Photoresponsive Aqueous Dissolution of Poly(N-Isopropylacrylamide

Apr 11, 2018 - Also, we examined application of the copolymers to the selective recovery of living cells from culture substrate under microscopic obse...
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Photoresponsive Aqueous Dissolution of Poly(N‑Isopropylacrylamide) Functionalized with o‑Nitrobenzaldehyde through Phase Transition Kimio Sumaru,* Toshiyuki Takagi, Kana Morishita, and Toshiyuki Kanamori Biotechnology Research Institute for Drug Discovery (BRD), National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba Central 5th, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8565, Japan S Supporting Information *

ABSTRACT: We report a sharp photoinduced aqueous dissolution of the copolymer through phase transition based on the photochemical reaction of o-nitrobenzaldehyde (NBA) and the principle of polymer effect. We synthesized the copolymers having poly(N-isopropylacrylamide) main chain and NBA side chain at 4, 7, and 10 mol % functionalizations and analyzed their photoresponsive characteristics. Light with 365 nm wavelength converted NBA groups at copolymer side chains to carboxylic acid efficiently at the rate of 7.3 cm2/J, and in the case of 10 mol % functionalization, the irradiation dosage no more than 56 mJ/ cm2 induced sharp aqueous dissolution of the copolymer thin layer in pH 7.4 at 25 °C. As example applications, we demonstrated on-demand release of polyethylene beads and fluorescent-labeled albumins, which had been immobilized on a substrate surface via the copolymers, by the precisely controlled light irradiation using a microprojection system. Also, we examined application of the copolymers to the selective recovery of living cells from culture substrate under microscopic observation. As a result, mild light irradiation at room temperature triggered immediate detachment of the cultured adherent cells only in the irradiated areas without critical influence on their viability.

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INTRODUCTION In recent years, the technology to control intracellular systems by light has been established as optogenetics and has been used widely to analyze the function of neural circuitry in detail.1,2 This situation has resulted from the implementation of spatiotemporally controllable light irradiation to small objects under microscopic observation as well as genetic engineering to express light-gated ion channels. Recently, Huang et al. reported the spatial control of gene silencing in human embryonic stem cells via light-controlled siRNA release from gold nanoshells by using tightly focused near-infrared light.3 On the other hand, many trials have been attempted to manipulate cultured cells via property change of photoresponsive materials for more than 30 years since the first proposal by Ishihara et al.4−12 Among them, the approaches based on a photocleavage mechanism have been examined actively,10−12 and the gel system with photocleavable cross-links has attracted attention as a powerful tool to control cellular function through its mechanical property change.11 Shin et al. demonstrated the cell © XXXX American Chemical Society

patterning and selective cell detachment based on photodecomposition of the gel layer.13 Photodecomposition based on the photocleavage, however, requires considerable irradiation dosage because the number of remaining crosslinks obeys the kinetics approaching 0 under constant irradiation. On the other hand, photocontrol of aqueous dissolution of polymer has been attempted on the basis of thermodynamic phase transition since a pioneering study reported by Irie et al.14−20 In clear contrast to the systems based on photocleavage, reaction of photoresponsive component introduced in a small proportion would trigger a phase transition through the physicochemical polymer effect; solvent affinity of a polymer reflects that of composing monomeric units proportionally to degree of polymerization.21,22 In practice, thermoresponsive polymers functionalized with a small amount of spiropyran have been reported to exhibit drastic and reversible photoReceived: March 19, 2018 Published: April 11, 2018 A

DOI: 10.1021/acs.biomac.8b00470 Biomacromolecules XXXX, XXX, XXX−XXX

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Biomacromolecules response.15−18 In these systems, visible light irradiation switched the spiropyran from a cationic hydrophilic state to a nonionic hydrophobic state in a stoichiometric manner.23 On the basis of this large property change of chromophore and the polymer effect described above, photoisomerization of spiropyran introduced at the rate of only 1 mol % triggered a drastic dehydration of the water-soluble polymer at room temperatures.15 Further, it was reported that the crosslinked hydrogels composed of this polymer exhibited the drastic shrinking in rapid response to light irradiation, and various architectures were examined for the photocontrollable microsystems in which the hydrogels were incorporated as lightdriven soft actuators.23−25 However, such a sharp photoresponse was available only in acidic conditions with pH 2 to 4,23 thereby limiting their applications to biorelated systems, which were near neutral in pH. Although the polymers surfaces functionalized with nitrospiropyran have been examined to implement photoresponsive cell adhesion in neutral culture environment,5−8 sharp and rapid photoresponse has still remained a challenge to practical application. In this study, we synthesized a novel photoresponsive copolymer (pNBANIPAAm) by functionalizing poly(N-isopropylacrylamide) (pNIPAAm) with o-nitrobenzaldehyde (NBA) to implement photoinduced aqueous dissolution in neutral conditions. NBA has been known to be converted irreversibly to o-nitrosobenzoic acid, which dissociates protons and becomes anionic in neutral aqueous systems in sharp response to near UV light.26−28 Unlike photoacid-generating copolymers, with which photoinduced cell killing were reported,29,30 photoreaction of pNBANIPAAm was expected to generate no strong acid or no low-molecular-weight component. These low-invasive characteristics would be favorable for the application to the photocontrol of cell culture. Here we report the photoresponsive characteristic of pNBANIPAAm in aqueous systems and discuss its applications with respect to several demonstrations of photocontrollable release of microscopic objects including living cells.

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For cell culture of NIH/3T3, Dulbecco’s Modified Eagle Medium (DMEM, no. 041-29775, Wako Pure Chemical Industries, Ltd.) and fetal bovine serum (FBS, no. 30-2020, ATCC) were used. For MDCK and HeLa, Minimum Essential Medium (MEM, no. 051-07615, Wako Pure Chemical Industries, Ltd.) and FBS were used. In the experiment using hiPS cells, culture medium (ReproFF2, ReproCELL Inc.), human recombinant FGF-2 (bFGF, ReproCELL Inc.), Rho-associated coiled-coil forming kinase (ROCK) inhibitor (Y-27632, Wako Pure Chemical Industries, Ltd.), and basal membrane matrix (Matrigel Matrix, human ESC-qualified no. 354277, Corning Inc.) were used. For fluorescent staining of HeLa cells in the demonstration of cell purification, CellTracker Red CMTPX (Thermo Fisher Scientific Inc.) and CellTracker Green CMFDA (Thermo Fisher Scientific Inc.) were used. Apparatuses. A UV−Vis spectrometer (V-560, Jasco Co.) having a temperature controller (ETC-505T, Jasco Co.) was used in the optical density (OD) measurement for polymer solutions. A spin coater (ASS-301, Able Co., Ltd.) was used for polymer coating. UVLED light source (Engineering System Co.) irradiating UV light with the center wavelength of 365 nm and the intensity of 40 mW/cm2 was used for the light irradiation to the solution sample in the OD measurement and BTB color change demonstration and for the evaluation of cell viability after photoinduced detachment. Micropatterned light irradiation onto the photoresponsive culture substrates was carried out by using a PC-controlled microprojection system (DESM-01, Engineering System Co.) installed in an inverted research microscope (IX70, Olympus Co.). UV light with a wavelength of 365 nm was irradiated onto arbitrary areas of the sample as observed through the same objective lens (UPlanFLN 10X, Olympus Co.) with the resolution of 725 pixels/mm. The light intensity (up to 120 mW/ cm2) was regulated by using ND filters. Bright-field images were taken with a cooled CCD camera system (VB-7000, Keyence Co.) installed on the same microscope. Fluorescent microscopic observation was carried out by using a confocal laser scanning microscope (FluoView300, Olympus Co.) installed in an inverted research microscope (IX71, Olympus Co.). Size and ζ-potential of the copolymer aggregation was measured in DTS1070 capillary cell with electrodes using a dynamic light scattering (DLS)/electrophoretic light scattering (ELS) apparatus with 633 nm He−Ne laser (Zetasizer Nano-ZS, Malvern Instruments Ltd.). Synthesis of pNBANIPAAms. First, we synthesized N-(3-(3-(1,3dioxolan-2-yl)-4-nitrophenoxy)propyl)methacrylamide as a polymerizable monomer having a NBA group protected by the acetalization with ethylene glycol. Then, the monomer was polymerized with Nisopropylacrylamide (NIPAAm) at the functionalization ratios ( f r) of 4, 7, and 10 mol % through the radical copolymerization with azobis(isobutyronitrile) as initiator in dry THF under N2 atmosphere at 65 °C for 24 h. After precipitation in diethyl ether, the copolymers were deprotected in a mixture of chloroform and aqueous HCl solution. Then pNBANIPAAms were precipitated in diethyl ether, collected, and dried under a vacuum. Composition ratios and molecular weight of the copolymers were estimated by 1H NMR analysis and by gel permeation chromatography, respectively. The detailed procedure is described in the Supporting Information. Observation of Photoresponsive Color Change of Coexisting BTB in Aqueous Solution of pNBANIPAAm. A BTB solution was prepared by dissolving 2.6 mg of BTB powder in a solvent prepared by mixing 0.75 g of 10 mM NaOH aqueous solution and 1.13 g of ethanol. A small amount (∼1%) of this BTB solution was added to 0.010 wt % pNBANIPAAm (f r: 10 mol %) aqueous solution in a glass cuvette with an optical path length of 10 mm. The color of the solution was conditioned by adding a small amount (∼1%) of 10 mM NaOH aqueous solution at 15 °C. The color change of the solution was observed in response to the irradiation of UV light from UV LED light source (center wavelength, 365 nm; intensity, 40 mW/cm2) to the lower part of the solution in the cuvette. pNBANIPAAm Coating. Polymer coating was carried out by loading the pNBANIPAAm (and pNIPAAm homopolymer) solutions in TFE on a bottom surface of polystyrene dishes and then annealing at 85 °C for 1 h. A typical coating condition of 1000 rpm rotation was

EXPERIMENTAL SECTION

Materials. Bromothymol blue (BTB, no. 027-03052, Wako Pure Chemical Industries, Ltd.) was used in the demonstration of photoresponsive color change of pNBANIPAAm solution. Nontreated polystyrene dishes (IWAKI no. 1000-035, AGC Techno glass Co., Ltd.) and tissue culture polystyrene dishes (IWAKI no. 3000−035, AGC Techno glass Co., Ltd.) were used as base substrates of pNBANIPAAm coated layers. Dulbecco’s Phosphate-Buffered Saline, pH 7.4 (DPBS, no. D8537, Sigma-Aldrich Co.), and phthalate pH standard solutions of pH 6.0 and 5.0 (no. B0166 and no. B0161, Tokyo Chemical Industry Co., Ltd.) and pH 4.0 (no. 028-03185, Wako Pure Chemical Industries, Ltd.) were used as aqueous buffer solutions in the experiments to examine the influence of pH to photoinduced dissolution of pNBANIPAAms. Polyethylene powder (UF1.5N, Sumitomo Seika Chemicals Co., Ltd.) was used in the experiment of their photoinduced release shown in Figure 5. In the experiment shown in the Supporting Information Movie, polyethylene powder (no. 332119, Sigma-Aldrich Co.) was also used. Albumin and tetramethylrhodamine isothiocyanate bovine (no. A2289, SigmaAldrich Co.) was used as a fluorescent-labeled bovine serum albumin (FBSA) in the experiment of their photoinduced release. Homopolymer of NIPAAm (no. 21458, MW ∼ 40000, Polysciences, Inc.) was used to prepare the polymer blend with pNBANIPAAm. For the cell manipulation experiments, MDCK (Madin−Darby canine kidney cells), NIH/3T3 (mouse embryonic fibroblasts), hiPS (human induced pluripotent stem) cells (no. 201B7), and HeLa cells were purchased from RIKEN Bioresource Center (Tsukuba, Ibaraki, Japan). B

DOI: 10.1021/acs.biomac.8b00470 Biomacromolecules XXXX, XXX, XXX−XXX

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Biomacromolecules started immediately after loading 8 μL of 1.5 wt % polymer solution onto the center of dish bottom under N2 atmosphere. The polymer density was estimated by the amount and concentration of used solution and the coated area. Evaluation of Photoresponse of NBA groups. To evaluate the photoresponse of NBA groups at side chain of pNBANIPAAm ( f r: 10 mol %), a copolymer thin layer with the areal density 37 μg/cm2 was prepared on a bottom surface of polystyrene dish. Being irradiated with UV light from UV LED light source (center wavelength: 365 nm), absorbance spectrum of the copolymer thin layer was measured with the UV−vis spectrometer in the wavelength range from 300 to 700 nm at prescribed irradiation dosages. Optical Density Measurement for Aqueous Solutions of pNBANIPAAms. pNBANIPAAm (0.010 wt %) ( f r: 0, 4, 7, and 10 mol %) aqueous solutions were prepared by diluting 1.0 wt % 2,2,2trifluoroethanol (TFE) solutions with aqueous buffer solutions of pH 4.0 or 7.4 or aqueous solution containing 20 mM HCl and 40 mM NaCl to 0.010 wt % in a glass cuvette with an optical path length of 10 mm. After being stirred in thermoregulated cell holder equipped in the UV−vis spectrometer at set temperature 1 °C, the optical density (OD, sum of absorbance and turbidity) spectra were measured in the wavelength range from 300 to 700 nm at temperatures from 1 to 60 °C. After saturated irradiation with UV light from UV LED light source (center wavelength: 365 nm) at 1 °C, where the solutions were transparent, OD spectra were measured for the irradiated solution at temperatures from 1 to 60 °C. Because no absorbance was observed at 700 nm in all the conditions, OD at 700 nm was defined to be turbidity at 700 nm (T700) as a measure of polymer dehydration. Size and ζ-Potential Measurement for pNBANIPAAm Aggregation in Aqueous System. pNBANIPAAm (0.010 wt %) with f r 7 mol % aqueous solutions were prepared by diluting 1.0 wt % 2,2,2-trifluoroethanol (TFE) solutions with 5.0 mN NaOH aqueous solution. The z-averaged size and ζ-potential of the copolymer aggregation were measured at 15.0 °C using a DLS/ELS apparatus. Then the solution was irradiated with UV light (center wavelength, 365 nm; dosage, 18 mJ/cm2), and the measurement was carried out at 23.1 °C. Photoinduced Dissolution of pNBANIPAAm Coated Layers. To examine the influence of NBA functionalization on the photoresponsive dissolution of pNBANIPAAm in neutral aqueous system, copolymer thin layers with the areal density of about 20 μg/ cm2 were prepared on a bottom surface of polystyrene dish for f r conditions of 4, 7, and 10 mol %. Using a PC-controlled microprojection system described above, UV light with 365 nm wavelength was irradiated in a polka-dot pattern to dissolve the thin layers locally in PBS of pH 7.4 at 25 °C. Then the thin layers thus patterned were irradiated with the uniform light at the intensity of 15 mW/cm2 under microscopic observation through a 10× objective lens and a 1.6× built-in zoom lens. The time required to dissolve the copolymer thin layer (td) was measured for each functionalization condition by observing that polka-dot pattern diminished completely. Also, to examine the pH dependence, micropatterned irradiation was carried out for the pNBANIPAAm ( f r: 10 mol %) thin layer in aqueous systems at several pH conditions (4.0, 5.0, and 7.4) by using the microprojection system. Under microscopic observation through a 10× objective lens and a 1.6× built-in zoom lens, UV light (wavelength, 365 nm; intensity, 120 mW/cm2) in a polka-dot pattern (diameter of dots: 100 μm) was irradiated to the copolymer thin layers from the bottom side at 25 °C. Photoinduced Release of Polyethylene Beads. Polyethylene beads (average diameter: 20 μm) were glued on the bottom surface of a polystyrene dish as aggregations by casting with a 2,2,2trifluoroethanol solution of pNBANIPAAm ( f r: 10 mol %) and then drying. After loading DPBS of pH 7.4, the dish was set on the stage of a PC-controlled microprojection system described above. Under microscopic observation through a 10× objective lens and a 1.6× builtin zoom lens at 25 °C, UV light (wavelength, 365 nm; intensity, 120 mW/cm2) was irradiated from the bottom side to the areas where targeted aggregations were immobilized.

Photoinduced Release of Fluorescent-Labeled Albumins. Two mL of DPBS of pH 7.4 were loaded onto the pNBANIPAAm ( f r: 7 mol %) layer coated on the bottom surface of a polystyrene dish, and then 0.05 mL of FBSA aqueous solution (0.004 wt %) was added to the DPBS. After incubation at 37 °C for 30 min, the loaded solution was substituted with DPBS, and the distribution of FBSA at the surface was observed by using a confocal laser scanning microscope through a 10× objective lens. Then micropatterned light irradiation was carried out for the corresponding area as in the experiments of photoinduced release of polyethylene beads. After the irradiation, fluorescent images of the corresponding view area were observed again. Photoinduced Detachment of NIH/3T3 Cells. Dispersed in DMEM containing 5 vol % fetal bovine serum (FBS) and penicillin− streptomycin, NIH/3T3 cells were seeded on pNBANIPAAm coated layer (f r, 10 mol %; density, 22 μg/cm2) prepared on the bottom surface of polystyrene dish. After 18 h incubation at 37 °C, the dish was set on the stage of the PC-controlled microprojection system. Under microscopic observation through a 10× objective lens and a 1.6× built-in zoom lens, UV irradiation (wavelength, 365 nm; intensity, 120 mW/cm2) was carried out for 2 s from the bottom side to a rectangular area of 700 μm × 530 μm at 25 °C. Photoresponsive Detachment of Human Induced Pluripotent Stem (hiPS) Cell Colony. Basal membrane matrix (Matrigel) was coated on a pNBANIPAAm coated layer (f r, 7 mol %; density, 13 μg/cm2) prepared on the bottom surface of polystyrene dish by loading its solution just after dilution of ice-cold liquid concentrate of Matrigel with DPBS (40-fold) with room temperature and by storing for 1 h at 37 °C. Dispersed in ReproFF2 culture medium supplemented with 5 ng/mL human recombinant FGF-2, ROCK inhibitor, and penicillin−streptomycin, hiPS cell clumps were seeded onto pNBANIPAAm coated layer and incubated for 6 days at 37 °C. The dish was set on the stage of the PC-controlled micro projection system. Under microscopic observation through a 10× objective lens at 25 °C, UV light (wavelength, 365 nm; intensity, 120 mW/cm2) was irradiated from the bottom side to the area where the targeted cell colony adhered. After irradiation for 10 s, the culture surface was flushed gently with the medium to remove the targeted cell colony, which shrank after the local irradiation. Photoresponsive Detachment of MDCK Cells. Photoresponsive polymer layer was prepared by coating the polymer blend of pNBANIPAAm (f r: 10 mol %) and 29 wt % pNIPAAm (density: 22 μg/cm2) on the bottom surface of polystyrene dish. Dispersed in MEM containing 5 vol % FBS, nonessential amino acid, and penicillin−streptomycin, MDCK cells were seeded on polymer coated layer. After 18 h culture at 37 °C, the dish was set on the stage of the PC-controlled microprojection system. Under microscopic observation through a 10× objective lens and a 1.6× built-in zoom lens, UV irradiation (wavelength, 365 nm; intensity, 120 mW/cm2) was carried out for 2 s from the bottom side to the areas where the targeted cells adhered at 25 °C. Demonstration of Cell Purification. HeLa cells were stained with CellTracker Red CMTPX or CellTracker Green CMFDA following the manufacturer’s instructions. Dispersed in MEM containing 10 vol % FBS, nonessential amino acid, sodium pyruvate, and penicillin−streptomycin, a mixture of equal parts of red-stained cells and green-stained cells were seeded on a pNBANIPAAm coated layer (f r, 10 mol %; density, 22 μg/cm2) prepared on the bottom surface of polystyrene dish. After 18 h incubation at 37 °C, fluorescent images of the cells stained in each color were observed by using a confocal laser scanning microscope through a 10× objective lens. According to the fluorescence images, UV irradiation (wavelength, 365 nm; intensity, 120 mW/cm2) was carried out for 2 s from the bottom side to the areas where the cells, stained in either of two colors, adhered by using the PC-controlled microprojection system at 25 °C. After gentle flushing with the culture medium, fluorescent images of the corresponding view areas were observed again. Evaluation of Cell Viability after Photoresponsive Detachment. Dispersed in DMEM containing 5 vol % FBS and penicillin− streptomycin, NIH/3T3 cells were seeded on a pNBANIPAAm coated layer ( f r, 4 mol %; density, 38 μg/cm2) prepared on the bottom C

DOI: 10.1021/acs.biomac.8b00470 Biomacromolecules XXXX, XXX, XXX−XXX

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Figure 1. Photoresponse of pNBANIPAAm. Chemical structures before and after photoreaction and proton dissociation (a), and light induced color change of an aqueous solution (f r: 10 mol %) containing small amount of bromothymol blue from a weakly basic condition (b). surface of polystyrene dish. After 2 days of incubation at 37 °C, UV light from UV LED light source (center wavelength, 365 nm; intensity, 40 mW/cm2) was irradiated for 10 s to the whole polymer coated area from the bottom side. All of the cells were removed and collected from the substrate by flushing gently with the medium and were dispersed to single cells with 1 mM EDTA in DPBS. As a reference sample, on the other hand, the cells were recovered from a tissue culture polystyrene dish after 2 day incubation by applying trypsin (0.05 w/v %)−EDTA (0.53 mM) solution for 2 min at 37 °C as treated in the general passage process. For each condition, the viability of the cells recovered from four samples was estimated by counting the stained and unstained cells after staining with Trypan blue.

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RESULTS AND DISCUSSION

Synthesis of pNBANIPAAm. A chemical structure of pNBANIPAAm was shown in Figure 1a. The main chain of the copolymers was pNIPAAm, well-known to show drastic thermoresponsive hydration change, and a photoresponsive NBA group was introduced at a side chain via amide and ether linkers. In the preliminary examination of the synthesis, polymerizable monomer having bare NBA was found to inhibit radical copolymerization, so we attempted the copolymerization using the protected NBA monomer and the deprotection afterward. As a result, copolymers with Mw 11000−13000 were obtained for all the functionalization ratios (f r) of 4, 7, and 10 mol % we tried (see Supporting Information). Photoresponsive Proton Dissociation of pNBANIPAAm in Aqueous Solution. As reported in many studies, NBA was known as a chemical actinometer showing irreversible structure change to o-nitrosobenzoic acid upon light irradiation.27 Therefore, copolymer structure after photoreaction and proton dissociation was considered to be as shown in Figure 1a. Actually, in 1H NMR analysis for the copolymer in CDCl3, we confirmed that a signal at 8.4 ppm, attributed to aldehyde in the NBA group at the copolymer side chain, disappeared after sufficient UV light irradiation (appearance of signal attributed to photogenerated carboxylic acid was unclear due to significant broadening). Figure 1b shows the light induced color change of an aqueous pNBANIPAAm (f r: 10 mol %) solution containing small amount of BTB, which had been weakly basic and had appeared bluish (left). In rapid response to UV light irradiation (center wavelength: 365 nm) to the lower part of the solution, the irradiated portion turned yellowish indicating the decrease in pH (right, see Supporting Information Movie). This result was a direct evidence of the photoinduced proton dissociation from pNBANIPAAm, suggesting that the copolymer became charged negatively and its hydration increased significantly. Sensitivity and Quantum Yield for Photoresponse of NBA groups. Figure 2a shows the absorbance spectra of a pNBANIPAAm ( f r: 10 mol %) thin layer with the areal weight density 37 μg/cm2 at various irradiation dosages. Irradiation of

Figure 2. Irradiation dosage dependence of absorbance spectra (a) and the absorbance at 365 nm (b) of pNBANIPAAm ( f r: 10 mol %) solid thin layer (40 μg/cm2).

the UV light with 365 nm wavelength increased the absorbance band with a maximum at 354 nm as photoreaction of NBA groups progressed. Sensitivity and quantum yield for the photoresponse can be estimated from the absorbance change. First, molar absorption coefficient of the NBA group ε (cm2/ mol) at 365 nm was estimated from the following relationship between absorbance at the wavelength A365 and the areal molar density d (mol/cm2): ε = A365 /d

(1)

After subtracting the contribution of polystyrene dish from the measured value, A365 was obtained to be 0.059. Using the d value (2.8 × 10−8 mol/cm2), calculated directly from the areal weight density, ε was estimated to be 2.1 × 106 cm2/mol. Because A365 was so small that the light intensity in the thickness direction was negligible, conversion of the photoreaction x at irradiation time t obeyed the following equation, D

DOI: 10.1021/acs.biomac.8b00470 Biomacromolecules XXXX, XXX, XXX−XXX

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Biomacromolecules x = 1 − exp( −2.303ΦεPt /NAhν)

facilitated significantly as f r increased; the LCST was not observed below 60 °C for 7 and 10 mol % functionalizations, and only a small amount of turbidity was observed at the temperatures above 50 °C for 4 mol % functionalization. Phase transition of the system was totally changed by the photoreaction of the NBA introduced even at only 4 mol %, suggesting the validity of the material design based on the polymer effect described above. Figure 3b shows the temperature dependent turbidity of the aqueous solutions containing 0.010 wt % pNBANIPAAm ( f r: 10 mol %) at several pH conditions. At all the examined pH conditions from 1.7 to 7.4, LCSTs of the solutions before irradiation were estimated to be in the temperature range from 11 to 15 °C. The variation in the LCST was considered to reflect the influence of the ionic strength of the solution rather than the pH. After irradiation, due to the pH dependent proton dissociation of the photogenerated o-nitrosobenzoic acid group,31 LCST of the solution was observed at 21 and 33 °C at pHs 1.7 and 4.0, respectively, and was not observed below 60 °C at pH 7.4 as described above. From a different point of view, the copolymer came to exhibit pH-dependent water solubility at room temperature in response to the light irradiation. The result indicated that the ionic o-nitrosobenzoate group facilitated the copolymer hydration much more strongly than nonionic o-nitrosobenzoic acid group, which was dominant at low pH conditions (Figure 1a). Size and ζ-Potential of pNBANIPAAm Aggregation in Aqueous System. For the pNBANIPAAm ( f r 7 mol %) in 5.0 mN NaOH aqueous solution at 15.0 °C, the aggregation size and the ζ-potential were estimated to be 45 nm and −0.9 mV, respectively, indicating that the copolymer was almost electroneutral before irradiation. After UV irradiation of 18 mJ/cm2, supposed to induce 12% photochemical conversion, similar size of aggregation (43 nm) was observed at 23.1 °C due to the facilitated hydration of the copolymer increasing LCST of the system and the ζ-potential was estimated to be −11 mV for the aggregations. As with the results of photoresponsive change in BTB color and the turbidity of pNBANIPAAm aqueous systems described above, the observed ζ-potential suggesting large negative charge of the aggregation supported the photoreaction scheme shown in Figure 1a. Photoinduced Dissolution of pNBANIPAAm Coated Layer. As expected from the experimental results above, pNBANIPAAm exhibited a sharp photoinduced aqueous dissolution from solid state at neutral conditions. Photoresponsive characteristics of pNBANIPAAm were tabulated in Table 1. Under the light irradiation with 365 nm wavelength and the intensity 15 mW/cm2 in pH 7.4 at 25 °C, the time required to dissolve the copolymer thin layer (td) was estimated to be 5.1, 4.2, and 3.7 s for each f r of 4, 7, and 10 mol %,

(2)

where Φ, P, NA, h, and ν were quantum yield of the photoreaction, light intensity (W/cm2), Avogadro’s number, Planck’s constant (J s), and the frequency of the irradiated light (s−1), respectively. Figure 2b shows the irradiation dosage dependence of A365. The kinetics agreed well with eq 2, and the sensitivity (r = 2.303Φε/NAhν) and Φ were estimated to be 7.3 cm2/J and 0.49, respectively. The estimation of Φ was consistent with the value 0.50, which had been reported previously for the solid crystal of NBA.26,27 Photoresponsive Turbidity Change of pNBANIPAAm Aqueous Systems. The ionization of NBA groups composing pNBANIPAAm was considered to influence much of the hydration of the copolymer. To discuss the influence quantitatively, the turbidity of pNBANIPAAm aqueous solution was analyzed as a measure of copolymer dehydration. Figure 3a

Figure 3. Temperature dependent turbidity of 0.010 wt % pNBANIPAAm aqueous solutions at several functionalization ratios (0, 4, 7, and 10 mol %) at pH 7.4 (a) and several pHs (1.7, 4.0 and 7.4) at 10 mol % functionalization (b) before (dashed lines) and after (solid lines) irradiation.

Table 1. Photoresponsive Characteristics of pNBANIPAAm shows the temperature dependent turbidity of the aqueous buffer solution of pH 7.4 containing 0.010 wt % pNBANIPAAm with f r of 0, 4, 7, 10 mol %. As is well-known, the pNIPAAm homopolymer (0 mol % functionalization) exhibited drastic dehydration at temperatures above 31 °C, a lower critical solution temperature (LCST) of the system. The pNBANIPAAm solutions also exhibited LCST behavior before irradiation, and the LCST decreased as f r increased. This result indicated that the unreacted NBA was hydrophobic and stabilized the dehydration of the copolymer. After sufficient irradiation, on the other hand, the copolymer hydration was

f ra 0.04 0.07 0.10

Φb

0.49

r (cm2/J)c

td (s)d

xde

Ed (mJ/cm2)f

7.3

5.1 4.2 3.7

0.43 0.37 0.33

77 63 56

a

NBA functionalization ratio of pNBANIPAAm. bQuantum yield of photoreaction at 365 nm. cSensitivity of photoreaction at 365 nm. d Time to dissolve copolymer thin layer by 15 mW/cm2 irradiation in pH 7.4 at 25 °C. eConversion of photoreaction required to dissolve copolymer thin layer in pH 7.4 at 25 °C. fDosage of UV irradiation required to dissolve copolymer thin layer in pH 7.4 at 25 °C. E

DOI: 10.1021/acs.biomac.8b00470 Biomacromolecules XXXX, XXX, XXX−XXX

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37 °C. Similar photoresponse was observed also at pH 5.0. On the other hand, the copolymer did not exhibit photoinduced dissolution at pH 4.0 even after long irradiation, suggesting that nonionic o-nitrosobenzoic acid group hardly facilitated copolymer hydration. Photoinduced Release of Polyethylene Beads and Fluorescent-Labeled Albumins. As a demonstrative application of the photoresponsive dissolution of pNBANIPAAm, we examined the photocontrolled release of small polyethylene beads from a substrate at arbitrary timing in arbitrary areas. The scheme and result of the experiment are shown in Figure 6. In DPBS, aggregations of polyethylene beads remained on the substrate stably, suggesting that pNBANIPAAm glued them firmly. Upon the local light irradiation in the areas where specifically two of the immobilized aggregations were placed (Figure 6c, fluorescence from the polystyrene substrate was observed), only targeted aggregations were released selectively from the substrate in a few seconds via photoinduced dissolution of pNBANIPAAm (Figure 6d). The others remained stably on the substrate, indicating that the effect of the irradiation was well localized and interference to neighboring aggregations was avoided in this spatial scale. Because of their low specific gravity, released polyethylene beads moved up in DPBS, disappearing from the microscopic viewing field. By the subsequent local irradiation, selective release was triggered for five additional aggregations (Figure 6e,f); other demonstrations of photoresponsive release of polyethylene beads can be seen in Supporting Information Movie). Also we attempted the photocontrolled release of FBSA with the molecular weight of 66 kDa. The scheme and result of the experiment are shown in Figure 7. By loading the solution and subsequent incubation at 37 °C, FBSA was immobilized onto the surface of a pNBANIPAAm coated layer stably (Figure 7c). Upon the micropatterned irradiation (Figure 7a), FBSA was released from the irradiated area in a few seconds via photoinduced dissolution of pNBANIPAAm (b,d) while no change in fluorescence was observed in the unirradiated area (e). Because the irradiation dosage was too small to induce photobleaching of FBSA, the result demonstrated that photoinduced release via pNBANIPAAm coated layer worked even for the objects such as albumins, which were much smaller than polyethylene beads. Photoresponsive Detachment of Cultured Cells. As a more practical application, we examined photoselective detachment of cultured cells by using pNBANIPAAm. The experimental results are shown in Figure 8. Seeded NIH/3T3 cells adhered and spread on the pNBANIPAAm coated layer as on a normal cell culture dish after overnight incubation (Figure

respectively. From these td values, the irradiation intensity and the value of r (7.3 cm2/J) estimated above, the necessary and sufficient x (conversions of the photoreaction, see eq 2) for the copolymer dissolution (xd) was calculated to be 0.43, 0.37, and 0.33 for each f r of 4, 7, and 10 mol %, respectively. Figure 4

Figure 4. xd of pNBANIPAAm coated layers at several functionalization ratios (4, 7, and 10 mol %) plotted on the conversion curve.

shows the xd value in each f r condition in the relationship between x and irradiation dosage. These xd values were sufficiently smaller than 1, suggesting the important advantage of the mechanism based on the phase transition of polymer system over that based on the number of crosslinks. Further, these xd values, which were much smaller than 1, indicating that the hydration effect of the photochemically generated ionic onitrosobenzoate group was very strong; when pNBANIPAAm with f r 10 mol % became dissolved from solid state, 67% of NBA groups remained unreacted. Because of this small xd and large Φ estimated above, irradiation dosage required for dissolution was as small as 56 mJ/cm2. This high sensitivity was favorable not only to speeding up the process of photocontrol but also to reducing the light exposure to the object. Also, pH of surrounding aqueous system was considered to influence much on the photoinduced dissolution of pNBANIPAAm. Parts a−c of Figure 5 show the appearance of coated thin layer of pNBANIPAAm ( f r: 10 mol %) after irradiation in a polka-dotted micropattern (diameter of dots: 100 μm) in pH 4.0, 5.0, and 7.4 at 25 °C. As described above, the pNBANIPAAm coated layer exhibited a sharp photoinduced aqueous dissolution from solid state at pH 7.4 (see Supporting Information Movie). This photoresponse was maintained even after being stored for more than 1 month in DPBS of pH 7.4 at

Figure 5. Photoinduced micropatterned dissolution of pNBANIPAAm coated layers (diameter of dots: 100 μm) at 25 °C in aqueous buffer solutions at pHs 4.0 (a), 5.0 (b), and 7.4 (c). F

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Figure 6. Photoinduced release of small polyethylene beads from a substrate via photoresponsive dissolution of pNBANIPAAm ( f r: 10 mol %) at pH 7.4. Schematic illustrations (a,b) and experimental result (scale bar: 200 μm, c−f).

Figure 7. Photoinduced release of FBSA from a substrate via photoresponsive dissolution of pNBANIPAAm (f r: 7 mol %) at pH 7.4. Schematic illustrations (a,b) and experimental results (c−e; scale bar: 100 μm).

pNBANIPAAm. With respect to the direct influence of the UV irradiation, we reported in our previous study that the viability of cultured cells were hardly damaged by the dosage of a few tens of J/cm2 and with the same wavelength.32 Then, we examined applying this photoinduced cell detachment to colony isolation for the hiPS cell culture system. The experimental results are shown in Figure 8d−f. The hiPS cell clumps, which had been seeded on the pNBANIPAAm coated

8a). Upon local light irradiation in a rectangular area, a wrinkle appeared immediately (Figure 8b), and then a flexible thin membrane came off of the polystyrene surface with the cells in a few seconds (c), indicating that pNBANIPAAm showed sharp photoresponse even in a cell culture environment (see Supporting Information Movie). On the other hand, the same irradiation (∼0.3 J/cm2) provided no influence to the cells cultured on a tissue culture polystyrene surface with no G

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Figure 8. Photoselective detachment of NIH/3T3 cells (a−c), hiPS cell colony (d−f), and MDCK cells (g−i) from pNBANIPAAm-coated culture substrates (scale bar: 200 μm).

Figure 9. Demonstration of cell purification through photoselective detachment using fluorescently stained HeLa cells on pNBANIPAAm-coated culture substrate. Fluorescence images before (a,c) and after (b,d) selective removal of the cells stained either red or green (scale bar: 100 μm).

layer, grew to mature colonies after 6 days of culture (Figure 8d) as steadily as on a normal cell culture dish. Upon local irradiation to the area where one of the colonies adhered, only the targeted colony shrank as a flexible thin membrane composed of the copolymer came off of the polystyrene surface (Figure 8e). Gentle flushing with medium easily broke the membrane, and only the targeted hiPS cell colony was removed from the substrate surface (Figure 8f). This result indicated that the photoinduced cell detachment was applicable also to the cell colonies and had a potential to reduce daily

burden of manual process of removing unwanted differentiated cells in the laboratories maintaining stem cells.33 As described above, we observed very thin and flexible, but insoluble membrane remaining after light irradiation onto the pNBANIPAAm thin layer, which had been in culture medium for more than half a day, while nothing remained in the case soaked in DPBS with similar pH. Considering aldehyde groups at pNBANIPAAm side chains, aggregation with some amino components contained in the culture medium was suggested as a possible mechanism for the remaining of thin membrane, so H

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Biomacromolecules we examined blending inert pNIPAAm homopolymer with pNBANIPAAm, expecting an intervening effect to prevent solid aggregation. Parts g−i of Figure 8 show the experimental result obtained with the coated layer of the polymer blend containing 29 wt % pNIPAAm. MDCK cells were observed to adhere, spreading well on the coated layer of the polymer blend after 12 h of culture (Figure 8g). Upon light irradiation, the cells started to shrink immediately in the irradiated area (Figure 8h) and then detached from the substrate surface spontaneously (i, see Supporting Information Movie). Unlike in the cases containing no pNIPAAm homopolymer, no insoluble thin membrane remained after sufficient irradiation and the independent detachment of the cells was implemented. However, the coated layer of the polymer blend soaked in the culture medium was found to lose its photoresponse just by being stored at temperatures lower than 25 °C for only half an hour. Practically in the experiment described above, we needed to carry out the light irradiation immediately after bringing the culture dish out of an incubator kept at 37 °C. This instability of the polymer blend was considered to be due to the fact that the room temperature was lower than the LCST of pNIPAAm homopolyer (∼31 °C) in aqueous systems. Demonstration of Cell Purification. To show the utility of the photoinduced cell detachment with a precision, we attempted the demonstration of cell purification using the cell culture having the same adhesive property. The HeLa cells, which had been stained in red or green, adhered and spread on a pNBANIPAm coated layer in a random distribution (Figure 9a). Upon the local irradiation with UV light to the areas where red cells adhered, the targeted cells shrank as the copolymer coated layer became hydrated. By flushing with the medium gently, all of these cells were removed (100% elimination) while most of other green cells remained on the culture surface (Figure 9b). Also, we succeeded in purifying red cells by removing green cells as well (Figure 9c,d). The result demonstrated that the spatial resolution of the photoinduced decrease of cell adhesion was enough to achieve cell purification. The cells remaining on the substrate after the purification process continued to grow in the subsequent incubation. Influence of Photoresponsive Detachment on Cell Viability. We examined the viability of the NIH/3T3 cells, which had been cultured on pNBANIPAAm coated layers for 2 days and then recovered from the substrate surface through photoinduced decrease of cell adhesion. As a result, we observed that 96% of the recovered cells maintained their viability well (Figure 10). Although pNBANIPAAm contained reactive aldehyde group, it was bound covalently to the side chain of the copolymer, which had been water-insoluble before light irradiation. As described above, various kind of adherent cells were cultured on pNBANIPAAm coated layer with no damage for some time, and applied UV irradiation (0.4 J/cm2) was considered to have a very small effect to their viabilities.32

Figure 10. Viability of the NIH/3T3 cells, recovered through the photoinduced decrease of cell adhesion of pNBANIPAAm coated layer ( f r, 4 mol %; density, 38 μg/cm2) and recovered from a tissue culture polystyrene surface by using trypsin−EDTA solution.

in aqueous systems, we demonstrated photocontrolled release of polyethylene beads and fluorescent-labeled albumin and cultured adherent cells from the substrates coated with the copolymers. Several experiments demonstrated that mild light irradiation at room temperature triggered immediate detachment of adhering cells from the culture substrate without loosing their viability, suggesting a feasible implementation of on-demand selective cell recovery. All the photodissoluble thin layers examined in this study were prepared simply by coating the dilute copolymer solutions on polystyrene substrates. Photoinduced decrease of cell adhesion was observed for the coated layer with the areal density of a few 10 μg/cm2. The preparation of the photoresponsive culture surface of 35 mm dish (∼10 cm2) required the copolymer to be as much as 1 mg. Further, the copolymer coated layer was confirmed to maintain the property of drastic photoinduced dissolution stably even after 6 months of storage in a dry and dark place at room temperature. Precisely controlled UV irradiation was achieved with a microprojection system just by designating the area to irradiate on the displayed microscopic field via a graphic user interface. This operation scheme was likely to be automated by the assist of imaging recognition, which has showed a rapid progress in recent years and was expected to contribute to qualitycontrolled mass production of standardized cells.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.biomac.8b00470. Description of synthesis procedures and supplementary characterization (PDF) Color change of pNBANIPAAm solution containing a trace of BTB in response to light irradiation (MOV) Micropatterned dissolution of pNBANIPAAm coated layer in response to light irradiation (MOV) Release of polyethylene beads in response to light irradiation (MOV) NIH/3T3 cells and from pNBANIPAAm coated layer in response to light irradiation (MOV) MDCK cells from pNBANIPAAm coated layer in response to light irradiation (MOV)

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CONCLUSIONS A photoresponsive copolymer of a new class has been developed by functionalizing pNIPAAm with NBA groups to implement a sharp and drastic photoinduced aqueous dissolution through phase transition. Dissolution of the copolymer coated layers was triggered immediately by UV light irradiation in wide pH and temperature ranges including neutral pH and room temperature. As example applications of the photoresponse to the manipulation of microscopic objects I

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(15) Sumaru, K.; Kameda, M.; Kanamori, T.; Shinbo, T. Characteristic phase transition of aqueous solution of poly (N-isopropylacrylamide) functionalized with spirobenzopyran. Macromolecules 2004, 37, 4949−4955. (16) Satoh, T.; Sumaru, K.; Takagi, T.; Takai, K.; Kanamori, T. Isomerization of spirobenzopyrans bearing electron-donating and electron-withdrawing groups in acidic aqueous solutions. Phys. Chem. Chem. Phys. 2011, 13, 7322−7329. (17) Moriyama, K.; Sumaru, K.; Takagi, T.; Satoh, T.; Kanamori, T. Dynamically controlled construction of microstructures based on photoinduced phase transition of a spirobenzopyran-modified polymer solution. RSC Adv. 2016, 6, 44212−44215. (18) Ziółkowski, B.; Florea, L.; Theobald, J.; Benito-Lopez, F.; Diamond, D. Self-protonating spiropyran-co-NIPAM-co-acrylic acid hydrogel photoactuators. Soft Matter 2013, 9, 8754−8760. (19) Akiyama, H.; Tamaoki, N. Polymers derived from Nisopropylacrylamide and azobenzene-containing acrylamides: Photoresponsive affinity to water. J. Polym. Sci., Part A: Polym. Chem. 2004, 42, 5200−5214. (20) Kröger, R.; Menzel, H.; Hallensleben, M. L. Light controlled solubility change of polymers: Copolymers of N, N-dimethylacrylamide and 4-phenylazophenyl acrylate. Macromol. Chem. Phys. 1994, 195, 2291−2298. (21) Flory, P. J. Thermodynamics of high polymer solutions. J. Chem. Phys. 1942, 10, 51−61. (22) Huggins, M. L. Some Properties of Solutions of Long-chain Compounds. J. Phys. Chem. 1942, 46, 151−158. (23) Sumaru, K.; Takagi, T.; Satoh, T.; Kanamori, T. Photoinduced reversible proton dissociation of spirobenzopyran in aqueous systems. J. Photochem. Photobiol., A 2013, 261, 46−52. (24) Szilágyi, A.; Sumaru, K.; Sugiura, S.; Takagi, T.; Shinbo, T.; Zrínyi, M.; Kanamori, T. Rewritable microrelief formation on photoresponsive hydrogel layers. Chem. Mater. 2007, 19, 2730−2732. (25) Sugiura, S.; Szilágyi, A.; Sumaru, K.; Hattori, K.; Takagi, T.; Filipcsei, G.; Zrínyi, M.; Kanamori, T. On-demand microfluidic control by micropatterned light irradiation of a photoresponsive hydrogel sheet. Lab Chip 2009, 9, 196−198. (26) Leighton, P. A.; Lucy, F. A. The photoisomerization of the onitrobenzaldehydes. J. Chem. Phys. 1934, 2, 756−759. (27) Galbavy, E. S.; Ram, K.; Anastasio, C. J. 2-Nitrobenzaldehyde as a chemical actinometer for solution and ice photochemistry. J. Photochem. Photobiol., A 2010, 209, 186−192. (28) George, M.; Scaiano, J. Photochemistry of o-nitrobenzaldehyde and related studies. J. Phys. Chem. 1980, 84, 492−495. (29) Sumaru, K.; Kikuchi, K.; Takagi, T.; Yamaguchi, M.; Satoh, T.; Morishita, K.; Kanamori, T. On-demand killing of adherent cells on photoacid-generating culture substrates. Biotechnol. Bioeng. 2013, 110, 348−352. (30) Sumaru, K.; Morishita, K.; Takagi, T.; Satoh, T.; Kanamori, T. Sectioning of cultured cell monolayer using photoacid-generating substrate and micro-patterned light projection. Eur. Polym. J. 2017, 93, 733−742. (31) Mallik, R.; Udgaonkar, J. B.; Krishnamoorthy, G. Kinetics of proton transfer in a green fluorescent protein: a laser-induced pH jump study. Proc. - Indian Acad. Sci., Chem. Sci. 2003, 115, 307−317. (32) Sumaru, K.; Edahiro, J.-I.; Ooshima, Y.; Kanamori, T.; Shinbo, T. Manipulation of living cells by using PC-controlled micro-pattern projection system. Biosens. Bioelectron. 2007, 22, 2356−2359. (33) ATCC Stem Cell Culture Guide: Tips and Techniques for Culturing Stem Cells; ATCC: Manassas, VA, 2015 https://www.atcc.org/ ∼/media/PDFs/Culture%20Guides/iPSCguide.pdf.

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Kimio Sumaru: 0000-0002-2583-0647 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Dr. Teruhiko Baba for his valuable advice on the DLS/ELS measurement. This research was supported by the KAKENHI Grant-in-Aid for Scientific Research B (25282148, 16H03845) from Japan Society of Promotion of Science (JSPS).

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