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Multifunctional biomaterial coating based on bio-inspired polyphosphate and lysozyme supramolecular nanofilm Xinyuan Xu, Dongyue Zhang, Shangwei Gao, Toshikazu Shiba, Quan Yuan, Kai Cheng, Hong Tan, and Jianshu Li Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.8b00002 • Publication Date (Web): 12 Feb 2018 Downloaded from http://pubs.acs.org on February 18, 2018

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Biomacromolecules

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Multifunctional Biomaterial Coating Based on Bio-Inspired Polyphosphate and Lysozyme Supramolecular Nanofilm

4

Xinyuan Xua,#, Dongyue Zhanga,#, Shangwei Gaoa, Toshikazu Shibab, Quan Yuanc, Kai

5

Chenga, Hong Tana, Jianshu Lia,*

1 2

a

6

College of Polymer Science and Engineering, State Key Laboratory of Polymer

7

Materials Engineering, Sichuan University, Chengdu, 610065, China

8

b

9

c

Regenetiss Inc., 1-7-20, Higashi, Kunitachi, Tokyo 186-0002, Japan

State Key Laboratory of Oral Diseases, West China School of Stomatology, Sichuan University, Chengdu, 610041, China

10

11

#

Authors contributed equally to this work.

12

ABSTRACT: Current implant materials have widespread clinical applications together

13

with some disadvantages, the majority of which are easily to induce infection and hardly

14

to exhibit biocompatibility. To efficiently improve their properties, the development of

15

interface multifunctional modification in a simple, universal and environment benign

16

approach becomes a critical challenge and appeals numerous attention of scientists. In

17

this study, a lysozyme-polyphosphate composite coating was fabricated for 1

ACS Paragon Plus Environment

Biomacromolecules 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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titanium(Ti)-based biomaterial in order to obtain multifunctional surface. This coating

2

was easily formed by sequentially soaking the substrate in reduced-lysozyme and

3

polyphosphate solution. Such a composite coating has showed predominant antibacterial

4

activity against Gram-negative bacteria (E. coli), and improved cell adhesion,

5

proliferation and differentiation, which are much better than the pure substrate. This

6

facile modification endows the biomaterial with anti-infective and potential

7

bone-regenerative performance for clinical applications of biomaterial implants.

8

Keywords: bio-inspired, lysozyme, polyphosphate, antibacterial, osteogenicity

9 10

INTRODUCTION

11

Implant materials have been widely used in orthopedic surgery and orthodontics,

12

especially titanium and its alloys due to their outstanding mechanical and chemistry

13

properties, high corrosion resistance and excellent biocompatibility.1 Nevertheless,

14

subsequent studies discovered several drawbacks of titanium implant materials (Ti),

15

including excessive mechanical stress due to elastic mismatch between implant and bone,

16

low cell and tissue responses on the account of implant surface’s bio-inertness,

17

post-operation infection caused by aggressive bacteria on the surface of implant, etc.,2-4

18

which restrict their clinical applications. Therefore, it is a vital significance that

19

modification on titanium implants to obtain the biocompatibility and bio-functionality.5, 6

20

For instance, a series of novel antibacterial systems were developed to overcome the risk

2


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of post operation infection7, 8. As for the improvement of implant surface’s bio-inertness,

2

there have contemporary modification mainly utilized physical and chemical methods to

3

modify surface topography and/or introduce certain functional molecules to enhance

4

biocompatibility between cell/tissue and Ti-implants, like polishing, plating, ion

5

implantation, chemical grafting, etc.9-12 However, there are still some drawbacks of these

6

modifications, especially complicated processing as well as toxic chemicals, which could

7

cause environmental pollution.13 Apart from that, recent implant modifications have been

8

concentrating on integrating multiple functions into one material to improve the

9

comprehensive performance of implants.14,

15

Generally, these integrations also face

10

additional challenges and difficulties, including economic consideration and rigorous

11

limitation on surface architecture and geometry. Therefore, the implant modification still

12

demands an environmental-friendly and facile process to overcome the existed

13

limitations and realize the multifunction of biomaterial surface.

14

Recently, Yang et al. reported an interesting phenomenon that lysozyme reduced by

15

tris (2-carboxyethyl) phosphine (TCEP) can be used to fabricate a phase-transition

16

lysozyme (PTL) nanofilm through a fast phase transition process.16 Moreover, the PTL

17

nanofilm

18

biocompatibility in vitro and in vivo, as well as transferrable robust adhesion to virtually

19

arbitrary materials.17 It is worth mentioning that this PTL nanofilm can be considered as

20

an ideal modification platform, where functional groups could be bonded via chemical

21

methods to fulfill the specific performance request. Besides, the PTL nanofilm is

possessed

broad-spectrum

antibacterial

activity,

and

remarkable

3



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demonstrated to be positively charged, which could support a robust adsorption of

2

negatively-charged

3

Meanwhile, as reported in the previous researches, polyphosphate exhibits excellent

4

properties of enhancing proliferation of normal human fibroblast cells18,

5

accelerating the initial attachment, proliferation, and differentiation of mouse

6

osteoblast-like cells (MC3T3-E1).20,

7

confirmed that artificial introduction of polyphosphate promotes the bone growth and

8

regeneration processes.22, 23 Based on these two aspect regards, integration of positive

9

charged PTL nanofilm with negative charged polyphosphate may achieve the fabrication

10

materials

through

21

straightforward

electrostatic

interaction.

19

and

More recently, in vivo experiments have

of multifunctional biomaterials coating.

11

In this research, a facile and efficient approach to modify titanium implants (Ti)

12

surface with lysozyme supramolecular nanofilm (Ly) and polyphosphate (Px) was

13

developed (Scheme 1). Just through the sequentially soaking the Ti substrates into

14

reduced lysozyme and polyphosphate solution, the targeted lysozyme and polyphosphate

15

composite coating (Ti-Ly-Px) was successfully constructed via the electrostatic

16

interaction, which was confirmed by the characterization of surface morphologies and

17

features. Meanwhile, the treated surface exhibits an outstanding antibacterial activity as

18

well as promotive performances on cell attachment, proliferation and differentiation

19

based on the results of the cell and bacterial assays. Through this work, we open a sight

20

into exploring a convenient and common modification strategy for improving the clinical

21

performance of biomaterial implants. 4


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Biomacromolecules

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Scheme 1. Preparation and biological evaluation of Ti-based biomaterials modified by

4

lysozyme and polyphosphate composite coating.

5 6

MATERIALS AND METHODS

7

Materials. 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) and tris

8

(2-carboxyethyl) phosphine (TCEP) were purchased from J&K Scientific Ltd. and

9

Energy Chemical Co. (Beijing, China), respectively. Polyphosphate was kindly provided

10

by RegeneTiss Inc. (Tokyo, Japan). Lysozyme and other chemicals were obtained from

11

Sigma-Aldrich (Shanghai, China). All the reagents were used without further

12

purification. Titanium sheets for biomedical application (0.2 mm in thickness) meeting 5



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the standard of ASTM (American Society Test & Materials, F67-2002) were purchased

2

from Baoji Shengze Metal Co., Ltd., China. Polished monocrystalline silicon sheets were

3

purchased from Lijing Silicon materials Inc., China. Before used, both titanium and

4

silicon sheets were sequentially rinsed with ultrapure water, alcohol and acetone under

5

the supersonic condition for 15 mins and then dried with nitrogen airflow.

6

Fabrication

of

lysozyme-polyphosphate

composite

coating.

First,

the

7

supramolecular nanofilm was prepared according to the method of Yang et al.24 The

8

lysozyme solution (20 mg/mL, pH=7.4) was blended with equal volume of TCEP

9

solution (50 mM, pH=6.2) to obtain a well-distributed phase-transition buffer, which was

10

subsequently added to a container. Then the pre-treated titanium sheets (Ti) were

11

immerged into the above buffer solution to incubate for 50 minutes. When the incubation

12

was over, these incubated titanium sheets were repeatedly rinsed with ultrapure water for

13

several times to remove those residues on the surface and then dried with nitrogen

14

airflow. Lysozyme supramolecular nanofilm modified titanium sheets (Ti-Ly) were

15

attained through this method. Subsequently, in a dark place, these Ti-Ly samples were

16

respectively immersed in polyphosphate solutions with different concentrations (1, 5 and

17

10 mg/mL) for 30 minutes, allowing the polyphosphates to adsorb onto the surface of

18

Ti-Ly samples. After removing all the residuals in the same treatment described above,

19

the targeted lysozyme-polyphosphate modified Ti samples (Ti-Ly-Px, x refers to the

20

concentration of polyphosphate solution) were finally obtained.

6


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Characterizations. The chemical composition of a series of coated samples were

2

determined by X-ray photoelectron spectroscopy (XPS, AXIS Ultra DLD, Kratos, Japan)

3

with a focused monochromatic Al Kα source (1486.6 eV) for excitation. The surface

4

morphologies of samples were visualized by field-emission scanning electron

5

microscopy (FESEM, FEI, USA) and atomic force microscopy (AFM, MFP-3D-BIO,

6

Oxford Instrument, UK). Due to the demand of flat surface for AFM device, we

7

substituted silicon sheets for titanium sheets with identical fabrication process, sicne the

8

supramolecular nanofilm could steadily adhere to any type of substrate and completely

9

cover the surface capacity of substrate with the same properties.25, 26 The rinsing solution

10

before and after the addition of polyphosphate were collected, and the size of granules

11

detached from sample surface in the solution were determined by dynamic light

12

scattering (Malvern Instruments Ltd, UK). Quartz crystal microbalance (QCM, 3T

13

GmbH & Co., Germany) was utilized to ensure the interaction between lysozyme

14

supramolecular (Ly) nanofilm and polyphosphate (Px) and quantify the adsorption mass

15

(ng/cm2) of polyphosphate, which is -4.3 folds of the frequency deviations. First, the

16

device was calibrated using pure quartz crystal Au. Second, some pure quartz crystal Au

17

were coated with Ly following the method mentioned above. Finally, the treated quartz

18

crystal Au were submitted to microbalance to test their vibrating frequency after injecting

19

samples and rinsing with ultrapure water. Hydrophilicity of the sample surfaces was

20

determined by water contact angle meter (Attension Theta, Biolin Scientific, Sweden).

21

Zeta potential was recorded by using electrokinetic analyzer (SurPASS, Anton Paar, 7



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1

Austria). Both characterizations also require level surfaces, thus silicon sheets were

2

employed again.

3

Antibacterial assays. This part of assay adopted previously reported methods2, 27 by

4

using Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) to evaluate the

5

antibacterial activity. All prepared samples were sterilized via 30-minutes immersion in

6

75% alcohol. Both bacteria solutions were adjusted to suitable concentrations (105

7

CFU/mL for E. coli and 106 CFU/mL for S. aureus) before use. 1 mL of the bacterial

8

suspension was added to the well with sample and co-incubated at 37 ℃ for 8 h, and then

9

these samples were rinsed thrice with PBS solution. After that, all samples with adsorbed

10

bacteria were divided into three groups. The first group was used to test the bacterial

11

adhesion by plate counting method, as described as follows. The adsorbed bacteria on

12

each sample were detached into 4 mL of fresh liquid culture medium by 5-minute

13

ultrasonic vibration (50 W). Subsequently, the resulting bacterial suspensions were

14

diluted to 104 folds, and each dilution was spread plated on the agar culture-medium to

15

count bacteria after 24-hour cultivation at 37 ℃. For the second group, scanning electron

16

microscope (SEM Quanta 250, FEI, US) was utilized to visualize the surface

17

morphology of the bacteria-adsorbed samples. The samples were soaked on the

18

glutaraldehyde solution for 12 h to immobilize bacteria. After that, these samples were

19

gradually dehydrated by alcohol (0, 30, 50, 70, 80, 90, 95 and 100%, 15 min for each

20

concentration) and then dried in air. The last group was stained using LIVE/DEAD™

21

BacLight™ Bacterial Viability Kit (ThermoFisher Scientific, USA) in the dark for 15 8


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min. Then these stained samples were imaged using an A1R MP ା confocal laser

2

scanning microscope (CLSM, Nikon Co., Japan) equipped with a 20× objective lens by

3

image channels set at excitation/emission maxima 488/500 nm for STYO 9 that stained

4

live cells, 488/620 nm for propidium iodide that stained dead cells. The antibacterial rate

5

and adsorption ratio was calculated by Image Pro Plus 6.0 software basing on the

6

Live/Dead staining assay. The untreated Ti sample was set as the control to calculate the

7

ratio. All tests were tested in triplicate.

8

Cell

culture.

Mouse

osteoblast-like

cells

(MC3T3-E1)

were

cultured

in

9

alpha-minimum essential medium (α-MEM) supplemented with 10% fetal bovine serum

10

(FBS) and 1% penicillin-streptomycin solution. The cells (passage 3) with a density of

11

3×105/mL were used in the following assays, and were cultured at 37 ℃ in a humidified

12

5% CO2 atmosphere.

13

Cell adhesion and proliferation assay. The MC3T3-E1 cells were seed on each

14

surface of samples placed in 48-wells plates and cultivated in the culture medium for 1,

15

3, 5 h and 1, 3, 5 days to estimate cell adhesion and growth behavior, respectively. The

16

culture medium for cell adhesion behavior had no addition of FBS, while there was 10%

17

FBS additive in the culture medium for the cultivation of proliferated cell. The cell

18

viability was detected by cell counting kit-8 (CCK-8, Mashikimachi, Kumamoto, Japan),

19

according to previously reported method.28 Briefly, these samples after culture were

20

rinsed thrice with PBS and then cultivated in fresh medium supplemented with 10% (v/v)

9



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CCK-8 for 3 h, the final absorbance at 450 nm of the culture medium was determined by

2

micro-plate reader (Kehua Bio-Engineering co., Ltd., Shanghai, China).

3

Determination of cell spreading area. The adhered cells on the samples were firstly

4

stained with 10 µg/mL fluorescein diacetate (FDA, Sigma-Aldrich, USA) for 5 min and

5

rinsed thrice with PBS, then observed under a fluorescence microscope (IX-71,

6

Olympus, Japan). The average cell spreading area was analyzed by the Image Pro Plus

7

6.0 software. At least 150 cells were analyzed for each sample.

8

Fluorescence labeling. The cells were seeded on the sample surfaces in the

9

FBS-free/containing culture medium for 3 h and stained by various dyes after three

10

rinsing in PBS. The stained method followed the work by Chen et al.28 The 4%

11

paraformaldehyde was utilized to immobilized the adsorbed cells at 4 ℃ for 15 min.

12

Subsequently, the samples were exposed in the Trition X-100 (0.1% in PBS) for 5 min,

13

phalloidin-TRITC (1 µg/mL in PBS) for 30 min and DAPI (5 µg/mL in PBS) for 10 min

14

in sequence. All samples were rinsed three times with PBS after each soaking. We

15

utilized fluorescence microscope (IX-71, Olympus, Japan) to observe the stained

16

samples.

17

Alkaline phosphatase (ALP) activity assay. For each sample, 3×105 MC3T3-E1 cells

18

were seeded on the surface in the FBS-containing culture medium at 37 ℃ for 3, 7, 14

19

days in a humidified 5% CO2 atmosphere. Then the cells adhered on the various surface

20

were individually collected in PBS buffer with 1% Trition X-100. The above solution

21

was supersonic broken and centrifuged to obtain the cell lysis buffer, and then the 10


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content of Alkaline Phosphatase (ALP) in the buffer was analyzed using ALP Kit

2

(Beyotime Biotechnology Co., China). The cell culture medium was refreshed every

3

three days during the assay.

4 5

RESULTS AND DISCUSSION

6

Fabrication of lysozyme-polyphosphate composite coating on titanium (Ti-Ly-Px).

7

Lysozyme, as a safe material approved by U. S. FDA25, can fabricate stable adhered

8

nanofilm on various substrates via simple one-step reduction, and the nanofilm can

9

provide a versatile platform to bond with functional groups or adsorb negatively charged

10

molecules. Thus the anionic polyphosphate, which can act as metabolic fuel for bone

11

regeneration,29 can form electrostatic interaction with lysozyme nanofilm to endow

12

potential osteogenesis capacity. To ensure that the modified coating was successfully

13

constructed, the chemical composition of the samples was firstly investigated by XPS.

14

As displayed in Figure 1 and Table 1, for pure titanium sheets, in addition to the

15

expected titanium and oxygen (532.5 eV, 43.21%), probably existing in the form of

16

titanium dioxide, carbon (284.4 eV, 52.84%) and traces of nitrogen (398.6 eV, 3.95%)

17

were also observed, which might be attributed to unavoidable traces of organics

18

adsorption from ambient air.30, 31 While for Ti-Ly sample, the relative nitrogen content

19

increased by about 15% and sulfur peaks (2s 229.7 eV, 2p 165 eV) were clearly

20

identified in the curve, as well as the absence of titanium peak. This phenomenon

21

demonstrated that the Ti sample was thoroughly coated by the supramolecular nanofilm, 11



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1

which was the only reason for nitrogen increase and sulfur presence. The mechanism for

2

the robust adhesion between the supramolecular nanofilm and titanium substrate can be

3

attributed to a bio-inspired adhesion from the amyloid structures,16,

4

substantially exist in the supramolecular nanofilm.24, 26, 34 All of Ti-Ly-Px samples have

5

similar XPS curves, presenting not only the existence of nitrogen and sulfur but also

6

phosphorus (2s 133 eV, 2p 191 eV). Apparently, the polyphosphate was successfully

7

adsorbed onto the supramolecular nanofilm. It was important to note that there was no

8

sign of phosphorus peak in Ti-Ly XPS curve, which means that TCEP did not remain in

9

lysozyme nanofilm after processing, corresponding to the report of Wang et al..25

32, 33

which

10 11 12

Figure 1. XPS curve of Ti, Ti-Ly, Ti-Ly-Px samples. A part of the graph (binding energy

13

ranging from 0 to 250 eV) was enlarged for detailed analysis and placed on the right.

14 15 16 12


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Biomacromolecules

1

Table 1. Relative atomic content of different samples determined by XPS analysis. Relative atomic content (%) Sample C

N

O

P

Ti

52.84

3.95

43.21

0.00

Ti-Ly

68.22

18.48

13.30

0.00

Ti-Ly-P1

63.82

12.79

22.27

1.12

Ti-Ly-P5

62.27

14.25

22.22

1.26

Ti-Ly-P10

63.90

13.41

21.30

1.38

2 3

Quartz crystal microbalance (QCM) was utilized to determine the actual absorbed

4

content of polyphosphate (Px) in the composite coatings, which were prepared with

5

various concentrations of Px solution. As for its well-known adhesive property, the strong

6

interaction between lysozyme and any substrate was described in detail by other group.26

7

Therefore, before investigating the interaction between composite coating and Au

8

substrate, the interaction between polyphosphate and Au was estimated primarily. Based

9

on the curve in Figure 2A, the obvious frequency decline occurred after injecting

10

different concentration of Px solution. The frequency of all the Px samples could come

11

back to the baseline after rinsing, which confirmed the negligible interaction between

12

polyphosphate and Au. As shown in Figure 2B-D, the ordinate value at zero of

13

lysozyme-treated Au was lower than that of pure Au, due to the reduction on wafer

14

waving, which was caused by the absorption of lysozyme onto the Au substance. 13



Page 14 of 38

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Furthermore, there existed a notable frequency change (as marked ∆f in Figure 2B-D),

2

indicating that polyphosphate had a strong interaction with Ly supramolecular nanofilm.

3

As mentioned above, the sample weights were calculated via the formula ∆m=-4.3*∆f

4

and the corresponding data were collected in Table 2. The same trend could be clearly

5

observed that the mass of adsorbed polyphosphate increased accompanying with the

6

increase of polyphosphate concentration, which was also concluded from the results of

7

XPS.

8 9 10

Figure 2. QCM curves obtained by injecting polyphosphate solution on (A) pure Au and (B, C, D) lysozyme treated Au.

11 14


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Biomacromolecules

1 2

Table 2. Frequency deviation and adsorbed mass of polyphosphate detected by QCM. 2

Sample

∆f (Hz)

∆m (ng/cm )

Pଵ

-3.13

13.46

Pହ

-18.31

78.73

Pଵ଴

-39.16

168.39

3 4

Morphology of Ti-Ly-Px composite coating. FESEM and AFM were employed to

5

directly observe the surface morphology of modified titanium sheets. From Figure 3A-Ti

6

sample, numerous irregular cracks on pure titanium surface were obviously seen, while

7

these cracks were covered by aggregates of lysozyme and became invisible in Ti-Ly and

8

Ti-Ly-Px samples. The morphology of modified Si sheets surface was also verified that

9

great amounts of lysozyme nanoparticles assembled on the surface, which were shown in

10

AFM images (Figure 3B). Especially at the enlarged AFM images on the left top of

11

Ti-Ly and Ti-Ly-Px samples, the structure of lysozyme nanofilm as reported in previous

12

report could be clearly distinguished from the flat background. Both FESEM and AFM

13

characterizations indicated that the supramolecular nanofilm was constructed as our

14

expectation. It was necessary to point out that there was no obvious morphological

15

distinction between Ti-Ly and Ti-Ly-Px owing to the low molecular weight (about 6

16

kDa) of polyphosphate. Compared the surface morphology of Ti-Ly and Ty-Ly-Px,

17

certain amounts of larger granules on the surface of supramolecular nanofilm

18

disappeared due to repeat rinsing process, especially after incubated with polyphosphate. 15



Page 16 of 38

1

Electrostatic interaction from polyphosphate molecules further decreased the adhesion of

2

lager granules to nanofilm, which made them readily being rinsed out by ultrapure water.

3

Dynamic light scattering (DLS) assay was also conducted to investigate the size of

4

granules that were detached from the samples surface after rinsing. The size of granules

5

dropped from Ti-Ly-Px surface was approximately 2000-2500 nm (Figure 3C and Table

6

3), which was corresponding to that of granules on the FESEM and AFM images of

7

Ti-Ly. All the characterization mentioned above confirmed that the compact

8

lysozyme-polyphosphate composite coating was efficiently constructed via soaking and

9

rinsing method.

10 11

Figure 3. Surface morphology characterization. (A) FESEM images taken from different

12

type of samples. (B) AFM images of different modified samples, inset shows

13

morphology in a bigger scale (scale bar: 500 nm). (C) DLS results of different rinse

14

solution after rinsing the samples. 16


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Biomacromolecules

Table 3. Granule size of different kind of rinse solution by DLS analysis Peak 1 Sample Size (d . nm)

Percenta

Rinse solution of Ti-Ly-P

2754±843.4

72.8


2284±1180.0

79.4


2485±668.4

100

1

5

10

1

a. The “percent” means the percentage of main peak in the spectra, calculated by DLS software.

2

Hydrophilicity and zeta potential. Since modification has great effects on the surface

3

performance, hydrophilicity change was also measured by water contact angle (WCA).

4

According to the results displayed in Figure 4A, the WCA of Ly supramolecular

5

nanofilm was around 75°, while those samples with Ly-Px composite coating displayed

6

gradual declines on WCA, which represents a better hydrophilicity. Meanwhile, the

7

WCA decline degrees were in according with the concentration increases of

8

polyphosphate solution. Referred to the previous report,35 disulfide bonds in lysozyme

9

could be broken down by TCEP accompanying with the conformation changes, which

10

lead to the explosion of the inner hydrophobic groups. As a result, the

11

conformation-changed

12

Subsequently, the introduction of polyphosphate onto the lysozyme nanofilm caused an

13

increase of hydrophilicity, which might be attributed to the interaction between the

14

anionic polyphosphate and those cationic hydrophobic groups in lysozyme nanofilm. The

lysozyme

presented

a

relatively

poor

hydrophilicity.

17



Page 18 of 38

1

hydrogen bonds between water molecules and anion groups in polyphosphate also

2

partially elevated the entire hydrophilicity of the Ly-Px composite coating.

3

In addition, we employed zeta potential (ZP) measurement to test the surface electronic

4

properties. The relative results were shown in Figure 4B. The untreated Si sample

5

exhibited electronegativity (ZP=-37 mV), which became electropositive (ZP=31.5mV)

6

after initially modified by lysozyme supramolecular nanofilm. As mentioned by Gu et

7

al.,17 lysozyme supramolecular nanofilm was rich of electropositive amino groups,

8

resulting in the high positive zeta potential. Once Ly-Px composite coating was formed

9

on the Si surface, all the samples displayed electronegativity again (ZP=-30 mV) due to

10

the adsorption of anionic polyphosphate. At the same time, the surface electronegativity

11

was related to the concentration of polyphosphate solution when fabricated the composite

12

coating.

13

18


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Biomacromolecules

1

Figure 4. Water contact angle (A) and zeta potential (B) data of pure silicon (Si),

2

supramolecular nanofilm-modified silicon (Si-Ly), and lysozyme-polyphosphate

3

composite coating silicon (Si-Ly-Px) surfaces.

4

Antibacterial activity. To explain the following results of antibacterial and

5

cell-related function, we summarize some relative articles about properties of

6

titanium-based materials. Pure titanium and its alloys without specific polishing process

7

have an oxide layer, which exhibits WCA of 78 ± 3° and negative zeta-potential (around

8

-8mV).36-38 The pure titanium sheets in this article were treated through washing without

9

any modification, thus we hypothesized that Ti sample possessed uniform fundamental

10

surface properties, including WCA and zeta potential.

11

Excellent antibacterial activity can effectively eliminate the incidence of

12

post-operation infection, which is one of the main reasons for implantation failure.39, 40

13

The anti-adhesive and bactericidal activity of all the samples were estimated by plate

14

count method and live/dead two color fluorescence assay, respectively. Modified

15

titanium surfaces, including Ti-ly and Ti-Ly-Px, displayed less adsorbed bacteria density

16

(E. coli) than that on the titanium surfaces, especially Ti-Ly-P10 sample showed

17

minimum number of adsorbed bacteria (Figure 5). According to the live/dead two color

18

fluorescence assay (Figure 6, Table 4), Ti-Ly sample exhibited the best bactericidal

19

activity (the most dots of red), however, a great amount of both live and dead bacteria

20

adhered to the surface. The excellent bactericidal performance of Ly supramolecular 19



Page 20 of 38

1

nanofilm was corresponding to the previous report,17 because its surface possessed lots of

2

hydrophobic and positively charged groups, which could interact with anionic bacterial

3

cells and perturb the cell wall via hydration effect, finally leading to the death of bacteria.

4

Compared to Ti-ly sample, an obvious decline of bacteria density (green dots) was

5

observed with the concentration increase of polyphosphate on the Ti-Ly-Px sample

6

surface as well as definite bactericidal activity (red dots) was obtained. The introduction

7

of polyphosphate can transform the positive surface into the negative one and enhance its

8

hydrophilicity, which were related to the effect of interaction with and perturbation on

9

the cell walls of microbes respectively.41,

42

The synergistic effect of both changes

10

resulted in the decrease on bactericidal activity.43,

44

11

negative-charged polyphosphate that inhibited E. coli adhesion via the electrostatic

12

repulsion towards the negatively charged cell wall.45 Hence, lysozyme-polyphosphate

13

composite coating exhibited both anti-adhesive and bactericidal activity against E. coli.

However, it was the

14 15

Figure 5. Antibacterial activity characterization. (A) Plate count assays and (B) FESEM

16

images of Ti, Ti-Ly, Ti-Ly-Px (X=1, 5, 10 mg/mL) surfaces after 8-hour co-incubation 20


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Biomacromolecules

1

with E. coli.. The bars connected by a horizontal line differed significantly for the

2

amount of cell adhesion, as assessed by one-way analysis of variance (**: p< 0.01).

3 4

Figure 6. Fluorescence images of live/dead assays for Ti, Ti-Ly, Ti-Ly-Px (X=1, 5, 10

5

mg/mL) surfaces against E. coli. Live bacteria were dyed green by STYO 9 and dead

6

bacteria were dyed red by propidium iodide.

Table 4. Antibacterial rate and adsorption ratio (E. coli)

7

Sample

Antibacterial rate (%)

Adsorption ratio

Ti

15.59

1

Ti-Ly

70.68

1.25

Ti-Ly-P1

56.24

0.70

21



Page 22 of 38

Ti-Ly-P5

53.92

0.65

Ti-Ly-P10

50.88

0.46

1

Nevertheless, the anti-adhesive performance of the composite coating against S. aureus

2

had no significant difference among all the samples (Figure S1, S2, Table S1), which was

3

ascribed to the structure difference of cell wall between Gram-negative and

4

Gram-positive bacteria. Referred to a number of previous studies,46-48 Gram-positive

5

bacteria (such as S. aureus) performed a higher phosphate removal activity than

6

Gram-negative bacteria (such as E. coli). It probably implied that S. aureus were prone to

7

harvest polyphosphate to regulate their internal phosphorus levels and support their own

8

metabolism.

9

Cell adhesive behavior. In order to study the cell adhesive behavior on the modified

10

Ti surface, we employed fluorescence labeling to observe the cell distribution and

11

calculate the average spreading area of osteoblast-like cells. Primarily, fetal bovine

12

serum (FBS) co-cultivation assays were operated and the relevant data was shown in

13

Figure S3 and Figure S4. The results displayed no obvious difference among the diverse

14

samples, because FBS had a great capability of promoting cell growth, which would

15

shadow the effect of modified surface to cell.49, 50 However, serum composed of various

16

undefined protein, hormones, antigens, etc., may cause immunes actions in vivo,51, 52

17

which is critical problem in clinical practice for biomaterials and medical devices.

18

Therefore, we evaluated the cell adhesion to samples in the culture medium without FBS. 22


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Biomacromolecules

1

After 3 hours’ co-cultivation without FBS, the number of adhered cells (blue pots) on

2

Ti-Ly-Px samples was significantly more than those on Ti and Ti-Ly samples, as well as

3

the lager spreading area of cell (red pots), which corresponded to the early study on

4

polyphosphate (Figure 7). 9a, 21

5 6

Figure 7. Fluorescence images of adherent cell on the Ti, Ti-Ly, Ti-Ly-Px (X=1, 5, 10

7

mg/mL) surfaces after 3h cultivation in culture medium without FBS. Cell nucleus were

8

dyed blue by DAPI and F-actin stress fibers were dyed red by phalloidin-TRITC.

9

The cell counting kit-8 (CCK-8) assay were utilized to determine the cell adhesion on

10

the sample surfaces. As shown in Figure 8A, the absorbance of Ti-Ly-Px samples after 1

11

h cultivation without FBS had no explicit increase than Ti and Ti-Ly samples; while the

12

value distinctly enhanced after longer cultivation (3 and 5 h).

23



Page 24 of 38

1

In addition, FDA labelled images (Figure S5) were directly utilized to calculate the

2

average cell spreading area (Figure 8B) through the computer software. At the early

3

cultivation stage (3 h) and longer cultivation time (about 24 h), all the samples shown no

4

significant differentiation. According to all the results listed above, the Ly-Px composite

5

coating with better hydrophilicity remarkably promoted the cell adhesive ability on the

6

substrate, which was similar to the previous reports.20, 53, 54

7 8

Figure 8. (A) Cell adhesion after 1, 3, 5 h cultivation and (B) the average cell spreading

9

areas of adherent cell after 3 and 24 h culture in culture medium without FBS on the Ti,

10

Ti-Ly, Ti-Ly-Px (X=1, 5, 10 mg/mL) surfaces. The bars connected by a horizontal line

11

differed significantly for the amount of cell adhesion, as assessed by one-way analysis of

12

variance (*: p < 0.05; **: p < 0.01).

13

Cell proliferation and differentiation. For purpose of determining the cell

14

proliferation and differentiation on various modified surface, the CCK-8 and ALP kit

15

assays were carried out to record the cell growth behavior, as shown in Figure 9. As time 24


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Biomacromolecules

1

went by, the number of cells were constantly increasing for all samples and composite

2

coating with high polyphosphate content displayed a relatively high cell proliferation

3

rate. (Figure 9A). Such positive effect on cell proliferation could be a consequence of

4

combination of multiple factors, as investigated by Maekawa et al.20 and Wang et al.21 It

5

was proposed that polyphosphate might serve as an important regulatory factor during

6

the cell mitosis process. Sufficient polyphosphate would greatly improve the cell

7

response to mitogens and highly accelerate its proliferation process.

8

It is broadly admitted that alkaline phosphatase (ALP) is one of the typical byproducts,

9

which can be considered as the early marker for assessing osteoblastic metabolic activity.

10

In this regard, ALP activity was investigated to estimate the differentiation capacity of

11

osteoblast-like cell. The results in Figure 9B indicated that cells on the Ti-Ly-Px

12

composite coating, especially Ti-Ly-P10 sample, showed highest alkaline phosphatase

13

activity after 7 days’ cultivation. The reason can be explained that polyphosphate

14

contributes to cell osteogenic differentiation, referring to the discovery that

15

polyphosphate is able to promote MC3T3-E1 cell maturation from a resting state to an

16

active osteoblastic cell phase.55 Moreover, it is also demonstrated that artificial

17

introduction of polyphosphate into the bone defect area will accelerate the bone growth

18

or regeneration10,

19

composite coating is able to induce osteoblastic differentiation of MC3T3-E1 cell and

20

potentially enhance osteogenicity.

23

Therefore, it can be concluded that our lysozyme-polyphosphate

25



Page 26 of 38

1 2

Figure 9. (A) Cell proliferation after 1, 3 and5 days cultivation and (B) ALP activity

3

after 3, 7 and 14 days in culture medium with FBS on the Ti, Ti-Ly, Ti-Ly-Px (X=1, 5,

4

10 mg/mL) surfaces. The bars connected by a horizontal line differed significantly for the

5

amount of cell adhesion, as assessed by one-way analysis of variance (*: p< 0.05; **: p

Multifunctional Biomaterial Coating Based on Bio ... - ACS Publications

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