Graphene Oxide-Mediated Protection from Photodamage - The

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Graphene Oxide-Mediated Protection from Photodamage Paulina Bolibok, Katarzyna Roszek, and Marek Wi#niewski J. Phys. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acs.jpclett.8b01349 • Publication Date (Web): 26 May 2018 Downloaded from http://pubs.acs.org on May 26, 2018

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The Journal of Physical Chemistry Letters

Graphene Oxide-mediated Protection from Photodamage Paulina Bolibok1, Katarzyna Roszek2, Marek Wiśniewski1,* 1. Faculty of Chemistry, Physicochemistry of Carbon Materials Research Group, Nicolaus Copernicus University in Toruń, Gagarina 7, 87-100 Toruń, Poland 2. Department of Biochemistry, Faculty of Biology and Environmental Protection, Nicolaus Copernicus University in Toruń, Lwowska 1, 87 – 100 Toruń, Poland

Corresponding Author *( e-mail:

[email protected] (Marek Wiśniewski)

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ABSTRACT: The article presents the unique properties of graphene oxide (GO) as a multitask material protecting from UVB-induced photodamage. Three mechanisms of GO action on fibroblast in vitro culture: physical – a barrier blocking UV radiation; chemical - antioxidative activity; biological - activation of cellular antioxidative defense were verified here. The changes in GO physicochemical properties appearing due to the UVB exposure underpin the observed UV-protection phenomena. The results reveal the simultaneous occurrence of two opposed processes i.e. under small doses of UVB, the tested material undergoes oxidation and sp2 network rebuilding. In the vicinity of GO surface, the locally triggered high temperature, is responsible for reduction process while strong oxidative agents as OH radicals cause parallel GO oxidation. This phenomena is enabled thanks to the exceptional properties of carbonaceous materials. In consequence, GO turns out as the multitask UV-protector increasing fibroblasts survival.

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The UV-mediated GO transformation was the aim of several works1-5. It was stated that the deoxygenation process of the graphene oxide sheets occurs without the requirement of photocatalysts, reducing agents or stabilizers. The comprehensive background could be found in works of Sun et al.6,7 However, during these studies the intense UV light, over 1 W∙cm-2, has been used without paying much attention to time-dependent changes. To the best of our knowledge there are no studies concerning the influence of low intensity UV on GO properties. It is widely accepted that the exposure to ultraviolet (UV) radiation is a major risk factor for most skin cancers8. Although human skin cells are equipped with a wide range of various protective and repairing systems, the current lifestyle and associated excessive exposure to UV radiation, have made it necessary to use external UV protection. UV radiation is both a mutagen and a non-specific damaging agent. As a result of direct absorption of UVB photons by aromatic groups of nucleotides in DNA, photoproducts are created8-10. Unrepaired lesions contribute to mutations in the cells of the epidermis, which in turn can lead to the cancer cells development8,11,12. UVB induces also reactive oxygen species generation that oxidize cell proteins and lipids. The literature on photoprotection is quite ample, however there is still no substance, compound or material that could be thought a perfect anti-UV protectant, i.e. very active, stable and non-toxic for human cells. Therefore, it is necessary to search for new photoprotective applications for both old and newly synthesized materials. Set of the results presented recently by Zhao et al.13 confirmed the possibility of using graphene oxide (GO) to protect proteins against photo-damage. However, their results are limited to one protein – lysozyme isolated from cells and tested “in the tube”. The described results turned out to be very promising, because the authors demonstrated that GO minimizes the UV-induced reactive oxygen species (ROS)


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generation process and thus reduces the amount of damaged proteins. Following this study, we decided to analyze the GO capability to serve as an universal, multi-functional photoprotectant in in vitro model cell culture. The thorough analysis of the literature indicates that the presented results shed the light to changes in the physicochemical properties of GO exposed to small doses UVB radiation. Moreover, our studies are the first in vitro tests towards the possible use of GO as a sunscreen. Our preliminary research hypothesis assumed that the concerted photoprotective action of GO should be realized through three mechanisms: (i) physical – a barrier blocking the UV radiation and therefore protecting the cultured cells during UV-exposure; (ii) chemical - anti-oxidative activity and ROS scavenging, when added after the UV-exposure; (iii) biological - activation of cellular anti-oxidative defense mechanisms, when added before the UV-exposure. The results presented in this communication are fully consistent with those by Zhao et al.13 and constitute their considerable complementation. The first requirement to be met during study on the UV-protective properties of the material requires the examination of its behavior after the UV exposure. Moreover, photoprotectant should be non-toxic. GO fulfills this condition in a very wide concentration range, i.e. its EC50 is much over 500 µg/mL (Fig. S1A), see the supporting materials for detailed description of experiments performed. It is worth noting, that even 1h exposure to UVB does not change its toxicity (Fig. S1B). The radiation does not change its structural properties what was proved by TEM and SEM analysis (Fig. S2). Interestingly, the results from Raman measurements are ambiguous. From the ID/IG ratio (ca. 1.14) analysis one can conclude that there were almost no changes in the sample as the ratio remains at the same level (Fig. S3). However, the quantitative analysis show that both G and D


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bands increase equally in intensity and this could be misleading and misinterpreted as the UVBexposure causes both oxidation and C=C network formation. The similar conclusions may be put forward from FTIR results. The spectral changes, presented as differential spectra registered during small doses (0.1 W∙cm-2) UVB radiation, revealed clearly that GO undergoes oxidation (Fig. 1). The increase in the exposure time causes the rise in the concentration of ⋅OH-radicals, present on the surface of GO. Due to presence of such strong oxidizing agent, the characteristic bands of C=O, and other surface oxygencontaining functionalities increase in the intensity. The appearance of the band, with a maximum near 1600 cm-1, usually attributed to C=C stretching vibration, together with rising of the background level, especially in high wavenumber region, means that the carbon surface undergoes reduction process. The observed changes coincide perfectly with the Raman results, and mean that the tested material surface rebuilds the sp2 network and parallelly has been oxidized. The mechanism of such phenomena should be further studied and need to be explained in the near future.


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Fig. 1. The differential FTIR spectra of GO after UVB exposure.

The absorption spectra of GO before and after UV exposure at various times (0 – 1h) are shown in Figure 2A. The UV-vis spectrum of GO is typical and exhibits two characteristic absorption bands, one with a maximum at 228 nm, corresponding to the ߨ-ߨ∗ transitions of C=C bonds, while the other is a shoulder at c.a. 300 nm and is ascribed in the literature to the n-ߨ∗ transitions of C–O–C and C–O–O–C linkages14. During UVB exposure the former band decreased in intensity and increased the half width, while the latter disappeared already after 2 min radiation. These observations are in agreement with the ones obtained by Li et al.15 and indicate that the spectral changes are caused by the reconstruction of the system of conjugated π bonds, what is in line with IR and Raman results, also. The generation of the sp2 network upon the GO radiation can also be verified through band gap (Eg) analysis. The values were determined from Tauc’s plot (inset in Fig. 2A) of (Aℎν)n for n=2 for indirect or ½ for direct transitions, versus photon energy (hν), wherein A means the


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absorbance, using the UV-vis spectra. The band gap for as received GO was calculated to be 3.60 eV for direct and 4.58 eV for indirect transitions (Fig. 2B). The Eg values decrease monotonically for UVB treated samples down to 1.12 and 3.96 eV for direct and indirect transitions respectively. As the decreasing is very well described with exponential function, it is obvious that the generation of the sp2 network is the first order reaction. According to the literature16 the decrease in the band gap means the reduction of GO to rGO. Moreover, it was stated that the band gap of GO and rGO is proportional to the oxygen functional groups on the basal planes and at the edge of the graphene sheet17. However, taking into consideration the above results this statements should be reconsidered. As the UVB radiation causes the C=C rebuilding, what was experimentally proven above, the reduction process, should be connected to rearrangement of the surface oxygen containing functionalities which cannot be typical for carbonaceous materials.


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Fig. 2. A) UV–vis absorption spectra of GO after UVB exposure (the insets are the Tauc plots) and B) the correlation of band gap vs. time of exposure.

From the above it is evident that GO has capability to be oxidized what justifies its use as


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photoprotective agent. We suppose that GO mediated protection can be realized through different mechanisms, thus studies on human volunteers would be necessary in the future. However, bearing in mind the law-regulations such as the European Union guidelines (2010/63/EU), we performed the in vitro studies as follows. Three selected non-toxic GO concentrations (10, 50 and 100 µg mL-1) were added to the growing 3T3 fibroblasts 24h before, during or after their exposure to UVB radiation. GO added before the UV-exposure reflected

biological mechanism i.e. activation of cellular anti-oxidative protection. During exposure GO acted as physical barrier blocking the UV radiation, while GO added after the UV treatment played the role of chemical anti-oxidant and ROS scavenger.

The most evident application of GO in anti-UVB protection is its capacity to block the radiation in a physical way during exposure. GO is supposed to make a mechanical barrier blocking the UVB, and therefore protecting the cultured cells. The results confirming this hypothesis are presented in Figure 3. Regardless the time of 3T3 fibroblasts exposure to UV, the number of metabolically active cells was reduced. However, cells exposed to UV in the presence of GO added to the cell culture in concentrations 10, 50 and 100 µg mL-1, survive the irradiation similarly or even better than control cells. For 4- and 5-minute exposure, the fibroblasts survival increases in dose-dependent manner (Fig. 3A). Undoubtedly, GO creates a mechanical barrier to protect cells from irradiation. Surprisingly, the 8-minutes irradiation seems to damage the cells predominantly at highest GO concentration. As has already been shown here, GO undergo the rearrangement due to UV radiation, the formation of oxygen-containing groups may be the explanation for lower viability of fibroblasts what was proven recently18. This phenomena confirms that GO-mediated influence and protection result from concerted action of different mechanisms. Addition of GO to the culture medium after the cell irradiation is supposed to reduce the


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amount of ROS formed and ultimately, to increase the percentage of cell survival. The obtained data (Fig. 3B) show that for the applied GO concentrations, this mechanism can work effectively only in the case of short exposure times to UV radiation. It is completely in line with results of Zhao et al.13 and suggests that with the increase of exposure time, the amount of reactive oxygen species, being formed, increases as much that exceeds the ROS neutralizing capacity of the GO used.


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Fig. 3. The influence of UV-exposure time and GO concentration on 3T3 fibroblasts proliferation rate using GO as (A) physical barrier, (B) chemical protector and (C) biological activator. Note that biological mechanism i.e. activation of cellular anti-oxidative protection is reflected when GO was added before the UV-exposure. During exposure GO acts as physical barrier blocking the UV radiation, while GO added after the UV treatment plays the role of chemical anti-oxidant and ROS scavenger.

The results from GO-mediated biological activation, which induces the cellular antioxidative defense mechanisms are shown in Fig. 3C. In each of the studied cases of GOmediated protection, an increase in the number of metabolically active cells is observed. GO added to cell culture 24h before irradiation enhances the activity of anti-oxidative compounds and UV exposure seems to be less harmful for the cells. In the literature, it was never mentioned that GO can activate the defense mechanisms of the 3T3 mouse fibroblast cell line. In addition, there is no data showing that UV-blocker with a similar mechanism of action is used on the cosmetics market. Thus, the issue should focus the scientific attention in the nearest future. The most satisfactory results were obtained when combination of mixed protection manners, that means GO supplementation 24h before UV exposure, 3T3 cells irradiation with GO solution added to the well and supplemented after the irradiation to the growing media for the next 24h, were applied (Fig. 4). The results obtained confirm a noticeable increase in the cell proliferation. The viability increases from close to 140 up to over 200% (relative to control) for the 4 min irradiation. The increase in the exposure time decreases the survival rate by about 20%, but it is still much over the control.


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Fig. 4. The effect of comprehensive GO-mediated protection of 3T3 fibroblasts exposed to UVB radiation. GO was supplemented before, during and after UVB irradiation.

To the best of our knowledge, the performed in this work in vitro assays are the first studies concerning GO as photoprotective agent. Moreover, the presented results experimentally prove the thesis suggested by Zhao et al.13 The possibility of proteins protection against photodamage is expanded here to whole cells. Their proliferation rate after UVB exposure with GO supplementation increases up to 200% of control cells. GO turns out as the multitask material working as a physical, chemical and biological protectant. Further studies seem to be necessary in order to understand the precise mechanism of GO influence. However, the obtained results allow to look forward hopefully to using GO in the cosmetics industry as a potential comprehensive UV filter.

Supporting Information Available: The Materials and Methods, and Figs. S1-S3 are enclosed as Supporting Materials (PDF)


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Notes The authors declare no competing financial interests.

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Graphene Oxide-Mediated Protection from Photodamage - The

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