LCST, proteins

The first elastomeric protein is elastin, this structural protein is one of the main components of the extracellular matrix, which provides stmctural integrity to the tissues and organs of the body. This highly crosslinked and therefore insoluble protein is the essential element of elastic fibers, which induce elasticity to tissue of lung, skin, and arteries. In these fibers, elastin forms the internal core, which is interspersed with microfibrils [1,2]. Not only this biopolymer but also its precursor material, tropoelastin, have inspired materials scientists for many years. The most interesting characteristic of the precursor is its ability to self-assemble under physiological conditions, thereby demonstrating a lower critical solution temperature (LCST) behavior. This specific property has led to the development of a new class of synthetic polypeptides that mimic elastin in its composition and are therefore also known as elastin-like polypeptides (ELPs). 

We have utilized thermoresponsive properties of PIPAAm and its gels as on-off switches for drug release [6,7], chromatography systems [9-11], and attachment/detachment of cells [12-14] (Scheme 1). Hydrophobic chains of collapsed PIPAAm above its LCST interact with cells and proteins. Although below the LCST, PIPAAms are highly hydrated flexible chains and... 

Thermoresponsive acrylamide co-polymers were also used to alter the physicochemical and biopharmaceutical properties of avidin. Similar to PEG, the acrylamide co-polymers with a lower critical solution temperature (LCST) of about 37 °C were conjugated to the protein amino groups. The polymers were conjugated either by polymer multipoint attachment using polyfunctional polymers or by single chain attachment using end-chain monoactivated polymer. In both cases, the polymer conjugation was found to produce bioactive derivatives with reversible thermal character (Fig. 11.12). 

Using this approach, hydrophilic (neutral or ionic) comonomers, such as end-captured short polyethylene oxide (PEO) chains (macromonomer), l-vinyl-2-pyrrolidone (VP), acrylic acid (AA) and N,N-dimethylacrylamide (DMA), can be grafted and inserted on the thermally sensitive chain backbone by free radical copolymerization in aqueous solutions at different reaction temperatures higher or lower than its lower critical solution temperature (LCST). When the reaction temperature is higher than the LOST, the chain backbone becomes hydrophobic and collapses into a globular form during the polymerization, which acts as a template so that most of the hydrophilic comonomers are attached on its surface to form a core-shell structure. The dissolution of such a core-shell nanostructure leads to a protein-like heterogeneous distribution of hydrophilic comonomers on the chain backbone. 

There are several recent examples of the switching of nonspecific protein binding on polymer surfaces by application of an external stimulus. Alexander and coworkers demonstrated that protein adhesion can be controlled on PNIPAM surface brushes [14, 181]. For instance, it was reported that the adsorption of FITC-labeled bovine serum albumin (FITC-BSA) on PNIPAM/hexadecanethiol micropatterned surfaces could be tuned by LCST. However, this effect was found to be less pronounced after prolonged incubation times or repeated heating/cooling cycles. The authors suggested that this behavior could be due to unspecific PNIPAM-protein interactions [14],... 

Recently, coatings composed of thermoresponsive side chain OEGs were employed for this purpose (Fig. 14) [44, 45], They offer the advantage of a better inherent biocompatibility than PNIPAM, show reduction of nonspecific protein adsorption even above the LCST, and exhibit effective control of cell adhesion by reducing the temperature from 37 to 25°C [191],... 

Fig. 15 Specific protein separation by a smart thermoresponsive polymer coating. Left PNIPAM with immobilized lactose and RCA 120 is below the LCST. The moieties are separated and, therefore, proteins from the mobile phase can bind to RCA120. Right PNIPAM below LCST. Polymer-bound lactose and RCA120 come into close contact and lactose displaces the protein. Reprinted, with permission, from [194]. Copyright (2003) American Chemical Society...

The adsorption of proteins onto such stimuli-responsive particles was found to be temperature dependent rather than pH and ionic strength (belowthe LCST of the corresponding homopolymer). At room temperature, the adsorption of various proteins revealed to be nil or low at room temperature in the case of poly(NIPAM) based particles [9,10]. 

PEG is well known to provide resistance to protein adsorption [117, 118], Thus, Gan and Lyon [119] tried to incorporate PEG chains into the thermoresponsive PNIPA microgels to minimize nonspecific interactions of the particles with biological environments. A reduced adsorption of BSA on the particle surface was observed as a result of incorporation of PEG chains into the particles, especially when the PEG chains were located in the shell of the particles. This effect is most pronounced when the PNIPA is phase-separated above the LCST, which indicates that the PEG side-chains may stretch outward from the particle surface as the particles collapse at temperatures above the transition temperature. Similar effects are also observed for particles where the PEG chains are localized in the particle core, which is then surrounded by a PNIPA shell. These results suggest that the PEG grafts can penetrate the PNIPA shell when it is in its phase-separated state. 

Teehnol., 44, pp. 79 4 (2005)]. Another process that exploits a phase transition to facilitate separation and recycle of solvent alter extraction utilizes ethylene oxide-propylene oxide copolymers in aqueous two-phase extraction of proteins [Persson et al.,/. Chem. Teehnol. Bioteeh-nol., 74, pp. 238-243 (1999)]. After extraction, the polymer-rich extract phase is heated above its LCST to form two layers an aqueous layer containing the majority of protein and a polymer-rich layer that can be decanted and recycled to the extraction. 

Hairy polymer colloids formed in this way might find application in several domains in the future. With a polymer brush exhibiting a LCST, the change of surface properties with temperature could be of high interest for appUcations based on adsorption-desorption processes, such as their use as stationary phases for bioseparation. Recently, PEG-based N-substituted acrylamide maeromonomers were grafted via SI-ATRP from the surface of polystyrene latexes. These PEG-based surfaces showed good protection against nonspecific protein adsorption from... 

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