N-Isopropylacrylamide and N-Acryloxysuccinimide Copolymer

Abstract

A copolymer capable of reaction with biologically active proteins was synthesized by a free radical polymerization in tetrahydrofuran (THF) under anhydrous conditions using azobisisobutyronitrile as the thermal initiator. Longer chain polymers were prepared using benzene instead of THF to avoid chain transfer. The thermally reversible phase transition properties of the copolymers suggested potential applications to the technologies of product isolation and pollutant removal. Conjugation of a monoclonal immunoglobulin to the copolymer is described and utilized in a novel antigen capture fluorescence immunoassay for human IgG.Poly(N-isopropylacrylamide) (polyNIPAAM), formed by a free radical polymerization of N-isopropylacrylamide, is a water soluble, temperature sensitive polymer. In aqueous solution, it exhibits a lower critical solution temperature (LCST) in the range of 30-35°C depending on the concentration and the chain length of the polymer. Thus, as the solution temperature is raised above the LCST, the polymer undergoes a reversible phase transition characterized by the separation of a solid phase which redissolves when the solution temperature is lowered below the LCST. Its physicochemical properties have been investigated by several laboratories (1-3).This thermally reversible precipitation suggested potential applications to the technology of reaction product isolation. It could be used as a tool to allow isolation of a specific product from a totally soluble reaction by raising the temperature.The first step in the process was to covalently incorporate biologically active protein molecules into this polymer. Methods analogous to previous reports (fr-6) involved first adding a functional group to the protein that would provide it with the ability to polymerize, such as a vinyl or substituted vinyl group, followed by copolymerization with the N-isopropylacrylamide monomer in aqueous solution using Ν,Ν,Ν',Ν'-tetramethylethylenediamine and

Probes of the Lower Critical Solution Temperature of Poly(N-isopropylacrylamide)

“…Moreover, the photon correlation spectroscopy of Yamamoto, Iwasaki and Hirotsu (5) has indicated that collapse in the hydrodynamic radius occurs several degrees lower than that of phase separation. Finally, the research group of Hoffman has observed that modification of PNIPAAM can result in an inability to visually detect aggregation above the LCST at all due to the formation of submicron-size particles (22). Therefore, cloud point measurements for determination of the LCST suffer as a result of dependence on instrumental sensitivity to scattering.…”

“…1.5 kcal/mol of repeating units, consistent with the loss of ca. 1 hy drogen bond per repeating unit upon phase separation (22). Studies of the endotherm associated with the LCST of this sample (at 0.4 mg/ml) show that the peak shape (transition width (ΔΤ1/2) = 0.4 ± 0.1°C, relative height (ACp) = 13 ± 1 cal/°C-g of poly mer) and maximum (LCST) are independent of heating rate over a range from 3.3 to 30°C/hr.…”

“…Use of a differential scanning microcalorimeter permits the precise measurement of thermodynamic parameters asso ciated with the transition; micropolarity-sensitive fluorescence probes report the LCST through solubilization in the precipitated polymer phase. These basic experiments are necessary for exploration of modified aqueous PNIPAAM systems that contain hydrophobic comomomers (22) or surfactants (28) that also lead to the formation of less polar sites (even below the LCST) that can competitively solubilize the fluorescent probes. For the binary mixture of PNIPAAM and water, we have demonstrated agree ment among the three techniques; this creates a sound foundation for study of more complex systems.…”

Synthesis and phase behavior of aqueous poly(N-isopropylacrylamide-co-acrylamide), poly(N-isopropylacrylamide-co-N,N-dimethylacrylamide) and poly(N-isopropylacrylamide-co-2-hydroxyethyl methacrylate)

“…The phase separation on heating is believed as a phase behavior with the lower critical solution temperature (LCST) [3] which is common to all polymer solutions, although it is stilll controversial [4]. This phase transition of PNIPAM has been used in attempts to control the enzymatic activity [5][6][7][8][9], affinity precipitation separation [10,11] and protein recycling systems [12][13][14][15]. Another growing area in which this property has been exploited is a targeted delivery of drugs and chemical agents such as anticancer drugs.…”

Complexation Between Poly(N-isopropylacrylamide) and Sodium n-Dodecyl Sulfate

“…Experimental Section) yields well-defined cloud points at least up to 5 mM SDS. The precipitated particles are apparently too small to cause visible turbidity at high surfactant concentrations (33). We can rationalize the retarded intermolecular aggregation as a consequence of the polymer-bound charged micelles leading to interparticle repulsion.…”