Bioinspired Dual Stimuli-Responsive Membranous System with

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Bioinspired Dual Stimuli-Responsive Membranous System with Multiple On-Off Gates Bom-yi Lee, Seung Hyun, Gumhye Jeon, Eun Young Kim, Jinhwan Kim, Won Jong Kim, and Jin Kon Kim ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b01788 • Publication Date (Web): 18 Apr 2016 Downloaded from http://pubs.acs.org on April 19, 2016

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ACS Applied Materials & Interfaces

Bioinspired Dual Stimuli-Responsive Membranous System with Multiple On-Off Gates

Bom-yi Lee,† Seung Hyun,† Gumhye Jeon,† Eun Young Kim,† Jinhwan Kim,‡ Won Jong Kim,‡ and Jin Kon Kim†,*

†National Creative Research Center for Block Copolymer Self-Assembly and Departments of Chemical Engineering, Pohang University of Science and Technology (POSTECH), 77 Cheongam-ro, Nam-gu, Pohang 790-784 Republic of Korea ‡Department of Chemistry, Pohang University of Science and Technology (POSTECH), 77 Cheongam-ro, Nam-gu, Pohang 790-784 Republic of Korea

* Corresponding author. E-mail: [email protected]

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ABSTRACT Stimuli-responsive polymers have been widely used for controlled release of several biomolecules. In general, a single stimulus among various stimuli, for instance, temperature, pH, or light, has been used for these polymers. Although some stimuli are applied together, one cannot control each stimulus independently at a given stimulus-responsive polymer. However, to mimic biological system like cell membrane, multiple on-off gates utilizing independent control of dual (or multiple) stimuli should be used. Here, we introduce a stimuli-responsive membrane controlled by two orthogonal stimuli. For this purpose, the top and the bottom parts of anodized aluminum oxide membrane walls are independently grafted by thermo-responsive poly(N-isopropylacrylamide) and pH-responsive poly(acrylic acid), respectively, by using surface-initiated atom transfer radical polymerization. The membrane clearly showed two independent on-off gates depending on temperature and pH. Furthermore, through light irradiation of two different wavelengths (near infrared and ultra-violet), temperature and pH were also controlled independently and promptly. Thus, this membrane shows two independent on-off gating of the transport of a model biomolecule of fluorescein isothiocyanate-labeled bovine serum albumin (FITC-BSA). This strategy suggests the potential of independently modified membrane in layers as stimuli-responsive on-off gates for the application of artificial cell membrane.

KEYWORDS: artificial cell membrane, biomimetics, hybrid materials, stimuli-responsive materials, surface modification, spatiotemporal and prompt gating, on/off gating

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1. Introduction The stimuli-responsive materials1-8 whose physicochemical properties dramatically depend on a subtle change of external stimuli, for instance, electric9-11 or magnetic field12-14, light15, 16, redox potential17, 18, ionic strength19, temperature20, 21, and pH22-25 have drawn great attention for their feasible applications to protein separation membrane26, sensors27,

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, and drug

delivery devices29-33. For the fabrication of those membranes, the surface of the membranes has been modified by various methods: layer-by-layer (LBL) assembly34-36, nanoparticle immobilization37 and grafting of polymer chains.38-43 In particular, the grafting of the stimuliresponsive polymer or block copolymer chains on the membrane has been widely used.38-40, 44-46

Most-frequently used responsive polymers are poly(N-isopropylacrylamide) (PNIPAM), and poly(acrylic acid) (PAA) or poly(methacrylic acid) (PMAA). PNIPAM chains in water at temperatures lower than the lower critical solution temperature (LCST) (~ 32 oC) show expanded coiled structures, whereas those become collapsed globule structure.47, 48 Thus, when PNIPAM chains are grafted on the pore walls of the membrane, one can easily control (or gating) water flux by changing temperature.44 Also, this membrane can be used as molecular valves49 where large species are effectively filtered, while small species are freely permeated. On the other hand, PAA chains are collapsed at lower pH (pH < 4.25), while those are expanded structures at higher pH.50 Thus, water flux is controlled by pH for membranes with PAA chains.38 Random copolymer of PNIPAM and PAA [P(NIPAM-co-PAA)] or PNIPAM-block-PAA copolymers have been used to obtain both temperature and pH-sensitive membranes.45 Wu et al. reported that nanofluidic device with cone-shaped nanopores grafted by P(NIPAM-co-MAA) brushes showed effective gating (or rectifying) of ions.51 But, those membranes operate as a single gate where “on” or “off” state can be achieved by temperature 3



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or pH. One of promising applications of stimuli-responsive membranes is to mimic biological cell membrane. An abiotic membrane based on polymeric or inorganic nanomaterials can be a proper choice, because it does not require any complicated bioconjugation and geometry which are encountered in real biological cell membrane.52, 53 Also, it shows good membrane stability against external contamination or enzymatic degradation compared with biological membrane, and pore size is easily controlled. To apply the stimuli-responsive membrane as a cell membrane, one should mimic the biological functions of plasma membrane which is driven by channel or transporter protein. Since the intracellular cytosol is distinctly separated from the external environment and the movement of substances is strictly regulated by a membrane transporter depending on the concentration gradient, plasma membrane requires a precise control in its orientation of transmembrane flux. That is, the gate of a membrane transporter should not be opened simultaneously to both extracellular and intracellular environment. Thus, the artificial cell membrane should have at least two gates, where the function of each gate is orthogonal. And, the inner gate should be opened when the outer gate is closed, and vice versa. In addition, a fast response under external stimuli is another important criterion to operate artificial cell membrane, because the carrier protein on the plasma membrane has to send its transporting materials quickly on demand of intracellular signal. Therefore, these two criteria are the prerequisite for mimicking the function of the plasma membrane and the carrier proteins: 1) independent modulation of the entrance and exit of the transporting materials, and 2) prompt response against external onoff signal. Zhang et al.54 reported bioinspired smart single ion pump having double-gate nanochannels responding independently to acid (lower pH) and base (higher pH) environments. But, the 4


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size and shapes of the pores are not uniform due to using a track-etched membrane. In addition, it is not easy to control the intrinsic pH inside cells remotely without secondary stimulus. Since light, as a stimulus, provides spatiotemporal controllability without any unwanted membrane penetration, it would be a safe and convenient tool for operating membrane gates. Thus, it needs to develop a new type of a membrane with well-defined pore size and shape capable of independently working as double gates depending on two orthogonal stimuli controlled by light. Here, we introduce a novel stimuli-responsive membrane having double gates which are controlled spatiotemporally and promptly by non-invasive light stimulus that could change effectively and remotely two orthogonal stimuli of temperature and pH. For this purpose, we employed anodized aluminum oxide (AAO) membrane and the upper and lower parts of the pores are grafted with PNIPAM and PAA chains on gold coated AAO membrane, respectively. Using both thermo-responsive PNIPAM chains and pH-responsive PAA chains on the membrane, we could independently control the “on” and “off” state of the pores by changing temperature and pH. Furthermore, we demonstrate that the independent membrane gating is fast achieved by using different wavelengths of lights as turn on switch. In the case of thermo-responsive PNIPAM, it responds to near infrared (NIR) light irradiation, because temperature is easily controlled the photothermal effect of gold deposition on AAO membrane. On the other hand, pH-responsive PAA responds to ultra-violet (UV) light irradiation, when an aqueous solution contains a photoacid generator, for instance, onitrobenzaldehyde (o-NBA). The fast transition (within 3 sec) of temperature and pH depending on NIR and UV lights opened (or closed) the pores of nanochannel promptly, demonstrating the promising potential of a nanochannel as a controllable abiotic carrier on the artificial cell membrane. 5



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2. Experimental Section 2.1.

Fabrication of dual-responsive membrane with two gates. The fabrication of

dual-responsive membrane with two gates is schematically shown in Figure 1. First, AAO membrane was prepared by two-step anodization (section 1 of SI). Ti was depo sited on the top surface AAO membrane and the upper parts of the pore walls, follo wed by the deposition of gold by thermal evaporation. A thin layer of Ti was necessa ry because the adhesion between gold layer without Ti layer and AAO wall was very poor. Then, NIPAM monomers were polymerized in a glovebox purged with nitrogen by surface-initiated atom transfer radical polymerization (SI-ATRP) from Br-initiated si tes of Au surface, forming tethered polymer brushes on pore walls. The sample was ri nsed with ethanol to terminate the reaction, and cleaned with dionized water (DI water) and methanol to remove ungrafted PNIPAM chains. Next, the lower parts of the pore walls were deposited sequentially by Ti and Au. Then, acrylic acid (AA) monomers in water solution with NaOH (pH = 10.2) were polymerized on the gold layer by SI-ATRP,55 resulting in PAA chains grafted onto the pore walls (section 2 of SI). 2.2.

Water Flux measurement. A flux test was carried out with a home-made flux cell,

as shown in Figure S3 in supporting information. Pure DI water flows from the reservoir to the cell and passes through the membrane at a nitrogen gas pressure of 0.1 bar. The penetrated water was collected at a beaker and weighed with a balance at a given time interval. The AAO membrane grafted with PNIPAM and PAA chains was placed to the bottom of the flux cell, and tightly sealed by O-ring to prevent any leakage. Temperature of water in the reservoir was adjusted with a temperature controller, while pH was controlled by adding HCl and/or NaOH. 6


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2.3. Release of FITC-BSA. We first loaded FITC-BSA (0.3 mg/ml in DMSO/water solution (1:4 v/v)) inside the pores of the AAO membrane at pH =3 and 40 oC, where both upper and lower pores are open in the solution. Then, both upper and lower pores were closed by putting the membrane into the solution maintained at pH =6 and 20 oC. The remaining FITC-BSA in the reservoir was completely removed by fresh DMSO/water solution without having FITC-BSA until there was no signal in fluorescence of the solution. The model biomolecule transport was tested depending on temperature and pH controlled by heating or cooling the reservoir, and adding HCl or NaOH into the reservoir, respectively. We also controlled temperature and pH change by irradiation of lights with two different wavelengths (NIR vs UV). Due to the existence of a gold layer below PNIPAM chains, the temperature of PNIPAM chains was easily increased above LCST of PNIPAM in water within very short times (less than 5 s) once a laser with NIR wavelength (here 808 nm) was irradiated. Also, when another laser with UV wavelength (303 nm) was irradiated on DMSO/water solution (1:4 v/v) with o-NBA (700 µM), pH was immediately decreased within very short times (less than 5 s) due to the acid generation from o-NBA by UV.

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3. Results and Discussion

Figure 1. (a) Scheme for the fabrication of dual-responsive membrane with two gates with PNIPAM and PAA chains grafted on AAO membrane. (b) Temperature-controlled PNIPAM gate located at the upper pores of the membrane. (c) pH-controlled PAA gate located at the lower pores of the membrane.

The fabrication of dual-responsive membrane with two gates is schematically shown in Figure 1. First, Ti and Au were consecutively deposited on AAO membrane prepared by twostep anodization (section 1 of supporting Information (SI)). Then, NIPAM monomers were polymerized on the gold layer of the membrane by SI-ATRP. Next, AA monomers were also polymerized on the lower parts of the membrane. (The details are explained in the experimental section and supporting information.) Figures 1b and 1c show open or closed 8


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pores located at upper and lower parts of AAO membrane, respectively, depending on pH and temperature.

Figure 2. FE-SEM images (Top views) of (a) Pristine AAO membrane. (b) Gold-coated AAO membrane. (c) Upper side pores with grafted PNIPAM chains after 6 h reaction of NIPAM, and (d) Lower side pores with grafted PAA chains after 8 h reaction of AA. Insets of (c) and (d) are cross-sectional FE-SEM images. (Scale bar: 1㎛)

Figure 2 gives top views of field emission scanning electron microscopy (FE-SEM) images of the nanoporous membrane corresponding to each fabrication step. The pristine AAO membrane made by two step anodization has pores of a diameter of 400 nm with excellent ordering of hexagonally packed cylinders (Figure 2a). Once gold was deposited, the pore diameter decreased to 380 nm (Figure 2b). Figures 2c and 2d gives top and cross-sectional views of FE-SEM images after NIPAM and AA are polymerized on the upper and lower pore walls, respectively by SI-ATRP. It is seen that PNIPAM and PAA chains are uniformly grafted onto the upper and lower parts of the pore walls. Also, the final pore sizes of these 9



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two parts are decreased to 190 nm and 195 nm, respectively.

Figure 3. The change of water flux depending on temperature and pH. Inner panels schematically show the state of the pores at the upper (PNIPAM chains) and lower (PAA chains) parts.

Figure 3 shows the change of water flux through the membrane depending on temperature and pH. It is well known that PNIPAM and PAA chains in water have their conformational change from coil to globule depending on environment conditions (for instance, temperature and pH), resulting in their volume change. Thus, PNIPAM and PAA chains can play a role as independent gates at both sides of the membrane. At 40 oC and pH = 3, both the upper gate containing PNIPAM chains and the lower gate containing PAA chains are open (“on” and “on” state). Thus, water flows freely through the membrane at a flux of 400 L/m2h. When pH is increased to 6, while maintaining temperature (40 oC), there is no flux through the 10


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membrane. This is because the lower PAA gate becomes closed, although the upper PNIPAM gate is still open (“on” and “off” state). Of course, when pH is decreased again to 3 at fixed 40 oC, the flux is recovered, showing good reversibility on pH. When the temperature is decreased to 20 oC at a fixed pH 3, there is no flux, because the upper gate is closed (“off” and “on” state). Also, good thermo-reversibility is obtained when temperature is again increased to 40 oC at a fixed pH =3. Finally, at 40 oC and pH =6, there is no flux (“off” and “off” state). We also fabricated another AAO membrane whose upper side was only grafted by PNIPAM chains, while the lower side was not grafted by PAA chains. As expected, this membrane also showed good thermo-reversibility when temperature was changed from 20 oC to 40 oC, or vice versa (see Figure S4). The flux of this membrane was 430 L/m2h, which is slightly larger than that (400 L/m2h) of the dual gated AAO membrane grafted with PNIPAM and PAA chains in the upper and lower sides, respectively (Figure 3). This indicated that good thermo-reversibility arising from PNIPAM chains was observed regardless of the presence of PAA chains, although the flux is a little decreased when PAA chains are grafted in the lower side of AAO membrane. Since the membrane fabricated in this study has two gates, and each gate is independently controlled by its own stimulus, it could mimic the channel or transporter on the surface of cell membrane. To demonstrate this possibility, we first loaded fluorescein isothiocyanate-labeled bovine serum albumin (FITC-BSA) as a model biomolecule inside the pores of the membrane.

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Figure 4. (a) Schematic of the loading of FITC-BSA inside AAO membrane for the release experiment. (b) The cumulative concentration of FITC-BSA in the upper (open/closed circles) and lower reservoirs (closed triangles) with time, depending on temperature at a fixed pH of 6. Open and closed symbols correspond to “on” and “off” states, respectively. (c) Illustration of thermo-responsive FITC-BSA release (Figure 4(b)). (d) The cumulative concentration of FITC-BSA in the upper (closed circles) and lower reservoirs (open/closed triangles) with time, depending on pH at a fixed 20 oC. (e) Illustration of pH-responsive FITC-BSA release (Figure 4(d)). Open and closed symbols in parts (b) and (d) correspond to “on” and “off” states of a gate, respectively. 12


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Figure 4a shows schematic of the loading of FITC-BSA inside AAO membrane for the release experiment. First, FITC-BSA was loaded inside the pores of the AAO membrane at 40 oC and pH =3 (“on” and “on” states). Then, both upper and lower pores were closed by putting the membrane into the solution maintained at 20 oC and pH =6 (“off” and “off states) and the remaining FITC-BSA in the reservoir was completely removed by fresh DMSO/water solution (The details are given in Section 4 in the Supporting Information). Figure 4b shows the release behavior of FITC-BSA from inside the pores of the membrane to the upper or lower reservoirs with time, depending on temperature at a fixed pH of 6. Both reservoirs are completely separated from each other by the membrane to demonstrate the directional controllability of the biomolecule transport of the membrane. The details of the release experiment is described in Figure S5 in supporting information. At 40 oC, the cumulative concentration of FITC-BSA in the upper reservoir is gradually increased because the upper gate containing PNIPAM chains operates as “on” state. But, at 20 oC, it does not change with time, indicating that no FITC-BSA penetrates through the upper part of the membrane. This is because of “off” state in the upper gate. When the temperature is increased again to 40 oC after reloading FITC-BSA in membrane, the cumulative FITC-BSA concentration in the upper reservoir increased again, showing good thermo-reversibility. Of course, the concentration of FITC-BSA in the lower reservoir is maintained to zero, because the lower gate becomes “off” state at pH=6. On the other hand, when pH is lowered to 3 at a fixed 20 oC, the cumulative concentration of FITC-BSA in the lower reservoir increases with time, while that in the upper reservoir is zero (Figure 4d). This corresponds to “off” (upper gate) and “on” states (lower gate). When pH is increased to 6, the cumulative concentration of FITC-BSA in the lower reservoir does not change with time, corresponding to “off” gate. The results given in Figure 4 indicated that 13



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we obtained one gate of “on” state and the other gate of “off” state, and vice versa. That is, the membrane employed in this study successfully controls the direction of the tranport of a model biomolecule, which is very similar behavior of the cavity in a channel protein of the cell membrane.

Figure 5. The cumulative concentration of FITC-BSA measured at upper (circles) and lower (triangles) reservoir with time. (a) Temperature-gating with near-IR wavelength (808nm) at a fixed pH of 6. (b) pH-gating with UV wavelength (303nm) at 20 oC. Open and closed symbols in parts (a) and (b) correspond to “on” and “off” states of a gate, respectively.

Finally, we showed remote directional controllability of the membrane by using different wavelenghts of light as stimuli. Figure 5 shows the cumulative concentration of FITC-BSA with time when the light was irradiated on the membrane. The release experiment is described in detail in Figure S6. When a laser with NIR wavelength of 808 nm was irradiated, the release of FITC-BSA in the upper reservoir was observed within very short times (less than 3 s) after turn on, while no FITC-BSA was detected in the lower reservoir (Figure 5(a)). This is because of photothermal effect of gold layer below PNIPAM chains.

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Also, when the laser turns off, the further release of FITC-BSA was not monitored within very short times, implying that the release was strictly controlled by NIR light stimulus alone. Figure 5(b) shows the cumulative concentration of FITC-BSA measured at the upper and lower reservoirs at 20 oC after turning on/off UV light with a wavelength of 303 nm. The excellent gating of FITC-BSA was achieved within very short times, and it is attributed to the photolysis of o-NBA inducing pH jump by UV irradiation. These results indicate that the membrane works promptly and orthogonally as two independent gates capable of controlling orientation of the biomolecule transport once a laser with different wavelengths (NIR vs UV) is used.

4.

Conclusion

We have successfully fabricated AAO membrane with PNIPAM and PAA chains, respectively, on upper and lower parts of the membrane, and these work as two independent gates depending on temperature and pH. By water flux test, we demonstrated that water flow was independently controlled as upward and downward direction depending on each stimulus. Using a model biomolecule, the direction of a model biomolecule transport was also successfully controlled. Furthermore, temperature and pH could be controlled by external trigger, i.e., light irradiation with different wavelengths and the response time for the gating was very short (less than 3 s). The membrane introduced in this study could be applied to investigate the transport of biological molecules, which could be helpful for developing new drugs for synthetic cellular systems.

Associated Content Supporting Information

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The Supporting Information is available free of charge on the ACS Publications website at https://pubs.acs.org. Fabrication of AAO membrane, polymerization of NIPAM and AA, flux measurement, the release of FITC-BSA, and the release of FITC-BSA by irradiation of light irradiation with two different wavelengths.

Author Information Corresponding Author *E-mail: [email protected] Phone: +82-54-279-2276

Acknowledgements This work was supported by the National Creative Research Initiative Program supported by the National Research Foundation of Korea (NRF) grant (no. 2013R1A3A2042196) funded by the Korean government.

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Bioinspired Dual Stimuli-Responsive Membranous System with Multiple On-Off Gates

Bom-yi Lee,† Seung Hyun,† Gumhye Jeon,† Eun Young Kim,† Jinhwan Kim,‡ Won Jong Kim,‡ and Jin Kon Kim†,*

SYNOPSIS TOC

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Bioinspired Dual Stimuli-Responsive Membranous System with

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