Reversible and Photoresponsive Immobilization of Nonadherent Cells

Jan 7, 2019 - Spatiotemporal control of cell–material interactions contributes to our understanding of cell biology and the development of cell engi...
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Cite This: ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX

Reversible and Photoresponsive Immobilization of Nonadherent Cells by Spiropyran-Conjugated PEG−Lipids Shin Izuta,† Satoshi Yamaguchi,*,‡,§ Takahiro Kosaka,† and Akimitsu Okamoto*,†,‡ †

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Department of Chemistry and Biotechnology, Graduate School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan ‡ Research Center for Advanced Science and Technology, The University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo 153-8904, Japan § PRESTO, Japan Science and Technology Agency (JST), 4-1-8 Hon-cho, Kawaguchi, Saitama 351-0198, Japan S Supporting Information *

ABSTRACT: Spatiotemporal control of cell−material interactions contributes to our understanding of cell biology and the development of cell engineering. Here, we first report the reversible and spatio-selective immobilization of nonadherent cells through the use of photoswitchable polymeric materials. The substrate coated with spiropyran-conjugated poly(ethylene glycol) (PEG) lipids, which bind to cell membranes via the lipid moiety only in their merocyanine form, enabled rapid cell immobilization and release in an on−off manner by irradiation with ultraviolet and visible light, respectively. Our work has the potential to improve the performance of cell manipulations on chips and to enable rapid cell arrangement/sorting on various surfaces. KEYWORDS: cell manipulation, photoswitchable materials, spiropyran, nonadherent cells, hydrophobicity

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control the adsorption of serum proteins, we used a novel approach to reversibly regulate the characteristics of a polymeric material that directly binds to the cell membrane. Spiropyran,19 a molecule that photoisomerizes leading to a substantial difference in water solubility, was attached to a poly(ethylene glycol) (PEG)−lipid conjugate (PEG−lipid), a nontoxic surfactant that rapidly immobilizes both nonadherent cells and adherent cells.7,20−23 The cell-immobilizing abilities of the spiropyran-conjugated PEG−lipid derivatives were quantitatively examined after irradiation with ultraviolet (UV) and visible light. From these experimental data, the optimal chemical structure for switching cell immobilization in an on−off manner was determined. This photoswitchable material enabled rapid and spatially selective in situ control of both the attachment and detachment of cells in a light-induced manner. This material extends the range of cells that can be reversibly immobilized and promotes cell manipulations on chips24 through its rapid responsiveness.

patiotemporal regulation of cell−material interactions has attracted a great deal of attention. First, it is crucial in furthering our understanding of cell biology since it enables the analysis of intercellular interactions by arranging the distance between cells1,2 and facilitates analysis by allowing cells to be sorted based on their characteristics.3 In addition, fine arrangements of cells are necessary in the field of cell engineering for applications such as regenerative therapies with artificial tissues4 and organ-on-a-chip techniques for drug evaluation.5,6 Although numerous methods have been developed for the spatiotemporal control of cell immobilization, most of these can only either activate or deactivate the cell−material interaction.7−9 Therefore, their applications are limited. Only a few methods enable spatiotemporal regulation of both cell attachment and detachment;10−15 however, all of these depend on cellular adhesivity for cell attachment. Thus, these methods required hours of incubation to immobilize cells and cannot be applied to cells with no adhesiveness. Rapid control of cells is essential for current “omics” analyses requiring high-throughput approaches.16 Nonadherent cells have also attracted growing attention in cancer immunotherapy,17 and detection of circulating tumor cells has significant benefits in the medical field.18 Here, for the first time, we report the development of photoswitchable surfaces for the quick and reversible immobilization of nonadhesive cells. Light, which has the best temporal and spatial resolution, was chosen as the external stimulus. Contrary to most conventional approaches that © XXXX American Chemical Society



MOLECULAR DESIGN Based on PEG lipids, we designed a photoswitchable material with cell-immobilizing properties that can be reversibly altered by the light of two different wavelengths. PEG−lipids are wellReceived: October 29, 2018 Accepted: December 28, 2018

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DOI: 10.1021/acsabm.8b00656 ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX

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ACS Applied Bio Materials known for their noncovalent interactions with lipid bilayer membranes via their lipid moiety. Using this interaction, PEG−lipid-modified surfaces have been employed for the rapid anchoring of cells and liposomes.7,20−23 On the surfaces, PEG−lipids decrease cell-immobilizing ability in accordance with increasing molecular density. A possible reason for this decrease is the formation of hydrophobic assemblies that appear to occur on the surface (Figure S1): at high density, PEG−lipids may form self-assemblies through tightly packing the lipid moiety toward the inside of the PEG layer, resulting in less access of the lipid moiety to cell membranes; conversely, at low density, the liberated lipid moiety may freely interact with cell membranes without self-assembly. Generally, the higher the hydrophobicity of the material, the lower the critical assembly density, similar to the critical micelle concentration of surfactants.25 Similarly, when using a more hydrophobic lipid moiety of PEG−lipid, the critical assembly density of PEG−lipid on the substrate can be lower. Therefore, an adequately hydrophobic lipid moiety may lead to little interaction between cells and the lipid moiety on the PEG− lipid-modified surface. From this working hypothesis, by reversibly changing the hydrophobicity of the lipid moiety, it appeared possible to reversibly switch the cell-immobilizing ability of the PEG−lipid-modified surface (Figure 1). As a

Scheme 1. Molecular Design of Spiropyran-Conjugated PEG−Lipids: (a) Chemical Structure of SpiropyranConjugated PEG−Lipids and (b) The Predicted Chemical Structures of Spiropyran in the Closed and Open Forms under Physiological Conditions

length between the main PEG−lipid chain and the spiropyran derivative (Scheme 1a, m). The designed spiropyranconjugated PEG−lipids were synthesized, and their photoisomerization was examined by optical analyses (Figure 2).

Figure 2. Optical analysis of synthesized spiropyran-conjugated PEG−lipids. (a) Schematic of photoisomerization. (b, c) Absorption spectra of spiropyran-conjugated PEG−lipids. Solid lines: open form, dashed lines: closed form.

Figure 1. Schematic illustration of the working hypothesis of reversible cell immobilization with spiropyran-conjugated PEG− lipid. In the hydrophobic closed form, PEG−lipid may form hydrophobic assemblies on the substrate surface, and its lipid moiety tends to localize in assemblies inside the PEG layer. Therefore, there is little interaction between cells and the lipid moieties. In the hydrophilic open form, the PEG−lipid may disperse in solution, and its lipid moiety tends to interact with the lipid bilayer of the cell. Therefore, there is strong interaction between cells and materials.

Details on the synthesis and photoisomerization are shown in the Supporting Information. After irradiation with UV light (360 nm), the spiropyran component of all six candidates converted to the open form, which exhibits absorbance at 540 nm (Figure 2b and 2c, solid lines). Conversely, irradiation with visible light (520 nm) converted the candidate compounds to the closed form, resulting in the disappearance of absorbance at 540 nm (Figure 2b and 2c, dashed lines). These optical changes were confirmed to be reversible (Figures S2 and S3), indicating that the direction of the photoisomerization of these PEG−lipids can be repeatedly switched by the two wavelengths.

component that alters in hydrophobicity in response to light, spiropyran was chosen for conjugation with the PEG−lipid at the position next to the lipid moiety (Scheme 1a). Spiropyran derivatives are reported to be hydrophobic in their “closed” spiro (SP) form and hydrophilic in their “open” merocyanine (MC) form.19 Therefore, we aimed to develop a spiropyranconjugated PEG−lipid that immobilizes cells in the hydrophilic opened form after UV irradiation and releases cells in the hydrophobic closed form after irradiation with visible light (Figure 1).



PHOTORESPONSIVE CELL IMMOBILIZATION ASSAY The cell-immobilizing ability of the spiropyran-conjugated PEG−lipids was evaluated in their closed and open forms (Figure 3). The PEG−lipid was used to modify the collagencoated substrate and then isomerized to each form by light irradiation. Here, collagen coating was selected as the basement coating which presented amine groups for modification of the PEG−lipid via the amide-coupling



OPTICAL ANALYSIS We investigated six candidates with diversity in the incorporation number of the spiropyran derivatives (Scheme 1a, n), the alkyl modification (Scheme 1a, R), and the linker B

DOI: 10.1021/acsabm.8b00656 ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX

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ACS Applied Bio Materials

Figure 3. Relative cell densities on the substrate surface modified with compound (a) [0Sp2]−NHS, (b) 2[0Sp2]−NHS, (c) [4Sp2]−NHS, (d) [0Sp6]−NHS, (e) [0Sp5]−NHS, and (f) [0Sp4]−NHS. Cell density values for the hydrophilic ring-opened form (“on” state) and the hydrophobic ring-closed form (“off” state) are indicated by the blue and red bars, respectively. Cell suspensions in PBS were used in this experiment. Values are the means ± standard error (n = 15−40, four independent trials for every condition).

Table 1. Predicted log P Values of Spiropyran-Conjugated PEG−Lipida,b

reaction. In our previous works, bovine serum albumin (BSA) was often employed as the basement coating for manipulation of nonadherent cells,7,20−23 but such manipulation on the collagen surface was experimentally easier to handle and more reproducible than on the BSA surface probably because collagen coating is more flat and stronger against washing the surfaces. Nonadherent Ba/F3 cells from the murine pro-B cell line were used, and the cell suspension was added to the modified substrate and washed away after 10 min of incubation. The density of the immobilized cells was determined from microscopic images. The relative cell density compared with that obtained with normal PEG−lipid without spiropyran was used to determine cell-immobilizing ability. The detailed experimental conditions are stated in the Supporting Information (Figures S4−S6). On the substrate modified with the prototype compound [0Sp2]−NHS, some differences in relative cell density were observed between the open and closed form as expected (Figure 3a). However, the on/off ratio was not ideal because cell immobilization occurred in the “off” state. According to the working hypothesis, this was thought to be caused by insufficient molecular hydrophobicity even in the ring-closed structure, such that hydrophobic assemblies might not have been sufficiently formed. Three second-generation compounds, compounds 2[0Sp4]−NHS, [4Sp2]−NHS, and [0Sp6]−NHS, were prepared to increase the hydrophobicity of the closed form. Here, to estimate the hydrophobicity of the compounds, we focused on their log P values, which is one of the indicators of hydrophobicity. The theoretically calculated log P values26 in their closed and open forms are listed in Table 1. Compared to the log P value of the prototype compound [0Sp2]−NHS, those of the second-generation compounds were confirmed to be clearly larger. These new candidates exhibited different behaviors in the cell-immobilizing experiments (Figure 3b−d). Compound 2[0Sp2]−NHS with two spiropyrans did not immobilize cells in either the open or closed form (Figure 3b).

compound

form A

form B

form C

form D

[0Sp2]−NHS 2[0Sp2]−NHS [4Sp2]−NHS [0Sp4]−NHS [0Sp5]−NHS [0Sp6]−NHS

10.00 12.87 11.21 10.56 10.85 11.16

8.37 10.90 9.79 8.97 9.33 9.69

9.02 11.85 10.34 9.58 9.90 10.23

9.32 11.72 10.53 9.87 10.17 10.48

a The predicted chemical structures are shown in Scheme 1b. The optical analysis data (Figure 2, the maximum absorption wavelength: 536 nm) clearly showed that the form B was the main form in the open ones. bBold: values exceeding 10.5, the boundary value for cell immobilization.

This result is also explained by the hydrophobicity, which was shown to be the highest among the candidate compounds from the calculated log P values (Table 1). Contrary to [0Sp2]− NHS, the too high molecular hydrophobicity might lead to hydrophobic assembly formation even in the ring-open structure. Compound [4Sp2]−NHS, with moderate hydrophobicity, showed differing cell-immobilizing ability in response to light irradiation and molecular density, which was partially consistent with our working hypothesis (Figure 3c). However, this compound did not also achieve a clear on/ off ratio as desired. On the other hand, compound [0Sp6]−NHS, with similarly moderate hydrophobicity, yielded a very good on/off ratio (Figure 3d). In the “off” state, cell immobilization was suppressed, while cells were immobilized in the “on” state. Accordingly, to achieve a more stringent photoresponse, compounds [0Sp5]−NHS and [0Sp4]−NHS, were prepared by decreasing the number of carbons in the linker moiety of compound [0Sp6]−NHS (Scheme 1a). Compound [0Sp5]− NHS similarly showed a good on/off ratio (Figure 3e); C

DOI: 10.1021/acsabm.8b00656 ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX

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ACS Applied Bio Materials however, the “off” state slightly worsened, probably because of decreased molecular hydrophobicity. Compound [0Sp4]− NHS, with further reduced hydrophobicity, drastically worsened, losing the desired “off” state (Figure 3f). From these results so far, we concluded that [0Sp6]−NHS is optimal for photoswitchable cell immobilization, as it exhibited good on/off responsiveness. In addition, it was found that most of the spiropyran-conjugated PEG−lipids with the log P values above 10.5 suppressed cell immobilization in their closed and open forms (Table 1 and Figure 3). Accordingly, the calculated log P value of 10.5 might be used as the boundary value for roughly estimating cell immobilization and release. This result suggests that cell-immobilizing ability can be controlled mainly by the strength of the hydrophobicity, although it may be also affected by other factors such as the isomerization efficiency which depended on the substituents of the dye (Figure S2) and steric effect of the PEG−lipid backbone.19



REVERSIBLE CELL IMMOBILIZATION Using this photoswitchable surface of [0Sp6]−NHS, lightinduced release of immobilized cells was confirmed by counting the number of cells remaining on the surface after visible-light irradiation and washing. In this experiment, BaF3 cells were once immobilized on the UV-irradiated surface and then exposed to visible light, followed with trypsin treatment before washing. In a preliminary experiment, trypsin treatment enhanced cell release from this surface probably due to disruption of the nonspecific interaction between spiropyranconjugated PEG−lipid and membrane proteins. After treated by this procedure, the relative cell density drastically decreased on the visible-light-irradiated surface (Figure 4a), while a minimal decrease was observed on the UV light-reirradiated surface (Figure S7). Thus, the immobilized cells were confirmed to be released from the surface in a wavelengthselective manner. In particular, under the optimal PEG−lipid modification conditions where the concentration of PEG−lipid was 100 μM, almost all immobilized cells were released by visible-light irradiation. This result showed that the present surface achieved reversible cell immobilization by successive irradiation of UV and visible light. Additionally, the reusability of the surface was then analyzed by repeating the reversible cell immobilization procedure. Cell immobilization and release were observed even after four on/off cycles (Figure 4b and Figure S8). On the present surface, the gradual decrease in cellimmobilizing ability was also observed over cycles in the “on” state, as observed in optical analysis in the solution (Figure S3), probably due to the well-known photofatigue of spiropyran.27,28 Although the gradual decrease should be improved by further structural optimization, the present photoswitchable surface was confirmed to be reusable several times through visible-light-induced cell release.

Figure 4. Sequential, reversible, and spatio-selective cell manipulation on the compound [0Sp6]−NHS-modified surface. (a) Relative cell densities on the substrate surface modified with [0Sp6]−NHS in the “on” state (blue bars) and in the “off” state (red bars). Cells were first immobilized on the surface in the ring-opened state (blue bars), followed by trypsin treatment and washing, which lead to cell detachment (red bars). Values are the means ± standard error (n = 15−40, four independent trials for every condition). (b) Repetitive cell immobilization ([0Sp6]−NHS: 100 μM). Blue dots indicate the “on” state, and red dots indicate the “off” state. Values are the means ± standard error (n = 72, three independent trials for every condition). (c) Confocal microscopic images of partial cell immobilization and (d) release of eGFP-expressing Ba/F3 cells. The merged fluorescence (green) and phase contrast images are shown. Scale bars = 100 μm.

visible light before culture (Figure S9i−l). Thus, the trypsinized adherent cells were also confirmed to be immobilized and released on this surface before they started to adhere to the basement coating (within 15 min at room temperature). Additionally, HeLa cells were observed to subsequently spread on the surface after 2 h of culture on the surface (Figure S9m). These cells therefore showed normal behavior after immobilization on the surface. In addition, the trypan blue-based cell viability assay confirmed that photoresponsive cell manipulation on the surface did not result in cytotoxicity (Figure S10). In the present system, UV light is irradiated on the surface before cell immobilization, and for cell release, visible light can be employed. Accordingly, cells are exposed to only safer visible light at 520 nm, while most of the methods for light-induced cell release required cell exposure to UV or blue light below 410 nm.7,29 Finally, spatially selective photoswitching was examined. The whole material-modified surface was irradiated with visible light to induce the “off” state. Then, only half the surface area was changed to the “on” state by irradiating it with UV light. Figure 4c clearly shows that the cells were immobilized only within the UV-light-irradiated area. Similarly, after UV-lightinduced cell immobilization on the whole surface, the immobilized cells could be selectively released from the visible-light-irradiated half area (Figure 4d). Thus, both lightinduced immobilization and release of cells could be carried out with high spatial selectivity (Figure 4c, 4d, and Figure S11).



APPLICATIONS TO VARIOUS CELLS AND SPATIAL SWITCHING To demonstrate the versatility of the present photoswitchable surface, we also applied it to the immobilization of other cell types. The other nonadhesive Jurkat (human T lymphocyte) cell was tested. HeLa (human epithelial carcinoma) cells, an adherent cell type, were also tested after harvesting by trypsinization. All the tested cell lines were reversibly immobilized on this surface (Figure S9). In the case of HeLa cells, the immobilized cells were released by irradiation with D

DOI: 10.1021/acsabm.8b00656 ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX

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ACS Applied Bio Materials



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CONCLUSION Spiropyran-conjugated PEG−lipids were designed and synthesized, and the compound [0Sp6]−NHS, which showed the best performance, enabled the reversible immobilization of three types of model cells without the need for their inherent cellular adhesion. This material could spatially control both cell attachment and detachment in situ without cytotoxicity. Furthermore, this light-guided on/off switching could be repeated for several cycles. This molecule will enable cell sorting on substrates and will help to elucidate intercellular communications and to construct artificial organs. In addition, with its ability for rapid cell immobilization, it has the potential to strongly advance the development of current single cell analysis techniques, cell-based sensor chips,30 and organ-on-achip in combination with MEMS technologies. Furthermore, in principle, the present photoswitchable surface can be applied to liposomes, exosomes, bacteria, and others that are coated with a lipid bilayer membrane. Therefore, the photoswitchable molecule is also a promising tool for a wide variety of research fields such as synthetic biology, molecular diagnosis, and bacterial biofuel production.



ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsabm.8b00656.



Materials and methods, synthetic details, optical analysis details, log P prediction of other possible candidate compounds, cell experiment details, additional supplementary figures and tables about absorbance spectra of the candidate compounds, schematic illustration of cell immobilization mechanisms, law data of cell-immobilizing experiments, microscopic images of cell-immobilizing experiments, cell viability assay, 1H and 13C NMR spectrometry, and MALDI TOF mass spectrometry (PDF)

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Satoshi Yamaguchi: 0000-0003-4822-7469 Akimitsu Okamoto: 0000-0002-7418-6237 Author Contributions

S.I., S.Y., and A.O. contributed to conception and design, and S.I. and T.K. contributed to data acquisition. S.I. and S.Y. drafted the manuscript, and A.O. and T.K. provided critical revisions. Accordingly, all four authors can claim authorship. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was supported by PRESTO, Japan Science and Technology Agency (JST), grant number 16815021. REFERENCES

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DOI: 10.1021/acsabm.8b00656 ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX