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A 65-year-old woman developed progressive apraxic agraphia, characterized by poorly formed graphemes, a kanji (Japanese morphograms) recall impairment, relatively preserved oral spelling of kanji characters, and incorrect stroke sequences on writing accompanied by micrographia over a 3-year period. She also showed minor degrees of rigidity, limb-kinetic apraxia, and ideomotor apraxia of the left hand. Although asymmetric rigidity and limb-kinetic apraxia strongly suggested corticobasal degeneration, (11)C-Pittsburgh compound B positron emission tomography (PiB-PET) showed the predominantly right-sided accumulation of amyloid β in the cortices and striatum. (18)F-fluoro-deoxy-glucose PET and single photon emission computed tomography with a (99m)Tc-ethylcysteinate dimer (ECD-SPECT) also revealed predominantly right-sided hypometabolism and hypoperfusion in the primary sensorimotor cortex, posterior cingulate gyrus, temporoparietal cortices, frontal cortices, thalamus, and basal ganglia, a pattern characteristic of both corticobasal degeneration and Alzheimer's disease. The findings suggest that progressive apraxic agraphia with micrographia presenting as corticobasal syndrome can show an Alzheimer's disease pathology. It is also suggested that ideomotor apraxia of the left hand can occur without a callosal lesion, and is caused by hypometabolism or hypoperfusion in the right frontal and parietal cortices, as revealed by PET and SPECT.

The hydrophobic monomer, n-butyl methacrylate (BMA) has been incorporated into thermoresponsive poly(N-isopropylacrylamide) (PIPAAm) to lower PIPAAm phase transition temperatures necessary for systematically regulating cell adhesion on and detachment from culture dishes at controlled temperatures. Poly(IPAAm-co-BMA)-grafted dishes were prepared by electron beam irradiation methods, systematically changing BMA content in the feed. Copolymer-grafted surfaces decreased grafted polymer transition temperatures with increasing BMA content as shown by water wettabilities compared to homopolymer PIPAAm-grafted surfaces. Bovine endothelial cells readily adhered and proliferated on copolymer-grafted surfaces above collapse temperature at 37°C, finally reaching confluence. Cell sheet detachment behavior from copolymer-grafted surfaces depended on the culture temperature and BMA content. In conclusion, cell attachment/detachment can be controlled to an arbitrary temperature by varying the content of hydrophobic monomer incorporated into PIPAAm grafted to culture surfaces. © 2004 Wiley Periodicals, Inc. J Biomed Mater Res 69A: 70–78, 2004

Fabrication of functional tissue constructs using sandwiched layers of cultured cells could prove to be an attractive approach to tissue engineering. Rapid detachment of cultured cell sheets is a very important recovery method that permits facile manipulation of the sheet and prevents functional damage. To accelerate the required culture substrate hydrophilic and hydrophobic structural changes in response to culture temperature alteration, poly(N-isopropylacrylamide) (PIPAAm) was grafted onto porous culture membranes by electron beam irradiation. Analyses by attenuated total reflection-Fourier transform IR and electron spectroscopy for chemical analysis revealed that PIPAAm was successfully grafted to surfaces of porous membranes. Atomic force microscopy (AFM) results showed that PIPAAm-grafted membranes had smoother surfaces than ungrafted controls while retaining their porous structure. The mean roughness of PIPAAm-grafted and -ungrafted porous membrane surfaces determined by digital AFM autocalculation was 4.40 +/- 0.4 and 5.9 +/- 0.4 nm, respectively. Tissue culture polystyrene (TCPS) dishes grafted with PIPAAm were compared with PIPAAm-grafted porous membranes in cell sheet detachment experiments. Approximately 75 min was required to completely detach cell sheets from PIPAAm-grafted TCPS surfaces compared to only 30 min to detach cell sheets from PIPAAm-grafted porous membranes. With porous membranes, the water accesses the PIPAAm-grafted surface from underneath and peripheral to the attached cell sheet, resulting in rapid hydration of grafted PIPAAm molecules and detachment of the cell sheet. With TCPS PIPAAm-grafted surfaces the water is supplied from only the periphery of a cell sheet, slowing detachment.

Lectin-like function was demonstrated in this study for a novel water-soluble polymer with phenylboronic acid residues (poly (AAPBA-DMAm)), which induced appreciable proliferation of murine spleen lymphocytes with an increased expression of interleukin-2 (IL-2) receptor on their surface. Consequently, boosted proliferation of lymphocytes with cytotoxic action to YAC-1 cells was achieved by concurrent addition of IL-2 with poly(AAPBA-DMAm) in the medium, indicating this boronate-containing polymer to be worked as an effective immuno-adjuvant for the induction of lymphokine-activated killer (LAK) cells. Flow-cytofluorimetry study revealed that poly(AAPBA-DMAm) competitively inhibited the cellular binding of N-acetylneuraminic acid-specific lectin (Limax Flavus Agglutinin) in a concentration-dependent manner, suggesting that phenylboronate moiety in the polymer may recognize N-acetylneur- aminic acid (sialic acid) residues existing on the plasma-membrane surface of lymphocytes to induce their proliferation.

To investigate the influence of molecular architecture of poly(acrylic acid) (PAAc)-graft-poly(N,N-dimethylacrylamide) (PDMAAm) (PAAc-g-PDMAAm) on its solution properties in aqueous media, graft copolymers were synthesized by free radical copolymerization of AAc with PDMAAm macromonomer. PAAc-g-PDMAAms were water-soluble at lower temperatures, while they showed a liquid−liquid phase separation from water above the characteristic transition temperatures. The phase transition behavior was affected by the copolymer compositions and pH of the solution. Below the transition temperature, hydrogen-bonding formation between AAc and DMAAm units was observed in the PAAc-g-PDMAAm aqueous solution by FT-IR−ATR measurement. Graft copolymers were thus formed spherical associates with a diameter of ca. 150 nm estimated from the dynamic light scattering measurement. At higher temperatures, the dehydrated regions and the weak dehydration of the PDMAAm chains induced cooperatively the hydrophobic aggregation, resulting in ...

Adriamycin (ADR) dimer was prepared and its antitumor activity was evaluated with mouse colon adenocarcinoma 26 (C 26). As compared with original ADR, the dimer did not show significant antitumor activity, either in vitro or in vivo. Furthermore, polymeric micelles containing varied ratios of the dimer to the original ADR were prepared. Polymeric micelles with a higher dimer/ADR ratio (9.7) showed significant antitumor activity, but the effective dose shifted higher. Effective doses were found to largely depend on the concentration of the original ADR, rather than that of the dimer at the tumor sites. Therefore, it was presumed that the original ADR played a major role in antitumor activity, and the dimer played a supplementary role to contribute selective delivery of ADR to the tumor sites.

To fabricate peptide delivery systems using polymeric drug carriers, an oligopeptide model drug, [Arg8]-vasopressin(AVP), was incorporated into nano-associates comprising poly(ethylene glycol)-( l -aspartic acid block copolymer (PEG-P(Asp)). Incorporation of the AVP was accomplished using a dialysis method. Static light scattering measurements revealed that the acid-type and mixture-type PEG-P(Asp)s formed nano-associates independently without AVP, while the salt form PEG-P(Asp) did not. High loading of AVP into acid-type PEG-P(Asp) was observed with loading levels controlled by changing the molar ratio of drug and block copolymer. Mixture-type PEG-P(Asp) did not show high loading and the salt-type PEG-P(Asp) form did not at all. Acid-type P(Asp) homopolymer formed associates including AVP, however, it was insoluble in aqueous medium. Dynamic light scattering measurements showed that the acid-type PEG-P(Asp) associates sizes narrowly clustered around 150 nm. This finding suggests that associates of acid-type PEG-P(Asp) effectively incorporates peptides possibly via a hydrogen bonding interaction between the block copolymer and the peptide.

Poly(6-(acryloyloxymethyl)uracil) (PAU) having uracil moieties as side chains was synthesized by ordinary radical polymerization to investigate its molecular discrimination for soluble nucleic acid bases in terms of phase transition changes of the polymer aqueous solution. PAU itself in distilled water formed a precipitate due to the polymer complexes at lower temperatures and changed drastically to become water-soluble above a characteristic transition temperature, showing an upper critical solution temperature (UCST). The phase transition behavior was shifted to lower temperatures with increasing concentrations of adenosine (Ado), which is the complementary nucleic acid base to uracil moiety. Ado might interact specifically with uracil moieties of the polymers, preventing the formation of the polymer complex at lower temperatures. Such changes were not observed in PAU aqueous solution containing guanosine (Guo). Additions of Ado and Guo exerted different effects to phase transition changes of the polymer. Moreover, addition of poly(adenylic acid) (Poly(A)) lowered remarkably the transition behavior of PAU solution as compared with Ado addition. It was conceivable that PAU and Poly(A) formed stable polymer complexes, assuming soluble states in even cold water. PAU changed its phase-transition temperature in response to species of the additive materials, demonstrating the specific molecular discrimination in aqueous milieu.

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.

WWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWW Poly(ethylene glycol-b-lactide) possessing a methoxygroup at the poly(ethylene glycol) (PEG) chain end anda polymerizable methacryloyl group at the poly(lacticacid) (PLA) chain end (MeO–PEG/PLA–methacryloyl)was prepared by an anionic ring-opening polymerizationof ethylene oxide and DL -lactide in tandem mannerinitiatedwithapotassium2-methoxyethanolate,followedby end-capping with an excess of methacrylic anhydride.The molecular weight of the obtained polymer wascontrolled by the initial monomer/initiator ratio, whichwas confirmed by the combination of gel permeationchromatography and nuclear magnetic resonance ana-lyses. The functionality of the methacryloyl–PLA endwas almost quantitative. The MeO–PEG/PLA–metha-cryloyl (38/35; these numbers in parentheses denote themolecular weights of PEG and PLA segments divided by100, respectively) formed a core–shell type sphericalmicelle in aqueous media obtained by a dialysistechnique, the cumulant diameter of which was ca.30nm with very low polydispersity factor.The methacryloyl group adjacent to the PLA waspolymerized in the PLA core of the micelle. Thepolymerizationproceededthermallywithradicalinitiatorand photochemically with photo-initiator to producecore-polymerized nanoparticles, which was found byspectroscopic and light-scattering techniques. Taxol-incorporated micelles were prepared to entrap Taxol intoMeO–PEG/PLA–methyacryloyl block copolymer mi-celles by the oil/water emulsion method. Copyright O1999 John Wiley & Sons, Ltd.KEYWORDS: polymeric micelle; stable nanoparticle;drugdeliverysystem;PEG/PLAblockcopolymer;micellestability; taxol

Adriamycin (ADR)-loaded thermally responsive polymeric micelles composed of poly( N -isopropylacrylamide- co - N , N -dimethylacrylamide)- b -poly( d,l -lactide) were prepared by dialysis from its dimethylacetamide solution against water. Microfiltration was successfully applied to removal of block copolymer associates, which were smaller than micellar structures. By this microfiltration polymeric micelles showing a lower critical solution temperature (LCST) at 40°C in phosphate buffered saline was obtained with a monodispersed size distribution of 69.2 nm in cumulant average diameter. ADR-loaded micelles released more ADR at 42.5°C (above the LCST) than at 37°C (below the LCST). ADR-loaded micelles did not show much cytotoxic activity against bovine aorta endothelial cells at 37°C, in contrast to high cytotoxicity at 42.5°C. On the other hand, free ADR expressed high cytotoxicity at both the incubation temperatures. Thus, thermally responsive polymeric micelles showed distinct control of ADR cytotoxic activity by temperature, while free ADR did not. From these results, an effective target therapy against solid tumors is feasible for these polymeric micelles by a combination of selective delivery to tumor sites based on stable micellar structures at 37°C and enhanced cytotoxic activity of these drug-loaded micelles at 42.5°C by local heating at tumor sites.

Four types of hydrophilic gel microcapsules containing water have been prepared by an interfacial polymerization method. Each type of microcapsules has a membrane of different composition. Using three kinds of monomers, N,N-dimethylacrylamide (DMAAm), 4-(aminomethyl)styrene (AmSt), and N,N-dimethylaminopropylacrylamide (DMAPAA), one type of aqueous copolymer having primary and tertiary amino groups was obtained. By the polymerization of three kinds of monomers, DMAAm, AmSt, and 2-[(methacryloyloxy)ethyl] trimethylammoniumchloride (METAC), another type of aqueous copolymer having primary and quaternary ammonium groups was also obtained. Two more types of copolymers were synthesized by copolymerization of α-acryloxy-ω-methoxy-poly(ethylene glycol) (a-PEG) with the above two kinds of monomer mixture. These copolymers were polymerized with terephthaloyldichloride at the water/oil interface to prepare four types of microcapsules containing water, i.e., poly(DMAAm-co-DMAPAA-co-AmSt-alt-terephthalic acid) microcapsules, poly(DMAAm-co-DMAPAA-co-AmSt-co-PEG-alt-terephthalic acid) microcapsules, poly (DMAAm-co-METAC-co-AmSt-alt-terephthalic acid) microcapsules, and poly (DMAAm-co-METAC-co-AmSt-co-PEG-alt-terephthalic acid) microcapsules, which will be abbreviated to MC 1, MC 2, MC 3, and MC 4, respectively. It has been predicted that the microcapsule membranes are hydrophilic and soft and have two-sublayer structures from electrophoretic mobility measurements and from the analysis of the data with Ohshima’s electrokinetic theory for soft particles. The outer sublayers of MC 1 and MC 2 are negatively charged and those of MC 3 and 4 are slightly positively charged. Also, the surfaces of MC 1 and MC 2 are harder than those of MC 3 and 4. By PEGylation, the surface charge density in the membranes decreases and the surface becomes softer. It has been found that the membrane of red blood cells (RBC) is also soft and is composed of two-sublayers, the outer sublayer of which is negatively charged and the inner one is positively charged. The interaction of four types of microcapsules with RBC has been studied. It was found that microcapsules with soft surfaces (MC 3 and MC 4) do not interact with RBC, even though the microcapsule surfaces are positively charged and the surface of RBC is negatively charged. On the other hand, microcapsules with negatively charged but harder surfaces (MC 1) interact with RBC to introduce hemolysis. The membrane surface of MC 2, which is obtained by PEGylation of MC 1, becomes softer than that of MC 1 so that the interaction with RBC was weakly suppressed. From these, it was concluded that the dominant factor to control the interaction between synthetic polymer surfaces and biological cell surfaces is not the surface charges carried by the polymer surfaces but the softness of the polymer surfaces.

Thermally-responsive culture surfaces were designed using copolymers of N-isopropylacrylamide, 4-(aminomethyl)styrene, and acrylic acid. These surfaces contained functional amine and carboxyl groups, which allowed biomolecules to be grafted by amide formation. Epidermal growth factor (EGF), and extracellular matrix (ECM) molecules (collagen type IV, and chondroitin sulfate) were investigated, as surface-grafted biomolecules, for their ability to stimulate cell attachment, proliferation, and function by signaling only from the basal side of cultured cells. Surface analysis of biomolecule-grafted porous inserts showed covalent binding of biomolecules to either amine or carboxyl groups. Multiple attachment to amine and/or carboxyl groups served as cross-linking points that made the polymer hydrogel permanently adherent to the culture surface. Immunofluorescence microscopy techniques gave positive identification of grafted biomolecules. Grafting of EGF improved cell proliferation versus that on nongrafted controls, or controls grafted only with ECM molecules. ECM grafting induced cell attachment on attachment-resistant surfaces. Analysis of trans-epithelial resistance, fluid transport, and polarized g-glutamyl transpeptidase activity indicated that simultaneous grafting of both EGF and ECM produced better polarized cell function than nongrafted controls, or controls grafted with only one type of biomolecule. Covalent grafting of biomolecules did not interfere with cells ability to detach from thermally responsive surfaces upon temperature decrease.

Interaction between protein and cell was discussed using bovine serum albumin (BSA) and yeast protoplast. Glutaraldehyde was used for crosslinking intracellular and intrinsic membrane proteins, which enabled experiments to be carried under various conditions such as pH, salt, and temperature. BSA bound to the surface of yeast protoplast mainly due to the electrostatic interaction, which was determined by investigating the surface potential of the protoplast and the binding isotherms at various pHs. The BSA binding demonstrated the modified Langmuir type and the binding constants and cooperativity parameters were calculated. At the isoelectric point of both BSA and protoplast, the amount of binding obtained the maximum, while at lower and higher pH the binding was significantly decreased. The binding was further discussed in terms of lipid fluidity of the protoplast.

Cross-linked poly(N-isopropylacrylamide) (PIPAAm) hydrogel-modified surfaces were prepared to investigate the effects of a three-dimensionally cross-linked structure of PIPAAm layers on both wettability changes and hydrophobic interactions with hydrophobic solutes in response to temperature changes. The temperature-responsive surface was prepared by polymerization of IPAAm in the presence of cross-linker on the substrates on which an azo polymerization initiator was covalently bonded. The PIPAAm hydrogel-modified surface showed temperature-responsive hydrophilic/hydrophobic surface property alterations as demonstrated by a large and discontinuous wettability changes in a range of 27−32 °C, a slightly lower temperature range than the phase transition temperature for soluble PIPAAm in aqueous media. This implies that the dynamic motion in response to temperature for PIPAAm segments in the modified hydrogel is restricted due to the cross-linked structure. The effect of the three-dimensional PIPAAm structure ...

We have developed a new cell culture substrate grafted with a temperature-responsive polymer, poly(N-isopropylacrylamide) (PIPAAm) using an electron beam irradiation method. These surfaces are hydrophobic in culture at 37 degrees C due to the hydration/dehydration changes intrinsic to PIPAAm at 32 degrees C, and they become highly hydrophilic below 32 degrees C. At 37 degrees C grafted and ungrafted surfaces showed no difference with regard to attachment, spreading, growth, confluent cell density, and morphology of bovine aortic endothelial cells. Stress fibers, peripheral bands, and focal contacts were established in similar ways. After the medium temperature was decreased to 20 degrees C, spread cells lost their flattened morphology, acquiring a rounded cell appearance similar to that of cells immediately after plating. After mild agitation cells floated free from the dish surface without trypsin treatment. Neither cell morphological changes nor cell detachment occurred on ungrafted surfaces. An ATP synthesis inhibitor, sodium azide, and a tyrosine kinase inhibitor, genistein, suppressed cell morphological changes and cell detachment while a protein synthesis inhibitor, cycloheximide, slightly enhanced cell detachment. An actin filament stabilizer, phalloidin, and its depolymerizer, cytochalasin D, also inhibited cell detachment. These findings suggest that cell detachment on grafted surfaces is mediated by intracellular signal transduction and reorganization of the cytoskeleton. While trypsinization causes damage to the cell membrane surface and extracellular matrix proteins, this alternative low temperature treatment is exceptionally noninvasive. The temperature-responsive cell culture surface also should prove useful for investigating the molecular machinery involved in cell-surface detachment.

Two types of thermo-responsive hydrogels are synthesized to obtain comb-type grafted gels with different lower critical solution temperatures (LCSTs) between graft chains and cross-linked backbone networks: these are poly(N-isopropylacrylamide) (PIPAAm) cross-linked hydrogels grafted with poly(N-isopropylacryl amide-co-N,N-dimethylacrylamide) (poly(IPAAm-co-DMAAm)) maintaining a freely mobile end and poly(IPAAm-co-DMAAm) cross-linked hydrogels grafted with PIPAAm chains. The effect of graft chain hydrophilic/hydrophobic balance as well as its mobility on deswelling kinetics of these grafted gels are investigated through the polymer LCST modulation and external temperature changes. The deswelling rate of poly(IPAAm-co-DMAAm)-grafted PIPAAm gel increases with increasing in temperature. This gel shows a discontinuous increase of the deswelling rate when the temperature is applied from below to above the graft chain LCST (37 degrees C). The deswelling rate of PIPAAm-grafted poly(IPAAm-co-DMAAm) gel increases continuously when the temperature is applied from below to above the graft chain LCST (31 degrees C). Due to the strong hydrophilicity of backbone network, the hydrophobic aggregation force weak. In contrast to the graft-type gels, normal-type poly(IPAAm-co-DMAAm) cross-linked gel without graft chains demonstrates the discontinuous decrease for the deswelling rate when the temperature is applied from below to above the polymer LCST (36 degrees C), entrapping water inside the gel due to the formation of an impermeable dense skin layer at the gel surface. These gel deswelling mechanisms are discussed in terms of gel structures.

Purpose. To determine aortic endothelial cells permeation ability and mechanisms of the aqueous block copolymeric micelles, poly(ethylene oxide)-poly ((β-benzyl L-aspartate) (PEO-PBLA) chemically conjugated with fluroescein isothiocyanate (FITC) by transport study and confocal laser scanning microscopy.

Block copolymer micelles (polymer−metal complex micelles) containing platinum(II) complexes in the core were prepared through the complexation of cis-dichlorodiammineplatinum(II) (cisplatin, CDDP) with poly(ethylene glycol)−poly(α,β-aspartic acid) block copolymer (PEG−P(Asp)) in an aqueous medium. Dynamic light scattering measurements revealed that CDDP-complexed micelles had diameters of approximately 20 nm with considerably narrow distribution. The critical substitution molar ratio of CDDP to Asp residues in PEG−P(Asp) (CDDP/Asp) to form a stable micelle structure was determined to be 0.5. The CDDP-complexed micelle, which was stable in distilled water at room temperature, started to dissociate in approximately 10 h of the induction period accompanying the sustained release of platinum(II) complex from the micelle in physiological saline (0.15 M NaCl solution) at 37 °C. The release rate of CDDP was inversely correlated with the chain length of P(Asp) segments in the block copolymer. This property of sus...

Four types of hydrophilic gel microcapsules containing water have been prepared by an interfacial polymerization method. Each type of microcapsules has membranes of different compositions. By the combination of three kinds of monomers, N , N -dimethylacrylamide (DMAAm), 4-(aminomethyl)styrene (AmSt), and N , N -dimethylamino-propylacrylamide (DMAPAA), two types of water-soluble copolymers having tertiary amino groups were obtained. Two more types of copolymers were synthesized by copolymerization of above three monomers with α -acryloxy- ω -methoxy-poly(ethylene glycol) (a-PEG). These four types of copolymers were polymerized with terephthaloy-dichloride at the water/oil interface to prepare four types of microcapsules containing water. Effects of the stirring rate during the preparation of microcapsules upon the membrane properties were studied. The effects of relative permittivity of dispersing media on microcapsule sizes were studied. It was found that the microcapsule sizes are controlled by the composition of the membranes and by the relative permittivity of the solvents in which microcapsules are redispersed. Depending on the stirring rates during the preparation procedure, the different changes in microcapsule size were observed during the exchanges of dispersing media of various relative permittivities. From these results, it is concluded that different microcapsules in their membrane compositions and in their properties are obtained depending on the stirring rate during the preparation of microcapsules, even if other preparation conditions are kept constant.

Deswelling mechanism for comb-type grafted poly( N -isopropylacrylamide) (PIPAAm) hydrogel was investigated to make clear the effect of gel architecture on the gel deswelling kinetics. Deswelling rate and mechanism were compared with those for conventional crosslinked PIPAAm gel and poly(ethylene oxide) (PEO)-grafted PIPAAm gel in response to temperature increase from below to above the gel-phase transition temperatures. The deswelling rate for the conventional PIPAAm gel are reciprocal to the gel size square, indicative of collective diffusion mediated manner for crosslinked network into water. Although PEO graft chains formed water release channels within the network to produce fast gel deswelling changes, deswelling change of PEO-grafted gel also demonstrated collective diffusion mediated manner of crosslinked backbone chains. On the other hand, one order faster rate of deswelling change of PIPAAm-grafted gel was observed than that of PEO-grafted gel with identical gel dimension. The deswelling change of PIPAAm grafted gel did not obey collective diffusion mediated manner, demonstrating reciprocal to the gel size to 1.58th power. This deswelling mechanism was explained in terms of rapid hydrophobic aggregation of freely mobile PIPAAm graft chains and an operation of intrinsic elastic forces of polymer network. The operation of both elastic force and strong hydrophobic aggregation force between the dehydrated PIPAAm graft chains released water inside the gel, causing more rapid deswelling changes of the PIPAAm-grafted gels.

Poly(ethylene oxide) (PEO) chains are introduced as graft chains maintaining freely mobile ends in thermo-responsive cross-linked poly(N-isopropylacrylamide) (PIPAAm) hydrogels by copolymerization of IPAAm with α-acryloyl-ω-methoxy-PEO. The deswelling response on raising the temperature of this gel above the gel phase transition temperature (TP) takes place within 10 min, whereas a conventionally cross-linked PIPAAm gel of the same dimensions requires 1 month for deswelling. This difference is due to the formation of water release channels within the skin layer by the hydrophilic PEO graft chains. The rapid deswelling of the grafted gel is compared with the deswelling changes of random copolymer gels composed of IPAAm and hydrophilic acrylic acid (AAc), which also accelerates gel deswelling. Deswelling is fastest in copolymers containing 1.3 wt % AAc and in grafted gels containing 13 wt % PEO. These results were interpreted as reflecting the gel structure.

A novel UV crosslinkable co-polymer of 4-(N-cinnamoylcarbamide)methylstyrene (CCMS) and N-isopropylacrylamide (IPAAm) was partially entrapped in traditional tissue-culture-treated polystyrene and crosslinked by UV light irradiation. Dishes modified by this method showed a change in contact angle with respect to temperature as compared to tissue culture polystyrene controls. Surface chemical analysis indicated that the crosslinked hydrogel does not detach from the surface after successive rinsing in ethanol and water, keeping the cells or cell construct free of unwanted soluble polymer after detachment. Cultures of both bovine endothelium and human retinal pigmented epithelium were confirmed to be able to attach and grow on the polymer-modified surfaces morphologically identical to that on control tissue culture polystyrene surfaces. Corresponding to a change in temperature, these cultures would detach and could be transplanted to another culture surface without functional and structural changes. These results show that the new, photo-crosslinkable hydrogel system can utilize the hydrophobic/hydrophilic change of the surface for cell culture detachment while being permanently applicable to any tissue culture geometry. © 1998 John Wiley & Sons, Inc. J Biomed Mater Res, 40, 631–639, 1998.

Interaction of rat lymphocyte subpopulations (B cells and T cells) with surfaces of poly ( N,N -dimethylacryl-amide- co -3-acrylamidophenylboronic acid) (DB copolymers) with varying composition was evaluated under a controlled shear stress by using a plane-parallel-type channel equipped with a polymer-coated bottom wail [field-flow fractionation/ adhesion chromatography (FFF/AC)]. This method enables to control the hydrodynamic shear stress at the channel wall resulting from the parabolic flow in the channel. The recovery profile of lymphocytes from DB channels was different among each subpopulation, and the selective recovery of T cells from the channel was achieved with most of B cells remaining in the channel. Phenylboronic acid residues in DB copolymers seem to work as binding sites for sugar residues, presumably sialic acid residues, existing on the membrane surface of lymphocytes. Yield and efficiency of the separation are in the practical range for the application in immunological field, allowing this system to be useful as a novel lymphocyte separator.

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.

Temperature-responsive hydration/dehydration changes in surface-grafted poly(N-isopropylacrylamide) (PIPAAm) were utilized for hydrophilic/hydrophobic surface property alterations in cell culture. In this report, we utilized PIPAAm-grafted surfaces to recover confluently-cultured vascular endothelial cells as coherent monolayers from this cell culture substrate and to transfer to new cell culture substrates. For this purpose, we used two different methods to recover and transfer cell monolayer cultures: (1) chitin membranes used as an apical side cell support during cultured cell transfer, allowing cell basal side reattachment to new culture substrates after transfer; and (2) a cell culture insert (porous PET) used as both a support as well as new substrate, allowing basal surfaces of cultured cells to be exposed to the medium after transfer. In both cases, all cells grown on PIPAAm-grafted surfaces detach completely with maintenance of basement membrane-like structure. Recovered cells attach to the second culture surfaces, covering more than 60% of the new substrate, and retain approximately 90% viability and their original function as judged from tissue-type plasminogen activator secretion. This technique could be utilized to prepare novel bioartificial organs as well as cell co-culture systems by multi-layering different cell types to mimic tissue structures for tissue engineering.

Novel hydrophilic-hydrophobic block copolymers - poly(2-hydroxyethyl methacrylate-block-4-bis(trimethylsilyl)methylstyrene) (poly(HEMA-block-BSMS) or BH polymer) - were prepared as materials potentially useful for blood-contacting devices. Among the BH(X) series including poly(HEMA) itself, platelet adhesion and activation are minimum on the surface of BH(10), the block copolymer composed of 10 mol% of the hydrophobic segment. The unique physicochemical characteristics of BH(10) such as the high free water content and the increased mobility of PHEMA segments seem to play an important role in the prevention of platelet adhesion and activation. The water content of BH(10) was twice that of poly(HEMA) itself, even in the presence of 10 mol% of the hydrophobic moiety [poly(BSMS)]. Further, an anomalous increase in free water content was observed for the BH(10) sample measured by DSC. The glass transition temperature of BH(10) in the dry state was determined to be c. 10 degrees C, which was much lower than that observed for each of the homopolymers (c.f.: Tg [poly(HEMA)] = 70 degrees C; Tg [poly(BSMS)] = 160 degrees C), suggesting an increased mobility of tethered PHEMA segments in the BH(10) film.

Purpose. To directly visualize and evaluate the aqueous block copolymeric micelles, poly(ethylene oxide)-poly(β-benzyl L-aspartate) (PEO-PBLA) chemically conjugated with pyrene fluorescence molecule, by nanotechnology of atomic force microscopy (AFM).

Copolymers (IABb) composed of N-isopropylacrylamide (I), N-(3-dimethylaminopropyl)-acrylamide (A), 3-acrylamidophenylboronic acid (B), and a hydrophobic comonomer, n-butyl methacrylate (b), were synthesized as cell culture substrata since we previously learned that bovine aortic endothelial cells (BAECs) cultured on the phenylboronic acid-containing copolymer (IAB) differentiated into capillary structures after 26 days culture. The synthesized IABb copo ymers contained higher boron and amine moieties than the IAB copolymer. The results of the dynamic contact angle measurement revealed that IABb copolymer-coated surfaces showed a relatively hydrophobic nature, changing to hydrophilic in response to the aqueous environment. BAECs cultured on the copolymer substrata developed into capillary networks after 7 days. This is probably due to the enrichment of boron and amine segments in the vicinity of the hydrophilic copolymer surface, enhancing more pronounced interaction of boronates with cell membrane glycocalyx. The introduction of n-butyl methacrylate into the polymers might enhance the diffusion of the hydrophobic segments to the bulk polymers and the concentration of relatively hydrophilic segments at the outermost polymer surfaces by contact with water. A copolymer (IAP) without boronic acid groups was also prepared using N-phenylacrylamide (P) as a comonomer instead of 3-acrylamidophenylboronic acid in the IAB copolymer to investigate the effect of boronic acid moieties on the capillary formation of the cultured cells. Although the endothelial cells seeded on the copolymer without boronic acid groups adhered during the early culture period, these cells showed neither proliferation nor differentiation and detached from the surface after 13 days. These results strongly support the opinion that the phenylboronic acid groups in the copolymers are responsible for the specific induction of tissue formation of BAECs through the interaction with glycoconjugates on the cell membranes.

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.

Alkyl-terminated PIPAAm was obtained by covalent coupling of alkyl groups of different chain lenghts (C3, C6, C8, C12 and C18) to one end of poly(N-isopropylacrylamide) (PIPAAm). At the molecular level, incorporation of the alkyl chains with PIPAAm chains promoted aggregation at temperatures below the LCST (lower critical solution temperature) of pure PIPAAm, reflecting the hydrophobic contribution of the alkyl chains to the LCST. The LCST changes were greatest for stearoyl (C18)-terminated PIPAAm (PIPAAm-C18H35). Interestingly, this polymer above its critical micelle concentration (cmc, 80 mg/L) in aqueous solution had the same LCST as unmodified PIPAAm, owing to formation of a core-shell micellar structure shielding the hydrophobic inner alkyl core from interactions with water. Micelle formation and the micellar structure of the alkyl-terminated PIPAAm aqueous dispersions were analyzed by fluorescence, surface tension and DLS (dynamic light scattering) measurements. Micelle formation is facilitated by both an increase in hydrophobicity of the incorporated group and suppression of PIPAAm hydrophilicity upon heating, closely relating to intramolecular hydrophilic/hydrophobic balance. PIPAAm-C18H35 micelles exhibit two heterogeneous microdomains; a hydrophobic inner core highly capable of solubilizing hydrophobic pyrene molecules, plus a hydrated outer shell that stabilizes this micellar structure below its LCST and shows reversible thermoresponsive aggregation/dispersion in heating/cooling thermal cycles through the LCST.

Poly ( α -hydroxy ethylene oxide-co- β -benzyl l -aspartate), α -hydroxy PEO/PBLA, block copolymers were used for the formulation of new functional polymeric micelles. They have a small diameter (about 30 nm) and a very low critical micellar concentration (cmc) in water, ca. 4 mg l −1 . Doxorubicin (DOX) was physically entrapped into the hydrophobic inner core of these functional micelles. The diameter of DOX-loaded α -hydroxy PEO/PBLA micelles was determined to be approximatively the same as the diameter of the corresponding empty micelles, ca. 25 nm. Moreover, the DOX-loaded functional micelles, as the corresponding empty micelles, were shown to be stable in 0.1 M phosphate buffered solution (PBS) pH 7.4 even in presence of proteins. The cytotoxicity of DOX-loaded functional micelles against P388D1 leukemia cells was studied and compared to the one of DOX-loaded α -methoxy PEO/PBLA micelles and to the cytotoxicity of both α -hydroxy and α -methoxy PEO/PBLA micelles. From this study it was concluded that the DOX-loaded functional micelles seem to have a slightly higher cytotoxicity than the DOX-loaded α -methoxy PEO/PBLA micelles, while both empty micelles were shown to be non-cytotoxic against P388D1 leukemia cells.

Purpose. To design glucose-responsive gels based on the complexation between polymers having phenylboronic acid groups and poly (vinyl alcohol). Specifically, high-glucose sensitivity at physiological pH was achieved through the interaction of phenylborate with amino groups.

The synthesis and characterization of comb-type grafted thermo-sensitive hydrogels is presented. These hydrogels were synthesized by polymerization of N-isopropylacrylamide (IPAAm) with a PIPAAm macromonomer. This process leads to a crosslinked IPAAm backbone polymer, copolymerized with highly mobile comb-type PIPAAm chains. These new thermo-responsive copolymers displayed higher equilibrium swellings at lower temperatures and rapid deswelling kinetics at elevated temperatures. The swelling/deswelling for comb-type gels is dependent on the graft chain lengths, in contrast to normal PIPAAm gel lacking the graft chains. As the temperature is increased above the critical temperature, the dehydrated graft chains aggregated due to hydrophobic attraction. Rapid and reversible kinetics of the graft-type gel were observed in response to stepwise temperature changes within short time cycles: phenomena not observed in normal crosslinked thermo-sensitive gels. The influence of freely mobile graft chains on both the equilibrium and dynamic properties of comb-type PIPAAm gel is demonstrated. Possible application of graft-type gel is discussed for actuator systems.

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.

Glucose-Sensitive Lower Critical Solution Temperature Changes of Copolymers Composed of N -Isopropylacrylamide and Phenylboronic Acid Moieties

We have prepared a copolymer containing both phenylboronic acid and tertiary amine moieties. The copolymer forms a stable complex with poly(vinyl alcohol) (PVA) since boronate moieties interact with PVA hydroxyl groups. The polymer-polymer complex changes its swelling degree with glucose concentration in Dulbecco's phosphate-buffered saline solution (PBS) at pH 7.4, due to the higher complex formation of boronic acid moieties with glucose hydroxyl groups over those in PVA. Glucose-responsive swelling changes of a membrane complex were then utilized to control glucose-responsive current changes with a membrane-coated platinum electrode. Glucose addition to PBS induces swelling of the cast gel membrane, leading to increased diffusion of ion species and thus increased measurable current changes. Since the addition of methyl α-d-glucoside has little influence on the current changes, the current change by the addition of glucose is indicative of the high selectivity of this system for glucose and its cis-hydroxyl groups in glucose. It is observed that current changes are proportional to glucose concentration in the range 0-300 mg/dL. This range corresponds well to physiological blood glucose levels. Current change rates determined from the slope of the time course immediately after glucose addition are also proportional to glucose concentration within this range, yielding even higher sensitivity to the change in glucose concentration. Reproducible signal output is also demonstrated by repetitive, stepwise glucose concentration changes. These results support the applicability of the platinum electrode coated with the gel membrane complex comprising a phenylboronic acid-containing polymer and PVA for a novel glucose-sensing device.

Changes in cytoplasmic free calcium levels and membrane fluidity of platelets in contact with poly(acrylamide-co-methacrylic acid) (PAAmMAc) particles were examined to analyze the mechanistic aspect of regulating platelet function. Our previous studies demonstrated interesting features of PAAmMAc particles during interaction with platelets: (1) PAAmMAc particles induce no calcium increase but enhance membrane fluidity of platelets: (2) thrombin induces no calcium increase in platelets when the platelets were mixed previously with PAAmMAc particles; and (3) PAAmMAc particles induce a calcium increase in platelets when they were treated previously with sodium azide (NaN3). These results suggest the possibility that PAAmMAc surfaces may regulate the calcium level by influencing platelet metabolism. In this study, non-cross-linked PAAmMAc solution with the same chemical composition as the particles showed a suppressive effect on thrombin-induced calcium increase, but, no influence on membrane fluidity. This result indicates that aggregated macromolecular surface assemblies of PAAmMAc may dominate the increase in membrane fluidity of platelets although the calcium change is induced by discrete molecular level interaction between the PAAmMAc and platelet membranes. It was also revealed that the suppression of thrombin-induced calcium increase and the membrane fluidity increase in platelets by PAAmMAc particles were reduced by albumin-treatment of the particles. This result suggests that such phenomena may be due to a decrease in any physicochemical interaction of PAAmMAc surfaces with albumin, rather than platelet metabolic change. PAAmMAc particle surfaces with higher carboxyl groups exhibited a more suppressive effect on thrombin-induced calcium increase, whereas those with lower carboxyl groups derived a higher calcium increase when the platelets were treated previously with NaN3. These results suggest the importance of electrostatic and any other physicochemical interaction of PAAmMAc chains on regulating cytoplasmic calcium levels.

Polymer-enzyme hybrid conjugates modified by a temperature-responsive polymer, poly(N-isopropylacrylamide) (PIPAAm), have been synthesized. We have investigated the molecular architecture of PIPAAm-enzyme conjugates by preparing two types of PIPAAm-trypsin conjugates, wherein PIPAAm chains are attached by either single-end or multipoint chemistry. A semitelechelic co-oligomer (IDc) was attached to trypsin by single-point conjugation (IDc-trypsin). A copolymer (PIDAAc) consisting of acrylic acid and IPAAm randomly linked in polymer chains was attached to trypsin using multipoint conjugation (PIDAAc-trypsin). Both conjugates exhibited reversible temperature-responsive phase separation. The IDc-trypsin conjugate exhibited phase separation at the same temperature as pure IDc, due to the highly mobile free polymer end group which remains sensitive to small temperature changes. The PIDAAc-trypsin conjugate precipitated at higher temperatures than pure PIDAAc, whose movement was restricted by multiple binding points. Enzyme stability in solution was improved after introduction of PIPAAm chains, which prevented autolysis attributed to conjugate steric hindrance. Stability under repeated temperature cycling was also dependent on the architecture of conjugates; the IDc-trypsin conjugate was more stable than the PIDAAc-trypsin. As a consequence, single-end conjugation of polymer to enzyme provides novel bioconjugate with novel functionality attributed to attached polymer while retaining native biological function with high stability.

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.

Amino semitelechelic poly(N-isopropylacrylamide)s (PIPAAm) with three different molecular weights were synthesized by telomerization of IPAAm monomer with 2-aminoethanethiol as a chain transfer agent, changing the molar ratio of monomer to chain transfer agent. Macromonomers of thermosensitive PIPAAm were synthesized by condensation reaction of amino semitelechelic PIPAAm with N-acryloxysuccinimide. The molecular weights of macromonomers determined by titration of the terminal amino groups were 2900, 4000, and 9000, respectively. The comb-type grafted PIPAAm hydrogels having different lengths of graft chains were synthesized by radical copolymerization of IPAAm monomer with PIPAAm macromonomer in the presence of N,N'-methylenebisacrylamide as a cross-linker. An important aspect of the graft-type gels is the construction of a molecular architecture different from PIPAAm normal type ofgel even though the composition is same. Higher equilibrium swellings at lower temperatures were observed in graft-type gels in contrast to the normal-type gel, and longer graft chains resulted in higher equilibrium swelling due to the freely mobile grafted chains. Both the normal-type and the graft-type gels exhibited reversible swelling-deswelling changes in aqueous milieu in response to an alteration of temperature. The deswelling kinetics at 40 °C changed from equilibrium swelling states at 10 °C, however, exhibited remarkable differences, and rapid responses were observed for graft-type gels. The rapid dehydration of graft chains during gel shrinking was confirmed by analysis of DSC measurements. These dehydrated graft chains strongly aggregated with hydrophobic intermolecular forces, inducing the rapid deswelling of the gels. The attractive forces operating between dehydrated chains were larger in the gel having longer grafted chains, resulting in faster deswelling. A deswelling mechanism distinct from polymer network collective diffusion was demonstrated with these comb-type grafted hydrogels having freely mobile grafted chains in the network.

This is a review of our recent electrokinetic studies of the surface of poly(N-isopropylacrylamide) hydrogel-coated particles and plates. Electrophoresis measurements were used for particles, while electroosmosis measurements were used for plate samples. It was found that the phase transition of a poly(N-isopropylacrylamide) layer is well reflected in the electrokinetic data. We found excellent correlation between the results obtained via these two different methods. The obtained data were found to be well described by an electrokinetic theory for “soft” surfaces.

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.

Temperature-responsive poly( N -isopropylacrylamide) and its copolymer gels with the hydrophilic comonomer, acrylamide and the hydrophobic comonomer, butyl methacrylate, all exhibit swelling-deswelling changes in response to external temperature changes. These hydrogels show negative swelling thermosensitivities, particularly swelling at lower temperature and complete deswelling over specific phase transition temperatures ( T p ). Shrinking kinetics of these gels from swollen to deswollen states at several different temperature changes have been investigated. When temperature changes are performed entirely below T p , the shrinking process is dominated by polymer network diffusion. On the other hand, shrinking kinetics for temperature changes from below to above T p , are dramatically influenced by gel surface structural changes and formation of a collapsed polymer skin layer. This surface skin formation prompts the accumulation of internal hydrodynamic pressure inside the gels upon shrinking by blocking the outflux of entrapped water. Both rate and magnitude of internal hydrodynamic pressure are modulated by the gel volume, the hydrophobicity of the gel polymer chains and the degree of external temperature changes. This internal pressure eventually causes convective outflow of water from the gel interior. Hydrodynamic internal pressure affects the drug release on-off pattern during gel shrinking.

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.

Temperature-responsive semitelechelic poly(N-isopropylacrylamide) (PIPAAm) bearing a carboxyl end group has been chemically immobilized on aminated polystyrene particle surfaces via condensation reaction. PIPAAm-grafted particles were uniformly suspended in aqueous media at lower temperatures. With increasing temperature, PIPAAm-grafted particles aggregated and precipitated. Such reversible changes in particle colloidal behaviour was correlated to temperature-modulated hydrophilic/hydrophobic changes of particle surfaces modified by PIPAAm hydration/dehydration with temperature changes. Interactions between platelets and PIPAAm-grafted surfaces were studied by monitoring cytoplasmic free Ca2+ concentration ([Ca2+]i) changes in platelets using intracellularly-trapped Ca2+ indicator dye, Fura 2, at various temperatures. Although changes in [Ca2+]i in platelets in contact with PIPAAm-grafted particles were not observed below the critical temperature of PIPAAm, significant changes in [Ca2+]i in platelets were induced by contact with particles above this critical temperature. Furthermore, temperature-modulated cell adsorption/desorption control by PIPAAm-grafted particles was investigated using a particle aggregation assay in the presence of lymphocytes. Below the critical temperature of PIPAAm, mixed suspensions were completely homogeneous due to minimal interaction between lymphocytes and hydrated particles. In contrast, aggregated precipitates were observed by increasing the suspension temperature above the critical temperature of PIPAAm resulting from strong hydrophobic interactions between particles with lymphocytes. These precipitates are reversibly resuspended in cold buffer. The feasibility of cell activation/inactivation or cell attachment/detachment control by temperature-modulated surface changes is attractive for suspension cell culture and drug delivery at targeted sites in vivo.

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)).

We have investigated temperature modulation of surface properties for hydrophilic/hydrophobic changes using poly(N-isopropylacrylamide) (PIPAAm). Two types of PIPAAm were used as surface modifiers: an end-functionalized PIPAAm with a carboxyl end group and a poly(IPAAm-co-acrylic acid) copolymer. By means of dynamic contact angle measurements in water, the wettability of terminally polymer grafted surfaces using end-functionalized PIPAAm with a carboxyl end group were compared with that of multipoint polymer grafted surfaces using PIPAAm copolymers containing carboxyl groups along the polymer chain. Each PIPAAm grafted surface showed completely hydrophilic properties under 20 o C

Chemical modification of proteins by use of functional polymers is expected to endow them with new properties without destroying their native functions, thus providing useful materials for application in different fields. We have synthesized poly(N-isopropylacrylamide) [poly(IPAAm)] co-oligomer with N,N-dimethylacrylamide (DMAAm) and reactive end groups by telomerization of IPAAm. This co-oligomer exhibits a lower critical solution temperature (LCST) at 37 degrees C. Using this temperature-responsive semitelechelic co-oligomer, we prepared polymer-enzyme conjugates of lipase by covalent coupling via carboxyl end-groups. This bioconjugate exhibits a LCST at 37 degrees C, having rapid, reversible hydration-dehydration changes due to highly mobile free polymer end groups. The conjugate retained its native enzymatic activity below this critical temperature, above which it precipitated and its catalytic function was shut off. This conjugate can be readily separated from reaction mixtures as a precipitate by simple temperature changes after reaction and reused in cycles without denaturation. Such a modulated system is attractive for application as a novel bioreactor system.

Well-defined poly(ethylene oxide)s with a primary amino group at one end and a hydroxyl group at the other terminus were synthesized with new sila-protected amino functionality initiator, potassium N-[2-(2,2,5,5-tetramethyl-1-aza-2,5-disilacyclopentyl)-ethyl]methyl amide [1b]. 1b initiated an anionic polymerization of ethylene oxide (EO) to form a polymer (PEO) without any side reactions such as a cleavage reaction of protective group and a chain transfer reaction. The molecular weights of the PEO determined from GPC and MALDI TOF-MS spectrometry agreed well with those from end group analysis using 1H and 13C NMR and TLC and also with the expected value from EO/initiator ratio. From these results, it was concluded that the polymers thus obtained had a primary amino group at one end and a hydroxy group at the other end and can be regarded as hetrobifunctional PEO.