Optoregulated biointerfaces to trigger cellular responses - Langmuir

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Invited Feature Article

Optoregulated biointerfaces to trigger cellular responses Yijun Zheng, Aleeza Farrukh, and Aranzazu del Campo Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.8b02634 • Publication Date (Web): 05 Nov 2018 Downloaded from http://pubs.acs.org on November 5, 2018

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Optoregulated biointerfaces to trigger cellular responses Yijun Zheng(1), Aleeza Farrukh(1) and Aránzazu del Campo(1,2)* (1)

INM-Leibniz Institute for New Materials, Campus D2 2, 66123 Saarbrücken. Germany

(2)

Chemistry Department, Saarland University, 66123 Saarbrücken, Germany

KEYWORDS: photoresponsive biomaterials, phototriggers, cell-materials interactions, light-guided cells

ACS Paragon Plus Environment

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ABSTRACT: Optoregulated biointerfaces offer the possibility to manipulate the interactions between cell membrane receptors and the extracellular space. This Feature Article summarizes recent efforts of our group and others during the last decade in the development of light-responsive biointerfaces to stimulate cells and elicit cellular responses using photocleavable protecting groups (PPG) as working tool. This article begins by providing a brief introduction to available PPGs, with a special focus on the widely used o-nitrobenzyl family, followed by an overview of molecular design principles for the control of bioactivity in the context of cell-materials interactions, and the characterization methods to follow the photoreaction at surfaces. We present various light guided cellular processes using PPGs, including cell adhesion, release, migration, proliferation and differentiation, in vitro and in vivo. Finally, this feature article closes with our perspective view on the current status and future challenges of this topic.

Introduction The extracellular matrix, the material surrounding cells in natural tissues, is a complex mixture of structural and signaling components that dynamically change in composition and assembly during its life time. The understanding and the emulation of this dynamics with artificial systems used for in vitro cell culture, for tissue engineering or in regenerative therapies represent a considerable challenge for biomaterials development.1 It is now recognized that static presentation of chemical, topographical or mechanical cues to cells, as classical biomaterials do in an attempt to replace the natural matrix, is not sufficient to recreate the natural environment and support cellular function with synthetic systems.2 Biomaterials for advanced biomedical applications need to incorporate dynamic elements in their designs.3 Responsive materials able to change properties under an external stimulus, such as heat, voltage, or light, are useful platforms for developing dynamic environments.4,5 Among them, optoregulated biomaterials are particularly interesting and will be the focus of this article. Light presents distinct advantages against other stimuli.6 It can be readily available and focused, allowing precise definition of the changes and responses over broad timeand lengthscales.7 It can be dosed by tuning exposure time and intensity, allowing modulation of the change and regulation of the strength of the response.8 By combining chromophores with selective responses to different wavelengths, multiplexed biomaterials and biointerfaces containing different optoregulable signals can be realized and applied to recreate sequential responses in cellular systems.9 There is a variety of molecular mechanisms by which the physicochemical properties of a biomaterial or a biointerface can be controlled by light. They include (i) molecular isomerisation (e.g., azo-derivatives),10-12 (ii) ionisation (e.g., spyrobenzopyran derivatives),13-14 (iii) dimerization (e.g., cinnamic acid derivatives),15 (iv) grafting (photoaffinity),16 (v) photoligation,17-19 or (iv) light-activation of chemical functionalities protected by photocleavable groups [also named photo protecting groups (PPGs) or “cages”] (Scheme 1). The latter is a particularly attractive approach, and will be the focus of this article, since it allows the generation of biointerfaces and materials with latent functional states that can be unlocked and regulated solely by light exposure at different wavelengths and doses.20

The first successful demonstration of photoactivatable surfaces for use in biology was in the field of photochemical patterning. PPGs were used for sequential and patterned coupling of nucleosides to a surface to build up high density DNA arrays for sequencing.21 Photochemically reactive patterns were also used to immobilize other molecules (polymers, proteins22) or small objects (colloids) in spatially structured geometries onto planar surfaces.23-25 Later, pioneering work in our group demonstrated how this chemical approach could be extended to directly control cell-surface interactions by introducing PPGs into cell adhesive ligands immobilized on the surface. In this way, dynamic biointerfaces, with the possibility of interacting with cells on demand by in situ regulation of relevant receptors for membrane proteins was realized. In subsequent steps this approach was extended to dynamic 3D cell cultures and in vivo26 applications. This progress has been possible by the development of novel chromophores27, allowing effective activation of the photochemical process at more benign wavelengths and doses, compatible with the presence of living cells.28 The development of PPGs with higher two-photon absorption cross-sections opened the possibility to activate biological processes within 3D materials at microscale resolution.29 PPGs have also been applied to modulate connectivity within hydrogels embedding cells.30-33 Dynamic gels with photoactivated depolymerization have become relevant models for the study of cellular responses to mechanical changes in their microenvironment.34 This Feature Article summarizes recent efforts of our group and others during the last decade in light-guided strategies to tune interactions between cells and surfaces and materials using PPG-based strategies. We will describe the established and recently synthesized new chromophores, the design principles and synthesis strategies for developing photocleavable biomolecular units, and most relevant examples for light-guided cell behavior using PPGs, in vitro and in vivo. Our perspective view on this topic will also be presented. Photo-cleavable protecting groups responsive to singleand two-photon activation Photocleavable protecting groups (PPGs) are used to temporally block a reactive functional group and can be removed upon light exposure. The photochemical reaction


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leads to irreversible removal of the chromophore and reactivates the chemical functionality. When the protected functional group is part of a biomolecule with specific biological function, this strategy can be used to photoregulate bioactivity of the molecule and eventually cellular responses. When the bioactive molecule is coupled to a material, light can be used to trigger and regulate cellmaterial interactions (Scheme 1). If the PPG is introduced within the molecular structure of a material, typically an

Activation

Multiplexing

1

organic polymer, light can lead to release molecules, to cleave polymer chains (i.e. depolymerization), or to activate reactive sites for further functionalization or property change (i.e. polarity or solubility).35-36 Using patterned illumination the photoactivated process can occur solely at the illuminated site. Combining PPGs with orthogonal response to different wavelengths,

Patterning

Release

Depolymerization

2

Scheme 1: Different possible uses of PPGs to regulate the chemical and physical properties of surfaces and materials. sequences of activated processes can be triggered in the same material (Scheme 1). Different families of PPGs have been used for photoregulating chemical activity.37 The o-nitrobenzyl (ONB) family is by far the most widely used PPG in the literature37, presumably due to the fact that most of these compounds have a comfortable photosensitivity for experimentation. They are not too photolabile and can be handled in normal laboratory conditions, with light protection measurements but not necessarily in the dark. On the other hand, they are photolabile enough to be photolyzed at doses compatible with living organisms. The photolysis reaction of o-nitrobenzyl esters and onitrophenylethyl esters of this family are represented in Figure 1a and 1b. O-nitrophenylethyl esters undergo photolysis and release nitrostyrene side product by betaelimination mechanism (Figure 1a). O-nitrobentyl esters undergo photolysis and release nitrobenzaldehyde (Figure 1b). The introduction of substituents at the aromatic ring or at the benzylic carbon leads to changes in the photochemical properties of the derived chromophores. ONB derivatives such as nitroveratryloxycarbonyl (NVOC), (4,5-dimethoxy-2-nitrophenyl)butan-2-oxycarbonyl (DMNPB) or p–dialkylaminonitrobiphenyl (ANBP) are relevant representatives of this family that have been used to derivatize biointerfaces and -materials (Figure 1b).37 NVOC is a commercially available PPG that can be readily incorporated to amines in one step reaction.37 The DMNPB caging group is an o-nitrophenethyl derivative that shows good photolysis efficiency and better hydrolytic stability than NVOC in experiments with cells.39

is the fact that they both require UV light to be activated. Attempts to develop o-nitrobenzyl PPGs activatable at visible wavelengths have been recently reported.38 In 1990, the development of two-photon ultrafast lasers enabled the exploitation of multiphoton processes for photoactivation and imaging, allowing the use of longer wavelengths for activation. In the context of biological systems, two-photon processes have two main advantages: lower photodamage and deeper penetration, allowing 3D resolution of the light-triggered process.40 This advance affected research in PPGs and their applications, since available o-nitrobenzyl PPGS had poor sensitivity to twophoton excitation. The two-photon sensitivity of a chromophore is described by the two-photon absorption cross section (σ2) and the quantum yield (φ).41 The twophoton cross section defines the probability of simultaneous absorption of two photons to reach molecular transition energy and it is measured in Goeppert-Mayer units, 1 GM = 10−50 cm4 s photon−1.42 The members of o-nitrobenzyl family show low two-photon absorption cross sections. However, by extending the conjugation, the effective o-nitrobiphenyl PPGs with improved two-photon photolysis properties were developed. The 3-(2-propyl)-4’-methoxy-4-nitrobiphenyl (PMNB) and p– dialkylaminonitrobiphenyl (ANBP) PPGs show twophoton absorption cross sections of 3.2 GM at 740 nm and 11 GM at 800 nm respectively when used to protect carboxylate groups. 44

The photolytic byproducts, o-nitrosobenzaldehyde or onitro phenyl alkene are mildly toxic. This fact imposes some concentration constraints in the use of PPGs in biological systems. However, when used for derivatization of biomaterials, the concentration of released photolysis by-products is well below the toxicity limit.37 Another limitation of NVOC and DMNPB for biological application


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attaching DMNPB to a carboxylic group.46 This peptide allowed α5β1-mediated adhesion of human umbilical vein endothelial cells (HUVECs) to surfaces after light exposure. Photoactivatable variants of the laminin peptidomimetic CASIKVAVSADR have also been designed and applied to trigger neuronal growth on biomaterials in vitro.47 In this case the PPG is introduced at the amine rest of the Lys, which is relevant for binding48 and allows facile coupling of the PPG. Three different variants of photoactivatable lysine were reported based on NVOC, DMNPB and 2,2’((3’-(1-hydroxypropan-2-yl)-4’-nitro-[1,1’-biphenyl]-4yl)aza-nediyl)bis(ethan-1-ol) (HANBP) chromophores (Fig. 2d). The photoactivatable variant with the HANBP group showed best performance in terms of photolysis efficiency and water solubility. Figure 1. a) Photolysis mechanism of o-nitrophenylethyl esters derivatives, b) Photolysis mechanism of onitrobentyl esters derivatives, c) Structure of reported onitroaryl groups.37 Design and synthesis of photoactivatable ligands to regulate specific cell-materials interaction The main challenge for an efficient design of a photoactivatable bioactive molecule is the adequate selection of the position at which the PPG will be attached. Previous knowledge of the structural characteristics of the binding site is necessary, in order to place the chromophore at a relevant position for target recognition. The interaction with the target is typically hindered either by distorting individual interactions at the binding site (H bonds, charge interactions), by imposing steric constraint close to the binding site, or by inducing conformational changes as a consequence of the presence of the PPG.

The 4-hydroxy-3-nitrophenyl acetyl (NP) hapten is specifically recognized by B1-8 antibody of B1-8-BCR– expressing B cells and the hydroxyl group in the phenol ring plays a relevant role in this interaction.49 To mask the antigenicity of NP, the UV-sensitive moiety 4,5-dimethoxy2-nitrobenzyl (NVOC) was conjugated to the -OH group to generate DMNB-NP (Figure 2e).50 This photoactivatable antigen was used to study the dynamics of B-cell activation process. In a different example, the peptide ANERADLIAYLKQATK (Figure 2f), an antigen of T cell receptor, was modified at the Lys position51 with a ONB group. The photoactivatable version was used to spatiotemporally regulate binding to T cell receptor and consequently light-regulated T-cell stimulation was achieved.52 A NVOC variant of this peptide was also synthesized in a later study.53

Take for example the pentapeptide cyclo(RGDfK), a relevant peptidomimetic for integrin binding used to derivatize interfaces to support cell attachment. It is known that the carboxylic group of the aspartate unit is relevant for complexing one of two key divalent cations required for integrin binding. By protecting the carboxylic group with the DMNPB PPG, the bioactivity of the peptide could be efficiently inhibited. Biomaterials modified with this peptide were unable to support cell adhesion. Upon light activation, the DMNPB group was released by photolysis, the peptide become active, supported integrin binding and mediated cell attachment to surfaces (Figure 2a).39 With a different design, the cell adhesive peptide YAVTGRGDSPASS was also synthesized.45 In this case a 2nitrobenzyl group was introduced at the amide bond between the Gly and Arg residues (Figure 2b). The presence of the PPG sterically hindered recognition of the RGD sequence by the integrins. After UV illumination, the PPG was cleaved and RGD became recognized by the cells. Similar design principles have been applied to photocontrol the bioactivity of other peptidomimetics. In a recent work, a photoactivatable variant of Kessler’s α5β1specific integrin ligand was synthesized (Figure 2c) by


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Langmuir inhibiting integrin recognition, cyclo[RGD(DMNPB)fK],39 (b) cell adhesive peptide YAVTGRGDSPASS with o-NB group intercalated between Gly and Arg residues that can be removed by UV irradiation,45 (c) cell adhesive peptide for α5β1 integrin specific binding,46 (d) different photoactivatable variants of the laminin peptidomimetic AASIKVAVSADR with NVOC, DMNPB and HANBP photoremovable protecting groups,47 (e) photoactivable antigen modified with 4-hydroxy-3-nitrophenyl acetyl (caged-NP),50 (f) T cell antigenic peptide ANERADLIAYLKQATK modified with ONB group at the 3amino group of Lys position,51 (g) photoactivatable biotin coupled to chitosan polymer to allow photoactivated biotin-mediated immobilization of Streptavidin-labelled partners,54 (h) working mechanism of a photocleavable oligohistidine for a light-mediated release from NTA affinity group.55 A general strategy for controlling bioactivity of surfaces with light is by using photoactivatable biotin54 or Histags.55 Upon light exposure, binding of cell ligands labelled with Streptavidin or nitrilotriacetic acid (NTA) is possible, eventually in a sequential fashion in order to immobilized more than one ligand type. The photoactivatable biotin requires two NVOC groups located at the N-1΄ and N-3΄ positions to render it non active (Figure 2g).54 The monosubstituted derivative keeps appreciable binding affinity for Streptavidin. The Histidine-tag (His-tag) consists of a polyhistidine sequence of at least 4 His residues in length. This tag allows protein immobilization to NTA-functionalized surfaces in the presence of transition metal ions. By inserting a photocleavable aminoacid within a His6 sequence, a photofragmentable tag was obtained (Figure 2h).55 This sequence showed high affinity for Ni(II)-loaded NTA surface and was used to block binding of His-tagged proteins. Upon light exposure in a later step, the NTA bound photocleavable His-tag is effectively fragmented into two His3 fragments with low affinity for NTA. These sequences rapidly dissociate from the NTA-Ni-His complex and allow coordination of other His-tagged molecules from solution. Using sequential illumination and incubation steps micropatterns with different His-tagged molecules can be prepared. Characterization of photolysis immobilized on biomaterials

Figure 2. The chemical structure and photolysis reactions of different photoactivatable molecules (a) cell adhesive peptide with DMNPB group attached to aspartic acid for

properties

of

PPGs

The chemical yield of photochemical reactions in solution is typically characterized by analyzing the photolytic products in the reaction vessel after separation. This methodology is not possible when the PPGs are coupled to molecules immobilized on surfaces or biomaterials. When the biomaterial is transparent characterization of the reaction parameters is possible by UV spectroscopy analysis of the film or of a thin film supported on a quartz substrate.56 The most favorable case is when the photolysis reaction cleaves the PPG from the material and a washing step allows removal of the photolytic product and, consequently, it leads to a decay of the UV absorbance of the material associated to the PPG. Studies performed at


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increasing exposure dose allow quantification of reaction yields and photolysis kinetics. Figure 3 shows the decay in the UV-Vis spectra observed on poly(acrylamide-co-acrylic acid) P(AAm-AA) hydrogel films modified the with laminin peptidomimetic CASIKVAVSADR derivatized with three different PPGs: NVOC, DMNPB and HANBP (see chemical structure in Figure 2). This analysis allowed quantification and comparison of the conversion of the photolysis reaction for the different PPGs at controlled conditions. Activation ratios of 17%, 14%, and 48% were obtained for NVOC, DMNPB and HANBP respectively after 15 minutes of exposure at λmax.58If the thickness of the swollen gel is known, the concentration of PPG, and therefore of bioactive molecule in the gel, can be calculated.56

Figure 3. Photodeprotection of CASIKVAVSADR (NVOC)/(DMNPB)/(HANBP) variants. UV-Vis of hydrogel films functionalized with the photoactivatable peptides, after irradiation and washing. Reprinted with permission from ref 47. Copyright 2018 Wiley-VCH. Photo guided cell contact to 2D surfaces Photo triggered cell adhesion to surfaces The first example of the application of PPGs to control the interactions between cells and materials was reported by our group using the photoactivatable cell adhesive peptide cyclo[RGD(DMNPB)fK].39 This peptide targets the integrin


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adhesive receptors of cells and is frequently used to mediate attachment of cells to synthetic biomaterials as a substitute for adhesive extracellular matrix proteins. In order to demonstrate the light-regulated bioactivity of the peptide and the molecular specificity of this approach to regulate cell-materials insteractions, it is important to use biomaterials with low nonspecific interactions with cells. For this purpose carboxy-terminated self-assembled monolayers (SAMs) of PEGylated thiols on gold substrates, Cyclo[RGD(DMNPB)fK] were functionalized with the peptide by reaction of the amine side group of the Lys rest with previously activated carboxylic acid of the SAM.58 Initial experiments performed on preirradiated substrates incubated with HUVECs showed that cells attached and spread preferentially on the irradiated areas of the substrate.58 where the cyclo[RGDfK] was exposed, and were not able to interact with the non-exposed areas, covered with cyclo[RGD(DMNPB)fK].58 Following experiments with improved surface layers showed micropatterns of HUVECs forming functional endothelial monolayers that could be maintained over ten days (Figure 4). By controlling the light dose, the density of RGD on the surface was regulated.58 HUVECs seeded on stripe patterns activated at increasing irradiation doses (25%, 50% or 100% photocleavage) selectively attach to the activated areas at different cell densities depending on the extent of the photoactivation. Fewer HUVECs were attached and spread on the 25% uncaged stripes (Figure 4A), and a larger number of cells were located on the 50% and 100% photocleaved surface (Figure 4B). On gold surface, it was found that the –NO2 group gets reduced to –NH2 in presence of excess electron which could be a limitation on gold surface patterning using o-nitrobenzyl PPG.59 PEGlated silicon substrates or P(AAm-AA) hydrogel films have been used as alternative low fouling surfaces. A usual way to compare the activity of the photoactivatable cell adhesive ligand vs the irradiated one is by checking cell density on patterned substrates after incubation with the cells. The reported selectivity of cells for the irradiated areas observed on cyclo[RGD(DMNPB)fK] derivatized substrates is significantly better than results reported for other photoactivation strategies, like the ones based on the azobenzene unit.10, 60-62 This is on one hand due to the higher affinity differences achieved between the latent and active form of cyclo[RGD(DMNPB)fK] vs. the azobenzene derivatives. Additional parameters that can contribute to maximize the contrast between activated and non activated regions is optimization of the surface density of the ligand for the cell type of interest and use surface which can retain low protein adsorption for long incubation times.

Figure 4. Pegylated SAMs modified with cyclo[RGD(DMNPB)fK] and irradiated were incubated with HUVECs. Cells attached to the exposed regions and formed clear patterns, as observed in the depicted microscopy pictures. In A) substrates were exposed through ha mask and the the images correspond to 24 h incubation. In B) the irradiation step was performed with a scanning laser. Stripes (150 µm width, marked with discontinuous lines) were scanned at different exposure doses to achieve 25% (I), 100% (II) and 50% (III) activation of the peptide. The increasing peptide concentration resulted in higher cell densities at the surface C) shows HUVECs on regions (II) and (III) after 24 h incubation. Reproduced with permission from ref 58. Copyright 2011 Wiley-VCH. A more flexible strategy to guide cell interactions with surfaces by means of specific ligands uses photoactivatable biotin to form streptavidin patterns on surfaces, onto which cell-specific biotinylated antibodies can be immobilized and use to guide attachment of cells (see chemical structure in Figure 2g).54 However, this method is not much different to other prepatterning strategies (i.e. microcontact printing) and does not allow in situ activation of the biological function. Chitosan thin films covalently modified with photoactivatable biotin were preirradiated and incubated with Streptavidin followed by biotinylated anti-CD31 which addresses an specific membrane receptor at endothelial cells. HUVECs seeded on this patterns attached selectively to the CD31functionalized areas (Figure 5). Cell patterns were clearly visible after 4 h of incubation, and cells remained confined to the areas functionalized with the specific antibody.54

Figure 5. Micropatterns of HUVECs on chitosan films formed by the selective recognition of immobilized CD31 antibodies. Scale bars: 100 μm. Reproduced with permission from ref 54. Copyright 2014 American Chemical Society. The real advantage of light-activation for biological processes is the possibility to do experiments in situ, i.e. in the presence of cells and at defined time points, and to be able to follow the cellular response in real time, under the microscope. In situ activation of cellular processes using cyclo[RGD(DMNPB)fK] is possible, without detectable signs of photodamage in the cells.57 Moreover, the precise control over the onset of adhesive ligand activation allows also indirect quantification of cell adhesion and spreading of a cell population using a surface detection technique like the quartz-crystal microbalance (QCM).63 QCM crystals modified with cyclo[RGD(DMNPB)fK] allowed monitoring


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of integrin binding events during early cell attachment of HUVECs (Figure 6a). RGD peptide activation was performed by illuminating the QCM crystal through the window of the QCM chamber. The immediate binding of the integrins can be detected by a drop in frequency which correlates with the expression levels of integrins at the cell membrane. Within 2 minutes, a more pronounced frequency drop and increase in the dissipation signals indicate membrane spreading (Fig. 6b). This is the first example of monitoring integrin binding separately from membrane spreading, only possible by the possibility to photoactivate the binding ligand in situ and at desired time point.

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RGDfK binds to many different integrins and does not allow dynamic integrin-selective studies. In a recent paper, photoactivatable peptidomimetic with high affinity and selectivity towards α5β1 integrin64 have been developed.46 A DMNBP introduced into the chemical structure allowed photocontrol of integrin recognition (see chemical structure in Figure 2c). Masked-irradiated substrates modified with caged ligand allowed sites selective attachment of HUVECs to the photoactivated areas (Figure 7). Cell adhesion and spreading levels and kinetics were similar to those of native α5β1 ligand.

Figure 7. Fluorescence images demonstrating siteselective attachment of HUVECs to PEGYlated substrates functionalized with photoactivatable α5β1 integrin specific ligand after masked irradiation. The mask had 600 μm chrome stripe patterns separated by 300 μm gaps. Cells selectively spread on the exposed areas. Images were taken 24 hours after seeding. Reprinted with permission from ref 46. Copyright 2018 Wiley-VCH.

Photo triggered cell release from surfaces

Figure 6. Demonstration of the application of c[RGD(DMNPB)fK] to quantify integrin-mediated cellular binding to surfaces using QCM-D technique (a) Schematic shows the sequential steps for synchronized cell adhesion and spreading on c[RGD(DMNPB)fK] modified QCM crystals. (b) Frequency and dissipation curves measured by QCM-D before and after light-activation of c[RGD(DMNPB)fK] on the crystal corresponding to steps marked in a). i- cells enter the QCM chamber; ii- cells sediment on the crystal; iii- UV exposure through the window of the QCM-D chamber and activation of the c[RGDfK] on the surface (magnified inset); iv-binding of membrane integrins and cell spreading on the substrate. Reproduced with permission from ref 63. Copyright 2015 Nature Publishing Group.

The strategy followed to trigger cell adhesion relies on a PPGs bound to the active site of the ligand, and activation of binding process occurs upon exposure.39 The activation of the reverse process, i.e. detachment, has also been achieved using PPGs, though in this case the PPG is intercalated in the spacer that anchors the ligand to the substrate.65 Figure 8 shows an example of such a design. An 4,5-dialkoxy 1-(ortho-nitrophenyl)-ethyl photolabile group was intercalated between the surface and the RGD which allows cell adhesion. The RGD ligand at the top of the surface mediates integrin binding (II). Light-induced photocleavage (III) of the linker effectively removes the RGD from the surface (IV), followed by detachment of the HUVECs. After illumination, over 85% of the original, adherent cells were removed from the surface by simple washing with saline, and the remaining cells displayed a round morphology (Figure 8). Similar strategy was also applied by Dong-Sik Shin et al to capture and release T-cells66-67. In their approach, PEG microwell arrays containing photolabile avidin terminated attachment regions were functionalized with anti-CD4biotin. These microwells were then incubated with CD4 antigen expressing T-cells. Cells started detaching immediately upon UV exposure and 90% of T-cells were detached after 1 min exposure followed by gentle rinsing.


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Figure 8. Controlled HUVECs release by photodegradation of light-sensitive linker.65 Reproduced after permissions. Reproduced with permission from ref 65. Copyright 2011 Wiley-VCH.

biology labs.74 Starting from cell patterns on preactivated substrates, a second exposure step will activate the areas between the patterned monolayers into which cells will move. The migration mode and velocity of the cells to close the activated space can be studied in these models. With the same assay one can check for the maximum distance that a cell can span in the absence of adequate adhesive receptors for binding.74 In such a “Jump the gap” assay, migratory lines scanned with the laser get interrupted by gaps of selected length. Cells migrating through the line will need to make the decision to go further and cross the non adhesive space, or turn back. Single cells (HeLa) migrated were able to surpass gaps shorter than 30 μm by stretching their front part to establish an adhesive front and then pulling the cell body over the non-adhesive gap (Figure 9d). Longer gaps were not crossed and cells either remained at the end of the line or moved back along the approach path (Figure 9e).

The photolysis reaction in PPGs is irreversible. This is a clear limitation of the PPGs approach to control biological processes. Reversible strategies based on reversible chromophores like azobenzene62, 68 or spiropyrans13 have been proposed to reversibly control cell attachment. These are interesting alternatives and the reader is referred to these literature12-13, 69-73 for more details. Photo guided cell migration on surfaces The possibility to in situ regulate the availability of adhesive ligands by scanning the surface of a pre-patterned cell culture with a laser beam allowed us to trigger cell migration processes on surfaces modified with photoactivatable adhesive ligands like cyclo[RGD(DMNPB)fK].74 In contrast to other light-based strategies to trigger migration,75 the use of photoactivatable peptidomimetics allows customized definition of the geometry of the migration pathways. In a typical experiment, a masked preexposure step allows confinement of cells within preactivated of cyclo[RGD(DMNPB)fK] modified substrate. Using a scanning laser, migration pathways of different geometries can be created in the previously non exposed areas. The dimensions of the patterned areas were observed to dramatically affect cell migration mode. Migration of single cells was observed on line patterns with width between 5 and 15µm (Figure 9a), whereas collective migration of connected cells out of the monolayer were observed on adhesive line patterns wider than 45 μm (Figure 9b). Migration velocity and persistence in single migration events was also dependent on adhesion line width.76 These line patterns of adhesive ligands are particularly interesting for migratory studies, as they can be regarded as simplified and experimental-friendly 1D models of natural 3D fibrillar spaces.77 Another interesting application of cyclo[RGD(DMNPB)fK] modified interfaces is in the field of wound assays, as clean alternatives of the scratch assay performed in most cell

Figure 9. Migration experiments with unconventional designs and geometries74. a,b) Migration of HUVECs on a) 15 and b) 45 μm sidelines of scanned cyclo[RGD(DMNPB)fK]. c) Time-lapse fluorescence confocal microscopy images of a migrating cell from a stripe pattern. d) “Jump the gap” assay performed on a caged RGD-modified NEXTERION slide with HeLa cells. Reprinted with permission from ref 74. Copyright 2013 Wiley-VCH.

Phototriggered cell differentiation in 2D cultures The possibility to in situ regulate the time point and concentration of ligand presentation in photoactivatable surfaces makes them ideal substrates to study how cells respond to dynamic changes in their natural environment, as it occurs during development, ageing or pathological processes. The first example of such use was the study of the proliferation and differentiation of C2C12 myoblasts with variable concentration of RGD ligand during cell culture.78 Cells were seeded on substrates modified with a mixture of active cyclo[RGDfK] and cyclo[RGD(DMNPB)fK]. This combination supports initial


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cell attachment mediated by a low basal concentration of active ligand, and dynamic increase of ligand concentration during cell culture by light exposure and activation of the latent cyclo[RGD(DMNPB)fK]. Myogenic differentiation of C2C12 cells was supported at high initial densities of surface-tethered RGD, or when the latent ligand was activated at early stages of cell culture, i.e. within the first 6 hours of incubation (Figure 10). Myogenic differentiation was significantly lower when cells were exposed to RGD at later time points. These results reflect how cell behavior can be influenced not only by the intensity of a certain signal (i.e. the concentration of adhesive ligand), but also by the time scale at which this signal occurs. Light-regulated biomaterials with the possibility to vary signal presentation in time are useful platforms to identify cellular time scales in vitro.

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observed (Figure 11a). Using masks or scanning lasers for site-selective activation, the oriented growth of neural stem cells (NSCs) dissociated from neurospheres was achieved (Figure 11b). The neuronal patterns on CASIK(HANBP)VAVSADR modified gels were maintained for at least 4 days.

Figure 11. (a) Behaviour of neural progenitor cells on nonirradiated and pre-irradiated hydrogels modified with photoactivatable IK-12 variants. (b)Dissociated neural stem cells (NSCs) from neurosphere (P-3) seeded on hydrogels functionalized with IK-12(HANBP) peptide, 24 h after seeding. Reproduced with permission from ref 47. Copyright 2018 Wiley-VCH.

Photopatterning of cells within 3D hydrogels

Figure 10. Presentation of uncaged cell-adhesive RGD peptide at different time points modulates (a) proliferation and (b) differentiation after 3 days in culture. Reproduced with permission from ref 78. Copyright 2013 Elsevier. In a recent study, the photo-triggerable laminin mimetic peptide CASIK(HANBP)VAVSADR was applied to direct neuronal growth and differentiation on hydrogels.47 After photoactivation, a significant increase in the morphological features of neuronal differentiation (number of processes and secondary branches, the length of the axon, and the number of dendritic filopodia) was

Over the past few decades, biomaterials for culturing cells in 3D have been developed, in order to better reconstruct the natural cellular microenvironment and circumvent the limitations posed by traditional 2D cell culture.70 To this end different hydrogel systems have been developed, with the ability to encapsulate cells in viable and functional way.31, 79-80 PPG-based strategies to regulate surface properties and biofunctionalization have also been transferred to 3D systems to obtain dynamic hydrogels with light-tunable properties able to transmit variable signals to embedded cells. Soichet’s Lab used a photoactivatable cysteine with a ONB group attached to the thiol group to modify agarose (Figure 12a).81 The photoactivatable agarose gels were able to react with maleimide-functionalized molecules, i.e. the GRGDS cell adhesive peptide, upon UV irradiation. The peptides were solely immobilized at the exposed regions where the ONB group was cleaved and the thiol group was available for reaction (Figure 12b). The functionality of the


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gel was tested with primary rat dorsal root ganglia (DRG) cells, which were able to grow into the gels at the exposed volumes (Figure 12c).

Figure 12. (a) Conjugation of o-nitrobenzyl-protected cysteine to agarose. UV irradiation liberates reactive thiols that can react with maleimide functionalized biomolecules. (b) Schematic of photopatterning within a 3D hydrogel. (c) Fluorescence image showing the green labelled immobilized GRGDS at the exposed volume, and migration of dorsal root ganglion cells (in red) into it. Reproduced with permission from ref 81. Copyright 2004 Nature publishing group. In order to improve the spatial resolution of the photoactivation process within a 3D environment, the activation of the PPG can be performed by two-photon excitation using femtolasers.82-84 This strategy reduces photodamage by using longer wavelengths (NIR), and allows 3D resolution in the activation step. A photoactivatable variant of cyclic[RGDfC] ligand containing the methoxynitrobiphenyl (PMNB) chromophore has been developed for a 3D-resolved activation of cellular processes in 3D gels using two-photon excitation. Cellular attachment and invasive behavior were photo-triggered in 3D cell cultures. For this purpose PEGthiol/maleimide based hydrogels functionalized with cyclo[RGD(PMNB)fC] were used (Figure 13A-B).85 This is a well established system for 3D cell cultures. A cell clot was encapsulated in the gel and a circular pattern was scanned with a two-photon laser close to the clot. Cells migrated from the spheroid into the illuminated region within 3 days and remained confined to the illuminated space (Figure 13C-F) without detectable photodamage.

Figure 13. (A-B) Chemical design of PEG maleimide-thiol hydrogels functionalized with cyclo[RGD(PMNB)fC]. (C-E) Immunofluorescence images of a fibrin clot containing fibroblast inside the 3D PEG hydrogel. The volume marked with yellow circles in front of spheroid was irradiated using a scanning laser. Within 5 days cells migrated into the irradiated region by recognizing the activated adhesive ligand.85 Photodegradable hydrogel The physical properties of hydrogels have been manipulated in the presence of live cells by incorporating PPGs in the polymer architecture. Kloxin and coworkers developed a photodegradable hydrogels based on cleavage of ortho-nitrobenzyl (oNB) derivatives.34 Initially, hydrogels were synthesized using a redox-initiated radical polymerization scheme to copolymerize mono- and diacrylated PEGs functionalized at each end with a nitrobenzyl ester functionality. Gel degradation was induced via light exposure (365, 405, or 740 nm), and spreading of encapsulated hMSCs was found to depend on the local polymer density. Furthermore, an adhesion peptide sequence, RGD, was tethered to the gel through a photolabile linker and subsequently released on demand. Photodegradable hydrogels have also been used to create gradients in the crosslinking density of initially uniform materials.87 When optically thick hydrogels were irradiated, the resulting gradient in light intensity led to a gradient in mechanical properties as a function of depth into the gel. Studies with hMSCs-laden photodegradable hydrogels revealed that cells near the surface (20 microns) adopted a


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more spread morphology compared to those deeper (100 microns) into the network.88 In a recent study, Yang et al.56 varied the culture time of hMSCs on stiff hydrogels before softening them in situ, and results showed that hMSCs retained a ‘‘mechanical memory’’ of the stiffness of past culture conditions and that there exists a threshold culture time upon which irreversible mechanical activation and differentiation occurs.89 Beyond control of biomechanical and biochemical properties, photodegradable hydrogels also enable the generation of topographical features, either on the surface or within the bioscaffold networks. Kirschner and colleagues90 studied the effect of such surfaces on the phenotype of cultured hMSCs. By exposing hydrogel surfaces through photomasks of rectangular patterns of increasing aspect ratio, it was found that higher aspect ratio surface patterns led to higher alignment of hMSCs. Three-dimensional topographies can be created within the bulk of hydrogel-cell scaffolds using focused laser light. This provides a precise means of guiding cellular interactions in 3D, and has been used to guide fibroblast migration32 and neurite91 extension in 3D environments.

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light-induced vascularization of these hydrogels. In conclusion, non-invasive, transdermal activation of the cell-adhesive RGD peptide on implanted biomaterials regulates in vivo cell adhesion and spatial patterning, inflammation, fibrous encapsulation, and vascularization of the material. Long-wavelength light is preferred for in vivo applications due to expected deeper penetration and lower photodamage. The incorporation of upconversion nanoparticles (UCNP) to the PPG-functionalized material is an interesting approach. Under the exposure of near-IR (NIR) light, the upconverted UV emission from the UCNP nanocarrier leads to the photocleavage of the PPGs and activates the reaction. This strategy has been applied to nanocarriers containing UCNP and drugs attached to the carrier by photocleavable anchoring units. Light-regulated intracellular release of drugs for controlled differentiation and long-term tracking of Human mesenchymal stem cells (hMSCs) in vivo was realized.100

By incorporation of photodegradable cross-linkers in PEG-based hydrogels, irradiation resulted in local degradation of the hydrogels to yield softened regions92 or to allow retrieval of specific cells for subsequent studies.67 Conversely, photochemistry can also be used to stiffen hydrogels, affecting the cellular morphogenesis particularly at the interface between the stiff and soft regions.93 The readers are referred to these literatures94-97 for more details. In vivo application The positive results obtained in in vitro cultures using PPGs to regulate cellular response motivated the testing of this approach in in vivo scenarios. An interesting question in tissue regeneration is whether the temporal presentation of bioligands on implanted materials can be exploited to guide reparative responses. In this context, the possibility to regulate inflammation and host response to implanted materials by means of controlling cell-materials interactions was tested in a prominent example.98 PEG gels modified with cyclo[RGD(DMNPB)fK] were implanted transdermally into mice and photoactivated at selected time points after implantation.99 The time-regulated presentation of the peptide modulated the chronic inflammatory response to the biomaterial. Hydrogels presenting RGD peptide immediately after implantation exhibited fibrous capsules significantly thicker than gels where the RGD was presented upon light exposure at day 7 or 14 after implantation (Figure 14). Using degradable PEG hydrogels presenting cyclo[RGD(DMNPB)fK] peptide and in the presence of VEGF, the post-implantation formation of functional blood vessels could be spatiotemporally controlled (Figure 14e). New vessels stained positive for the endothelial cell marker CD31 and smooth muscle cell marker SMA (Fig. 14d), indicating

Figure 14. Light-triggered activation of cell-materials interactions in vivo (a) Setup used for masked, transdermal UV light exposure of implanted PEG gels modified with photoactivatable cell adhesive ligand, (b) Explanted hydrogels showing cells attaching to the exposed areas of the hydrogels, and no attachment without light irradiation. (c) Photographs of histology sections stained by Mason’s trichrome showing fibrous capsule formation around implanted hydrogels at 28 days (scale bar, 100 m). (d) Immunostaining images of hydrogels explanted at 14 days showing angiogenic response when the RGD peptide was


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activated with light at days 0 and 7 after implantation. (e)Fluorescent images of blood vessel ingrowth (green) into PEG-maleimide hydrogels implanted subcutaneously for 14 days (scale bar, 100 µm). Reproduced with permission from ref 99. Copyright 2015 Nature publishing group.

Conclusion and outlook PPGs combined to biomaterials can be used to guide the interactions between cells and artificial materials. The PPGs can be attached to ligands relevant for binding, or to the material itself and allow light-mediated binding or release of biomolecules. By regulating the exposure dose or the exposure wavelength, sequential and different stimuli can be realized on the same system. Based on these molecular tools, we have triggered and studied various dynamic cellular processes, including cell adhesion, release, migration, proliferation and differentiation. Most reported demonstrations of light-activated cellular responses deal with adhesive peptides and guiding of cell attachment itself or cell-attachment related processes. However, the same approaches might be transferable to many other ligands for membrane receptors. Work in this area requires joining organic chemists for the development of efficient and orthogonal PPGs and coupling chemistries, molecular biologists understanding the biological function of the activated biomolecules. We foresee the urgent need to address the issues of the biocompatibility and the efficiency of the irradiation step,

(1) Tam, R. Y.; Smith, L. J.; Shoichet, M. S., Engineering cellular microenvironments with photo- and enzymatically responsive hydrogels: toward biomimetic 3D Cell Culture Models. Acc. Chem. Res. 2017, 50 (4), 703-713. (2) Rosales, A. M.; Anseth, K. S., The design of reversible hydrogels to capture extracellular matrix dynamics. Nat. Rev. Mater. 2016, 1 (2), 15012. (3) Dhowre, H. S.; Rajput, S.; Russell, N. A.; Zelzer, M., Responsive cell-material interfaces. Nanomedicine (London, U. K.) 2015, 10 (5), 849-871. (4) Gooding, J. J.; Parker, S. G.; Lu, Y.; Gaus, K., Molecularly Engineered Surfaces for Cell Biology: From Static to Dynamic Surfaces. Langmuir 2014, 30 (12), 32903302. (5) Kuroki, H.; Tokarev, I.; Minko, S., Responsive surfaces for life science applications. Annu. Rev. Mater. Res. 2012, 42, 343-372. (6) Brieke, C.; Rohrbach, F.; Gottschalk, A.; Mayer, G.; Heckel, A., Light-Controlled Tools. Angew. Chem., Int. Ed. 2012, 51 (34), 8446-8476. (7) Alge, D. L.; Anseth, K. S., Bioactive hydrogels Lighting the way. Nat. Mater. 2013, 12 (11), 950-952. (8) Wang, G.; Zhang, J., Photoresponsive molecular switches for biotechnology. J. Photochem. Photobiol., C 2012, 13 (4), 299-309.

by developing chromophores that can be excited at longer wavelengths or weaker doses. Design of visible and infrared sensitive chromophores instead of UV-sensitive ones and preparation of novel photoswitches including multi-photo-responsiveness will be further explored. Up to now most studies have been performed in 2D biointerfaces, but the envisioned potential of PPG-based approaches for 3D dynamic cell cultures, and the few impressive experimental demonstratinos, open new possibilities for biological experimentation in better mimics of the natural microenvironment.

AUTHOR INFORMATION Corresponding Author *Tel: +49 (0) 681-9300-510. E-mail: [email protected]

Present Addresses INM - Leibniz Institute for New Materials gGmbH Campus D2 2 D-66123 Saarbrücken, Germany

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT

REFERENCES

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14 ACS Paragon Plus Environment

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Adhesion to Surfaces. ChemBioChem 2011, 12 (17), 26232629. (59) Han, X.; Pradeep, S. N. D.; Critchley, K.; Sheikh, K.; Bushby, R. J.; Evans, S. D., Supported Bilayer Lipid Membrane Arrays on Photopatterned Self-Assembled Monolayers. Chem. Eur. J. 2007, 13 (28), 7957-7964. (60) Liu, D.; Xie, Y.; Shao, H.; Jiang, X., Using Azobenzene-Embedded Self-Assembled Monolayers To Photochemically Control Cell Adhesion Reversibly. Angew. Chem., Int. Ed. 2009, 48 (24), 4406-4408. (61) Vaselli, E.; Fedele, C.; Cavalli, S.; Netti, P. A., "OnOff" RGD Signaling Using Azobenzene PhotoswitchModified Surfaces. ChemPlusChem 2015, 80 (10), 1547-1555. (62) Gong, Y.-H.; Li, C.; Yang, J.; Wang, H.-Y.; Zhuo, R.-X.; Zhang, X.-Z., Photoresponsive “Smart Template” via Host–Guest Interaction for Reversible Cell Adhesion. Macromolecules 2011, 44 (19), 7499-7502. (63) Iturri, J.; García-Fernández, L.; Reuning, U.; García, A. J.; Campo, A. d.; Salierno, M. J., Synchronized cell attachment triggered by photo-activatable adhesive ligands allows QCM-based detection of early integrin binding. Sci. Rep. 2015, 5, 9533. (64) Carlos, M. M.; Roberta, F.; Florian, R.; Stefanie, N.; G., K. T.; Horst, K., αvβ3‐ or α5β1‐Integrin‐Selective Peptidomimetics for Surface Coating. Angew. Chem., Int. Ed. 2016, 55 (25), 7048-7067. (65) Wirkner, M.; Alonso, J. M.; Maus, V.; Salierno, M.; Lee, T. T.; García, A. J.; del Campo, A., Triggered Cell Release from Materials Using Bioadhesive Photocleavable Linkers. Adv. Mater. 2011, 23 (34), 3907-3910. (66) Shin, D.-S.; Hyun Seo, J.; Sutcliffe, J. L.; Revzin, A., Photolabile micropatterned surfaces for cell capture and release. Chem. Commu. 2011, 47 (43), 11942-11944. (67) Shin, D.-S.; You, J.; Rahimian, A.; Vu, T.; Siltanen, C.; Ehsanipour, A.; Stybayeva, G.; Sutcliffe, J.; Revzin, A., Photodegradable Hydrogels for Capture, Detection, and Release of Live Cells. Angew. Chem., Int. Ed. 2014, 53 (31), 8221-8224. (68) Bian, Q.; Wang, W.; Wang, S.; Wang, G., LightTriggered Specific Cancer Cell Release from Cyclodextrin/Azobenzene and Aptamer-Modified Substrate. ACS Appl. Mater. Interfaces 2016, 8 (40), 27360-27367. (69) van Dongen, S. F. M.; Maiuri, P.; Marie, E.; Tribet, C.; Piel, M., Triggering Cell Adhesion, Migration or Shape Change with a Dynamic Surface Coating. Adv. Mater. (Weinheim, Ger.) 2013, 25 (12), 1687-1691. (70) Bai, T.; Sinclair, A.; Sun, F.; Jain, P.; Hung, H.-C.; Zhang, P.; Ella-Menye, J.-R.; Liu, W.; Jiang, S., Harnessing isomerization-mediated manipulation of nonspecific cell/matrix interactions to reversibly trigger and suspend stem cell differentiation. Chem. Sci. 2016, 7 (1), 333-338. (71) Kadem, L. F.; Holz, M.; Suana, K. G.; Li, Q.; Lamprecht, C.; Herges, R.; Selhuber-Unkel, C., Rapid Reversible Photoswitching of Integrin-Mediated Adhesion at the Single-Cell Level. Adv. Mater. (Weinheim, Ger.) 2016, 28 (9), 1799-1802. (72) Zhang, J.; Ma, W.; He, X.-P.; Tian, H., Taking Orders from Light: Photo-Switchable Working/Inactive Smart Surfaces for Protein and Cell Adhesion. ACS Appl. Mater. Interfaces 2017, 9 (10), 8498-8507.


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Langmuir

TOC

Light-guided biointerfaces with photocleavable protecting groups were fabricated to manipulate the cellular behaviors, including cell adhesion, release, migration, proliferation and differentiation, in vitro and in vivo.


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Aránzazu del Campo studied chemistry at the Universidad Complutense in Madrid. She received her doctorate in 2000 at the Instituto de CyT de Polímeros, also in Madrid. Following her post-doctoral work at the Max Planck Institute for Polymer Research in Mainz, and at the University of Urbino in Italy, she had her own research group at the Max Planck Institute for Metals Research in Stuttgart. From 2007 to 2008, she led the Functional Surfaces program at the Leibniz Institute for New Materials. Since 2009, she has worked as a Minerva research group leader at the Max Planck Institute for Polymer Research in Mainz, where she led the Dynamic Biointerfaces research group. Since September 2015, she has joined Leibniz Institute for New Materials as scientific director and program area manager. Her current research interests focus on developing cell-engineering materials that can communicate with cells and control their behavior.

Yijun Zheng graduated from the department of chemistry, Beijing Normal University in China and defended her Ph.D. thesis in 2011 at Peking University in China. During a postdoc stay from 2011 to 213 at Max Planck Institute for Polymer Research in Mainz, Germany, she worked on the design of thiophene supermolecular 2D materials for energy storage, displays, and biosensing. Presently, she is a postdoc research fellow at the department of Dynamic Biomaterials at Leibniz Institute for New Materials in Saarbrücken, Germany. Her current research interest is focused on photo responsive materials and cell material interactions.


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Aleeza Farrukh received her degree in Organic Chemistry from University of Punjab in Pakistan and defended her Ph.D. thesis in 2017 at Max Planck Graduate School, Mainz, Germany. Currently she is a postdoctoral fellow in department of Dynamic Biomaterials at Leibniz Institute for New Materials in Saarbrücken, Germany. She is working on development of and hydrogels. Her research focus encompasses the design and synthesis of photo-responsive and bio-orthogonal chemistries for controlling cell-biomaterials interaction.


Optoregulated biointerfaces to trigger cellular responses - Langmuir

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