and lowering temperature, ADR was successfully conjugated to PEO-P(Asp) without side reactions. The obtained ADR-conjugated poly(ethylene oxide)-poly(aspartic acid) block copolymer [PEO-P(Asp(ADR))] was observed to form a micellar structure with a narrower distribution in size than the PEO-P(Asp(ADR)) synthesized by the previously reported method. Unconjugated ADR remained in the inner core of the PEO-P(Asp(ADR)) micelles was removed by dialysis in organic solvents to obtain the micelle without free ADR. Regulated amount of free ADR was then successfully entrapped into the inner core of these purified micelles. In vitro cytotoxicity against P388D1 cells of PEO-P(Asp(ADR)) micelle without free ADR was assayed to estimate the contribution of physically entrapped ADR on anti-tumor activity of the micelle-forming polymeric drug, PEO-P(Asp(ADR)).

A new concept for inflammation responsive drug delivery systems using biodegradable hydrogels of crosslinked hyaluronic acid (HA) has been proposed in our recent studies [ l-3 1. HA is well known to be degraded specifically b;r the presence of hydroxyl radicals, which are produced by phagocytic cells such as leukocytes and macrophages, locally at inflammatory sites [ 41. Our system was established by constructing a heterogeneous-structured device of drug microreservoirs (lipid microspheres) dispersed into degradable matrices of crosslinked HA gels. The most attractive characteristics of this device are: (1) to release lipid microspheres (LM) in response to surface-controlled degradation of crosslinked HA gels and (2) to be quantitatively inflammation-responsive and degradable. These unique features of the device are favorable to regulate drug delivery in several inflammatoryrelated diseases, where LM are released in response to inflammation-induced degradation. The heterogeneous structured device of our design has much potential in drug delivery for severe inflammation. It is well recognized that dysfunction induced by free radicals may be a

A novel polymer complex system sensitive to glucose was studied as a candidate material for formulating a chemically regulated insulin delivery system. A phenylboronic acid (PBA) moiety was incorporated in poly(N-vinyl-2-pyrrolidone) [poly(NVP-co-PBA) ] as a glucose sensor molecule by the radical copolymerization of N-vinyl-2-pyrrolidone with m-acrylamidophenylboronic acid. Complex formation as well as dissociation was estimated by viscosity changes in the complex solution. A positive viscosity change was observed for the poly (vinyl alcohol) (PVA)/poly(NVP-co-PBA) complex system due to complex formation between PVA diol units and poly(NVP-co-PBA) boronate units; minimal or no change in viscosity was observed for either PVA/boric acid or PVA/poly(NVP). The viscosity of the PVA/poly(NVP-co-PBA) system is able to be controlled through changes in polymer molecular weight and/or polymer concentration. The maximum point in viscosity was observed at a molar ratio of PVA to poly (NVP-co-PBA) of 4:1 in this system. The diol-boronate complex interaction was further investigated by following decreased complex viscosity with the addition of a competitive polyol: glucose, tris(hydroxymethyl)aminomethane (TRIS) or (±)-3-amino-1,2-propanediol (APD). The viscosity decreased steeply when glucose was added while minimal change in viscosity was observed when either TRIS or APD was added. This suggested that the chemical structure of the diol compound had an important role in exchange with the diol-boronate complex. These results point to the promising use of PVA/poly(NVP-co-PBA) complex systems in the development of a novel glucose responsive insulin release system.

The swelling behaviour of interpenetrating polymer networks (IPN) composed of poly (acrylic acid) (PAAc) and poly (acrylamide(AAm)-co-butyl methacrylate (BMA)) in water was studied and compared to crosslinked poly(AAm-co-BMA) and crosslinked poly(AAm-co-AAc-co-BMA). The IPNs were prepared by a sequential IPN method in which crosslinked PAAc chains were formed inside of three-dimensional networks ofpoly(AAm-co-BMA) as initial gels. Crosslinked poly(AAcco-AAm-co-BMA) gels (random gels) were also synthesized with the same monomer compositions as the IPNs to elucidate the effect of gel first-order structure on swelling behaviour. Positive swelling changes, i.e. lower swelling ratio at lower temperature and higher swelling ratio at higher temperature, were observed with temperature changes for both the IPNs and the random gels, while no change in swelling was observed for crosslinked poly(AAm-co-BMA) without AAc. In particular, IPN swelling showed a transition point at a certain temperature as a function of temperature change, while random gels showed simple and dull swelling changes with temperature change. The transition temperature of the IPNs shifts to higher temperature with increasing BMA content in IPNs. The positive temperature dependence is suggested to be linked to the formation and dissociation of hydrogen-bond complexes between AAc and AAm with temperature changes in IPNs and random gels. The observed transition in swelling changes in the IPN series is hypothesized to result from concurrent dissociation of continuous ladder-like polymer complexes between PAAc and PAAm matrices in the IPN. The shift of transition temperature induced by incorporation of BMA was due to the stabilization of the complex at higher temperature by increasing hydrophobic interactions. Reversible swelling changes, i.e. higher swelling ratio at high temperature (20°C, 30 °C, 40 °C) and lower swelling ratio at 10°C, in response to step-wise temperature changes between low and high temperature, were observed only for BMA-incorporated IPNs, while irreversible swelling was observed for IPNs without BMA. This result suggests that the presence of hydrophobic BMA had an important role in reversible swelling change of IPNs in response to temperature change. To evaluate the feasibility of IPNs for drug delivery systems, the release of ketoprofen from IPNs was performed with fluctuation of temperature between 10°C and 30 °C. The release rate of drug was reduced at 10°C, while it was increased at 30 °C. The difference of release rate between 10°C and 30 °C was considered to be due to different drug diffusivities between the temperatures induced by swelling changes of IPNs.

Drug release from the monolithic devices of segmented polyether-poly(urethane-urea) (PEUU)s based on both poly (tetramethylene oxide) (PTMO) and poly (ethylene oxide) (PEO) as their soft segments were thoroughly investigated in terms of the relationship between their microdomain structure and the drug release profiles. These PEUUs exhibited diverse microdomain structure depending upon the differences in the solubility parameter of constituting segments and their weight fractions in the polymers. The drug release profiles of these PEUUs were closely connected with the mode of microdomain structure composed of the PTMO, PEO, and hard segments. That is, the formation of distinct microdomains of the PTMO and hard segments dispersed in the PEO matrices allowed the definite regulation of both release rate and transport mode of drug release in the monolithic devices of highly water-swollen PEUUs. These results indicate that the design consisting of microdomains has distinct function as a drug reservoir and transport channel and is a promising way for regulating the release profile of drugs with a variation of solubility parameters from highly water swollen polymeric formulations.