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Construction of functional coating with durable and broadspectrum antibacterial potential based on mussel-inspired dendritic polyglycerol and in-situ-formed copper nanoparticles Mingjun Li, Lingyan Gao, Christoph Schlaich, Jianguang Zhang, Ievgen S. Donskyi, Guozhi Yu, Wenzhong Li, Zhaoxu Tu, Jens Rolff, Tanja Schwerdtle, Rainer Haag, and Nan Ma ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b10541 • Publication Date (Web): 15 Sep 2017 Downloaded from http://pubs.acs.org on September 17, 2017

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ACS Applied Materials & Interfaces

Construction of functional coating with durable and broad-spectrum antibacterial potential based on mussel-inspired dendritic polyglycerol and in-situ-formed copper nanoparticles Mingjun Li,1 Lingyan Gao,*1 Christoph Schlaich,1 Jianguang Zhang,1 Ievgen S. Donskyi,1 Guozhi Yu,3 Wenzhong Li,1 Zhaoxu Tu,1 Jens Rolff,3 Tanja Schwerdtle,4 Rainer Haag,*1 Nan Ma*1,2 1

Institut für Chemie und Biochemie, Freie Universität Berlin, Takustrasse 3, 14195 Berlin, Germany 2 Institute of Biomaterial Science and Berlin-Brandenburg Center for Regenerative Therapies, Helmholtz-Zentrum Geesthacht, 14513 Teltow, Germany 3 Institut für Biologie, Freie Universität Berlin, Königin-Luise-Str. 1-3, 14195, Berlin, Germany 4 Institute of Nutritional Science, Department of Food Chemistry, University of Potsdam, Arthur-Scheunert-Allee 114-116, D-14558 Nuthetal, Germany

Abstract A novel surface coating with durable broad-spectrum antibacterial ability was prepared based on mussel-inspired dendritic polyglycerol (MI-dPG) embedded with copper nanoparticles (Cu NPs). The functional surface coating is fabricated via a facile dip-coating process followed by in-situ reduction of copper ions with a MI-dPG coating to introduce Cu NPs into the coating matrix. This coating has been demonstrated efficient long term antibacterial properties against Escherichia coli (E. coli), Staphylococcus aureus (S. aureus) and kanamycin-resistant E. coli through an “attract-kill-release” strategy. A synergistic antibacterial activity of the coating was shown by combination of two functions of the contact killing, reactive oxygen species (ROS) production and Cu ions released from the coating. Further, this coating inhibited biofilm formation and showed good compatibility to eucaryotic cell. Thus, this newly developed Cu NP-incorporated MI-dPG surface coating may find potential application in the design of antimicrobial coating, such as implantable devices. Keywords: Cu NP-incorporated MI-dPG coating • universal coating • in-situ chemical reduction • antibacterial effect • drug resistant bacteria

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Introduction In the last decade, bacterial infections at the site of an implanted device have been given more and more attention, because of chronic and recurrent infections, which can be lifethreatening to patients.1 At the cellular level, an implant-associated infection is caused by adhesion of the bacteria on the implant surface and subsequent formation of a biofilm at the implant-tissue interface. Once the biofilms are established, it is extremely difficult to treat the infection with conventional antibiotic therapies. Also, local infections could further result in severe systemic infections that occur in the bloodstream.2 Therefore, preventing bacterial adhesion on the implant surface is an efficient solution to prevent these complications.1, 3-5 The ongoing occurrence of drug resistance in pathogens due to over- and misuse of antibiotics has led to another big challenge.6,

7

Therefore, current research is focused on designing

alternative antibacterial agents that reduce the risk of bacterial resistance evolution.3,

8-13

Given this context, it is of considerable interest to develop implant materials embedded with novel antibacterial activity that would offer an effective local antibiotic concentration at the implantation site for preventing the emergence of implant-related infection. Metal nanoparticles, such as silver (Ag),14, 15 copper (Cu),16, 17 gold (Au),18 and zinc (Zn),19 have attracted much attention due to their low cost and excellent antimicrobial properties against a wide range of microorganisms. The small size and high surface-to-volume ratio in comparison with metal salts allow them to closely interact with the membranes of microorganisms, which enhances their antibacterial effect. Besides, considering their antibacterial mechanisms, nanometal-based antibacterial therapies would be an alternative approach to deal with the threat of multiple drug resistance in pathogenic bacteria.20-22 Among them, copper nanoparticles (Cu NPs) have shown huge potentials as antibacterial materials, however, they are not fully exploited so far.23 They have been used as outstanding antibacterial materials attributed to their unique physicochemical characteristics, the induction of oxidative stress by free radical formation and other properties. Thus, extensive research effort has been made to design copper-based antibacterial materials. For instance, excellent antibacterial ability against the gram-negative (E. coli) and gram-positive (S. aureus) bacterial species was shown by starch hydrogel incorporated with Cu NPs.17 The substrate of implants and other medical devices varies from metal materials to ceramic materials and polymers, and require antibacterial coatings. Therefore, substrateindependent coatings with antibacterial properties have recently received great attention.24 In some attempts, polydopamine (PDA) representing the most prominent mussel-inspired adhesive was employed as a universal surface coating for various antibacterial techniques,11,


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25-28

which can be used as an initial polymeric layer that can be further post-modified with

various external antibacterial agents to produce antifouling surfaces. According to our previous studies, mussel-inspired polyglycerol (MI-dPG) effectively mimics the mussel-foot protein 5 (Mfp-5) and can serve as an efficient universal coating.25-27 Similar to the natural protein, one can obtain a really strong adhesion to nearly every kind of substrate through a combination of catechol and amine groups together with the multivalent character of the polymer core.4 In this work, MI-dPG was chosen as the foundation layer for Cu NPincorporated coatings for several reasons. First, in contrast to PDA-based coatings, a porous base layer in the micrometer range can be directly obtained using the MI-dPG, which contributes to the stability of incorporated NPs. Furthermore, the high amount of catechol functions in the coatings fulfills the functions of the metal coordination, which anchors the surface and also the crosslinking of the polymer as shown in Figure 1. As a result of substrateindependent coatings that highly stabilize the Cu NPs by chelation, durability of antibacterial coating can be easily achieved. In addition, the MI-dPG coating with amine groups shows affinity to negatively charged microorganisms,28,

29

which will help to improve the

interactions between antibacterial Cu NPs in the coating and the bacteria in solution. Here, a rapid and efficient approach for antibacterial surface coating was developed based on MI-dPG and Cu NPs, which exhibited excellent, durable and broad-spectrum antibacterial properties (Scheme 1). Although ionic copper is less toxic than ionic silver, the Cu NPs are less stable than Ag NPs, which is the main reason that the Cu NPs are less studied than Ag NPs for their use in the development of antibacterial field.30 To solve this problem, a simple and environmental friendly method was used in this work. Cu NPs were prepared by in-situ chemical reduction of exposed reactive groups of the MI-dPG coating and further stabilized by the coating matrix. Based on the literature, the exposed reactive groups of the MI-dPG coating, the catechol groups, have strong affinity to metal surfaces due to the formation of stable complexes and indeed stabilize the Cu NPs.31, 32 The resulting coating was quite stable when immersed in aqueous solutions or bacterial suspension, and the Cu NPs embedded in the coating were able to constantly release antibacterial Cu ions per day for more than 40 days. In this work, the MI-dPG coating, which acted as an active surface to attract bacteria on the interface, integrated with the antibacterial agent Cu NPs in the coating matrix. Together they worked as a new antibacterial platform via an “attract-kill-release” strategy. The system showed a long-term and highly antibacterial effect against a gram-positive bacterium, Staphylococcus aureus, and a gram-negative bacterium, Escherichia coli, including a drug-resistant strain without significant toxicity toward mammalian cells. The



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contact kill mechanism, ROS dissolved in bacterial suspension, and Cu ions released to the bacterial suspension showed a synergistic effect in the antibacterial behavior. In addition, it was demonstrated that this modified surface could also inhibit the formation of biofilms.

Scheme 1. Schematic illustration of the contact killing of bacteria on a Cu NP-incorporated MI-dPG surface coating via the “attract-kill-release” route.

Results and discussion According to the literature, Cu NPs could be prepared through chemical reduction by using catechol or amine ligands, while MI-dPG with catechol and amine groups could be used to prepare stable and highly efficient hierarchical multilayer coatings.31-33 In order to obtain a stable and efficient Cu NP-incorporated surface coating, three different methods were employed as depicted in Figure 1 and the supporting information. By the first method (Figure 1c, I): the Cu NPs and MI-dPG surface coating was formed by a one-pot synthesis, while the second and third methods were both based on the prefabricated MI-dPG surface coatings. In the second way (Figure 1c, II), a Cu NPs aqueous solution that was synthesized according to the reported procedure in literature34 was used to prepare a Cu NP-loaded surface coating by the diffusion method. In the third approach (Figure 1c, III), the coating was immersed in a CuCl2 solution overnight, in which the residual catechol groups in the MI-dPG coating were oxidized to quinones and thus acted as a reducing agent to generate the Cu NPs. During the redox and chelation reaction, the Cu ions were in situ reduced to Cu NPs and embedded into the thin film. After the Cu NP-incorporated MI-dPG surface coatings were fabricated by these different preparation methods, the stability of all these coatings were investigated by immersion in a bacterial suspension for 72 h. The samples by the third approach demonstrated the best stability (Figure S1). The coatings on the surfaces by method III were intact, while


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the other coatings by method I and II were damaged. Thus, the third method was employed to prepare the Cu NP-incorporated MI-dPG surface coating for further study.

Figure 1. Chemical structure of MI-dPG (a), the mechanism of catechol anchoring and crosslinking (b), and preparation of Cu NP-incorporated MI-dPG surface coatings through three different ways (c). All substrates were prepared on 1.0×1.0×0.1 cm3 samples of glass slides. Scanning electron microscope (SEM) was used to observe the morphologies of bare MIdPG coating and Cu NP-incorporated MI-dPG coating, which demonstrated the formation of a MI-dPG surface coating on the glass slide and the successful introduction of Cu NPs into the coating. For the bare MI-dPG coating shown in Figure 2a and c, the MI-dPG polymer formed multilayer structures on the glass slide, which was consistent with the reported paper.25 After the prefabricated coating was further immersed in CuCl2 solution for overnight, some nanoparticle agglomeration could be observed on the coating surface with the sizes ranging from 50 to 100 nm (Figure 2b and d). In order to identify the subsequent embellished 5 ACS Paragon Plus Environment


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nanoparticle clusters on the coating were Cu NPs formed through the Cu (II) reduction, samples were further characterized by X-ray photoelectron spectroscopy (XPS). In Figure 2e, survey spectra show presence of carbon, nitrogen, oxygen, sodium and copper ions (0.6%) in the Cu NP-incorporated MI-dPG surface coating. In addition, highly resolved Cu 2p spectra clearly indicate the presence of Cu (0) and Cu (II) at 933.2 eV and 935.0 eV, which was a direct evidence for the formation of Cu NPs in the coating. Moreover, the spectra revealed that the ratio of the content of Cu (0) and Cu (II) was 1:2. In Figure 2e, peaks of C (1s), N (1s), and O (1s) attributed to the MI-dPG polymer can be observed. In addition, a peak in the Cu (2p) at 934.1 eV and 943.8 eV corresponding to Cu (0) and Cu (II) was observed, which gave direct evidence for the formation of Cu NPs in the coating. The spectra in Figure 2f also revealed that the ratio of the content of Cu (0) and Cu (II) was 8:1 and that the atomic percent of the Cu was 0.6%. These XPS results indicated that Cu (II) ions in the solution could be successfully in situ reduced to Cu (0) by the remaining catechol groups on the prefabricated coating, which led to the formation of Cu NPs and further stabilization of the coating surface. Furthermore, according to the ratio of Cu (0) and Cu (II) content, the overwhelming majority of the existing atomic copper in the coating was Cu NPs. Cu (II) ions were introduced into the coating matrix by chelating with the surrounding phenol hydroxyl groups and amine groups on the polymer.

Figure 2. Scanning electron microscopy (SEM) images of bare MI-dPG surface coating (a and c), Cu NP-incorporated MI-dPG surface coating (b and d) whereby the particles in red circles represent the Cu NPs, and X-ray photoelectron spectra of Cu NP-incorporated MI-dPG surface coating (e and f). After SEM and XPS measurements were carried out to investigate the morphologies and


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chemical composition of the bare MI-dPG coating and the Cu NP-incorporated MI-dPG coating, the wetting properties of these coatings were tested. The water contact angles of bare MI-dPG and Cu NP-incorporated MI-dPG coatings were shown in Figure S3. The original MI-dPG coating presented a value of 52.9°, whereas the water contact angle value of the Cu NP-incorporated MI-dPG coating demonstrated a significantly lower contact angle of 28.2°. The sharp decrease in the water contact angle can be ascribed to the introduction of hydrophilic Cu (II) ions and Cu NPs. Overall, the SEM, XPS, and water contact angle results indicated that the MI-dPG surface coating embedded with Cu NPs by the in-situ reduction method was successfully prepared. Since Cu NPs have excellent antibacterial properties, the MI-dPG surface coating embedded with in-situ-formed Cu NPs was treated with representative gram-negative bacterium, E. coli, to determine whether the Cu NPs introduced through in-situ reduction by catechol groups in the coating matrix maintained their antibacterial ability. When the samples were incubated with E. coli suspension for 24 h, it was found that the Cu NP-incorporated MI-dPG surface coating exhibited a remarkable antibacterial behavior against E. coli with more than 99.99% efficiency, while the bare MI-dPG surface coating and simple glass slides did not show effective antibacterial activities (Figure 3a). Subsequently, taking a deeper look at the feasibility of long-term antibacterial ability of the Cu NP-incorporated MI-dPG surface coating, the same sample underwent a continuous antibacterial test against E. coli for three times. From Figure 3a, we could see that the antibacterial efficacy gradually decreased from 99.99% to 99.52% (second time) and 93.50% (third time). Although the antibacterial efficacy showed a slight decrease, the coating could still be considered to be highly antibacterial for all three repeats. To gain a deeper understanding of the process and the durability of the antibacterial Cu NP-incorporated MI-dPG surface coating, inductively coupled plasma mass spectrometry (ICP-MS) was performed to monitor the amount of Cu released per day. Figure 3b shows the amount of released Cu ions from the coating in 40 days. First, there was a sharp release of ions on the first (502.9 ppb) and second day (39.1 ppb), which could be ascribed to the Cu ions loaded into the coating matrix during the preparation process by chelation with surrounding phenol hydroxyl groups and amine groups on the polymer. Then, the leaching rate of ions became stable within 38 days, approximately 3 to 5 ppb per day, which arises from the hydrolysis of Cu NPs when immersed in the aqueous solution. After the Cu ion release profile was obtained by ICP-MS for 40 days, complementary XPS and SEM measurements were subsequently employed to monitor the remaining Cu NPs in the coating matrix after the Cu NP-incorporated MI-dPG surface coating was immersed in



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PBS or MilliQ water for one month to evaluate its durable antibacterial feasibility (Figure S6 and S7). As shown in Figure S6 and S7, the peak in the highly resolved Cu 2p spectra at 933.2 eV and 934.9 eV ascribed to Cu (0) and Cu (II) could still be observed in the XPS spectra, although the coating material was immersed in aqueous solutions for one month. In addition, copper contents in the coating surfaces after immersion in PBS or MilliQ water for one month decreased compared with that in the initial coating proving data of ICP-MS profile of the release of Cu ions. These results demonstrated that the Cu NP-incorporated MI-dPG surface coating was quite stable and that the effective antibacterial agent (Cu NPs) in the coating could be preserved for 40 days. Considering the above-mentioned antibacterial results and a previously reported possible antibacterial mechanism of Cu NPs35, 36 that Cu NPs are able to generate reactive oxygen species (ROS) in solution, which can damage the cell wall of the bacteria, break the bacterial cell, and finally lead to the cell’s death, a peroxidase activity assay was further performed to assess the production of ROS with the corresponding aqueous extract (the first day, the second day, the third day, and the thirtieth day) of the Cu NP-incorporated MI-dPG coating (Figure 3c and Supporting Information).37 Fluorescence measurement showed that the significant oxidation of Amplex Red (AR) and formation of fluorescent resorufin occurred in the presence of Horseradish Peroxidase (HRP) and aqueous extract of Cu NP-incorporated MI-dPG coating in all the samples. In addition, the fluorescence intensity proportionally rose with increased volumes of Cu NP-incorporated MI-dPG leachate. Besides, the similar peroxidase activity observed for the sample taken on the thirtieth day indicated this coating was highly stable for antibacterial application and that there was no activity loss after one month. After fluorescence measurement confirmed the ROS production of this system, the Cu NP-incorporated MI-dPG leachate was further incubated on the first, second, and third days with E. coli suspension to test the antibacterial ability and to get a deeper understanding of this antibacterial coating. As shown in Figure S8, the antibacterial rates of leachates were all less than 90%, which indicated that the released Cu ions and corresponding ROS production were not efficient enough to kill bacteria in solution. However, from the antibacterial result demonstrated in Figure 3a, the Cu NP-incorporated MI-dPG surface coating exhibited excellent antibacterial ability within three subsequent test times. According to previous report, the MI-dPG coating with the remaining amine group possessed sticky properties to the negatively charged proteins and microorganisms.38, 39 Thus, when this Cu NP-incorporated MI-dPG surface coating was immersed in bacterial suspension, bacterial cells were attracted


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to the coating interface, in which the effective local antibacterial concentration at the surface site actively killed the bacteria. Once the bacteria cells were destroyed, the fragments were gradually released from the coating surface. This “attract-kill-release” route and cycles propelled the sterilization process, as shown in Scheme 1. Taking into consideration the results of the antibacterial test against E. coli, ICP-MS and XPS measurements, and ROS production study, it could be speculated that the released Cu ions, corresponding ROS production, and the “attract-kill-release” route ascribed to MI-dPG all contributed to the antibacterial ability of this newly prepared Cu NP-incorporated MI-dPG surface coating. Moreover, the third antibacterial result demonstrated that the integral of released amount of Cu ions (3 to 5 ppb), corresponding ROS production, and the sticky MIdPG surface was efficient to kill the bacteria in solution. Therefore, the consistently released amount of Cu ions over long periods and the XPS outcome for the stability of the coating material in solution for one month suggested that this functional coating material is a good candidate for a durable antibacterial material.

Figure 3. Antibacterial ability of different samples against E. coli (a), release profile of Cu ions (b), peroxidase activity tests by using different volumes of Cu NP-incorporated MI-dPG leachate and the corresponding controls in the absence of Cu NPs or HRP (c). Data are presented as mean ± SD, n = 5. Statistically significant differences at the same period are indicated by **p < 0.01 compared with Blank Control.



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To demonstrate that Cu NP-incorporated MI-dPG coating could not only efficiently kill a gram-negative bacterium but also serve as a broad-spectrum antibacterial platform, grampositive bacteria (S. aureus) and drug resistant bacteria (kanamycin-resistant E. coli) were subsequently employed to investigate its bactericidal properties. Figure 4a showed the antibacterial results against gram-positive bacteria S. aureus and the drug resistant bacteria, kanamycin-resistant E. coli. From the result, we could see that Cu NP-incorporated MI-dPG also exhibited an excellent antibacterial effect against S. aureus and kanamycin-resistant E.coli with more than 99.99% efficiency, while bare MI-dPG surface coating and simple glass slides did not show effective antibacterial activity. The antibacterial results against gramnegative bacteria, gram-positive bacteria and drug-resistant bacteria demonstrated that Cu NPincorporated MI-dPG coating was a durable and effective broad-spectrum antibacterial material. Furthermore, the in vitro cytotoxicity of this material was evaluated via the MTT assay using NIH3T3 cells. As shown in Figure 4b, the viabilities of NIH/3T3 cells treated with the extract of Cu NP-incorporated MI-dPG surface coating were around 80% after 24 h of incubation, which suggested that Cu NP-incorporated MI-dPG surface coating had low cytotoxicity toward mammalian cells and could be used as a biocompatible, durable, and effective antibacterial surface coating.

Figure 4. (a) Antibacterial ability of different samples against S. aureus, kanamycin-resistant E. coli: Cu NP-incorporated MI-dPG coating, MI-dPG coating, glass slides, and blank control (without materials). (b) In vitro cytotoxicity of Cu NP-incorporated MI-dPG coating leachate 10 Environment ACS Paragon Plus

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determined by MTT assay against NIH/3T3 cells after 24 h incubation. (c) Typical photographs of the agar plate testing results: Cu NP-incorporated MI-dPG coating, MI-dPG coating, glass slides, and blank control (without materials). Data are presented as mean ± SD, n = 5. Statistically significant differences at the same period are indicated by **p < 0.01 (S. aureus ) or ##p < 0.01 (kanamycin-resistant E. coli) compared with Blank Control. As mentioned before, implant-associated infections are caused by the bacterial adhesion and the following biofilm formation at the site of devices. Even worse, once the biofilm has formed, the conventional antibiotics treatment is not efficient enough for combating the exist biofilm. So it would be quite valuable if the medical device coating exhibits the ability to inhibit biofilm formation on the surface of the device. In order to evaluate the anti-biofilm activities of the Cu NP-incorporated MI-dPG coating, E. coli suspension was used as a model bacteria strain to assess the biofilm formation on the surface.40-42 After incubation with an E. coli suspension for 24 h, live/dead staining was performed to observe the bacteria growth on the surfaces. The images shown in Figure 5 displayed the double staining (SYTO 9 and PI, green and red, respectively) which highlights viable (green) and membrane-damaged (red) cells. Figure 5a−d represents the bacteria growth on the bare MI-dPG surface coating, which shows that the surface was covered with biofilm with an average height of 5−10 µm. This meant the bare polymer coating could not efficiently resist bacterial adhesion, which also gave an evidence for the “attract-kill-release” route for the above-mentioned antibacterial process. While Figure 5e−f demonstrates the bacterial adhesion on Cu NP-incorporated MIdPG surface coating, we could see that most of the bacteria adhere to the surface were dead and that the bacteria density was much lower than that on bare MI-dPG coating. These results indicated that the Cu NP-incorporated MI-dPG coating could effectively kill the adherent bacteria and inhibit the biofilm formation due to the embedded antibacterial agent Cu NPs. Therefore, the Cu NP-incorporated MI-dPG coating has the potential to prevent the bacterial proliferation on it and provide an effective local antiseptic concentration for mitigating the emergence of implant associated infections.

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Figure 5. Biofilm observation by confocal laser microscopy via live/dead staining (green: viable bacteria, red: dead bacteria): MI-dPG coating [(a) live bacteria, (b) dead bacteria, (c) merged image of the live and dead bacteria, (d) merged image of bacteria and coating], Cu NP-incorporated MI-dPG coating [(e) live bacteria, (f) dead bacteria, (g) merged image of the live and dead bacteria, (h) merged image of bacteria and coating]. Scale bar, 50 µm. Conclusion In conclusion, we have successfully developed a macrobiotic MI-dPG surface coating embedded with Cu NPs, in which the antibacterial metal nanoparticles were efficiently introduced into the coating matrix by in-situ chemical reduction of MI-dPG. This surface coating exhibited excellent, durable and broad-spectrum antibacterial properties against grampositive bacteria, gram-negative bacteria, and drug-resistant bacteria through “attract-killrelease” route without significant toxicity toward mammalian cells. The contact kill mechanism, ROS dissolved in bacterial suspension, and Cu ions released to the bacterial suspension showed synergistic effect in the antibacterial behavior. Moreover, anti-biofilm studies revealed that the Cu NP-incorporated MI-dPG coating could also effectively inhibit biofilm formation on the surface. Thus, this work may have significant implication in designing implantable delivery devices in the future.

ASSOCIATED CONTENT Supporting Information The supporting information is available free of charge on the ACS Publication website at DOI: 10.1021/acsami.6bxxxxx. The materials and methods description; synthesis of Cu NP-incorporated MI-dPG coating; micromorphologies of Cu NP-incorporated MI-dPG, SEM images, TEM images, 12 Environment ACS Paragon Plus

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XPS images; water contact angle measurement; in vitro Cu ions release experiments; antibacterial analysis; cell cytotoxicity; ROS production of Cu NPs (PDF)

AUTHOR INFORMATION Corresponding authors *E-mail: [email protected] *E-mail: [email protected] *E-mail: [email protected]

NOTES The authors declare no competing financial interest.

ACKNOWLEDGMENTS Mingjun Li and Lingyan Gao gratefully acknowledge financial support from China Scholarship Council and DRS-POINT-2016 Fellowship, respectively. The authors thank the core facility BioSupraMol, Felix Gerke (Freie Universität Berlin, Germany) for his support with the SEM measurements, Dr. Sören Meyer (University of Potsdam, Germany) for his support with ICP-MS measurements, Falko Neumann (Freie Universität Berlin, Germany) for his support with confocal test, and Dr. Pamela Winchester (Freie Universität Berlin, Germany) for language polishing this manuscript.

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