Journal of Materials Chemistry B This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online PAPER View Journal | View Issue Stable anchoring of bacteria-based protein nanoparticles for surface enhanced cell guidance† Cite this: J. Mater. Chem. B, 2020, Open Access Article. Published on 29 April 2020. Downloaded on 12/14/2020 3:29:29 PM. 8, 5080 Marc Martı́nez-Miguel, ab Adriana R. Kyvik,ac Lena M. Ernst, a Albert Martı́nez- Moreno,a Olivia Cano-Garrido, cd Elena Garcia-Fruitós, ‡cd Esther Vazquez,cd Nora Ventosa, ac Judith Guasch, *abc Jaume Veciana, ac Antonio Villaverde cd and Imma Ratera *ac In tissue engineering, biological, physical, and chemical inputs have to be combined to properly mimic cellular environments and successfully build artificial tissues which can be designed to fulfill different biomedical needs such as the shortage of organ donors or the development of in vitro disease models for drug testing. Inclusion body-like protein nanoparticles (pNPs) can simultaneously provide such physical and biochemical stimuli to cells when attached to surfaces. However, this attachment has only been made by physisorption. To provide a stable anchoring, a covalent binding of lactic acid bacteria Received 13th March 2020, (LAB) produced pNPs, which lack the innate pyrogenic impurities of Gram-negative bacteria like Accepted 23rd April 2020 Escherichia coli, is presented. The reported micropatterns feature a robust nanoscale topography with DOI: 10.1039/d0tb00702a an unprecedented mechanical stability. In addition, they are denser and more capable of influencing cell morphology and orientation. The increased stability and the absence of pyrogenic impurities represent a rsc.li/materials-b step forward towards the translation of this material to a clinical setting. Introduction study the influence of different surface topographies.6–8 Other approaches such as self-assembly and soft-lithography enabled Cell behavior is influenced by numerous factors, ranging from versatile chemical functionalizations and immobilizations of biological and chemical to physical cues, such as the influence biological materials on surfaces.9–13 Among the techniques of nearby cells and soluble molecules or the surface stiffness of used to fabricate these soft materials, microcontact printing the extracellular matrix (ECM).1 Indeed, cells are able to receive (mCP) stands as a cost-effective and fast soft-lithography tech- and integrate these inputs, resulting in responses such as nique to control surface chemistry at the microscale.14 mCP adhesion, proliferation, migration, and apoptosis.2,3 Using this allows the use of a broad range of materials to coat surfaces knowledge, tissue engineering aims to mimic the physiological such as proteins,15,16 nucleic acids,17 genetically modified and environmental conditions of biological systems to provide viruses,18 and electroactive ligands with dynamic surfaces.19,20 better strategies for regenerative medicine.4 Several surface Recently, mCP has been used to print proteins and peptides to engineering approaches have been used to design and produce generate micropatterned surfaces for cell guidance and mechano- materials able to interact with cells and study their behavior.5 transduction studies.21–23 For example, micro- and nano-structured surfaces produced Our group applied the same technique to pattern protein by hard lithography techniques have been of great utility to nanoparticles (pNPs) on surfaces. These pNPs consisted of bacterial inclusion bodies (IBs) of green fluorescent proteins a Department of Molecular Nanoscience and Organic Materials, Institut de Ciència (GFPs) or fibroblast growth factors (FGFs).27,55–58 Such pNPs de Materials de Barcelona (ICMAB-CSIC), Campus UAB, Bellaterra 08193, Spain. were produced through biological synthesis in recombinant E-mail: iratera@icmab.es b bacteria obtaining highly pure aggregates,24 which preserved Dynamic Biomimetics for Cancer Immunotherapy, Max Planck Partner Group, ICMAB-CSIC, Campus UAB, Bellaterra 08193, Spain the function of the protein of interest.25 They were roughly c Networking Research Center on Bioengineering, Biomaterials and Nanomedicine spherical and showed diameters ranging from 50 to a few (CIBER-BBN), Spain hundred nanometers, although their sizes and geometries d Institut de Biotecnologia i de Biomedicina and Departament de Genètica i de can be tuned.26 In these studies, negatively charged pNPs were Microbiologia, Universitat Autònoma de Barcelona (UAB), anchored through electrostatic interactions on amino termi- Cerdanyola del Vallès 08193, Spain † Electronic supplementary information (ESI) available. See DOI: 10.1039/d0tb00702a nated self-assembled monolayers (SAMs), resulting in the pre- ‡ Present address: Department of Ruminant Production, Institut de Recerca i ferential adhesion of fibroblasts in pNP-rich micropatterned Tecnologia Agroalimentàries (IRTA), 08140 Caldes de Montbui, Spain. areas.27 pNPs were thus proved to be useful nanomaterials for 5080 | J. Mater. Chem. B, 2020, 8, 5080--5088 This journal is © The Royal Society of Chemistry 2020 View Article Online Paper Journal of Materials Chemistry B cell culture and for the development of biomaterials for regen- SAMs and used to study the adhesion and spreading of human erative medicine. Nevertheless, some challenges still have to be osteosarcoma U2-OS cells. Thus, we demonstrate the suitability of overcome in order to achieve wider applicability, such as the LAB as safe cell factories and the possibility to produce endotoxin- lack of a strong and specific binding of the pNPs to surfaces free nanomaterials in a cost-effective and scalable process, while This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. as well as their safety issues related to their production in still sharing many of the positive features of pNPs derived from Escherichia coli (E. coli), which are bacteria rich in endotoxins. E. coli for cell culture studies.41 There are several strategies for protein attachment to surfaces that are compatible with mCP, such as affinity-based biological reactions28 and surface chemistry strategies such as Experimental the thiol–gold,15 amine-reactive crosslinking after the silaniza- Bacterial strains, plasmids and IB-like protein tion of glass,29,30 or thiol–maleimide interfacial reactions,31 Open Access Article. Published on 29 April 2020. Downloaded on 12/14/2020 3:29:29 PM. nanoparticle production among others. The interfacial maleimide–thiol reaction is an interesting option due to its resulting covalent interaction IB-like pNPs were produced both in the E. coli strain MC4100 and compatibility with many biological functional groups of (StrpR), transformed with the expression vector pTV1GFP (ApR), proteins.31 Moreover, the natural presence of thiol groups in and in L. lactis subsp. cremoris NZ9000, clpP htrA (clpP-htrA; cysteine residues32 allows a strong binding with the maleimide EmR) (kindly provided by INRA, Jouy-en-Josas, France; patent of a wide range of proteins into specific micropatterns, without Nr. EP1141337B1) strain44 transformed with the expression prior modification. To our knowledge, the covalent attachment vector pNZ8148 (CmR) (MoBiTech). E. coli was grown in lyso- of pNPs to surfaces has not yet been reported in contrast with geny broth (LB) rich medium at 37 1C with its respective the well-known anchoring of single proteins32 and the immo- antibiotic supplementation (ampicillin, 100 mg ml1; and bilization of other nano-objects, such as liposomes,33 gold streptomycin, 30 mg ml1) at 37 1C and 250 rpm. L. lactis was nanoparticles,34 and molecularly imprinted polymeric nano- grown in M17 medium enriched with 0.5% glucose at 30 1C particles.35 The covalent binding of pNPs on surfaces through with antibiotic supplementation (chloramphenicol, 5 mg ml1; the maleimide–thiol reaction is interesting to prevent the pNP and erythromycin, 2.5 mg ml1) at 30 1C without shaking. The displacement caused by the exerted forces of cells in long lasting production of pNPs in E. coli was induced when reaching an cell culture studies. Such covalent bindings are also expected to OD550 = 0.5 with 1 mM isopropyl b-D-1-thiogalactopyranoside enhance the life time of the pNP-decorated surfaces. (IPTG), while the production of IB-like pNPs in L. lactis was On the other hand, the production of IB-like pNPs in E. coli induced by 12.5 ng ml1 nisin when OD550 = 0.5. After 3 h, cell as a cell factory represents a major drawback due to the cultures were harvested for NP purification. presence of lipopolysaccharides (LPS) in their outer cell membrane, which are also known as endotoxins. LPS are able Production of GFP IB-like pNPs in Escherichia coli to over-activate the immune system at only mg per kg of LPS/ All the materials used were sterile and the procedure was body mass ratios36,37 and its removal from proteins produced carried out in a laminar flow hood under sterile conditions. in Gram-negative bacteria, such as E. coli, is tedious and The E. coli MC4100 pTV1GFP strain was grown in LB rich expensive.36,38 Among the alternatives, Gram-positive lactic medium supplemented with 300 ml of ampicillin and 300 ml acid bacteria (LAB) stand out,39 which are LPS-free and have of streptomycin in 300 ml shake flasks at 37 1C and 250 rpm been classified as generally recognized as safe (GRAS) micro- until it reached an OD550 = 0.715. The culture was induced to organisms by the Food and Drug Administration (FDA),40 thus produce GFP pNPs using 1 mM IPTG for 3 h. Purification allowing their development as biologically-safe production started when the induced culture optical density reached platforms. In spite of the high solubility of LAB-protein 4.38 units. Protease inhibitors were then added to the broth products, fully functional IB-like pNPs composed of relevant (one small complete EDTA-free pill). 3 ml of lysozyme at proteins in humans and veterinary medicine were produced 0.5 mg ml1 was also added to the medium and the culture and purified.41 Since then, LAB-produced IB-like pNPs have was incubated for 2 h at 37 1C and 250 rpm. After this step, the continued to be developed42 and used for applications such as culture was frozen overnight (ON) at 80 1C. Afterwards, 1.2 ml therapeutic protein delivery.43 of Triton X-100 was added to the media when the culture In this work we aimed at covalently attaching pNPs derived defrosted. The culture was then incubated with agitation at from LAB to pre-functionalized surfaces, thus improving both room temperature (RT) for 1 h. A first sterility control was the robustness and biocompatibility of pNP-decorated surfaces performed by spreading 100 ml of the culture broth in an for cell guidance. Our objective was to increase the adhesion of antibiotic-free LB Petri dish and incubating it ON at 37 1C. large pNPs to their supporting substrate through covalent The rest of the culture was frozen ON at 80 1C. The freezing/ bonding to produce dense and well-defined pNP patterns that defrosting process was repeated by spreading 100 ml of the would promote cell adhesion with resistance to cell-applied culture in an antibiotic-free LB plaque after each cycle, until forces. At the same time, we explored the efficacy of the non- no organism was grown in the dish after 24 h. After 3 cycles pyrogenic pNPs produced in LAB compared to those produced in of freezing/defrosting, the broth was sterile. 75 ml of Nonidet E. coli. With these objectives in mind, pNPs produced in Lactococcus NP-40 was then added to the mixture and kept under agitation lactis (L. lactis) were micropatterned onto maleimide-terminated for 1 h at 37 1C and 250 rpm. Afterwards, the culture was This journal is © The Royal Society of Chemistry 2020 J. Mater. Chem. B, 2020, 8, 5080--5088 | 5081 View Article Online Journal of Materials Chemistry B Paper centrifuged at 3900 rpm for 30 min and the pellet was resus- silicon chips. Substrates were left to air dry for 2 min and then, pended with 15 ml of lysis buffer and Triton X-100. The mixture were dried completely with filter paper. Samples were observed was frozen ON and the culture was defrosted and centrifuged at in a FEI Magellan 400L XHR SEM (Nanolab technologies, 3900 rpm for 15 min at 4 1C. The supernatant was discarded Milpitas, CA) operating at 2 kV. Images were obtained using a This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. and the pellet was resuspended with 15 ml of phosphate- vCD back-scattered electron detector. buffered saline (PBS). The mixture was transferred to a 1 ml Eppendorf tube (each one containing 10 ml of the original Colorimetric quantification of accessible cysteine residues of broth). Samples were then centrifuged and the supernatant was GFP IB-like pNPs discarded. GFP pNPs were finally purified and ready to be used. The comparative quantification of accessible cysteine residues They were kept frozen at 80 1C until their use. between the different IB-like pNPs produced was carried out by following a previously described protocol using 4 0 4-dithio- Open Access Article. Published on 29 April 2020. Downloaded on 12/14/2020 3:29:29 PM. Production of GFP IB-like pNPs in Lactococcus lactis pyridine (DTDP), which interacts with accessible thiol groups All the materials used were sterile and the procedure was carried of cysteines and yields a product that strongly absorbs light at out in laminar flow hoods always under sterile conditions. The 324 nm.45 Briefly, IB-like pNP samples were diluted in PBS up to L. lactis NZ9000 clpP htrA pNZ8148-VP1GFP strain was cultured a concentration of 4 mg ml1 and sonicated 10 min before the in M17 medium enriched with 0.5% glucose and supplemented addition of the DTDP reagent. The progress of the reaction was with 150 ml of erythromycin and 75 ml of chloramphenicol in monitored on a spectrophotometer (JASCO V-780) for 5 min at 300 ml shake flasks at 30 1C and 250 rpm until it reached an 324 nm. The increment of absorbance was compared in each OD550 = 0.37. Recombinant gene expression was induced to sample after 5 min. produce GFP pNPs using 12.5 ng ml1 nisin for 3 h. Purification started when the optical density reached 3.8 units. The culture Preparation of functional terminated SAMs was distributed in 50 ml sterile falcons and centrifuged at Glass substrates were placed in piranha solution [concentrated 3900 rpm for 30 min. The supernatant was then discarded and H2SO4, aqueous H2O2, 3 : 1] for 40 min, rinsed with ultrapure the pellet was resuspended in 30 ml PBS/falcon, which was frozen water and dried under a nitrogen stream. A 3 nm layer of ON at 80 1C. Afterwards, the sample was defrosted and titanium was deposited on the glass substrates in order to disrupted for three rounds using a French Press at 15 000 PSI. improve the gold adhesion using a vapor deposition equipment The resulting solution was frozen ON at 80 1C and 600 ml of (Edwards Auto 306; Edwards, Crawley, UK). On top of that, an lysozyme/falcon was added to the sample and kept under agita- 8 nm layer of gold was deposited. Substrates were cleaned tion for 2 h at 37 1C and 250 rpm when defrosted. Then, the through 5 min sonication consecutively in dichloromethane, mixture was frozen ON at 80 1C and 120 ml of Triton X-100/falcon acetone, and ethanol, and then dried under a nitrogen stream. was added after defrosting. Falcons were agitated for 1 h at RT. Prior to the SAM formation, substrates were activated in an After, a first sterility control was performed by spreading 100 ml of ozone cleaner (UVO-cleaner: model 42 series; Jelight company, the culture broth in an antibiotic-free 0.5% glucose M17 broth USA) for 20 min. Substrates were then immersed in 1 mM Petri dish and leaving it ON at 30 1C. The rest of the culture was ethanolic solutions containing either an HO-terminated molecule frozen at 80 1C ON. A freezing/defrosting process was repeated [HO-EG3-C11-S-S-C11-EG3-OH, where EGx is poly(ethylene)glycol, spreading 100 ml of culture in an antibiotic-free M17 broth plaque and Cx is –(CH2)x–] or a maleimide-terminated molecule [a 98 : 2 after each freezing cycle, until no organism was grown in the mix of HO-EG3-C11-S-S-C11-EG3-OH and S-S-C4CONH-C3-EG3- media. After 3 cycles of freezing/defrosting, the broth was sterile. C-NHCO-C2-Mal (lipoic acid–poly(ethylene)glycol-maleimide) Then, 75 ml of NP-40 was added to the mixture and kept under (Mal = maleimide) molecules] (Prochimia Surfaces, Poland) in a agitation for 1 h at 4 1C. 18 ml of 1 M MgSO4 and 18 ml of controlled argon atmosphere for 24 h (Scheme 1). 1 mg ml1 DNAse were added to each falcon and kept under agitation for 1 h at 37 1C and 250 rpm. The mixture was Contact angle measurements of deposited IB-like pNPs centrifuged at 3900 rpm for 30 min and the supernatant was IB-like pNPs were resuspended in PBS up to a final concen- discarded. The pellet was resuspended with 5 ml lysis buffer and tration of 20 mg ml1. Freshly prepared HO- and maleimide- 0.5% Triton X-100. Falcons were then frozen ON at 80 1C. Tubes terminated SAMs were immersed in pNP suspensions for 2 h were centrifuged at 3900 rpm for 30 min after defrosting and the and gently rinsed with ultrapure water before measuring the bacterial pellet was resuspended with 5 ml of sterile PBS. The wettability of the substrates with a Drop Shape Analyzer – a resulting solution was transferred to 1 ml Eppendorf tubes (each DSA100 (Krüss, Germany) contact angle goniometer. Three sets one containing 10 ml of the original broth). Aliquots were of static contact angles at different positions in each sample centrifuged at 1300 rpm for 15 min and the supernatant was were measured and the average values were obtained. discarded. GFP pNPs were finally purified and ready to be used. They were kept frozen at 80 1C and defrosted for immediate use. Microcontact printing Polydimethylsiloxane (PDMS) stamps were fabricated using a High resolution scanning electron microscopy of GFP IB-like pNPs commercial kit (Sylgard 184 Silicone Elastomer kit; Sigma- IB-like pNPs were resuspended in ultrapure water and soni- Aldrich, USA), mixing the PDMS precursor and the curing agent cated for 10 min before the deposition of a few microdrops on together in a 10 : 1 (w/w) ratio, and degassing the mixture to 5082 | J. Mater. Chem. B, 2020, 8, 5080--5088 This journal is © The Royal Society of Chemistry 2020 View Article Online Paper Journal of Materials Chemistry B Optical and confocal microscopy pNP-printed SAMs were observed using a fluorescence micro- scope (Olympus BX51 with a U-RFL-T reflected fluorescence system; Olympus, Japan) in order to assess the result of the pNP This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. deposition by mCP. To study cell adhesion and morphology, confocal microscopy images were obtained using a Leica TCS SP5 AOBS spectral confocal microscope (Leica Microsystems, Mannheim, Germany) with a HCX PL APO lambda blue Scheme 1 Molecular structures used in the formation of –OH and 63 objective. maleimide terminated SAMs. (a) HO-EG3-C11-S-S-C11-EG3-OH and (b) S-S-C4CONH-C3-EG3-C-NHCO-C2-Mal. EGx = polyethylene glycol, Open Access Article. Published on 29 April 2020. Downloaded on 12/14/2020 3:29:29 PM. Image analysis Cx = –(CH2)x–, and Mal = maleimide. Focal adhesion (FA) quantification as well as cell orientation and morphology data were extracted from microscopy images remove bubbles in a vacuum desiccator for 20 min. The mixture and treated with the ImageJ software (National Institute of was then poured into a previously silanized photolithographi- Health, USA). The treatment of the obtained data and the cally patterned silicon master, degassed again, cured for 2 h at statistical Kruskal–Wallis ANOVA test were performed using 60 1C, and released. Before soaking the clean stamps in the pNP the Origin software (OriginLab, USA). To determine the FA suspension, the stamps were sonicated for 5 min in acetone mean area, images were treated with contrast enhancement and dried in vacuum for 10 min. Then, the stamps were and a mean filter to improve structure resolution. Afterwards, sonicated for 5 min in a sodium dodecyl sulfate (SDS) solution captures were turned into binary using the threshold tool, and [10% in ultrapure water, w/w] and conditioned in the same the ‘‘Analyze Particles’’ function was used to measure the area media for 5 min. Afterwards, stamps were dipped in ultrapure of the FAs in the cell. The mean FA area per cell was determined water to remove the excess of SDS and dried under a nitrogen and plotted using Origin. The ‘‘Analyze Particles’’ function was stream. After the latter pre-treatment, stamps were immersed also used to approximate the whole cell area into ellipses to in previously sonicated E. coli- or L. lactis-pNP solutions in PBS determine cell orientation. The angle between the long axis of for 1 h. Stamps were then gently rinsed with ultrapure water, the ellipses and the stripped pattern was calculated. Then, dried under a nitrogen stream, and placed on the SAM-coated orientation data were classified into 101 segments, ranging substrates under a 13 g weight. After 1 h of contact, stamps from 0 to 1801, and represented using radial plots. Values were carefully removed in the presence of PBS. between 1801 and 3601 are a symmetric repetition of the data Cell culture, seeding, and immunostaining to improve the plot visualization. Similarly, upon orientation analysis, images were turned into binary using the threshold Human osteosarcoma cells (U2-OS) were obtained from the tool to calculate the cell area, which were approximated into American Type Culture Collection (ATCC; Manassas, VA, USA). ellipses, and their elongation was calculated as their aspect Cells were routinely cultured in Dulbecco’s Modified Eagle’s ratios minus one. Medium (DMEM) (Thermo Fisher Scientific, USA) supple- mented with 10% fetal bovine serum (FBS) and 1% penicillin/ streptomycin in a humidified atmosphere containing 10% CO2 Results and discussion at 37 1C. U2-OS cells were seeded on IB-like pNP micropat- terned surfaces at a concentration of 5000 cells per cm2. Plates GFP IB-like pNPs produced by E. coli and L. lactis were were incubated at 37 1C and under 10% CO2 for 24 h. There- covalently attached to maleimide-terminated SAMs through after, cells were fixed by the addition of 4% paraformaldehyde their cysteine aminoacids by means of a Michael addition in PBS for 20 min. After fixation, cells were permeabilized by interfacial reaction using the mCP technique, as recently shown adding 0.1% Triton in PBS and treated with a blocking solution for other nano-objects.46–48 (1% bovine serum albumin in PBS) for 30 min to prevent non- More specifically, the exposed thiol groups of the cysteine specific binding. After blocking, substrates were incubated for residues of GFP at the surface of the pNPs reacted with the 1 h at RT with a mouse monoclonal anti-paxillin antibody maleimide groups of the SAM forming covalent bonds, thus (Sigma-Aldrich, USA) diluted at a ratio of 1 : 400. After incuba- providing a more stable binding in comparison with a simple tion with the primary antibody, samples were washed with PBS physisorption procedure (Fig. 1). using a shaker at 50 rpm for 10 min and then they were In order to assess the effect of the covalent bond, the same incubated for 45 min at RT with the secondary antibody Alexa pNPs were also printed on HO-terminated SAMs, in which pNPs Fluor 647-labeled goat anti-mouse IgG (1 : 100; Thermo Fisher are expected to be only physisorbed. Moreover, to compare Scientific, USA) and with Hoechst (1 : 1000; Thermo Fisher the viability of LAB produced pNPs (Lactis-pNPs) with E. coli Scientific, USA). Primary and secondary antibodies were diluted produced pNPs (Coli-pNPs), both are produced and printed in the blocking solution. Finally, samples were washed with onto the different substrates using suspensions with the same PBS using a shaker for 10 min and mounted with ProLong Gold mass concentrations. Thus, the seeding of cells on these Antifading Mountant (Thermo Fisher Scientific, USA). substrates and the study of their morphology and orientation This journal is © The Royal Society of Chemistry 2020 J. Mater. Chem. B, 2020, 8, 5080--5088 | 5083 View Article Online Journal of Materials Chemistry B Paper Total integrated intensity was weighted against the total area of the field to obtain the average percentage of coverage. This analysis indicates that surfaces decorated with Lactis-pNPs show a higher surface coverage in comparison with those This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. decorated with Coli-pNPs. Furthermore, the maleimide-terminated SAMs with Lactis-pNPs exhibit the highest surface coverage. These results were further validated by water contact angle measurements of the surfaces decorated with pNPs. A higher contact angle was detected on the SAMs decorated with Lactis-pNPs in comparison with those decorated with Coli-pNPs (Fig. S2, ESI†) in agreement Open Access Article. Published on 29 April 2020. Downloaded on 12/14/2020 3:29:29 PM. with the higher quantity of deposited material on the surface. Afterwards, the pNP retention was assessed by the integra- tion of the GFP fluorescence intensity of microscopy images after rinsing the surfaces with ultrapure water, thus eliminating any interference from non-covalent interactions (Fig. 2c). The maleimide-terminated surface decorated with Lactis-pNPs showed a higher fluorescence intensity than the HO-terminated surface, indicating that covalent bonds between maleimide groups on the surface and the cysteine residues of GFP were properly formed. Thus, the density of attached pNPs and their resistance to being washed away increased. Even though there is a slight increase of fluorescence for the Coli-pNPs decorated maleimide-terminated SAM with respect to the HO-terminated SAM, there is no statistically significant difference between Fig. 1 Scheme of the strategy for culturing cells on micropatterns of them. To compare the remaining density of attached Coli-pNPs pNPs. (a) HO- and maleimide-terminated SAMs were prepared. (b) A and Lactis-pNPs after washing with water on a given surface, it micropatterned PDMS stamp was inked in a pNP suspension, produced should be taken into account that the specific fluorescence of GFP either in E. coli or in L. lactis, and put gently in contact with the SAM to Coli-pNPs is approximately 200% higher than that of Lactis-pNPs form the stripped pattern of pNPs. (c) In the maleimide terminated SAM, a covalent bond was formed between the maleimide and the exposed (see the ESI,† Section S4). According to this result, the higher cysteine groups of proteins, while in the HO-terminated SAM, pNPs were integrated fluorescence intensities based on the images of merely physisorbed on the surface. (d) Cells were seeded on the surfaces surfaces decorated with Lactis-pNPs can be correlated with a and studied using confocal microscopy. Scale bar = 20 mm. relevant increase in the interaction of the particulate material with the surface in comparison with that of Coli-pNPs. To understand if the physicochemical characteristics of the allowed us to measure the effect of the covalently attached pNPs pNPs influence the differential formation of covalent bonds and the influence of the host organism in which these particles between the external cysteine residues of GFP-pNPs and the were produced (Fig. 1). Finally, we also explored mechano- maleimide groups of the surface, we compared the average transduction by the formation of focal adhesions (FAs), given sizes and Z-potential of the pNPs produced in either E. coli or that stronger applied forces were expected from cells seeded on L. lactis. The Z-potential of both particles is very similar the covalently attached pNPs compared to the physisorbed (37 mV) (see Section S1 of the ESI†), while the average sizes ones. Indeed, FAs are protein complexes that are formed on of Coli-pNPs and Lactis-pNPs are 350 and 520 nm (Fig. S1, the cell membrane, which communicate cells with their ECM ESI†), respectively. The observed differences in the size of the and are responsible for cell attachment and migration. two types of pNPs could be at least partially responsible for the larger formation of covalent bonds on the Lactis-pNPs.41 Interactions between IB-like pNPs and functionalized surfaces In addition, the number of accessible thiol groups of the First, we analyzed the surface coverage of the pNPs attained cysteine residues on the pNP surfaces was investigated. Speci- upon mCP of Coli-pNP and Lactis-pNP suspensions with similar fically, we performed a colorimetric assay in which pNPs were mass concentrations on the two functional terminated SAMs, incubated with 4,4 0 -dithiopyridine (DTDP). This reagent inter- HO-SAM and maleimide-SAM, without any further treatment. acts with thiol groups and yields a product that absorbs light Thus, 20 mm wide stripped patterns of pNPs were printed on at 324 nm, allowing the quantification of cysteine residues of the SAMs and analyzed without rinsing using fluorescence proteins in a fast and simple manner.45 To comparatively microscopy to determine the pNP surface coverage and to quantify the amount of thiol groups in each sample, we diluted compare the number of pNPs adhered on the different termi- the pNPs to equal concentrations and monitored the reaction nated surfaces (Fig. 2a and b). The quantitative surface coverage with DTDP over 5 min. Lactis-pNPs showed an approximately analysis was performed by the integration of the GFP fluorescence 25% higher amount of accessible thiol groups than Coli-pNPs intensity of microscopy images in 30 random fields per sample. (Fig. S3, ESI†). Therefore, enhanced accessibility to thiol groups 5084 | J. Mater. Chem. B, 2020, 8, 5080--5088 This journal is © The Royal Society of Chemistry 2020 View Article Online Paper Journal of Materials Chemistry B This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. Open Access Article. Published on 29 April 2020. Downloaded on 12/14/2020 3:29:29 PM. Fig. 2 (a) Representative fluorescence microscopy images of 20 mm striped mCP patterns of E. coli (up) and L. lactis (down) GFP pNPs on functionalized SAMs with HO- (left) or maleimide (right) terminated groups. Scale bar = 20 mm. Images were taken immediately after the removal of the PDMS stamp. (b) Percentage of E. coli and L. lactis pNPs coverage area after being adhered to maleimide or HO-terminated SAMs through mCP. Higher coverage area relates to higher pNP density in the pattern. The information was extracted from fluorescence microscopy images. Nexperiments = 3; and Nimages = 30. Statistical analysis was performed using a Kruskal–Wallis ANOVA test. **p o 0.001, and ***p o 0.0001. (c) Fluorescence intensity retention after rinsing with ultrapure water on HO- and maleimide-terminated surfaces with deposited GFP pNPs produced in E. coli and L. lactis. in Lactis-pNPs could also be another explanation for the their ECM.49–52 Through these anchoring points, cells are able improved interactions. to transmit and receive mechanical forces and regulatory It is important to note that the proposed interfacial pNP- signals (Fig. 3a).53 Here, the FA areas were measured and their SAM reaction requires the presence of accessible thiol groups. mean value per cell was determined (Fig. 3b), since their size is Although the tendency of the thiols to dimerize, no reducing related to adhesion and migration processes.50 U2-OS cells agents were applied during the printing process, which cultured on surfaces with Coli-pNPs showed slightly larger FA indicates that the possible dimerization of thiols on the pNPs mean areas per cell than those cultured with Lactis-pNPs. did not impede the interaction with the surface. Another These results are significant for both HO- and maleimide- indication of the absence of dimerization is that size distribu- terminated SAMs. As discussed before, the role of the host tions, as measured by DLS,41 showed clearly defined intensity organism in which the pNPs are produced can play an impor- peaks, as well as a negative Z-potential which supports its tant role in the properties of the resulting material. In this case, colloidal stability by the action of the electrostatic repulsion Coli-pNP decorated surfaces show higher FA areas in compar- between NPs. Furthermore, the DTDP assay confirmed the ison to surfaces decorated with Lactis-pNPs, even though these presence of accessible thiol groups without scattering for as yielded a higher coverage and retention (Fig. 2). This increase long as 2 h after the resuspension of the pNP samples. More- could be justified by a higher roughness of the particulate over, the resulting patterns are stable and well formed, and in material,54 which was observed on Coli-pNP decorated surfaces. SAMs featuring maleimide groups these patterns are denser In addition, larger FAs were observed when the pNPs, either and more resilient to washes, reinforcing the hypothesis that produced in E. coli or L. lactis, were covalently bound to the the interfacial maleimide–thiol reaction successfully occurred. surface, although no significant differences were obtained. In the following sections, we analyzed the potential relation Interaction between cells and pNP-decorated surfaces between such higher FA areas and the morphology of the U2-OS cells were seeded on substrates micropatterned with adhered cells. 20 mm wide stripes made of either Coli-pNPs or Lactis-pNPs using hydrophilic HO-terminated or maleimide-terminated SAMs. The Cell elongation interaction of cells with the four types of pNP-decorated surfaces Cell morphology was assessed on the four decorated surfaces by was studied using an immunofluorescence staining technique measuring their elongation through the AR-1 values, where AR after 24 h of U2-OS cell seeding (Fig. 3a). The quantification is the aspect ratio (Fig. 4a). U2-OS cells seeded on Coli-pNP of the averaged FAs, cell elongation, and cell orientation was decorated surfaces with OH- and maleimide-terminated groups performed. did not show significant differences in elongation. This could be due to the particular physicochemical properties of the Cell adhesion quantification Coli-pNPs, which might impair the covalent union between Cell adhesion on surfaces can be studied by imaging the FAs, the maleimide-group and the cysteine groups from GFP. On the which are macromolecular complexes that connect cells with other hand, cell elongation was significantly higher on the This journal is © The Royal Society of Chemistry 2020 J. Mater. Chem. B, 2020, 8, 5080--5088 | 5085 View Article Online Journal of Materials Chemistry B Paper Lactis-pNP decorated maleimide-terminated surface in compar- ison with the HO-terminated one. This difference is most likely due to the higher adhesion of pNPs to the maleimide-terminated surface, and the subsequent increase in pNP density and robust- This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. ness of the pattern. Thus, the increased interaction of pNPs with the surface can be relevant for cells when following a pattern, since non-covalently bound pNPs are able to be displaced by cells. The formation of covalent bonds therefore enhances the robustness of the system and allows cells to adhere and elongate through the pattern without disrupting it. Open Access Article. Published on 29 April 2020. Downloaded on 12/14/2020 3:29:29 PM. Cell orientation The orientation of the U2-OS cells in E. coli and L. lactis produced pNP striped micropatterns was assessed by mea- suring the angle between the long axis of cells and the axis of the striped pattern (Fig. 4b and c). This data correlates with the elongation data previously reported, and also provides information about how cells are able to follow the pattern when adhering to the surface. When evaluating the alignment of cells on surfaces decorated with Coli-pNPs, we could not appreciate a relevant difference between the surfaces that can establish covalent bonds and the others. However, on surfaces decorated with Lactis-pNPs, an alignment increase with the pattern was observed for the maleimide-terminated SAM in comparison with the HO-terminated one. In the latter, cells can probably displace the pNPs due to their low interaction with the surface, while the covalently bound pNPs form a denser and more robust pattern, providing more stable anchoring points for cells. Thus, we demonstrated the utility of the maleimide– thiol interfacial reaction for anchoring pNPs on surfaces, which is of high importance for longer cell culture experiments and Fig. 3 (a) Representative confocal fluorescence microscopy images of applicability. immunofluorescence staining against paxillin for FAs (red) and DAPI for the nuclei (blue) of U2-OS cells seeded on the (left) HO- and (right) maleimide- terminated SAMs with (top) E. coli and (bottom) L. lactis GFP pNP patterns (green). Scale bar = 20 mm. (b) FA mean area per cell in E. coli and L. lactis pNP Conclusions patterned substrates. Nexperiments = 3; and Ncells = 45. Statistical analysis was performed using the Kruskal–Wallis ANOVA test. *p o 0.05. Well-defined stripped micropatterns were prepared using novel IB-like pNPs by mCP. These pNPs were produced by E. coli and Fig. 4 (a) Elongation of cells, measured as aspect ratio-1, cultured on HO- and maleimide-terminated surfaces micropatterned with E. coli and L. lactis produced pNPs. Nexperiments = 3; and Ncells Z 140. Statistical analysis was performed using the Kruskal–Wallis ANOVA test. *p o 0.05. ***p o 0.001. (b and c) Radial distribution plots of cell orientation versus the frequency of appearance of cells cultured on surfaces with striped micropatterns decorated with E. coli and L. lactis-produced pNPs, respectively, printed onto HO- (red line) and maleimide-terminated SAMs (blue line). The spindle- shaped plots show the strong orientation of cells along the pattern. Nexperiments = 3; and Ncells Z 140. 5086 | J. Mater. Chem. B, 2020, 8, 5080--5088 This journal is © The Royal Society of Chemistry 2020 View Article Online Paper Journal of Materials Chemistry B L. lactis and resulted in average sizes of 350 and 520 nm, and Nanostructuring Unit (U6), Unit of the CIBER in Bio- respectively. Both pNPs were successfully attached to surfaces engineering, Biomaterials & Nanomedicine (CIBER-BBN) through covalent bonding, due to the interaction between the located at the Institute of Materials Science of Barcelona accessible thiol groups of exposed cysteine residues of pNPs (ICMAB-CSIC). This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. and the maleimide groups of the surfaces. Patterns that fea- tured covalent bonds were denser and allowed stronger cell adhesion and larger cell spreading than those where pNPs were Notes and references only physisorbed to the HO-SAM surface, especially for Lactis-pNP patterns. Moreover, LAB produced pNPs present other advan- 1 B. 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