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Redox state of PDA directs cellular responses through pre-adsorbed protein Yifei Zhu, Lili Yao, Wen-Jian Weng, and Kui Cheng ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/ acsbiomaterials.8b00987 • Publication Date (Web): 27 Nov 2018 Downloaded from http://pubs.acs.org on November 27, 2018

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ACS Biomaterials Science & Engineering

Redox state of PDA directs cellular responses through pre-adsorbed protein Yifei Zhu, Lili Yao, Wenjian Weng, Kui Cheng* School of Materials Science and Engineering, State Key Laboratory of Silicon Materials, Cyrus Tang Center for Sensor Materials and Applications, Zhejiang University, Hangzhou 310027, China

*Correspondence

and

requests

for

materials

should

be

addressed

to:

[email protected]

Abstract Polydopamine (PDA) is capable of adhering on nearly all kinds of surfaces and shows good biocompatibility. Moreover, its surface state could be switched between oxidation and reduction states under different electrical stimulation. In this work, the effects of PDA redox states on protein adsorption, as well as the subsequent effects on cellular responses were characterized and evaluated. It was found that the electrical treatment changed the redox states of PDA, which, in turn changed state of preadsorbed protein molecules, and eventually affected the cellular responses. BSA preadsorbed PDA film was found to be beneficial for cell proliferation when PDA was changed into reduction state (RPDA), while BMP-2 pre-adsorbed PDA film showed promotion on cell differentiation when PDA was changed into oxidation state (OPDA).

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It was found that the transitions of PDA to RPDA and OPDA greatly changed the secondary structure of protein pre-adsorbed on it. This work provides a deeper insight on changes of protein molecules between cells and PDA surfaces with different redox states, that is important for both optimization of cell-materials interactions and application of PDA as a functional coating for biomedical engineering. Keywords: Polydopamine, redox state, protein, electrical stimulation, cell response

1. Introduction Polydopamine

(PDA),

which

is

polymerized

from

dopamine

(3,4-

dihydroxyphenethylamine), has attracted quite a lot of attention due to its special properties.1-3 It is reported to have an analogous structure to the pivotal composition of mussels’ secreted adhesive proteins, which makes it be able to adhere on different kinds of inorganic and organic surfaces.4-6 Since surface coatings were found to be one of the most efficient approaches to improve the biocompatibility and bioactivity of materials,7-10 much efforts have been made to exploit PDA as bioactive coatings for direct cell regulation in biomedical engineering applications. PDA coatings were found to be able to facilitate endothelial cell adhesion11-13 and promote the crystallization and mineralization of hydroxyapatite.14-15 PDA also improved cell adhesion, spreading and viability on well-known anti-adhesive polymers,16 it can activate integrin α5/β1, phosphatidylinositol-3-kinase signaling pathways and further promote osteogenic induction of periodontal ligament stem cells.17 Moreover, PDA film promoted early adhesion and subsequent differentiation of

2


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stem cells on different kinds of materials.18-19 It has been demonstrated to not only regulate the adhesion, proliferation, differentiation and multipotency of bone marrow stromal cell, but also enhance reprogramming of human somatic cells into human induced pluripotent stem cells and maintain its growth under defined conditions.20-21 Owing to its abundant functional groups, such as quinone group，phenolic hydroxyl group, amino group and so on, PDA has also been used as intermediate linker to immobilize bioactive molecules and drugs onto all kinds of surfaces and potentially beneficial for medicine technology and industry.22-24 The ZnO nanorod arrays were covalent immobilized with RGD-cysteine peptide through modification of polydopamine, which enhanced the osteo-inductivity and effectively killed bacteria simultaneously.25 It provides a convenient and easy pathway for surface functionalization. Moreover, as a conductive modification film, PDA was also used in the condition of electrical stimulation. For instance, Xie et al. made a conductive and electro-responsive polydopamine-polypyrrole microcapsules on titanium surface for electrical therapy through on-purpose drug delivery.26 PDA could also be fabricated on conductive substrates with complicated three-dimensional structure via electro-polymerization method.27-28 Recently, Tan et al. found that the spreading and proliferation properties of MC3T3-E1 cells were promoted by oxidized polydopamine with more quinone groups and phenolic groups on reduced polydopamine enhanced cell differentiation.29 That actually means electrical stimulation could directly influence cellular responses. Nevertheless, in many cases, extracellular matrix protein molecules and other 3



functional protein molecules act as intermediates between surface and cells.30 The variations of surface characteristics, such as surface potential, morphology, wettability, and chemical states,31-34 have great impacts on the adsorption and secondary structure of proteins. That eventually influences the cellular responses of the surface. Therefore, how PDA redox states affect the protein adsorbed, and then influence cellular responses become an interesting topic. In this work, electrical stimulation was utilized to regulate the state of PDA, then the influence of PDA with different redox states on pre-adsorbed protein was investigated, moreover, cellular responses on PDA with different proteins were further studied.

2. Materials and Methods 2.1 PDA fabrication and characterization Before composing the modified film, titanium substrate was purified by mixed acid (the volume ratio of HF : HNO3 : H2O was 1 : 2 : 4.2), deionized water, ultrasonic and dried. Dopamine hydrochloride (DA-HCl, 2 mg mL-1, Aladdin) was dissolved in Tris(hydroxymethyl)aminomethane solution (Tris, 10 mM, pH 8.5) to obtain dopamine buffer solution (DA-Tris). After that, clean Ti substrate was soaked in DA-Tris buffer solution for 18 h under slow stirring. When the polymerization was done, the modified substrate was carefully taken out from the container and washed by deionized water to clear away the extra liquid on the substrate, samples were further dried in the oven under 37 °C for subsequent experiments. The surface morphology of PDA films treated by different electrical stimulation were investigated by means of scanning electron microscopy (SEM, SU-70, Hitachi, working 4


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voltage at 3 kV). The qualitative change of surface groups on PDA film with different electrical stimulation was measured by Raman spectroscopy (Thermo Fisher Scientific, DXR532, 532 nm laser), the analysis of Raman spectra was utilized at room temperature. Further investigation of surface composition’ change under electrical stimulation was determined by X-ray photoelectron spectroscopy (XPS, Kratos AXIS Ultra DLD) using Al Kα (1486.6 eV) source. A sessile drop method was used to investigate the water contact angles (WCAs) by a contact angle meter (Dataphysics, OCA20). In addition, surface roughness and surface potential were studied by Scanning Near-field Optical Microscopy (AFM, KFM, SNOM, NTEGRA Spectra, NTMDT). 2.2 Protein adsorption Bovine serum albumin (BSA) was dissolved in phosphate-buﬀered saline (PBS, 0.01 M, pH 7.2) to form 5 mg mL-1 BSA solution. Before immersed in BSA solution, with a Pt electrode as counter electrode, Ti-PDA samples were treated by +0.5 V and -0.2 V with electrochemical workstation (CHI660E, CH) for 20 min, respectively, to get oxidation (OPDA) and reduction (RPDA) state (C-V curve of the PDA was shown in Fig. S1). The PDA substrate was immersed in BSA solution for 24 h and washed by deionized water gently for 3 times. After that, PDA samples were immersed in 300 L sodium laurylsulfonate (SDS, 1mg mL-1) solution to remove BSA from the surface. The amount of BSA adsorbed on PDA substrate was investigated by MicroBCA kit with microplate reader (562 nm). The secondary conformation of BSA adsorbed on these substrates was tested by attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR, Thermo Fisher, Nicolet 5700), the FTIR data was fitted and 5



analyzed by PeakFit. 2.3 Cell culture MC3T3-E1 cells (pre-osteoblastic cells, CRL-2594, ATCC) were used as model cell. Cells were seeded and cultured on different PDA substrates with 1640 cell culture medium (Cellmax, RPMI1640) which used 10% fetal bovine serum (FBS, Cellmax, Australia) as a supplement. Cells were cultured in a standard environment with an atmosphere of 5% CO2 at 37 °C. After cultured for specific time, 0.25% trypsin/EDTA (Gibco) solution was use for cell harvest, cells were then suspended in fresh culture media prepared for further experiments. 2.4 Cell viability The prepared samples with PDA film were sterilized in clean bench under UV light for 30 min, before cell culture, samples were immersed in bacteria free BSA solution for 24 h (5 mg mL-1). Osteoblasts (MC3T3-E1) with a density of 5 × 104 cells cm-2 were then seeded on PDA substrate (control group) in different redox state. Samples were carefully transferred to new 24-well plate at prescribed time, rinsed three times gently by adding 500 L phosphate buffered saline (PBS) along the wall, then culture media and CCK-8 reagent were added to the plates at a specific volume ratio (10:1). When the reaction time reacted 3 h, 96-well plate was shocked for a while, then the optical density (OD) of the reacted solution was examined at 450 nm after cultured for 1day and 5 days. 2.5 Immunofluorescent staining The MC3T3-E1 cells at a density of 5 × 104 cells cm-2 were cultured on PDA films 6


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with three states (PDA, OPDA, RPDA). Samples were immersed in bacteria free BMP2 solution (10 g mL-1) for 24 h before cell culture. After cultured for 1 day, 4% paraformaldehyde was used to fix the samples for 15 min, 0.05% Triton X 100 in PBS were utilized for further permeabilization, and these samples were then blocked in PBS solution. The nuclei and F-actin were stained by DAPI and Alexa-Fluors 594 phalloidin (Sigma), respectively. Cell states on different samples were observed by using a confocal laser scanning microscopy (LSM780, ZEISS, Germany) after staining completed. Cell morphologies were quantitatively analyzed by ImageJ software. 2.6 Alkaline phosphatase (ALP) activity The MC3T3-E1 cells at a usual density of 5 × 104 cells cm-2 were seeded on PDA films with three states (PDA, OPDA, RPDA) under a suitable atmosphere of 5% CO2 at 37 °C. When incubation time reached 14 days, different PDA samples were transferred to brand new 24-well culture plates after removing culture medium with pipettes and rinsed gently with PBS for three times along the wall. CelLytic Buffer (Sigma) were then used for lysing cells on samples at 0 °C, lysate was centrifuged to remove cell residue. The LabAssayTM ALP Kit (Beyotime Institute of Biotechnologe, Shanghai, China) was used to assess ALP (ab65834, abcam) expression at a wavelength of 405 nm, total amount of protein was determined by a BCA protein assay. 2.7 Statistic analysis All data were conducted in triplicate (or more) and expressed as mean ± standard deviation (SD). Statistical analyses were performed by SPSS software. Differences were considered statistically significant for *p < 0.05. 7



3. Results and discussion 3.1 Redox states of PDA As shown in Fig. 1, uniform PDA nanoparticles covered Ti substrate and surface morphology hardly changed after electrical stimulation (The morphology of Ti substrate was shown in Fig. S2), the surface roughness are 53.1±3, 37.8±2 and 47.4± 3 nm for PDA, OPDA and RPDA, respectively.

Fig. 1 SEM and AFM images of PDA (a-b), OPDA (c-d), RPDA (e-f), data represent mean ± SD (n = 3). To study the changes of chemical composition after electrical stimulation, the prepared PDA, OPDA and RPDA were investigated by Raman spectroscopy and XPS. 8


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The qualitative analysis of surface group changes was shown in Fig. 2a. The peaks at 1440 cm-1 and 1573 cm-1 represent the stretching vibration and deformation vibration of benzene ring in PDA, respectively. It also proves that polydopamine has been deposited on Ti substrate successfully. The proportion of the intensity changes when the electrical stimulation was applied to PDA film, which illustrated that redox reactions came to pass during the stimulation.

Fig. 2 Raman patterns of Ti, PDA, OPDA and RPDA substrates (a); XPS spectra of PDA (b), OPDA (c), RPDA (d), respectively. Table 1. C1s binding energy and relative area (%) Samples

C-C/C-H

C-N

C-O

C=O

PDA

58.9

14.9

21.5

4.7

OPDA

56.6

18.9

18.5

6.0

9



RPDA

62.0

10.4

23.3

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4.1

The XPS detail scan results (C1s) of PDA surfaces were shown in Fig. 2b, Fig. 2c and Fig.2d, the quantitative results were tabulated in Table 1. For all the samples, peaks of C=O (288.6 eV), C-H (284.6 eV), C-N (286.0 eV) and C-O (286.6 eV) were identified. The proportion of quinone group increased from 4.7% to 6.0% after positive voltage processing (+0.5 V) and decreased to 4.1% after negative voltage processing (0.2 V); while the proportion of phenolic hydroxyl decreased from 21.5% to 18.5% after positive voltage processing (+0.5 V) and increased to 23.3% after negative voltage processing (-0.2 V). The C=O/C-O ratios were 34% and 17%, respectively. That was contributed to redox reactions under electrical stimulation, during which the phenolic hydroxyl group was converted to quinone group under positive voltage, and vice versa. Such results indicate that PDA could be easily converted to oxidation (OPDA) and reduction (RPDA) state by different constant voltage processing.

Fig. 3 The amount of BSA adsorbed on PDA, OPDA and RPDA, respectively; data represent mean ± SD (n = 3), ***p < 0.001.

10


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3.2 Protein adsorption and conformation Protein adsorption on surfaces is important in determination of the performance of biomaterials. Protein layers formed at the bio-interface can greatly influence cellular responses and even biological responses of materials’ surfaces. In order to understand the effects of electrical driven redox state variations on protein behaviors, BSA was used as a model protein. As shown in Fig. 3, the quantity of BSA adsorbed on OPDA was 2.2 times that of BSA adsorbed on PDA, moreover, the quantity of BSA adsorbed on RPDA was 3 times that of BSA adsorbed on PDA. The changes of redox state caused by electrical stimulation had greatly enhanced the protein adsorption capacity of PDA, which may affect the subsequent cellular responses. It was well-known that the behavior of protein adsorption on samples was influenced by changes of surface performance, such as surface topography, water contact angle, surface potential and so on. It was mentioned earlier that surface topography and roughness did not change much after electrical stimulation, and the change of water contact angle was within 5 degrees (As shown in Table. S1). As for surface potential (Fig. 4), it was measured by calculating the average of 5 different points, the surface potential decreased from 468±8 mV to 357±11 mV after PDA changed into OPDA, it decreased even more to 122±5 mV after it transformed to RPDA. Combined with protein adsorption conditions, the lower the surface potential is, the more powerful the protein adsorption capability is.

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Fig. 4 KFM images of PDA (a), OPDA (b), RPDA (c); Second derivative resolution enhancement and curve-fitted amide I region (1700–1600 cm-1) for BSA on PDA (d), OPDA (e) and RPDA (f), respectively. Table 2. Secondary structure content of BSA adsorbed on PDA, OPDA and RPDA β-sheet

random coil

α-helix

β-turn

PDA

36

10

20

34

OPDA

42

16

24

18

RPDA

36

17

36

11

Secondary structure (%)

12


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Moreover, the changes of surface properties, especially surface potential, had a considerable effect on the secondary conformation of BSA. As shown in Fig. 4, a quantitative analysis of the secondary conformation for BSA on PDA, OPDA and RPDA was further carried out. As tabulated in Table 2, the calculated results showed that BSA on PDA was 20% α-helix (1653 cm-1), 36% β-sheet (1618, 1626 and 1635 cm-1), 34% β-turn structure (1672 and 1680 cm-1), and 10% random coil (1644 cm-1). An obvious amount decrease of β-turn structure from 34% (PDA) to 18% (OPDA), 11% (RPDA) and a slight increase in random coil from 10% (PDA) to 16% (OPDA), 17% (RPDA) were observed, indicating that some β-turn structure converted into random coil when surface chemical groups changed along with surface potential. It also made a dramatic increase of α-helix from 20% (PDA) to 24% (OPDA), 36% (RPDA), indicating that the chemical environment of RPDA somewhat enhanced the hydrogen bonding networks and induced the significant structural change of BSA, causing the increase of α-helix of BSA. Combined with previous study on surface group changes of PDA, OPDA and RPDA, the phenolic hydroxyl groups on RPDA were effective on changing the secondary structure of BSA adsorbed, especially α-helix and β-turn structure, these changes may further affect the responses of cells. 3.3 Cell responses on redox states mediated protein conformation In order to explore the regulatory influence of conformational changes of proteins on cells, the OD value of osteoblasts on PDA samples (PDA, OPDA, RPDA) with BSA pre-adsorbed were obtained after cultured for 1 and 5 days (as shown in Fig. 5a). RPDA 13



showed the best cell adhesion property compared with other states. After cultured for 5 days, compared with PDA film, the OD value of OPDA and RPDA increased, and RPDA was the best one among them for cell adhesion and proliferation.

Fig. 5 Biocompatibility of BSA pre-adsorbed (a) and ALP activity of BMP-2 preadsorbed (b) PDA, OPDA and RPDA, respectively; Cell morphologies of MC3T3-E1 cells on PDA (c), OPDA (d) and RPDA (e) with BMP-2 pre-adsorbed after 1 day of cell culture. F-actin was stained by Alexa-Fluor 594 phalloidin (red), and cell nuclei were stained by DAPI (blue), scale bar are 100 um (left three columns) and 20 um (right). Data represent mean ± SD (n = 3), *p < 0.05, **p < 0.01, ***p < 0.001. As we all know, BSA is a blocking protein that limits cell adhesion.35-37 Nevertheless, OPDA and RPDA showed better cell adhesion and proliferation with more BSA preadsorbed on the surface. In view of the PDA regulated BSA amount and conformation changes discussed previously, such cellular responses could be ascribed to that the secondary structure variations of pre-adsorbed BSA further influenced the following

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adsorption of other biomolecules, and eventually enhanced osteoblasts proliferation. It is noteworthy that such tendency of cell proliferation was different from Tan’s work,29 in which electrically stimulated PDA was directly used for cell culture. However, considering the complex composition of the culture medium and possible competitive adsorption of various biomolecules, that is actually a different situation. Since it is reported that surface can regulate the conformation and function of proteins38-39 and eventually altered biological activities of subsequent molecules,40 pre-adsorbed proteins regulated by different redox states of PDA may surely affect the subsequent biomolecules adsorption and consequent cellular responses. Such results imply the regulation of pre-adsorbed biomolecules may lead the PDA surface to show different regulation effects on cells.

Fig. 6 Schematic illustration of different cell responses on OPDA and RPDA. 15



In order to further evaluate the effects of such regulation, bone morphogenetic protein 2 (BMP-2), which is an important growth factor for bone regeneration, was chosen for further investigation. BMP-2 was pre-adsorbed on PDA, OPDA and RPDA before cell seeding. As shown in Fig. 5c-e, after cells were cultured for 1 day, the spreading area of cells on OPDA was significantly larger than PDA and RPDA, and more filopodia was observed (cell spreading areas were shown in Fig. S3). Such early state of cells on OPDA is reported to be able to promote osteogenesis-related signal transduction.41 Moreover, it was found that OPDA showed the best ALP activity after 14-day cell culture, as shown in Fig. 5b. Since the combination with biomaterial matrix makes BMP-2 achieve maximal functional efficacy,42 and BMP-2 in associated to the surface can better imitate the native physiology and enhance its impactful biological effects,43-44 such effects of OPDA on BMP-2 might imply that BMP-2 conformation on OPDA was more suitable for binding to receptors that related to osteogenic differentiation on the cell surface. In view of that the phenolic hydroxyl groups on RPDA were effective on cell adhesion and proliferation by changing the secondary structure of BSA pre-adsorbed, the quinone groups on OPDA also have important effects on cell differentiation by changing the state of BMP-2 pre-adsorbed (Fig. 6). That means that electric regulated PDA redox states can further affect the state of adsorbed protein molecules and eventually direct osteoblasts proliferation and differentiation.

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Conclusions In this study, it was found that PDA with different redox states could regulate the amount and secondary structure of pre-adsorbed proteins, which in turn influenced the subsequent interactions between different protein molecules, and eventually affected cellular responses. Both the reduced state and oxidized state of PDA could affect cellular responses through changing the secondary structure of different adsorbed protein molecules. This research provides more insights of regulation of PDA on cellular responses and demonstrates that PDA could be a more versatile coating materials for biomedical application.

Acknowledgements This work is financially supported by the National Natural Science Foundation of China (51872259, 51772273 and 51472216) and the 111 Project (B16042).

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For Table of Contents Use Only Redox state of PDA directs cellular responses through pre-adsorbed protein Yifei Zhu, Lili Yao, Wenjian Weng, Kui Cheng*

27



Supporting Information Page S2. Fig. S1 Cyclic voltammogram of Ti- PDA in 0.1 M PBS (pH 7.0) at 50 mV s-1 scan rate. Page S3. Fig. S2 SEM (a) and AFM (b) images of Ti. Page S4. Table S1. Water contact angles of PDA, OPDA, RPDA. Page S5. Fig. S3 Spreading area of MC3T3-E1 cells on PDA, OPDA and RPDA with BMP-2 pre-adsorbed after 1 day of cell culture.

28


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Redox State of PDA Directs Cellular Responses ... - ACS Publications

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