Poly(N-isopropylacrylamide) (PNIPAAm)-based temperature-responsive fluorescence polymer probes were developed using radical polymerization, with 3-mercaptopropionic acid as the chain-transfer agent, followed by activation of terminal carboxyl groups with N-hydroxysuccinimide and reaction with 5-aminofluorescein (FL). The lower critical solution temperatures (LCSTs) of the resulting fluorescent polymer probes differed depending on the copolymer composition, and had a sharp phase-transition (hydrophilic/hydrophobic) boundary at the LCST. The cellular uptakes of the fluorescent polymer probes were effectively suppressed below the LCST, and increased greatly above the LCST. In particular, the cellular uptake of a copolymer with N,N-dimethylaminopropylacrylamide, P(NIPAAm-co-DMAPAAm2%)-FL (LCST: 37.4 °C), can be controlled within only 1 °C near body temperature, which is suitable for biological applications. These results indicated that the cellular uptakes of thermoresponsive polymers could be accurately controlled by the temperature, and such polymers have potential applications in discriminating between normal and pathological cells, and in intracellular drug delivery systems with local hyperthermia.

IntroductionPoly(N-isopropylacrylamide) (PNIPAAm) has the sharpest phase transition of the class of thermosensitive N-alkyl acrylamide polymers. This feature makes it the most suitable for studies and practical applications. PNIPAAm exhibits a thermally reversible soluble-insoluble change in response to temperature changes across a lower critical solution temperature (LCST) at 32˚C in aqueous solutions. Polymer chains of NIPAAm hydrate to form expanded structures in water at lower temperature. At higher temperature, however, the chains form compact structures by dehydration. The remarkable phase transition of PNIPAAm in aqueous media is due to rapid hydration and dehydration changes of the polymer chain.PNIPAAm hydrogel has been utilized for drug delivery systems, 1,2 and as a cell culture substrate. 3,4 PNIPAAm has also been utilized in thermo-responsive bioconjugates, 5,6 which can be applied to reversible bioreactor systems. However, there have been few reports concerning the use of these thermoresponsive polymers in chromatographic separation. We have studied a new HPLC method using packing materials modified with these temperature-responsive polymers, PNIPAAm and its copolymer. [7][8][9][10][11][12] A novel chromatography system may be achieved using stimuli-responsive polymers, which alter their structure and physical properties in response to an external environmental change. These methods are expected to be applicable to the separation of pharmaceuticals, nucleotide, peptides and proteins. In this study, we prepared a temperature-responsive hydrogel-modified surface in order to enhance the density of PNIPAAm chains on the surface of packing materials. Using a PNIPAAm hydrogel-modified column, we have now achieved the successful separation of steroids and amino acid phenylthiohydantoins (PTH-amino acids) using only an aqueous solution as a mobile phase without an organic solvent. ExperimentalMaterials N-Isopropylacrylamide (NIPAAm) was kindly provided by KOHJIN (Tokyo, Japan) and was purified by recrystallization from hexane and dried at 25˚C in vacuo. N,NDimethylformamide (DMF) and N,N′-methylenebisacrylamide (MBAAm) were obtained from Kanto Chemicals (Tokyo, Japan).4,4′-Azobis(4-cyanovaleric acid) (Wako Pure Chemicals) was used after drying at 25˚C in vacuo. 1-Ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) was purchased from Tokyo Chemical Industry (Tokyo, Japan). Aminopropyl silica was obtained from Nishio Industry (Tokyo, Japan). Other chemicals were of analytical reagent grade. Preparation of PNIPAAm hydrogel-modified silica4,4′-Azobis(4-cyanovaleric acid) (3.50 g, 12.5 mmol) and EEDQ (6.18 g, 25.0 mmol), an initiator and a condensing agent, respectively, were dissolved in 250 ml of DMF in a separable flask. The solution was bubbled with N2 gas for 30 min. Aminopropyl silica beads (5 µm) were then immersed in the solution, and the mixture was degassed again for 30 min before the reaction was started. The reaction was carried out at 25˚C for 6 h under an N2 gas atmosphere. Modified silica beads ...

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Extensive research has been carried out on functional polymers which are currently playing important roles in various fields such as medicine and engineering. Such functional polymers which respond to various kinds of stimuli are termed 'intelligent materials'. Poly(N-isopropylacrylamide) (PNIPAAm), a temperature-responsive polymer, was utilized as a chromatography column matrix modifier for a novel chromatographic approach in which only aqueous media are used as a mobile phase. The ability of the developed temperature-responsive chromatography system to separate solutes without using an organic solvent is advantageous from the point of view of maintaining the structure and activity of bioactive compounds. Recently, we designed and synthesized a new pH- and temperature-responsive copolymer as a representative of such environment-responsive polymers and grafted it onto aminopropyl silica beads. The products were evaluated as HPLC packing materials for separation systems based on a new concept, according to which the properties of the stationary phase surface are altered by external stimuli such as pH and temperature. This chromatography system utilizing the PNIPAAm copolymer is very useful for the separation of bioactive substances, such as proteins and peptides, because separation in the aqueous mobile phase is controlled solely by changing the temperature. This analytical system reduces organic waste because no organic solvent is used to separate the solutes and can therefore be classified as environmentally friendly. Future medical and pharmaceutical applications are expected.