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Poly(L-glutamic acid) carrying a terminal disulfide group at one end of the chain was self-assembled on a porous membrane. The rate of water permeation through the surface-modified porous membrane depended on pH. In the region of low pH, poly(L-glutamic acid) chain is protonated and folded to form -helical structure; in the region of high pH, it is de-protonated to have extended random structure. The ionic strength also regulate the rate of water permeation. Increasing ionic strength reduced the pH dependence of permeation due to shielding effect. The permeation through the porous membrane, the surface of which was covered with the self-assembled polypeptide brush, was sensibly regulated by changing pH because of direct contact of brush chains with environment.
A variety of nano- or micro-structured materials have been prepared to apply for catalysis, separation, and host compound for template synthesis of other nanoscopic materials. These materials have found many potential applications in the areas of device technology and drug delivery systems. We have synthesized various signal-responsive polymers-grafted porous membranes to regulate substance permeations (Ito et al, 1992; Ito et al., 1997a; Ito et al., 1997b).
In the present investigation, we synthesized a polypeptide brush on a nanoporous polymeric membrane to have stimulus-sensitive gating of channel. It is known that the conformation of polyelectrolyte chain is dependent upon the environmental conditions such as pH and ionic strength. In the region of low pH, poly(L-glutamic acid) chain is protonated and folded to form -helical structure; in the region of high pH, it is de-protonated to have extended random structure. The permeation through the porous membrane, the surface of which was covered with the self-assembled polypeptide brush, was sensibly regulated by changing pH because of direct contact of brush chains with environment.
Poly(L-glutamic acid) carrying a disulfide group at the amino terminal was synthesized. The oxidation of 11-mercaptoundecanoic acid (Aldrich) was attained by 24 h treatment in DMSO/1N HCl. The product (216 mg) was dissolved in 15 mL of dimethylformamide (DMF). 380 mg of O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HATU, PerSpective Biosystems, Hamburg, Germany) and 210 microlitter of N,N-diisopropylethylamine (DIEA, Aldrich) were added to the solution to make disulfide:HATU:DIEA molar ratio 1:2:2.4. After addition of poly(g-benzyl-L-glutamate) (PBLG, 1.0 g), the solution was stirred for 1 h in ice bath. PBLG was purchased from Sigma Chemical Co. which provided the following MW data: (viscosity) 22,000; (light scattering) 17,400. The mixture solution was stirred for 24 h at room temperature. The product, poly(benzyl-L-glutamate) carrying a terminal disulfide group (PBLG-SS), was isolated by twice precipitation with methanol, and then dried overnight in vacuo at room temperature.
In order to hydrolyze PBLG, 500 mg of PBLG-SS were treated in dioxane (6 mL)/ methanol (2 mL)/ 4N NaOH (2 mL) mixture for 2 h at room temperature. The precipitate was again dissolved in 500 mL of distilled water, and then concentrated to 50 mL by ultrafiltration using AMICON model 2000 (mw cut-off = 10,000). The concentrated solution was lyophilized to obtain poly(L-glutamic acid) carrying a terminal disulfide group (PLGA-SS).
Track-etched porous polycarbonate membrane (DuPont Nuclepore membrane: average pore diameter, 200 nm) was coated with gold. The Au-coated membrane was immersed in aqueous solution of PLGA-SS (2.5 mM, pH = 3.0) for 24 h. The surface-modified membrane was washed with deionized water until the pH of the washing liquid became neutral.
FT-ATR-IR spectra were measured on Perkin-Elmer infrared spectrometer using KRS-5 prism.
Water permeation through the membrane was investigated using an apparatus previously reported (Ito et al, 1997a). The prepared membrane was mounted on a ultrafiltration cell (Toyo Roshi UHP-25), and placed 200 cm below a water reservoir. The reservoir was filled with an aqueous solution and adjusted to different pHs using NaOH and HCl. The aqueous solution was allowed to flow under a constant hydraulic pressure. The permeation rate was calculated by measuring the mass of water permeating through the membrane every minute.
The presence of immobilizated PLGA on the surface of Au-coated membrane was confirmed by infrared spectroscopy. The FT-ATR-IR spectrum of PLGA-immobilized membrane shows the amide I (1650 cm-1) and amide II (1550 cm-1) absorptions, and the ester carbonyl absorption (1730 cm-1), which are characteristic of PLGA.
The rate of water permeation through an nonimmobilized membrane was independent of pH. The rate of water permeation through the grafted membrane was dependent upon pH, which was high at low pH, but low toward neutral pH. It is expected that in the region of low pH, the poly(L-glutamic acid) chain is protonated and folded to form a-helical structure, while in the region of high pH, it is de-protonated to have extended random structure. This type of conformational change of polypeptide chain may have affected the porosity of the membrane, leading to the pH-dependent permeability of water.
The permeation rate also depended on the ionic strength. With increasing ionic strength, the pH dependence decreased. In the high pH region, permeability was strongly dependent on the ionic strength. The high concentration of ions should moderate charge-to-charge interactions of polypeptide brush leading to conformational change.
With the PLGA-grafted membrane, the change in permeability in response to pH change occurred within a few minutes. The PLGA brushes were put in direct contact with the media of different pH; this situation allowed the brushes to quickly respond to the environmental change. In the case of hydrogel-based system, the fastest response so for reported is 20 min, and it usually takes several hours to a day.
A reversible change of permeation rate with pH change between 2 and 7 was observed. The conformational change of self-assembled PLGA brush reversibly regulates the pore size of the membrane.
Previously we devised some porous membranes having polyelectrolyte brush on the surface, which was prepared by plasma polymerization. It was difficult to control the length and density of polymer brush. The present method enabled us to fabricate intelligent membrane by combination of porous membrane and polymer brush. This technique should be useful to prepare micro-device and nano-devices in the near future.
Ito, Y.; Inaba, M.; Chung, D. J.; Imanishi, Y. (1992) Macromolecules 25, pages 7313-7316. Control of water permeability by pH and ionic strength through a porous membrane having poly(carboxylic acid) surface-grafted.
Ito, Y.; Ochiai, Y.; Park, Y.S.; Imanishi, Y. (1997a) J. Am. Chem. Soc. 119, pages 1619-1623. pH-sensitive gating by conformational change of polypeptide brush grafted on porous polymer membrane.
Ito, Y.; Park, Y. S.; Imanishi, Y. (1997b) J. Am. Chem. Soc. 119 , pages 2739-2740. Visualization of critical pH-controlled gating of porous membrane grafted with polyelectrolyte brushes.